Halász, Norbert

Joó, Ferenc

Párducz, Árpád

English

Halász Norbert

Párducz Árpád

Joó Ferenc

University of Szeged

Acta Biológica Szeged 17 ( 1 — i ) , pp. 1-13—152 (1971) C O N T R I B U T I O N TO T H E PROBLEM OF T H E SPECIFICITY OF T H E ZIO IMPREGNATION" N . H A L Á S Z , Á . P Á R D U T Z a n d F . J o ó Electron Microscope Laboratory, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged (Received July 21 , 1970) Introduction As the results of the ultrastructural investigations accepted a!most_ univer-sally, in the later years evermore perfect varieties of the electron microscopic methods of fixation, embedding and impregnation have been elaborated. Never-theless, as compared with the number of visualizing procedures tried and found suitable for light microscopes, there are comparably few methods adapted to electrón microscopic examinations. Some fundamental methods ( 0 s 0 4 , fCMnO.,, aldehydes, negative ways of staining, etc.) are suitable for fixing all the com-ponents of the animal and plant tissues and cells recognized so far with a by and large unequivocal result. In the later decades electron histochemistry became a wide-spread way of investigation in order to reveal the chemical structure of the components of cells. Having recognized many components of tissues and tells fundamentally, the importance of histochemistry is recently growing for the methods often enable us to study the structures biochemically, enzymatically, pharmacologically, etc. The procedure of zinc iodide — osmium tetroxide impregnation (ZIO) investigated by us is similarly of light microscopic origin (MAILLET, 1959; 1962: J A B O N E R O , F A B R A , M O Y A a n d J A B O N E R O , 1 9 6 1 ; J A B O N E R O , 1 9 6 4 : T H I E S , 1 9 6 4 : R O D R Í G U E Z - P É R E Z , Í 9 6 4 ) , applied lately also on electron microscopic level ( A K E R T a n d S A N D R Í , 1 9 6 8 ; P E L L E G R I N O D E I R A L D I a n d G U E U D E T , 1 9 6 8 ; 1 9 6 9 ; M A R T I N , B A R L O W a n d M I R A L T O , 1 9 6 9 ; N I E B A U E R , K R A W C Z Y K , K I D D a n d W I L G R A M , 1 9 6 9 ; L A M P A R T E R , S T E I G E R , S A N D R I a n d A K E R T , 1 9 6 9 ; K A W A N A , A K E R T a n d S A N D R I , 1 9 6 9 ) . Since the beginning of the application of ZIO impregnation, the researches have been interested in the problem, what is in fact, impregnated by ZIO, resp, in wich sense this method may be considered to be specific. The most literary data report only on ZIO impregnated nerve components (nerve fibers, synaptic vesicles), but there are also results that have not supported this kind of spe-cificity of the reaction. In our present work we want to make known our results obtained in various nerve structures concerning the specificity of ZIO impreg-nation. ^ This investigation was performed while the Electron Microscope Laboratory was in the framework of the Faculty of Sciences, Attila József University, Szeged. 144 n. halAsz . A. pardutz and f. jo6 P l a t e I Light microscopic picture of a rat iris after 2 1 0 impregnation for five hours. The arrows point to the varicosities of the nerve fibers. (* 520) Materials and Methods For determining the specificity of ZiO reaction we have performed structural investigations «n the corpus pineale and iris of full-grown rats, as well as on the superior cervical ganglion (SCG) of the cat. The investigations were carried out by impregnating with Z I O solution, pre-pared according to AKEKT and SANDRJ'S (1968) method at a temperature of 0—4°C for different lengths of time (1, 2, 5 and T hours). The way of preparing the ZIO solution is as follows: the reaction of 7,5 p.c. Zn + 2,5 p.c. J j being realized in a wet medium, the solution obtained is mixed with the 2 p.c, diluted solution of OsO* in a ratio of 4 to 1. immediately before appli-cation. Here we note that in earlier investigations there were generally longer impregnations <16—20 hts) applied and this method is followed by MADARASZ and co-workcrs, too, in the C O N T R I B U T I O N T O T H E . P R O B L E M OR TH^ ZIO I M P R E G N A T I O N 1 4 5 Anatomical Institute of the University Medical School of Budapest during their similar investi-gations. The matter prepared for the electron microscopic study after the usual alcoholic dehyd-ration was embedded in Araldit (PEASE. 1964), It was contrasted in block (during dehydration) with 3 — 4 p.c. uranyl acetate dissolved in 70 p.e. ethanol, then on the section accord in i* to REYNOLDS'S (1963) method and our pictures were taken by electron microscope Tesla BS 242 D, as well as by Tcsla BS 413. W e have carried out light microscopic examinations on iris removed from rat in physiological NaCl solution and impregnated in ZIO for five hours. Results After impregnating the iris of the rat with ZIO — separating sharply from the adjacent elements of the connective tissue — we could reproduce light mic-roscopically the staining of nerve fibers (Fig. 1). The nerve fibers run in bundles, in some piaccs we could well observe the ZIO positivity of the so-called varixes in the nerve fibers, as in the case of silver impregnation. After a ZIO impreg-nation for 2 hours in the electron microscopic pictures of the corpus pineale of the rat, the electron-dense granules that demonstrated the site of Z I O positivity were found mainly in the vesicular regions of the nerves (Fig. 2). The average size of the ZIO-positive granules is 300—500 A, often — but not always — localized in the wall or interior of the synaptic vesicles. Also in case of the two-hours impregnation we have observed the mitochondria being moderately swollen, supposedly owing to the low pH of the impregnating medium. An impregnation lasting for a longer period of time considerably decreases the specificity of ZIO reaction and its elective axonal localization, changing even its granularity of usual size. In this case we often get reaction product of finer distribution of appearing in larged spots. After an impregnation of three hours, in the superior cervical ganglion of the cat the ZIO positivity has appeared, apart from the nerve fibers, on the nuclear membrane of Schwann cell, too (Fig. 3). Besides the granules, however, positivity also appears in spots, mainly eytoplasmatically. The size of ZIO-granules that can sometimes be observed in the perinuclear sites, as well, approaches that of the granules which arc connected with the clear synaptic vesicles. At the same time, activity may be observed in the axolemma and in the myelin sheath, as well, appearing in the form of minor clods and being expressed particularly- on the internal lamellae (Fig. 4) . After being impregnated for seven hours, the reaction of the endoplasmatic reticulum in the ganglion is remarkable (Fig. 5). It is well discernible in photo-graphs of higher magnification (Fig. 6) that the ZIO-granules that are situated in the cysternae of the endoplasmatic reticulum do not fill compactly the tubules but there are ZIO-reactivc elementary units of about 80 A in diameter sur-rounding non-reacting units that may sometimes have 300—400 A diameters. We want to notice that earlier Csillik and KNYIHAR (1968), studying the elect-ron microscopic localization of AChE similarly in the ganglion of the cat, des-cribed inactive endoplasmatic units. On the basis of the similar morpholo-gical pictures, it can be imagined that the same endoplasmatic units were visua-lized with the ZIO impregation, as well. In these preparations of ours also the perinuclear ZIO positivity is strong, appearing on the membrane forming both walls of the cysterna and in the gap itself, too. Also some mitochondria got stained and there may be observed in the cytoplasm also a large number of ZIO-positive granules that cannot be localized in a structural element. 1 0 A c t a B i o l o g i e s 146 N . H A L A S Z . A . P A R D O T Z A N D F. J 0 6 D i a c u s s i o n Several literary- data are available to evaluate the specificity of ZIO reac-tion. The method of impregnation used, after the original KJ—OsOj staining (CHAMPY, 1 9 1 3 ) was modified, in the form of Z n J 2 — 0 s 0 4 first on light microscopic level ( M A I L L E T , 1 9 5 9 ) , proved to be suitable mainly for visualizing the nerve fibers and nerve terminals (MAILLET, 1959, 1962 : J A B O N E R O , F A B R A , M O Y A a n d J A B O N E R O , 1 9 6 1 ; J A B O N E R O , 1 9 6 4 ) . J A B O N E R O ( 1 9 6 4 ) emphasises that he has not succecded in staining the neurons of the su-perior cervical ganglion. RODRIGUEZ—PEREZ ( 1 9 6 4 ) , however, seems to have already stained in his adrenal gland preparation not only pericellular plexuses but cytoplasm, as well. T H I E S ( 1 9 6 4 ) showed the so-called dendrite cells and the melanocytes, too, with Z I O in the human skin. After applying the Z I O impregnation electron microscopically, A K E R T and SANDRI ( 1 9 6 8 ) reveal nerve impregnations. Later PELLEGRINO D E IRALDI and G U E U D E T ( 1 9 6 8 , 1 9 6 9 ) discuss the Z I O positivity observed also in the layer of rods and cones in the photoreceptor cells in the retina of the rat. N I E B A U E R , K R A W C Z Y K , K I D D and WILGRAM ( 1 9 6 9 ) stained the granules of the epidermal LANGERHANS cells, G O L G I zone and the nuclear membrane at patiens suffering from the L E T T E R E R - S I T E disease. Simultaneously with the above investigations, the question of the specificity of Z I O was also raised. M A I L L E T ( 1 9 5 9 , 1 9 6 2 ) and, following him, N I E B A U E R , K R A W C Z Y K , K I D D and W I L G R A M consider to be probable that the lipid-like matters are stained. According to A K E R T and SANDRI ( 1 9 6 8 ) the cholinergic mechanism is supposedly indicated by Z I O positivity and, essentially, this supposition is supported also by the work of M A R T I N , B A R L O W and M I R A L T O ( 1 9 6 9 ) , opposite to the more recent investigation of K A W A N A , A K E R T and SANDRI ( 1 9 6 9 ) . It is worth noticing that, the dense-core vesicles do not take up the stain while reserpin inhibits the Z I O positivity of the nerve fibers ( P E L L E G R I N O D E IRALDI and G U E U D E T , 1 9 6 8 ) . In our present investigations, with an impregnation lasting only for a short time, we have succeeded in showing electively Z I O positivity localized in the nerve fibers and mainly in the clear synaptic vesicles of the corpus pinealc of the rat. With the help of that, apart from the impregnation of nerve fibers by a long-lasting staining, we have demonstrated the ZIO activity appearing in the ganglion cells of the superior cervical ganglion of the cat that presented Abbreviations: A\ axon N nucleus Coll collagen M mitochondrium Cy cytoplasm My myelin E r endoplasmic reticulum Sch Schwann eel! P l a t e I I Corpus pinealc of the rat. In the dilated gap of the connective tissue that separates the pincalo-cytes from each other several nerve fibers can he observed. ZIO positivity can be found exclusi-vely in the nerve fibers, (x 32 000) P ! j I e 1 1 1 Superior cervical ganglion of the cat after ZIO impregnation for three hours. The ZIO positivity can he found in „aspectfic" sites, too: in the perinuclear cysterna of Schwann cell, on the axo-Icmma and, non-bound to structure elements in the cytoplasm (arrows), (x 40 000) t C O N T R I B U T I O N T O T H E P R O B L E M O F TILE ZIO I M P R E G N A T I O N 1 4 T P l a t e I I 1 10* ш N. H A L Á S Z , Á . [ ' Á R D U T Z A N D F. J O Ó P l a t e I I I C O N T R I B U T I O N T O THE. PROBLEM OR T H ^ Z I O I M P R E G N A T I O N 149 itself strongly in the perinuclear cysterna and in the endoplasmatic reticulum, and it could be observed also mitochondrially, as well as in the cytoplasm, top, sporadically. In the some place, also nuclear membrane and processes of Schwann cells gave a strong reaction, and also the lamellae of the myelinated nerve fibers showed themselves active. As compared with the literary data we may summarize the results of our investigations so that the ZIO method stains not only the nerve fibers and synaptic vesicles specifically, but after an impregnation for a longer period of time, it stains, in addition to the above mentioned ones, also several other ele-ments of cells and nerve fibers. At any rate, further investigations are needed for recognizing the similarity of the chemical structure wich equally results in the ZIO positivity of the structures of different functions, * * * Special thanks of the authors are due to Prof. Dr. B. CsiLLiK, Director of the Anatomical Institute, University Medical School, Szeged, for having kindly granted the use of electron microscope Tesla BS 413. References L AKERT, K. and SANDRI, C. (1968) : An electron-microscopic study of zinc iodide-osmium impreg-nation of neurons. I. Staining of synaptic vesicles at cholinergic junctions. — Brain Res. 7, 286—295. CuAMPY. Ch. (1913) ; Granules et substances réduisant l'iodure d'osmium. — J , Anat. physiol. 4. 323. CSILLIK, B. and KNYIHÁR, E. (1968) : Endoplasmic units. — Acta Biochim. Btophys. Hung. 3. 165—170. JABONERO, V. (1964) ; Über die Brauchbarkeit der Osmiumtetroxyd-Zinkjodid-Mcthode zur Ana-lyse der vegetativen Peripherie. — Acta Neuroveg. (Wien) 26, 184—210. JABONERO, V., FABRA. L., MOYA, J . and JABONERO, R. M. (1961) : Resultados del método ácido ósm ico-yod uro de zinc para la demonsr ración de los elementos nerviosos periféricos. — Trab. Inst. Cajal, Madrid 53. 1. KA^ANA, E., AKERT, K. and SANDRI, C. (1969) ; Zinc iodide osmium tetroxide impregnation of nerve terminals in the spinal cord. — Brain Res. 16. 325—332. L.AMPARTER, H. E., STEIGER, U., SANDRI, C. and AKERT, K. (1969) : Zum Feinbau der Synapsen im Zentralnervensystem der Insekten. — Z. Zellforsch. 09, 435—442 . MAILLET, M. (1959) : Modifications de la technique de Champe au tetraos ¡de d'osmium- tod ure de potassium. Résultats de son application a l'étude des fibres nerveuses. — C.omt. rend. Soe. hiol. Paris 153, 939. MARTIN, R., BARLO1*', J, and MIRALTO, A. (1969) : Application of the zinc iodide-osmium tet-roxide impregnation of synaptic vesiclcs in Cephalopod nerves. — Brain Res. 15. 1 — 1 6 . NIEBAUER, G.. KRAIFCZYK. W. S.. KIDD, R. L. and WJLCIÙM, G. F. (1969) : Osmium zinc iodide reactive sites in the epidermal Langerhans cell. — Cell Biol. 43, 80—89. P l a t e I V SCG of the cat. after ZIO impregnation For three hours. The ZIO-positive electron-dense pre-cipitate is hound, besides the synaptic vesicles of the axons, to several structure elements (arrows), (x 40 000) P l a t e V A : Ganglion cell from a cat's SCG, after ZIO impregnation for seven hours. ZIO positivity can be observed freely in the perinuclear cystcrna (arrows), the endoplasmatic reticulum of the ganglion cell, as well as in the cytoplasm (arrow marked with an asterisk), (x 2 1 0 0 0 ) B : Picture of a SCG ganglion cell of the cat after ZIO impregnation for seven hours. The gra-nules of ZIO positivity having, on the average, 80 A diameter surround in some places non-rcacting units of a size 300—350 Â (arrows), (x 130 000) C O N T R I B U T I O N T O THE. P R O B L E M OR TH^ Z I O I M P R E G N A T I O N P l a t e V 1 5 1 1 5 2 K H A L A S Z , A. P Ä R D U T Z A N D F. JOÖ PFASE, D . C. (1964 : ) Histological techniques for electron microscopy. — Acad. Press: New York and London. PELLEGRINO DE IitALDl. A. and GUEUDET, R. (1968) : Action of rcserpine on the osmium t et roxi de zinc iodide reactive site uf synaptic vesicles in the pineal nerves of the rat. — Z. Zellforsch, 91, 178—185 . PEI.LEGRINO »£ IKALIM. A. and GUEUDET, R. (1969) ; Osmium tetroxide-ztnc iodide reactive sites in the fotoreceptor cells of the retina of the rat. — Z. Zell forsch. iOl, 2 0 3 — 2 1 1 , REYNOLDS, E, S, (196!)): The use of lead citrate at high pH as an elect ton-opaque stain in electron microscopy. — J . Cell Biol. 17, 208—212. RODRIGUEZ-PEREZ. A. P. ( 1 9 6 4 ) : Über die Ncrvcne lernen re der Epididymis, des Ductus deferens, des Corpus pineale, der Pars intermedia hypophyseos cerebri und der Substantia corticalis des Corpus suprarenale (mittels der Oimiumtctroxid-Zinkj od id-Methode nach Mailler). — Acta Neuroveg. 26. 211—222 . TKIES, W . (1964) : Vergleichende neuroh istologische Untersuchungen an gesunder und kranker menschlicher Haut sowie bei weichen Zcllnacvi, Pigmentnaevi, Melanoma I ignora und Vitiligo mit Hilfe der Osmiumwnkjodid Methode. — Acta Neuroveg. 26. 2 2 3 — 2 4 7 . Address of the authors; D r . N . HALASZ Dr. A. PÄKL>UCZ Dr, F, J o o Electron Microscope Laboratory. Institute of Biophysics. Biological Research Center. Hungarian Academy of Sciences. Süeged. Hungary Odcsszai krt. 62 

Contribution to the problem of the specificity of the zio impregnation

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Contribution to the problem of the specificity of the zio impregnation

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