A CHOLINERGIC COMPONENT IN THE INNERVATION OF THE LONGITUDINAL SMOOTH MUSCLE OF THE GUINEA PIG VAS DEFERENS : The Fine Structural Localization of Acetylcholinesterase

Acetylcholinesterase (AChE) has been detected on the plasma membrane of about 25% of the axons in the longitudinal smooth muscle tissue of guinea pig vas deferens. These axons are presumably cholinergic. No enzyme was detected in the remaining 75% of axons. These axons are presumably adrenergic. The plasma membrane of the Schwann cells associated with the cholinergic axons also stained for AChE. Some axon bundles contained only cholinergic or adrenergic axons while others contained both types of axon. When a cholinergic axon approached within 1100 A of a smooth muscle cell, there was a patch of AChE activity on the muscle membrane adjacent to the axon. It is suggested that these approaches are the points of effective transmission from cholinergic axons to smooth muscle cells. Butyrylcholinesterase activity was detected on the plasma membranes of all axons and smooth muscle cells in this tissue.     

THE ELECTRON MICROSCOPIC LOCALIZATION OF CHOLINESTERASE ACTIVITY IN THE CENTRAL NERVOUS SYSTEM OF AN INSECT, PERIPLANET A AMERICANAL.

The distribution of esterase activity in the last abdominal ganglion, the connectives and the cereal nerves of the cockroach Periplaneta americana has been investigated cytochemically. Activity of an unspecific eserine-insensitive esterase (or esterases) has been found in glial elements in these regions of the nerve cord. In addition, sites of cholinesterase (eserine-sensitive) activity have been found in association with (a) the glial sheaths of the axons in the cereal nerves and connectives, (b) the glial folds encapsulating the neuron perikarya in the ganglion, and (c) in localized areas along the membranes of axon branches within the neuropile, often flanked by focal clusters of synaptic vesicles. These results are discussed with particular reference to the previously reported insensitivity of the insect nerve cord to applied acetylcholine, and to the probable existence of a cholinergic synaptic mechanism in the central nervous system of this insect.     

Autonomic innervation of the arteriovenous anastomoses in the dog tongue

Summary The innervation of the arteriovenous anastomoses in the dog tongue has been investigated. At the lightmicroscopic level, the vessels were found to be densely supplied with adrenergic and AChE-positive nerve plexuses and less densely with the quinacrine-binding nerve plexus. At the electron-microscopic level, at least two apparently different types of axon profiles were identified: 1) Small vesicle-containing axons, characterized by many small granular vesicles, variable numbers of small clear vesicles and large granular vesicles. Storage of endogenous amines and uptake of exogenous amines into most small granular vesicles and many large granular vesicles was demonstrated. These axons stained only lightly with reaction products for AChE activity and thus seemed to be adrenergic in nature. Some axons contained numerous large granular vesicles, whose cores occasionally stained with uranyl ions; this suggests a co-localization of ATP or peptides as neurotransmitters. 2) Small granular vesicle-free axons, containing small clear vesicles and large granular vesicles in variable ratio. Most cores of these large granular vesicles were heavily stained with uranyl ions. No storage or uptake of amine into the synaptic vesicles was detected. Some axons appeared to be typically cholinergic, some, typically non-adrenergic, noncholinergic, and the rest, intermediate between the two. All axons stained heavily with reaction products for AChE activity, suggesting their cholinergic nature.     

Evidence for a cerebral cholinergic system and suggested pharmacological patterns of neural organization in the prostomium of the polychaete Nereis virens (SARS)

The histological visualization of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) on frozen sections of prostomia of Nereis virens indicate a concentration of cholinergic activity in the anterior brain. Components are probably sensory epithelial cells with cholinergic axons entering the brain in cephalic nerves and efferent cholinergic axons to prostomial muscle leaving the brain in the same nerves. There are also subepidermal cholinergic cells that may be second order motor neurons serving epidermal mucous cells. The smaller, second lobe of the corpora pedunculata and its associated vertical fibre tract are CAT⁴ and appear continuous, on each side of the cerebral ganglion, with a dorsal and a ventral longitudinal bundle of AChE⁺ fibers. This system tapers to nothing at the level of the posterior eyes. There is a small AChE⁺ component to each optic nerve and AChE is present in the nuchal epithelium. These observations are discussed in relation to earlier studies on aminergic and neurosecretory activity in the same ganglion.     

Acetylcholinesterase in the ocellus of Apis mellifica

The ultrastructural localization of the enzyme acetylcholinesterase (AChE) in the ocellus of the honey bee (Apis mellifica) was studied by electron microscopy. High AChE activity was found both in the receptor-cell axons and in the surrounding glial cells. Second order neurones exhibited a remarkably lower anzyme activity. AChE was also detected in the intercellular spaces between the receptor-cell axons and the second order neurones. These results provide additional support to the assumed cholinergic nature of the photoreceptor cells in the insect ocellus.     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.     

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.     

THE FINE STRUCTURE OF THE TRANSITIONAL EPITHELIUM OF RAT URETER

The fine structure of the transitional epithelium of rat ureter has been studied in thin sections with the electron microscope, including some stained cytochemically to show nucleoside triphosphatase activity. The epithelium is three to four cells deep with cuboidal or columnar basal cells, intermediate cells, and superficial squamous cells. The basal cells are attached by half desmosomes, or attachment plates, on their basal membranes to a basement membrane which separates the epithelium from the lamina propria. Fine extracellular fibres, ca. 100 A in diameter, are to be found in the connective tissue layer immediately below the basement membrane of this epithelium. The plasma membranes of the basal and intermediate cells and the lateral and basal membranes of the squamous cells are deeply interdigitated, and nucleoside triphosphatase activity is associated with them. All the cells have a dense feltwork of tonofilaments which ramify throughout the cytoplasm. The existence of junctional complexes, comprising a zonula occludens, zonula adhaerens, and macula adhaerens or desmosome, between the lateral borders of the squamous cells is reported. It is suggested that this complex is the major obstacle to the free flow of water from the extracellular spaces into the hypertonic urine. The free luminal surface of the squamous cells and many cytoplasmic vesicles in these cells are bounded by an unusually thick plasma membrane. The three leaflets of this unit membrane are asymmetric, with the outer one about twice as thick as the innermost one. The vesicles and the plasma membrane maintain angular conformations which suggest the membrane to be unusually rigid. No nucleoside triphosphatase activity is associated with this membrane. Arguments are presented to support a suggestion that this thick plasma membrane is the morphological site of a passive permeability barrier to water flow across the cells, and that keratin may be included in the membrane structure. The possible origin of the thick plasma membrane in the Golgi complex is discussed. Bodies with heterogeneous contents, including characteristic hexagonally packed stacks of thick membranes, are described. It is suggested that these are "disposal units" for old or surplus thick membrane. A cell type is described, which forms only 0.1 to 0.5 per cent of the total cell population and contains bundles of tubular fibres or crystallites. Their origin and function are not known.     

On the issue of innervation of dorsal longitudinal musculature of <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> with inferior cervical nerve

Using the method of the anterograde dextran tetramethylrhodamin transport, there is obtained the topographic picture of branching of inferior cervical nerve axons on fibers of the dorsal longitudinal muscle in Lymnaea stagnalis (L.). Using the retrograde staining, the neuronal bodies sending their processes into this nerve are marked. Manifestations of asymmetry in distribution of neurons stained through the right and left nerves are described. The electron microscopic studies have shown that the main number of the inferior cervical nerve axons is represented by thin fibers presumably belonging to the sensory cells. A part of the nerve fibers and their endings show imunoreactivity to serotonin and acetylcholine. The serotoninergic fibers predominate quantitatively over the cholinergic ones and account for a half of the fibers stained with dextran. A possible functional role of the serotoninergic and cholinergic innervation of the dorsal longitudinal muscle in Lymnaea stagnalis is discussed.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 40, No. 6, 2004, pp. 569–578.     

The development of innervation in the rat atrioventricular node

Summary The problem of development of the innervation of the rat atrioventricular node has been investigated by electron microscopy. Nerve bundles appear in relation to the node as early as the second postnatal day and vesiculated axons are seen throughout the entire node by the fourth day. Intimate contacts between nodal cells, axons and terminal varicosities are frequently observed.Use of the 5-hydroxydopamine tracer technique has enabled the identification of both cholinergic and adrenergic axons. It is concluded that the node has a dual innervation although cholinergic endings far outnumber those classified as adrenergic on the sixth postnatal day.These results are quite different to earlier findings made at the light microscope level and the discrepancies are discussed with respect to the histochemical techniques used. The suggestion that nodal differentiation is induced by nerves is considered in relation to the differences in cholinesterase activity exhibited by nodal cells during normal development and following neonatal sympathectomy.     

Ultrastructure of the basiepithelial nerve plexus of the sea urchin,Centrostephanus longispinus

Summary In submandibular glands of rabbits both adrenergic and cholinergic axons are intimately associated with parenchymal cells of the intercalary ducts and the granular tubules, lying beneath the basement membrane and often in the space between the parenchymal cell and an associated myoepithelial cell. The submandibular acini receive a less intimate and less plentiful innervation by adrenergic and cholinergic axons which remain outside the basement membrane and are still associated with Schwann cells. Occasional axons of both adrenergic and cholinergic type occur beneath the basement membrane of submandibular striated ducts in intimate association with basal parts of the cells.In the parotid glands numerous adrenergic and cholinergic axons are found beneath the basement membrane of acini and intercalary ducts in intimate association with the cells.This work has been helped by the technical assistance of Mr. P.S.A. Rowley     

Membrane properties of cultured rat sympathetic neurons: Morphological studies of adrenergic and cholinergic differentiation

Dissociated neurons from the newborn rat superior cervical ganglion were grown under conditions which lead to either adrenergic or cholinergic differentiation. Lectins and toxins were used to detect differences in the cell membrane associated with transmitter status, age of the neurons, or location on the neurons. These ligands were made visible in the light or electron microscope by coupling to rhodamine or colloidal gold. The density of binding sites for concanavalin A (Con A), ricin (RCA₆₀), and wheat germ agglutinin (WGA) increased with age in culture on both adrenergic and cholinergic cells. Soybean agglutinin (SBA) binding increased about threefold on adrenergic axons, but failed to increase on neurons induced to become cholinergic by medium conditioned by rat heart cells (CM). The effect of CM on SBA binding paralleled previously described effects of CM on transmitter production; the CM binding pattern developed slowly and was not readily reversible. Mature adrenergic neurons also appeared to bind more WGA than neurons in CM cultures. Tetanus toxin gold binding was uniform, but low, on axons of adrenergic and cholinergic neurons at all ages. In contrast, cholera toxin binding decreased with age on adrenergic axons. Binding sites for SBA and tetanus toxin were found to be less numerous on the cell body surface than on the axonal surface. Thus growth in CM induces fundamental changes in the phenotype of developing sympathetic neurons involving the cell membrane as well as transmitter choice. Differences also appear with maturation and between axonal and somatic cell surface membranes.     

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick,Dermacentor variabilis (Say)

Summary Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.     

ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.     

Calelectrin, a Calcium-Dependent Membrane-Binding Protein Associated with Secretory Granules in Torpedo Cholinergic Electromotor Nerve Endings and Rat Adrenal Medulla

Calelectrin, a calcium-dependent membrane-binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine beta-hydroxylase, although the two antisera did not cross-react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vesicles and also self aggregates in a calcium-dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30-80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.     

Ultracytochemical demonstration of cholinesterase activity in the atrium of the guinea-pig heart

Summary The ultrastructural distribution of cholinesterase (ChE) activity was examined in the atrium of the guinea-pig heart using Karnovsky's method.The reaction products showing the ChE activity were found mainly in the neural elements in the atrium. There were axons showing no reaction products intermingled with axons showing the positive enzymatic activity. In the enzymatically positive axons, the reaction products were observed in the axon-Schwann interspace, the interspace between neighboring axons and the corresponding plasma membranes in nerve fiber bundles as well as preterminal axons. It was of interest to find that not only axons containing ordinary agranular vesicles, but also axons with larger granular vesicles showed the ChE activity.In terminals the enzymatic activity was found also in the axon-Schwann interspace, the interspace between the axolemma and the sarcolemma and the corresponding plasma membranes of the enzymatically positive areas. In the neuron the enzymatic activity was positive in the endoplasmic reticulum and the interspace between the neuronal plasma membrane and the corresponding plasma membrane of the satellite cell. Plasma membranes facing to these enzymatically positive spaces were also positive for the enzymatic activity.This investigation has been supported by a research fund from Takeda Chemical Industries, Ltd., Osaka, Japan.A part of this work has been presented at the 8th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Kyoto on Nov. 30–Dec. 1, 1967, and also at the 24th Annual Scientific Meeting of the Japanese Society of Electron Microscopy held in Kofu on May 11–12, 1968.     

Fine structure and innervation of the avian adrenal gland

Summary Apart from cholinergic nerve fibers, which make up the main part of efferent fibers to the avian adrenal gland (Unsicker, 1973b), adrenergic, purinergic and afferent nerve fibers occur. Adrenergic nerve fibers are much more rare than cholinergic fibers. With the Falck-Hillarp fluorescence method they can be demonstrated in the capsule of the gland, in the pericapsular tissue and near blood vessels. By their green fluorescent varicosities they may be distinguished characteristically from undulating yellow fluorescent ramifications of small nerve cells which are found in the ganglia of the adrenal gland and below the capsule. The varicosities of adrenergic axons exhibit small (450 to 700 Å in diameter) and large (900 to 1300 Å in diameter) granular vesicles with a dense core which is usually situated excentrically. After the application of 6-hydroxydopamine degenerative changes appear in the varicosities. Adrenergic axons are not confined to blood vessels but can be found as well in close proximity of chromaffin cells. Probably adrenergic fibers are the axons of large ganglion cells which are situated mainly within the ganglia of the adrenal gland and in the periphery of the organ and whose dendritic endings show small granular vesicles after treatment with 6-OHDA.A third type of nerve fiber is characterized by varicosities containing dense-cored vesicles with a thin light halo, the mean diameter (1250 Å) of which exceeds that of the morphologically similar granular vesicles in cholinergic synapses. Those fibers resemble neurosecretory and purinergic axons and are therefore called p-type fibers. They cannot be stained with chromalum-hematoxyline-phloxine. Axon dilations showing aggregates of mitochondria, myelin bodies and dense-cored vesicles of different shape and diameter are considered to be afferent nerve endings. Blood vessels in the capsule of the gland are innervated by both cholinergic and adrenergic fibers.Supported by a grant from the Deutsche Forschungsgemeinschaft (Un 34/1).     

Close apposition of photoreceptor cell axons in the house fly

Transmission electron microscopy of the first optic ganglion of the house fly Musca domestica reveals that retinular (R) axons branch and interdigitate throughout the external plexiform layer. Axonal membranes of neighboring cells are in close apposition to each other, usually without junctional modifications. In some cases R axonal membranes exhibit greater electron density in the areas of contiguity. Adjacent axons may show a near confluency of axoplasm at points where membrane boundaries are interrupted or obscured. Physiological implications of these ultrastructural findings are discussed.     

AChE synthesis in cholinergic neurons: electron histochemistry of enzyme translocation

Summary Endoplasmic reticulum of cholinergic nerve cells exhibits acetylcholinesterase activity. In central neurons that exert an ephemeric acetylcholinesterase activity only during some ontogenetical states, enzyme reaction product is present also in the Golgi system. Neurotubular and neurofilamentar structures exert an acetylcholinesterase in terminal axons of young animals. From these electron histochemical studies it is concluded that enzyme protein molecules, synthesized in the endoplasmic reticulum, are translocated to the active sites (surface membranes) via axonal filaments (or tubules), or will be extruded from the neuron via the Golgi system.Our thanks are due to Dr. P. Röhlich, head of the Budapest Electron Microscope Laboratory, for his generous help in various aspects of the electron microscopic work.     

THE FINE LOCALIZATION OF NUCLEOSIDE TRIPHOSPHATASE ACTIVITY IN THE RETINA OF THE FROG

Nucleoside triphosphatase (NTPase) activity was demonstrated at the submicroscopic level in the frog retina by the Wachstein-Meisel method utilizing various purine and pyrimidine nucleosides. Under the electron microscope magnesium-activated NTPase was localized in the outer and inner segments, and in the plexiform layers. NTPase active sites in the cones were localized diffusely in the 70 to 80 A interspaces between the double membranes of the stacked lamellae and in the investing cytoplasm. In the rods, on the other hand, sites of activity were observed at the periphery of the stacked lamellae as discrete electron opaque deposits measuring 1000 to 1500 A which interdigitated between the lamellae for short distances. Deposits of reaction product appeared more numerous in rods of dark-adapted frogs stimulated with monochromatic light with a wave length of 510 mµ. Enzyme activity was also observed in mitochondria of the rod and cone ellipsoids. In the outer and inner plexiform layers NTPase active sites were present on and between the membranes of axons and the plasma membranes of some of the neurons.