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.     

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.     

Characterization of "fetal-type" acetylcholinesterase in hemin-treated K562 cell culture

The alteration of acetylcholinesterase (ACHE) activity, a marker enzyme of erythroid differentiation, was studied during the hemin-induced erythroid differentiation of K562 human leukemia cells in suspension culture. The kinetics of postinduction differentiation was followed by determining the hemoglobin (Hb) content and the ACHE activity of cells. Embryonic hemoglobins as well as small quantities of fetal Hb (HbF) were synthetized by stimulated cells. The peaks of ACHE activity preceded the highest level of Hb content and, following induction, reached their pinnacles at 72 and 120 hours, respectively. These data indicate that ACHE activity is an earlier and more sensitive marker for hemin-induced erythroid differentiation of K562 cells than is elevated Hb content. Electrophoretic mobility of ACHE from hemin-treated cells proved to be the fetal type, but after incubation with neuraminidase, the rate of migration decreased to the level of the adult type enzyme.     

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     

A possible sensory-motor neuron in Aplysia californica

A granule-containing cell is described in the secretory epithelia of the accessory genital mass in Aplysia californica. The ciliated apical process of this cell protrudes into the lumen of the oviduct. The granule-containing basal region of the cell is drawn out into fine processes that resemble axons. Granule-filled dilatations of these axons are found directly under large secretory cells. On this basis, it is suggested that these cells fulfill the morphological criteria for sensory-motor cells, and this data will be used as a basis for microelectrode studies to confirm or deny the above suggestion.     

The characteristics of cholinesterase distribution in the development of the human and mammalian brains]

Histochemical studies have been made on the distribution of acetyl- and butyrylcholinesterases (ACHE and BCHE) in various parts of the human and rat brain. Statistical analysis showed that at the 8th week, the highest ACHE activity in the human foetus is observed in the intermediate and plexiform layers of the cerebral cortex. The highest BCHE activity was found in the ependymal layer of various cerebral regions. High BCHE and ACHE activities were noted in the dorsal thalamus and epithalamus. In 10-week human foetuses, total high level of ACHE and BCHE was revealed in various nuclei of the thalamus and subcortical structures of the forebrain (Meynert nucleus, nucleus caudatum). In rats, the highest ACHE activity at the 14th day of prenatal life was found only in subcortical structures of the forebrain. Accumulation of BCHE activity in some of the thalamic nuclei of rats begins at the 10-17th day of postnatal life.     

Immunochemical identification of vasopressin in neurons and in granule-containing cells of the human cerebral blood vessels

By means of indirect immunofluorescent method vasopressin localization has been defined in neurons of the locus coeruleus, in terminals, converging to the substantia nigra cells and the sylvian aqueduct area, as well as in the granule-containing cells of the human cerebral blood vessels. The granule-containing cells are identified as melanocytes, when investigated by comparative light-optic and electron microscopical technique. The melanocytes of the cerebral vessels have various size and form in accordance to their different functional state. In some melanocytes peptide vesicles are revealed. This demonstrates a possible synthesis of vasopressin. It is supposed that local endocrine regulation of the cerebral hemocirculation is performed by cooperation of granule-containing cells, producing hormones with oppositely++ directed constrictor and dilatator action to the vessels.     

Development of prespore vacuoles inDictyostelium cells differentiating in a liquid shake culture

Summary When shaken in a glucose-albumin-cyclic AMP medium, dissociated aggregative cells form small clumps in which prespore cells differentiate fairly synchronously (Okamoto 1981). Formation of prespore vacuoles (PSVs) in differentiating prespore cells was examined in these culture conditions, by electronmicroscopy and immunocytochemistry.After 6 hours of culture, a typical Golgi apparatus composed of vesicles and stacked flat cisternae develops near the nucleus. FITC-conjugated antispore serum stains a crescent-shaped region in the cells which seems to correspond to the Golgi area. After 9 hours, flat sacs which contain electron dense lining membrane similar to that of PSVs appear alongside Golgi cisternae. Later, partially and fully round PSVs are observed in this region, suggesting that flat sacs round up to become mature PSVs. After 12 hours, as mature PSVs increase in number, they become dispersed throughout the cytoplasm and a typical Golgi apparatus with cisternae disappears. When cultured in a medium devoid of cyclic AMP, cells develop neither Golgi cisternae nor PSVs. These results strongly suggest that PSVs form from Golgi cisternae.     

Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL

Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.     

Fine structure of the sensory epithelium of guinea-pig organ of Corti: Subsurface cisternae and lamellar bodies in the outer hair cells

The fine structure of subsurface cisternae and lamellar bodies in the outer hair cells of the guinea-pig organ of Corti was studied with thin sections and freeze-fracture replicas. Subsurface cisternae in the outer hair cells consist of multilayers along the lateral plasma membrane of the cell. The outermost layer is a flattened cistern in the upper part of the supranuclear region, but comprises a series of tubules in the lower part. Deeper layers are fenestrated cisternae in which disc-like areas are found in the upper part of the supranuclear region. Lamellar bodies consist of concentric layers of fenestrated cisternae and are located in the apical cytoplasm beneath the cuticular plate. They are continuous with the subsurface cisternae. In the supranuclear cytoplasm, bulges of the subsurface cisternae and the lamellar bodies are found. Dilated cisternae are also present. Some dilated cisternae contain many small vesicles, which display acid phosphatase activity. The dilated cisternae are considered as forms of the bulges undergoing transformation into multivesicular bodies. The possible role of the lamellar bodies, and the origin and fate of the subsurface cisternae are discussed.     

The Golgi apparatus in the acinar cells of the developing embryonic pancreas: I. Morphology and enzyme cytochemistry

The Golgi apparatus of pancreatic acinar cells of rat embryos was studied during development from day 13 through day 20 of gestation. The morphological and enzyme cytochemical patterns varied characteristically in the course of cell differentiation. A pronounced system of "rigid lamellae" characterized the area near the trans face of the Golgi stacks in the protodifferentiated and early phases of the differentiated states; by contrast, "rigid lamellae" were sparse in the terminal period of gestation. Reaction product of acid phosphatase labeled the "rigid lamellae" in the protodifferentiated state, was extended across the majority of the stacked cisternae in the early differentiated state, but was restricted to the trans side again in the later periods of cell differentiation. The early phase of the differentiated state was characterized by the tight association of the endoplasmic reticulum and Golgi cisternae on the trans side; the close spatial relationship of the two compartments was lessened after production of secretion granules had started. The findings are in line with those of recent studies on the Golgi organization in some other types of cells in different functional states, and they present the embryonic pancreatic tissue as another model for demonstrating the high flexibility of the Golgi complex. In agreement with the patterns previously found in the absorptive cells of the small intestine, the present results show that the close associations of the endoplasmic reticulum and cisternae of the trans Golgi side predominate in the early stages of cell differentiation.     

Histochemical localization of acetylcholinesterase in the cerebral ganglia of Fasciola hepatica, a parasitic flatworm

Acetylcholinesterase activity was found in the cell bodies and extracellularly in the neuropile of the cerebral ganglia of the adult trematode parasite, Fasciola hepatica. Within neuronal cell bodies of the cerebral ganglion, acetylcholinesterase reaction product was found in the endoplasmic reticulum, in the cisternae of the Golgi apparatus, and in secretory vesicles near the inner (releasing face) cisternae. Acetylcholinesterase reaction product was not seen intracellularly within any nerve processes. The reaction product was found around the somatic cell membranes and in the extracellular space between closely apposed nerve processes in the neuropile. Acetylcholinesterase reaction product was associated with synaptic endings that contained clear spheroidal synaptic vesicles, and the reaction product was localized at the site of synaptic contact between the zone of apposition of the pre- and postsynaptic terminals. This intracellular and extracellular distribution of the enzyme is consistent with its function as the degrading enzyme in cholinergic transmission.     

OBSERVATIONS ON THE RELATIONSHIP OF THE GOLGI APPARATUS TO WALL FORMATION IN THE MARINE CHRYSOPHYCEAN ALGA, PLEUROCHRYSIS SCHERFFELII PRINGSHEIM

The role of the Golgi apparatus in wall formation of vegetative cells of a marine chrysophyte, Pleurochrysis scherffelii, is described. Wall fragments are synthesized within the cisternae of the Golgi apparatus. A single Golgi apparatus is always located at the cell periphery, and the distended cisternae are oriented toward the cell surface. A highly-ordered body found near the inflated cisternae is associated with spherical, membrane-bounded bodies which may be involved in the progressive degeneration of cisternal membranes which release wall fragments. Protoplast movement has been detected by time-lapse cinephotomicrography and is correlated at the ultrastructural level with change in positions of the Golgi cisternae. Wall-synthesizing capacity is greatest during transverse wall formation. Senescent cells lack a Golgi apparatus with inflated cisternae. In addition, wall fragments are not present in the Golgi cisternae at this stage. Zoosporogenesis results in a temporary loss of the wall-forming capacity of the Golgi apparatus; this activity then resumes with the formation of a different morphological entity, the scale. Preliminary quantitative measurements of the turnover capacity of the Golgi apparatus have been made. From these data it has been determined that between 41 and 82 Golgi generations are required to synthesize the cell wall of an actively growing cell; this estimate indicates that approximately one cisterna is produced every 2 min, provided the cell generation time is 3 days. The time-lapse cinephotomicrographic data confirm that the rate of production of Golgi cisternae is at least one cisterna every 2 min.     

Evidence for a transcellular cisternal route across the caecal epithelium of an insect

Summary The cells of the mesenteric caeca in the midgut of certain insects possess a labyrinth of transepithelial cisternae. Their existence can be seen in thin sections of lanthanum-incubated tissue, where the tracer enters not only the intercellular clefts but also membranous cisternae which are inpocketings from, and, in continuity with, both the lateral clefts and basal membrane. These infoldings, which are numerous, run from the basal or lateral surfaces into the perinuclear region of the cells, where they are found, laden with lanthanum, as smooth cisternae or vesicles in the peripheral cytoplasm near the plasma membrane. These can be followed in serial sections and are quite distinct from other sub-surface cisternae of the lateral borders which are studded with ribosomes on the cytoplasmic surface. Near the luminal surface, tracer-laden structures in the form of vesicles and granules become increasingly predominant over those in the form of cisternae. Freeze-fracture replicas confirm the above observations, in that the plasma membrane of the intercellular cleft can be characterized as such unequivocally, since it exhibits smooth septate junctional E face grooves and P face ridges. Lateral infoldings, cisternae and vesicles can be seen arising directly from these junction-bearing membranes. The transepithelial cisternae and vesicles may be the morphological basis of an insect transcellular transport system, comparable to the tubulocisternal endoplasmic reticulum present in the transporting secretory and absorptive epithelia of vertebrate tissues. However, in insect midgut caecal epithelia, the cisternae appear to be, albeit presumably transiently, in direct continuity with the extracellular space, forming a plasma membrane reticular system which seems not to be the case with the tubulo-cisternal endoplasmic reticulum which terminates in subsurface cisternae.     

Retracted: Upregulation of acetylcholinesterase caused by downregulation of microRNA-132 is responsible for the development of dementia after ischemic stroke

MicroRNA-132 (miR-132) has been shown to participate in many diseases. This study aimed to understand the correlation between the level of miR-132 and the severity of dementia post-ischemic stroke. An online tool ( www.mirdb.org ) was used to find the miR-132 binding site in acetylcholinesterase (ACHE) 3′-untranslated region (UTR), followed by a luciferase reporter assay to validate ACHE as a miR-132 target. A similar relationship between miR-132 and ACHE was also established in cerebrospinal fluid samples collected from human subjects. A negative correlation was established between ACHE and miR-132 by measuring the relative luciferase activity. Meanwhile, Western blot analysis and real-time polymerase chain reaction were also conducted to compare the levels of ACHE messenger RNA and protein between two groups (dementia positive, n = 26 and dementia negative, n = 26) or among cells treated with miR-132 mimics, ACHE small interfering RNA, and miR-132 inhibitors. As shown in the results, miR-132 can reduce the expression of ACHE. Further experiments were also carried out to study the effect of miR-132 and ACHE on cell viability and apoptosis, and the results demonstrated that miR-132 enhanced cell viability while suppressing apoptosis. In addition, ACHE reduced cell viability while promoting apoptosis. miR-132 targeted ACHE and suppressed its expression. Additionally, miR-132 and ACHE have been shown to affect the cell viability and apoptosis in the central nervous system.     

Granule-containing cells in the human superior cervical ganglion.

The fine structure of granule-containing cells in the human superior cervical ganglion is described. These cells are larger than the typical SIF cells in mammals and exhibit green-yellow fluorescence. They are characterized by numerous granular vesicles (80-140 nm in diameter) in the cytoplasm, but have many features in common with ordinary ganglion cells. They emit several long processes which form bundles together with ordinary nerve fibers. No synapses are found where the cells are presynaptic, although a few synapses are observed there where nerves are prosynaptic on the perikarya and processes of the cells. No close topographical relations are seen between the cells and blood vessels. It is suggested that the granule-containing cells are a special type of postganglionic aminergic neurons.     

Multiplication of the dictyosome during the formation of autospores in the green alga Chlorococcum infusionum

Numerical changes in dictyosomes during the formation of autospores in a green alga, Chlorococcum infusionum, were investigated by electron microscopy. Two dictyosomes were seen near the nucleus in young vegetative cells. Four dictyosomes were seen in large mononucleate cells which appeared to enter mitosis soon. Binucleate cells contained 4 or 8 dictyosomes, the latter number being found in the large binucleate cells. Large tetranucleate cells contained 16-25 dictyosomes in each cell. Dictyosomes consisted of about 4 cisternae with a diameter of 0.4-0.6 micron in mono- to tetranucleate cells. Cytokinesis began with the formation of the septa during the third nuclear division, and 16 cells were finally formed. Dictyosomes did not increase in number in 8- and 16-nucleate cells. In septum-forming cells, dictyosomes were 0.6-1.0 microns in diameter, with 6-9 cisternae. A single dictyosome was included in each of the 16 resultant cells. These observations suggest that the dictyosomes multiply in association with the multiplication of the nuclei without correlation with formation of the cell wall or cytokinesis.     

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.     

Structural organization of the normal and anoxic heart of Scyllium stellare

Summary The general and ultrastructural organization of the heart of the elasmobranch, Scyllium stellare, was studied in normal and in anoxic animals. The rich coronary supply was revealed three-dimensionally by the use of corrosion casts, showing a thebesian system of coronary arterioles and capillaries in the thin, outer compact layer as well as in the predominant, inner spongy layer of trabeculae.Only the sinus venosus received a neuronal input of large bundles of granule-containing axons terminating at fenestrated regions of the endocardium and suggesting a neurohormonal function.A simple, tubular sarcoplasmic reticulum with flattened junctional cisternae was present in myocardial cells of 1–5 m diameter, which contained one or two bundles of myofibrils. The latter were closely apposed to the inner aspect of the plasmalemma. Mitochondria were located centrally in the cells, which were joined by unfolded desmosomes involving Z-band material.Long periods of anoxia were tolerated without loss of heart function, but at the expense of cytoplasmic glycogen. Lipid granules were abundant in all layers and chambers, notably in animals prepared in the summer. The lipid granules displayed a marked increase in electron density when the heart was incubated in a buffered oxalate solution prior to fixation. A glycogen-sparing effect of the lipids during anoxia was observed.     

A light and electron microscopic study on the embryonic development of the rat carotid body.

The embryonic development of the rat carotid body was studied with electron microscopy. In the 11 mm embryo a cell aggregation consisting of undifferentiated cells and unmyelinated nerve fibers appears on the anterior wall of the third branchial artery. Granule-containing cells appear in the 12 mm embryo and continue to increase in number as the cellular aggregation increases in size and becomes separated from the wall of the third branchial artery. Synapse formation and the appearance of fenestrated capillaries occur almost simultaneously at the 17 mm stage. There are two types of synapses, one with membrane densification and vesicles clustered inside the nerve endings, the other with dense material and vesicles inside the granule-containing cells. At the 20 mm stage the undifferentiated cells send enveloping cytoplasmic processes toward adjacent granule-containing cells and the carotid body anlage displays rudimentary lobules.