Article

The Fine Structural Localization of Acetylcholinesterase at the Myoneural Junction

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Abstract

A study of the cytochemical localization of acetylcholiriesterase activity, combining histochemistry with electron microscopy, showed that the final product of the reaction, which was deposited at or near enzyme sites, occurred at four places in the myoneural junction. These included: plasma membrane of the muscle covering the junctional folds, the primary and secondary synaptic clefts, parts of the plasma membrane covering the axon terminal, and vesicular structures in the terminal axoplasm. No reaction occurred in the presence of 10(-4) eserine or DFP, whereas 10(-5) DFP inhibited the reaction at all sites except in the vesicles of the terminal axon. These findings are discussed with reference to the histochemical method used and to the occurrence of esterolytic activity in the vesicles, as well as to some of the current hypotheses concerning the relationship of the site of acetylcholinesterase and synaptic transmission.

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... These reagents provide excellent cytological fixation, and also add density to some of the reactive components of the cells. However, their use in cytochemistry is very limited since they are heavy metal-containing, oxidizing reagents that completely destroy enzymatic activities except after very short periods of fixation (5)(6)(7)(8)(9). ...
... (d) Carboxylic acid esterase activity was demonstrated with a naphthyl acetate as substrate (30) and diazo blue B or garnet GBC as the reagent for light microscope studies. (e) In addition, the activity of esterases was also demonstrated with thiolacetlc acid as substrate, incubating in the cold (4°C) in the presence of lead ions (9). Separation of the various enzymatic ac-tivities able to split thiolacetate was in part obtained by use of inhibitors; for example, activity present at motor end-plates which was sensitive to eserlne or DFP (10 -~ ~) was considered acetylcholinesterase. ...
Article
The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.
... One hopes that finer localization will come from EM histochemistry which potentially has a much higher resolution than does EM radioautography. Unfortunately, however, problems of diffusion of reaction product have prevented, in several studies with EM histochemistry, the utilization of this higher resolution and the reaching of an agreement on whether the enzymes are located only on the membranes (Davis and Koelle, 1967 ;Bergman et al ., 1967) or in the clefts as well (Barrnett, 1962 ;Zacks and Bloomberg, 1961 ;Lehrer and Ornstein, 1959 ;Csillik, 1965) . Recent studies have even suggested a postjunctional cytoplasmic localization for AChE (Teravainen, 1967) . ...
... Many histochemical studies suggest that there are (e .g . Davis and Koelle, 1967 ;Barrnett, 1962) . ...
Article
The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP-3H. Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per µ3 of cleft tissue or 12,000 sites per µ2 of postjunctional membrane surface area. A considerable concentration is also present in the teloglial cap. It has previously been demonstrated (Rogers et al., 1966) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)—a compound which selectively reactivates phosphorylated acetylcholinesterase. In the present study, it was found that this ratio of 1:2 holds also on a fine-structural level. Muscle mast cells were found to have a heavy concentration of bound DFP.
... It is known that in the neuromuscular synaptic cleft, there is an acetylcholinesterase which rapidly cleaves the neurotransmitter acetylcholine [62]. It was shown that when the temperature of the rat diaphragm preparation was reduced from 37 to 17°C, the activity of acetylcholinesterase decreased by 34% [63]. ...
... Wilson (14) introduced the use of thiolacetic acid as a substrate for esterase and indicated that an esterase enzyme had split this molecule into acetic acid and hydrogen sulfide which could be precipitated in the presence of lead or copper. Barrnett adapted this technique for electron microscopy (15). Koelle and Foroglou-Kerameos (16) used aurous ions as the heavy metal and found that this procedure yields a finer reaction product (17). ...
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The esterases of rabbit lung have been investigated from two viewpoints, the cytochemical and the biochemical. To accomplish this objective, we designed and synthesized a series of ester substrates which provide both a cytochemical indicator of the location of the enzyme and a means of following the enzymatic activity in tissue homogenates and subfractions. The substrates are p-nitrophenylthiol esters which yield, upon hydrolysis, carboxylic acid and p-nitrothiophenol. The latter can react with aurous ions to give an electron-opaque deposit; in addition, the strong absorption of p-nitrothiophenol at 410 mµ permits continuous kinetic measurements. Thus, it is possible to correlate the intracellular site of action and the biochemical behavior of the esterases. The new substrates are the thiol analogues of the p-nitrophenyl esters frequently employed as esterase substrates. The rates of hydrolysis of the two series of esters are compared in vitro. During tissue fractionation, most of the esterase activity sediments with a particulate fraction. The effects of a number of common esterase inhibitors, such as diisopropyl phosphorofluoridate and eserine sulfate, are examined, and the effects of enzyme concentration and heat inactivation are shown with the use of the partially purified preparations. The cytochemical work shows that the esterase activity is most prominent in the lamellar bodies of the giant alveolar (type II, septal, or granular pneumatocyte) cells of the lung and to a lesser extent in squamous (type I, or membranous pneumatocyte) epithelial and endothelial cells. In both the cytochemical and biochemical studies, the enzymes are inhibited by diisopropyl phosphorofluoridate and phenyl methylsulfonyl fluoride but are insensitive to eserine sulfate.
... Wilson (14) introduced the use of thiolacetic acid as a substrate for esterase and indicated that an esterase enzyme had split this molecule into acetic acid and hydrogen sulfide which could be precipitated in the presence of lead or copper. Barrnett adapted this technique for electron microscopy (15). Koelle and Foroglou-Kerameos (16) used aurous ions as the heavy metal and found that this procedure yields a finer reaction product (17). ...
Article
The esterases of rabbit lung have been investigated from two viewpoints, the cytochemical and the biochemical. To accomplish this objective, we designed and synthesized a series of ester substrates which provide both a cytochemical indicator of the location of the enzyme and a means of following the enzymatic activity in tissue homogenates and subfractions. The substrates are p-nitrophenylthiol esters which yield, upon hydrolysis, carboxylic acid and p-nitrothiophenol. The latter can react with aurous ions to give an electron-opaque deposit; in addition, the strong absorption of p-nitrothiophenol at 410 mµ permits continuous kinetic measurements. Thus, it is possible to correlate the intracellular site of action and the biochemical behavior of the esterases. The new substrates are the thiol analogues of the p-nitrophenyl esters frequently employed as esterase substrates. The rates of hydrolysis of the two series of esters are compared in vitro. During tissue fractionation, most of the esterase activity sediments with a particulate fraction. The effects of a number of common esterase inhibitors, such as diisopropyl phosphorofluoridate and eserine sulfate, are examined, and the effects of enzyme concentration and heat inactivation are shown with the use of the partially purified preparations. The cytochemical work shows that the esterase activity is most prominent in the lamellar bodies of the giant alveolar (type II, septal, or granular pneumatocyte) cells of the lung and to a lesser extent in squamous (type I, or membranous pneumatocyte) epithelial and endothelial cells. In both the cytochemical and biochemical studies, the enzymes are inhibited by diisopropyl phosphorofluoridate and phenyl methylsulfonyl fluoride but are insensitive to eserine sulfate.
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The localization of thiolacetic acid esterase activity in plasmodia of the slime mold, Physarum confertum Macbr., permitted the visualization of a system of esterolytic invaginations of the plasma membrane which appeared closely related to the arrangement of fibrillar structures. Fibrils running perpendicularly to the long axis of tubes and channels of the conducting system were found to be attached to relatively large lateral infoldings, while rows of smaller invaginations seemed to follow the course taken by the fibrils. In living and fixed specimens, constrictions in the diameter of tubes and channels were observed at the level of fibrils and linearly arranged rows of infoldings. These findings suggest that esterase-positive infoldings may result from the contraction of fibrillar structures which are attached to the plasma membrane at multiple points along their length.
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The localization of acetylcholinesterase (AChE, EC 3.1.1.7.) in the motor end-plate region has been investigated for almost three decades with light and electron microscopy and with several biochemical techniques. The extensive literature has been critically reviewed by Silver [44] and by Friedenberg and Seligman [17] with special emphasis on the cytochemical methods used. Although data in the literature vary with the different methods used, there is general agreement on the localization of the enzyme in the synaptic cleft of the neuromuscular junction. However, activity related to other structures, e.g., the subneural endoplasmic reticulum, the L-tubules of the sarcoplasmic reticulum, the sarcolemmal T-system, the teloglial Schwann cells, the axolemma and the axonal terminal, have been mentioned occasionally but questioned or even denied by other investigators [2, 3, 7, 12, 27, 49, 50, 51].
Chapter
The development of a single cell (the fertilized egg) into the many specialized cells comprising the tissues of a multicellular animal is one of the most intriguing subjects in biology. The progress of cells toward increased specialization in structure and function is known as differentiation. Differentiation is the basis of development and, as its consequence, various parts of an organism become restricted and acquire the ability to perform special functions.9 For example, heart muscle cells are highly specialized to beat rhythmically and continuously throughout life. Melanocytes in the skin are differentiated to produce melanin pigment granules in their cytoplasm. These granules in turn function as a barrier to filter out harmful and excessive rays of sunlight. These differentiated cells still carry out the normal metabolic processes necessary to maintain homeostasis, but, in addition, they are specialized to perform certain functions that other cells are incapable of performing. All these specialized cell types existing in a single animal developed from the same fertilized egg.
Chapter
Der Impuls zur Kontraktion wird durch Nervenfortsätze des motorischen Neurons an die Fasern des quergestreiften Muskels herangebracht, und die Kontaktzone dieser beiden Zelltypen ist mit speziellen Einrichtungen ausgestattet, welche die Überleitung vollziehen. In der vorliegenden Aufnahme ist die quergestreifte Muskelfaser durch einige Myofibrillen (*) dargestellt. Wo das terminale Axon an den Muskel herantritt, liegt es in einer Vertiefung, die man Trog oder Rinne (Ri) genannt hat und die in die Oberfläche der Faser einschneidet. Tiefe Einfaltungen des Sarkolemms, die man als Faltenapparat (FA) bezeichnet, erstrecken sich vom Boden der Rinnen in das darunterliegende Zytoplasma oder in das Grundplasma der Faser. In dieser Region der Faser können auch Mitochondrien (M) und der Kern (N) der Muskelzelle gefunden werden. Der subneurale Apparat, das heißt die Rinnen und der Faltenapparat sind von einem mäßig dichten, amorphen Material angefüllt, das der Bedeckung in anderen Zonen der Muskeloberfläche ähnlich ist und in diese übergeht; im wesentlichen handelt es sich um eine Basalmembran. Andererseits liegen die Nervenendigungen (NE) frei in dem Trog; keinerlei zelluläre Hüllschicht ist zwischen sie und die Muskelfaser geschaltet. Vielmehr sind die beiden Zellarten nur durch eine Lage des oben beschriebenen amorphen Materials voneinander getrennt. Die Schwannsche Zellschicht, welche das Axon bis in die Nähe der motorischen Endplatte umhüllt, läßt die Endigung des Axons frei und bleibt nur als lidförmige Bedeckung der Endplatte bestehen. Teile von Schwannschen Zellen (SZ) sind in der Abbildung sichtbar. Sie werden von einer Basalmembran (BM) und Bindegewebsfasern (BG) des Endomysiums umgeben. Diese strukturellen Beziehungen werden schematisch in Textabbildung 27 a dargestellt.
Chapter
Die OsO4- und Kaliumpermanganat-Fixation bewirken außer einer Fixation auch gleichzeitig eine Kontrastierung durch Ablagerung von Schwermetallen. Dies demonstrieren vor allem die Fixationsmethoden ohne Metalleinbau — Gefriertrocknung oder Aldehydfixation. Aber auch bei der Aldehydfixation wird Aldehyd bei der Vernetzung miteingebaut und die Dichte der Substanz verändert. Bei einer OsO4-Nachbehandlung von aldehydfixiertem Gewebe wirkt diese vorwiegend als Kontrastierungsmittel, obwohl dabei auch noch einige Fixationsprozesse ablaufen. Eine klare Trennung zwischen Fixation und Kontrastierung läßt sich daher nicht ziehen. Jede Kontrastierung beruht auf chemischen Anlagerungsprozessen, die zu einer weiteren Denaturierung führen.
Chapter
Die Narkose, mit anderen Worten die reversible Herabsetzung von Lebensfunktionen, ist ein Problem der allgemeinen Biologie. Im Prinzip ist alles Lebendige narkotisierbar; die Existenz eines differenzierten Zentralnervensystems ist keine Voraussetzung.
Chapter
During recent years cell membranes have moved to the center of biological research: (1) They are the site of some of the most vital cellular functions, such as, e.g., energy supply, photosynthesis, vision, nerve impulse conduction and others. (2) It has been realized that there is a strong impact of structure and organization on chemical reactions. Whereas classical biochemistry generally analyzed chemical reactions in solution, it has become apparent that structural factors such as microenvironment, cooperativity, allosteric effects, allotopy, regulatory effects, protein-protein, protein-lipid interactions and many others may modify and drastically change the reactions observed in solution. A vast amount of information about the chemical events in membranes has accumulated. Perhaps the most important result has been a conceptual change. Since the turn of the century lipids were considered as the essential component acting as barriers and preventing the exchange of compounds between the inside of the cell and its outer environment. Today it is well established that proteins are the essential components responsible for the function of membranes. Many cell membranes are formed by two-thirds of proteins, and only one-third of phospholipids. From some membranes 30 different proteins (or more) have been extracted and identified.
Chapter
The discovery, in the latter part of the 18th century, that the powerful shock of certain fish is an electric discharge immediately raised the question as to the mechanism by which living cells produce electricity. The importance of this problem for biology in general became apparent when it was firmly established during the 19th century that nerve impulses are propagated by electric currents. Thus, the understanding of one of the most vital functions of the organism became linked to the knowledge of the mechanism of bioelectricity. At the turn of this century, two notions were widely accepted. First, in a fluid system, such as the living cell, ions must be the carriers of electric currents. Since it was known that Na+ ions are highly concentrated in the outer environment of cells whereas K+ ion concentrations are high in the interior, Overton (1902) proposed, on the basis of simple experiments, that during activity Na+ ions move into the cell interior and an equivalent number of K+ ions flow to the outside. Overton’s assumption was borne out when the availability of radioactive ions after World War II made it possible to measure the ion movements during rest and during activity. The second notion was concerned with the control of these ion movements; it was postulated that the cell membranes surrounding nerve and muscle fibers must be able to change their permeability to ions during activity.
Article
A discussion of the factors involved in the physiological synthesis of acetylcholine in nerve endings, and of the action thereon of hemicholinium No. 3.
Chapter
With the development of the copper thiocholine (CuThCh) histochemical method for acetylcholinesterase (AChE) and pseudocholinesterase (ChE), it became possible to determine the localization of these enzymes in fresh frozen sections with high degrees of specificity and sensitivity and to a reasonable degree of accuracy within the limits of resolution afforded by light microscopy. In order to investigate the cytological localization of AChE in the superior cervical, ciliary, and other autonomic ganglia of the cat, beyond what could be seen by direct light-microscopic observation of stained sections, additional procedures were employed. Prior to staining, tissues were treated in vitro and in vivo, with various anticholinesterase (anti-ChE) agents, including reversible and irreversible, and lipidsoluble and -insoluble compounds, administered alone and in combination with both normal and preganglionically denervated cats. Electron microscopic studies have been performed with modifications of the CuThCh method and the related thiocholine- copper-ferricyanide method. However, this approach has inherent limitations to the accuracy of localization obtainable, such as the quaternary, and hence poorly penetrating nature of the substrate (acetylthiocholine) and the relatively large dimensions and low electron-density of the primary reaction products.
Article
The digestive cycle following reabsorption of hemoglobin by cells of the proximal convoluted tubules in mouse kidney and the uptake of ferritin by glomerular mesangial cells in the kidney of normal and nephrotic rats were investigated by electron microscopical histochemical procedures. Mouse kidneys, sampled at closely spaced time points between 1 to 48 hours after intraperitoneal injection of hemoglobin, and rat (normal and nephrotic) kidneys, sampled at 30 minutes, 2 hours, and 48 hours after intravenous injection of ferritin, were fixed in glutaraldehyde, cut at 50 µ on a freezing microtome, incubated for acid phosphatase and thiolacetate-esterase, and postfixed in OsO4. Satisfactory preservation of fine structure permitted the localization of the enzymatic reaction products on cell structures involved in uptake and digestion of exogenous proteins. The latter were identified either by their density (hemoglobin) or their molecular structure (ferritin). It was found that lysosomal enzymic activities and incorporated exogenous proteins occur together in the same membrane-bounded structures. In the cells of the proximal convolution, lytic activities become demonstrable within 1 hour after hemoglobin injection, appear first in apical vacuoles filled with hemoglobin, and persist in fully formed protein absorption droplets. At the end of the lytic cycle (∼48 hours post injection), the cells have an increased population of polymorphic bodies which exhibit lytic activities. In smaller numbers, identical bodies occur in controls. It is concluded that they represent remnants of previous digestive events. The means by which the resorptive vacuoles acquire hydrolytic activities remain unknown. Fusion of newly formed vacuoles with residual bodies was not seen, and hemoglobin incorporation into such bodies was only occasionally encountered. Acid phosphatase activity was found sometimes in the Golgi complex, but enzyme transport from the complex to the resorbing vacuoles could not be established. Autolytic vacuoles containing mitochondria or mitochondrial remnants were frequently found during the early stages of hemoglobin resorption, but no definite conclusions about the mechanism involved in the segregation of endogenous material were obtained. In nephrotic rats ferritin was segregated in membrane-bounded bodies mainly in the mesangial cells and to a lesser extent in epithelial and endothelial cells. Most of these sites were marked by the reaction products of acid phosphatase and organophosphorus-resistant esterase and therefore identified as lysosomes connected with the digestion of incorporated exogenous proteins.
Article
Tritiated diisopropylfluorophosphate (DFP) was used to phosphorylate acetylcholinesterase (AChase) in the motor end plate of mouse sternomastoid muscle, and its distribution within the end plate was evaluated quantitatively by electron microscope radioautography. With the use of emulsion layers whose sensitivity to tritium had been calibrated, the density of AChase in different components of the end plate was calculated. The AChase was primarily localized (85%) in the junctional fold region. The concentration of AChase there was more than 20,000 active sites per cubic micron of tissue. The resolution of the technique was not sufficient to determine whether there was some AChase in the nerve end bulb; however, if there is any there, the concentration must be less than 10% of that at the junctional fold region.
Article
Histochemical studies, using the light and electron microscopes, have been made of carboxylic esterase activity in the tegumental cells and surface tegument of the frog lung fluke Haplometra cylindracea. Regional differences in reactivity for esterases were found. The tegumental cells and tegumental lining of the two suckers and tegumental cells connected to the tegument surrounding the oral sucker stained intensely for esterases, but the tegumental cells and associated tegument in the rest of the body stained only weakly or not at all. The use of specific substrates or eserine sulfate showed that most of the activity was due to acetylcholinesterase. With the electron microscope, enzyme reaction product was found at the nuclear membranes and endoplasmic reticulum of esterase-positive tegumental cells and, in large concentrations, on the membranes of numerous platelike inclusion bodies present in the cells and connecting tegument. No structural differences relating to enzyme activity were found between reactive and nonreactive tegumental cells and tegument. The acidophilic gland cells present in the oral region of the worm were at all times negative for esterase activity.
Article
Mit einer 5×10−3molaren stabilen Emulsion von α-Naphthylacetat läßt sich die unspezifische Esterase in jeder Nervenzelle des Meerschweinchengehirns nachweisen. Sie ist mit Ausnahme weniger Kerngebiete vorwiegend im Perikaryon lokalisiert, die Zellfortsätze sind wesentlich geringer aktiv. Das differente Verhalten gegen verschiedene Inhibitoren und die völlig unterschiedlichen Verteilungsmuster beweisen, daß sich mit den angewandten histochemischen Methoden die unspezifische Esterase und die Acetylcholinesterase ohne wesentliche Überlagerung nebeneinander nachweisen lassen. Die Verteilungsunterschiede werden beschrieben. Der größte Teil der Esterase wird durch E 600 gehemmt und gehört deshalb zum Typ B. Nach vergleichender Untersuchung einer Reihe histochemisch nachweisbarer Enzyme wird ein Modell der Enzymverteilung im Zentralnervensystem zur Diskussion gestellt. In vielen dendritenreichen telencephalen Kerngebieten gibt es deutliche Aktivitätsunterschiede zwischen dem Perikaryon und den Zellfortsätzen. Während der größte Teil der am Energiestoffwechsel beteiligten Enzyme in der Masse der Zellfortsätze lokalisiert ist, sind die beiden Hydrolasen unspezifische Esterase und saure Phosphatase im Perikaryon konzentriert.
Article
The fine structure of normal and denervated motor end-plates of gastrocnemii from albino mice was examined in this study. Changes following nerve section are described and include a decrease in the number of synaptic vesicles and axonal terminal retraction from subsynaptic sarcolemma. Counts on the number of synaptic vesicles and the degree of axonal terminal retraction in terms of percentage of total sub-neurilemmal cross-sectional area were made with the aid of a compensating polar planimeter.The events (decrease in the number of synaptic vesicles and axonal terminal retraction) observed here are compared with previously reported loss of neuro-muscular transmission in cat gastrocnemius and decrease in acetylcholine concentration in the distal portion of sectioned popliteal nerves (12). It is suggested from this study that causes for neuromuscular transmission failure following denervation are multiple and due as much to retraction of axolemma from the subsynaptic sarcolemma as to any other single factor.
A preliminary electron microscope study of human neuromuscular junction is presented. The biopsy material was taken from the palmarus longus, and fixed routinely in osmium tetroxide and embedded in methacrylate. The structure of the motor endings and the relationship of the synaptic vesicles to the axolemmal membrane are described. The synaptic clefts are filled with an homogeneous material in continuity with the basement membrane covering the muscle fiber. The subneural apparatus is described, and special attention is paid to a vesicular component present in the sarcoplasm of the junctional area, which differs from synaptic vesicles and is presumed to be a derivate of the sarcoplasmic reticulum.
Experiments which combined histochemistry and electron microscopy were performed in studying the sites of enzymatic hydrolysis of thiolacetic acid in the presence of lead ions in diaphragmatic and cardiac muscle. It was found that in these striated muscles the electron opaque, final product of the histochemical reaction (PbS) was discretely deposited on the swelling of the thick elemental filaments that occurs at the M band. Additional sites of enzymatic activity occurred in mitrochondria and in round sarcoplasmic bodies. A reaction, probably non-enzymatic, also occurred in contraction bands in the area of the Z bands and in the sarcoplasmic reticulum. To ascertain the enzymatic nature of the reaction and to define the enzyme involved, control experiments were carried out and the effect of various esterase inhibitors was assayed. It is suggested that the M band enzyme is a cholinesterase, but the enzymes in the mitochondria and the sarcoplasmic bodies that hydrolyze the substrate appear to be different. A possible role of the M band enzyme is discussed.
Article
This chapter presents the results of the studies of the synaptic junction carried out during the past several years using the electron microscope. The electron-microscope study of synaptic regions reveals a highly differentiated and specific submicroscopic organization suitable to carry out the transmission of the nerve impulse. Inspite of differences in morphology, distribution, and geometry, synaptic regions have several basic similarities. First, a definite discontinuity between the cytoplasm of the two apposed cellular elements of the synapse, confirming that the individuality of the neuron applies to the finest submicroscopic expansions. Second, a direct contact of the presynaptic and subsynaptic surface membranes separated only by an interspace of 100 to 500 A, indicating that at the junction no other cellular material alien to the two synaptic elements is interposed. Third, the presence of a special microvesicular material—the synaptic vesicles—on the presynaptic side of the synapse. These structural similarities suggest that an essentially analogous physiological mechanism may be involved in all synaptic junctions.
Article
The localization of acetylcholinesterase activity in the fine structure of mouse and human neuromuscular junctions was studied by the method of Barrnett and Palade. A formolsucrose solution was used for primary fixation followed by osmification. It was found that deposits of lead sulphide, the product of the histochemical reaction, were found in axonal and sarcoplasmic mitochondria and most prominently in the primary and secondary synaptic clefts of the subneural apparatus. Rarely were reactive sites found near the M-band. Control experiments utilizing inhibitors and high concentrations of Pb(NO 3 ) 2 were performed. The possible significance of acetylcholinesterase activity in the areas described is discussed.
Electron micrographs are presented of synaptic regions encountered in sections of frog sympathetic ganglia and earthworm nerve cord neuropile. Pre- and postsynaptic neuronal elements each appear to have a membrane 70 to 100 A thick, separated from each other over the synaptic area by an intermembranal space 100 to 150 A across. A granular or vesicular component, here designated the synaptic vesicles, is encountered on the presynaptic side of the synapse and consists of numerous oval or spherical bodies 200 to 500 A in diameter, with dense circumferences and lighter centers. Synaptic vesicles are encountered in close relationship to the synaptic membrane. In the earthworm neuropile elongated vesicles are found extending through perforations or gaps in the presynaptic membrane, with portions of vesicles appearing in the intermembranal space. Mitochondria are encountered in the vicinity of the synapse, and in the frog, a submicroscopic filamentary component can be seen in the presynaptic member extending up to the region where the vesicles are found, but terminating short of the synapse itself.
The presence of cholinesterase at the myoneural junction of intercostal muscle has been demonstrated in both light and electron microscopic preparations. A new simultaneous diazo coupling technique using alpha-naphthyl acetate as substrate and "hexazonium pararosanilin" as coupler has been applied to cold formalin-fixed tissues. After postfixation in buffered osmium tetroxide the sites of esterase activity are faithfully demonstrated at a high level of resolution. The details of cholin-esterase distribution and some technical aspects of the procedure are discussed.
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