The ultrastructural ionic fixation technique localizing acetylcholinesterase activity, reveals simultaneously acetylcholine-like cation in the synaptic vesicles of the motor nerve terminals

A new cytochemical technique is proposed for side by side localization of acetylcholine and of acetylcholinesterase activity of motor end-plate at ultrastructural level. The technique is based on the simultaneous "ionic fixation" of vesicular acetylcholine and of histochemical copper thiocholine precipitate with phosphomolybdic acid: the molybdic heteropolyanion forms insoluble salts with these two quaternary ammonium cations, providing in situ "acetylcholine phosphomolybdate" and "copper thiocholine phosphomolybdate". Both of them are osmium resistant; the electron dense precipitates allow for a fine localization of acetylcholine and acetylcholinesterase activity at electron microscopic level.     

Koelle's copper thiocholine method performed with a low-pH phosphate buffer,followed by osmification of the precipitate: revival of two abandaned procedures

Summary Using a glutaraldehyde-fixed mouse neuromuscular junction, a fine precipitate of copper thiocholine was obtained with Koelle's medium prepared by a mixture of phosphate buffer (pH 5.6–5.9) and copper glycine solution (strongly acidic). The final pHs of these incubation media were very low, being situated between 3.8 and 4.2, respectively. It is well known that phosphate buffer, at such a low pH value, has no buffering effect on the acetic acid of enzymatic hydrolysis. This probably caused a sharp drop of the pH value in the vicinity of the enzymatic site and allowed a fine localization of copper thiocholine, the precipitation of which is pH dependent. Furthermore, the osmification of copper thiocholine in the same phosphate buffer provided a finely localized electron dense product. The chemical nature of the osmified copper thiocholine is discussed.     

Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds

Summary Several compounds of osmium^VIII, including potassium osmiamate and coordination complexes of OsO₄ with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO₄ in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation.The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO₄, has a negligible vapor pressure of OsO₄. It has the molecular formula C₆H₁₂N₄.2OsO₄ and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO₄) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO₄, and the odor of OsO₄ becomes apparent.Such solutions of osmeth are (0.25%) considerably less concentrated with respect to OsO₄ than solutions (1–2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO₄ appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO₄ solutions of equivalent concentrations. Our findings indicate that OsO₄ solutions of less than 1% may be satisfactorily utilized in many cytochemical studies.Osmeth is safer and more convenient to handle than OsO₄ because small amounts may be solubilized as needed. It should be considered as a substitute for OsO₄ in ultrastructural cytochemistry.This investigation was supported by NIH research grant number DE 02668 from the National Institute of Dental Research and by NIH grant number RR 05333 from the Division of Research Facilities and ResourcesVisiting Professor, Dental Research Center, University of North Carolina at Chapel Hill, Jan.-May, 1975. Supported in part by USPHS Grant HD 09209     

羊水胆碱酯酶凝胶圆盘电泳改良法及其产前诊断神经管缺损的应用

本文在羊水胆碱酯酶凝胶圆盘电泳中,改用亚铁氰化铜的棕色显带沉淀反应,使显带比原法清晰,温培时间由12小时以上缩短为3小时。测定了210例正常的和19例神经管缺损畸胎的妊娠羊水,其阴性和阳性期望率分别达97.1％和100％。     

Oxidation of ruthenium red for use as an intercellular tracer

Summary When ruthenium red (RR) is combined with OsO₄, an electronopaque complex forms which readily binds to the cell surface coat. However, the RR-OsO₄ complex is often excluded from intercellular spaces in many cell types, and thus is not dependable as a tracer of regions continuous with the extracellular space. Postfixation of erythrocytes agglutinated by the lectin helix (Helix promatia) and intact carotid artery endothelium with a freshly prepared mixture of 1% OsO₄ containing 0.1% ruthenium red (RR) resulted in a dense surface deposit of these cells, but intercellular regions were penetrated to a minimal degree by the stain. When a similar mixture of RR-OsO₄ was allowed to stand 3 h before use, RR is oxidized by OsO₄ to yield a ruthenium compound that has a spectrophotometric absorbance maximum at 365 nm. This RR molecule has a reduced number of cationic sites due to binding with osmium dioxide OsO ₂ ⁼ . Postfixation of agglutinated RBCs and carotid artery endothelium with this oxidized ruthenium-OsO₄ mixture resulted in a 50–80% decrease in surface deposition but markedly enhanced penetration into intercellular regions. The enhanced penetration is attributed to decreased binding affinity of the oxidized ruthenium for anionic surface membrane components, permitting effective stain penetration into regions of cell-to-cell contact rather than extensive surface deposition. These studies indicate that the ruthenium compound formed by OsO₄ oxidation of ruthenium red may be a useful tracer for ultrastructural visualization of intercellular spaces and junctions.     

Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.     

Electron microscopic demonstration of cholinesterases in nervous tissue

Summary Acetylcholinesterase was demonstrated at ultrastructural level in the motor nerve cells of rat's spinal cord using the Karnovsky-Roots modification of Koelle's thiocholine method. Selective inhibitors were employed to check the validity of the reaction.Prolonged formaldehyde fixation improved the poor penetration of the reactive agents and diminished the relatively large crystal size of the end product, which were the two main difficulties of the method. The preservation of ultrastructure was highly improved, when thin sections were made without freezing using a tissue chopper.Acetylcholinesterase was localized in the nuclear envelope, in the rough-surfaced endoplasmic reticulum, in medium-sized vesicles of the Golgi apparatus, and around synaptic terminals. Synaptic vesicles were found negative.     

The ultrastructural ionic fixation technique localizing acetylcholinesterase activity,reveals simultaneously acetylcholine-like cation in the synaptic vesicles of the motor nerve terminals

Summary A new cytochemical technique is proposed for side by side localization of acetylcholine and of acetylcholinesterase activity of motor end-plate at ultrastructural level. The technique is based on the simultaneous ionic fixation of vesicular acetylcholine and of histochemical copper thiocholine precipitate with phosphomolybdic acid: the molybdic heteropolyanion forms insoluble salts with these two quaternary ammonium cations, providing in situ acetylcholine phosphomolybdate and copper thiocholine phosphomolybdate. Both of them are osmium resistant; the electron dense precipitates allow for a fine localization of acetylcholine and acetylcholinesterase activity at electron microscopic level.     

Quantitative evaluation of the trapping reaction of copperthiocholine histochemical procedures for localization of cholinesterases

Summary The aim of the present study was to investigate whether the trapping reaction of the histochemical procedure for the localization of ChE of Koelle and Friedenwald (1949) and its modification by Brzin and Pucihar (1976) proceeds quantitatively. The weight of the precipitate formed in the tissue sample during the histochemical procedure was compared with enzyme activity of an equal sample. The differential magnetic microbalance was used for measurements of reduced weight and for previous determination of density of the precipitate. The evidence for the composition of the final product was drawn from the quantitative analysis of copper and iodine and from the infrared spectra.Tsuji's statement (1974) that cuprous copper thiocholine iodide is the final product of histochemical procedures investigated was confirmed. It was found that the trapping reaction of the original as well as of the modified procedure under our experimental conditions in tissue sections proceeds quantiatively which means that one of the basic conditions for reliable localization is fulfilled.This work was supported by the grant of Research Association of Slovenia and NIH Grant No 02-008-1, Z-ZF-6     

Eine Methode zur Lokalisation14C-markierter Assimilate im Blatt vonVicia faba L. mit Hilfe der elektronenmikroskopischen Autoradiographie

Zusammenfassung Für die elektronenmikroskopische Autoradiographie wasserlöslicher¹⁴C-markierter Assimilate wird eine neue Technik beschrieben. Die notwendigen Präparationsschritte werden systematisch auf Erhaltung der Feinstruktur und Verlust an markierten Assimilaten untersucht. Als erfolgreicher Kompromiß stellten sich in den wichtigsten Phasen die Vorfixierung mit 40%igem OsO₄ in CCl₄, eine Gefriersubstitution mit getrocknetem Diäthyläther bei –78 °C, die Nachfixierung mit Acrolein und das Auffangen der Ultradünnschnitte auf 1,3-Propandiol heraus. Besonderer Wert wurde auf einen mit wenigen technischen Hilfsmitteln sicher reproduzierbaren Präparationsgang gelegt.

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Autoradiographical method for the localization of¹⁴C-labelled assimilates in leaf cells ofVicia faba L. using electron microscopy

Summary To evaluate the compartmentation of synthesis and transfer of assimilates within the leaf tissue a technique for ultrastructural histoautoradiography of¹⁴C-labelled assimilates was developed. All essential steps were examined in detail as far as preservation of fine structure and minimal loss of¹⁴C-substances are concerned. The following phases proved to be the most important and successful compromises: prefixation with 40% OsO₄ dissolved in CCl₄ at –20 °C, freeze substitution with dry diethylether at –78 °C, postfixation in acrolein as 10% solution in diethylether, and collecting of ultrathin sections on 1,3-propandiol. The procedure is compared with data given so far to the literature and results in reproducible data even if a normal equipment for ultrastructural work is provided only.

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Teil einer Dissertationsschrift, angefertigt unter Leitung von Prof. Dr. J.Willenbrink und vorgelegt der Math.-nat. Fakultät der Universität Bonn.     

Fine structural localization of acetylcholinesterase in electroplaque of the electric eel

The electroplaques composing the electric organ of the eel, Electrophorus electricus, have been utilized for the dual purpose of demonstrating the subcellular sites of acetylcholinesterase activity and as a model for comparison of the several cytochemical methods available. Fresh tissue and tissue fixed by immersion in formalin, hydroxyadipaldehyde, or glutaraldehyde was reacted with the Cu-thiocholine method, the Cu-ferrocyanide thiocholine method, or the thiolacetic acid (TAA) method using Pb, Ag, or Au as capture reagents. Controls were obtained by omission of substrate, or by addition to complete media of varying concentrations of different cholinesterase inhibitors. Reactions were run at 0–5°C at a pH range of 5.0–7.1 for 0.25 to 120 min. Regardless of the capture metal, the localization obtained with TAA as substrate was identical with that observed with acetylthiocholine, the majority of precipitate being deposited on or near the external innervated surface of the plaque and within the tubulovesicular organelles opening onto the innervated surface. Both of the thiocholine methods and the Pb-TAA method showed reaction product in synaptic vesicles of the nerve endings innervating the plaque which was uninhibitable by 10^-4 M physostigmine. All methods also showed some inhibitor-sensitive deposition of reaction product in the mucoid material forming the immediate extracellular environment of the innervated surface.     

Iodide,thiocyanate and cyanide ions as capturing reagents in one-step copper-thiocholine method for cytochemical localization of cholinesterase activity

Summary The necessity of the presence of iodide in Cu-ThCh reaction was investigated by following the precipitate formation in vitro and by evaluating the ultrastructural localization of the precipitate in sympathetic ganglion cells of the frog and in the end-plate regions of the rat diaphragm.It was found that thiocyanate or cyanide is the only anion that can be substituted for iodide as the capturing agent in precipitation. The optimal concentration in the preincubation and incubation media of any one of the three anions is from 2 to 5 mM. At a concentration below 1 mM precipitation in vitro is considerably delayed as a result of which in electron microscopy diffusion artefacts appear in tissue sections.The unconverted primary precipitate obtained in the presence of iodide had been used for ultrastructural localization of ChE activity and now this use has been extended to precipitates obtained in the presence of CN^– or CNS^–. Better-quality localization in the presence of either one of the latter anions suggests that they, and particularly CN^–, should be substituted for I^– in the one-step Cu-ThCh method for the cytochemistry of cholinesterases.This work was supported by the grant of Research association of Slovenia and by the NIH grant PL 480 N° 02-008-N     

The determination of inorganic orthophosphate in biological systems

A simple and rapid method is described for determining P_i by spectrophotometric measurement of a soluble complex of phosphomolybdic acid and Cirrasol ALN-WF, a non-ionic detergent formerly known as Lubrol W. The measured complex has a molar extinction coefficient of 4.59 · 10³ at 390 nm and little interference is found with relatively high concentrations of chelating agents, salts, and other compounds which interfere with most other P_i assays. Linearity is observed in the range 0–1.2 μmoles P_i and developed assay samples are stable for 8 h at 20 °C or 24 h at 4 °C. The method is suitable for use in the presence of moderate concentrations of protein or ATP.After suitable modification the assay can be used at pH 4.0. Sensitivity is reduced at this pH (ε_{M, 390nm} = 2.79 · 10³) but linearity is maintained up to 1 μmole P_i and the coloured complex is stable for 4 h at 20 °C. The pH-4 procedure is suitable for measurement of P_i in the presence of very labile phosphate esters such as creatine phosphate.The phosphomolybdic acid-Cirrasol complex can be reduced at ambient temperature in both the above systems. A blue complex results with ε_{M, 820nm} of 9.9 · 10³ at pH 4.0, and 1.8 · 10⁴ under more acidic conditions.     

THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO₄ with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO₄. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.     

Quantitative aspects of cytochemical methods for acetylcholinesterase studied with a cytochemical model system

A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974). Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.     

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal

Summary Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO₄) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO₄ darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO₄, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.     

Uptake of iodide in the marine haptophyte Isochrysis sp. (T.ISO) driven by iodide oxidation

Uptake of iodide was studied in the marine microalga Isochrysis sp. (isol. Haines, T.ISO) during short‐term incubations with radioactive iodide (¹²⁵I^?). Typical inhibitors of the sodium/iodide symporter (NIS) did not inhibit iodide uptake, suggesting that iodide is not taken up through this transport protein, as is the case in most vertebrate animals. Oxidation of iodide was found to be an essential step for its uptake by T.ISO and it seemed likely that hypoiodous acid (HOI) was the form of iodine taken up. Uptake of iodide was inhibited by the addition of thiourea and of other reducing agents, like L‐ascorbic acid, L‐glutathione and L‐cysteine and increased after the addition of oxidized forms of the transition metals Fe and Mn. The simultaneous addition of both hydrogen peroxide (H₂O₂) and a known iodide‐oxidizing myeloperoxidase (MPO) significantly increased iodine uptake, but the addition of H₂O₂ or MPO separately, had no effect on uptake. This confirms the observation that iodide is oxidized prior to uptake, but it puts into doubt the involvement of H₂O₂ excretion and membrane‐bound or extracellular haloperoxidase activity of T.ISO. The increase of iodide uptake by T.ISO upon Fe(III) addition suggests the nonenzymatic oxidation of iodide by Fe(III) in a redox reaction and subsequent influx of HOI. This is the first report on the mechanism of iodide uptake in a marine microalga.     

Heterologous expression,purification, and biochemical characterization of a greenbug (Schizaphis graminum) acetylcholinesterase encoded by a paralogous gene (ace‐1)

Acetylcholinesterase is a critical enzyme in the regulation of cholinergic neurotransmission in insects. To produce Schizaphis graminum acetylcholinesterase‐1 for structure–function analysis, we constructed a recombinant baculovirus to infect Sf9 cells, which secreted the soluble protein at a final concentration of 4.0 mg/L. The purified enzyme had an apparent M_r of 70 and 130 kDa in the reducing and nonreducing SDS‐polyacrylamide gels, respectively, indicating that it formed a dimer via an intermolecular disulfide bond. The fresh enzyme had a specific activity of 245 U/mg, which stabilized at a lower level (115 U/mg) in storage. The Michaelis constant and maximum velocity were 88.3 ± 9.6 μM and 133.2 ± 1.6 U/mg for acetylthiocholine iodide, 113.9 ± 12.5 μM and 106.4 ± 3.0 U/mg for acetyl(β‐methyl)thiocholine iodide, 68.9 ± 7.8 μM and 76.7 ± 1.0 U/mg for propionylthiocholine iodide, and 201.1 ± 21.0 μM and 4.4 ± 0.1 U/mg for S‐butyrylthiocholine iodide, respectively. The IC₅₀ values (5 min, room temperature) of ethopropazine, BW284C51, carbaryl, eserine, malaoxon, and paraoxon were 102, 1.66, 0.94, 0.20, 0.061, 0.016 μM, respectively. The bimolecular reaction constants (k_i) were (6.50 ± 0.40) × 10⁴ for carbaryl, (1.00 ± 0.16) × 10⁵ for eserine, (4.70 ± 0.13) × 10⁵ for malaoxon, and (9.06 ± 0.23) × 10⁵ M^?1 min^?1 for paraoxon. The enzyme was also inhibited by one of its products, choline, at concentrations higher than 20 mM, suggesting that choline bound to an anionic site and regulated the enzymatic activity. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:51–59, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20311     

Relative speed of fixation of glutaraldehyde and osmic acid in plant cells measured by grana appearance in chloroplasts

Summary Brief fixation in a mixture of glutaraldehyde and OsO₄ caused stacked chloroplast grana membranes in leaf cells of wheat, barley, tobacco, maize, cowpea, pigweed or bean plants to distend and vesiculate. Fixation with glutaraldehyde followed by OsO₄ prevented this fixation artifact. In a fixative mixture, OsO₄ apparently reacted with cell contents before glutaraldehyde.     

Osmium tetroxide as a histochemical and histological reagent

Summary From the evidence discussed it can be concluded that osmium tetroxide (OsO₄) would be reduced to black OsO₂ (or an equivalent compound) by the ethylene bonds of liquid or solid cis-unsaturated lipids or by the ⁵-double bond in cholesterol in solid state in tissues. No evidence has been obtained to suggest that OsO₄ is either reduced or bound by proteins and polysaccharides in tissue-sections.