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Labeling of electrical surface charges at synaptosome membrane: an electron cytochemical and biochemical study
214
Brain Research, 112 (1976),214-220 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Labeling of electrical surface charges at synaptosome membrane: an electron cytochemical and biochemical study*
ALFREDO FERIA-VELASCO, SALVADOR S/~NCHEZ-DE-LA-PEiqA and VICTOR MAGDALENO Section of Neurobiology, Departamento de Investigacidn en Medicina Experimental, Instituto Mexieano del Seguro Social, Mdxieo, D.F. (Mexico)
(Accepted April 28th, 1976)
The glycocalix plays an important role in cell physiology, particularly related to cell aggregation zl and contact recognition 11. Glycoproteins are essential components of glycocalix in most of the cells, including the neurons, whose membranes are rich in sialoglycoproteins and gangliosides~, 1°. In the central nervous system, the neuronal membrane, its glycocalix and the elements present in the extracellular space act together in the regulation of the ionic transport involved in membrane excitation and recovery12. Electrophoretic mobility of brain synaptosomes depends upon their negative surface charges 26, conferred either by sialic acid residues, present in sialoglycoproteins and gangliosidesS,2aY, or by acid mucopolysaccharides present in the synaptosome membrane6, 25. Because of the importance of the synaptic plasma membrane and of sialoglycoproteins and sialogangliosides at nerve cell surface coat, the present communication deals with the cytochemical identification of electrical charges at synaptosome surface by means of electron cytochemical and correlative biochemical techniques. Brains of adult albino mice were obtained quickly after decapitation and pooled in a chilled homogenizer for subcellular fractionation. A 10 ~ (w/v) homogenate in 0.32 M sucrose was prepared in a Teflon-glass homogenizer under gentle conditions. The primary fractions (nuclei, mitochondria, microsomes and supernatant) were obtained by following the procedure of De Robertis et al. 7 and the mitochondrial fraction was subfractionated in a discontinuous sucrose density gradient 2°. The synaptosomal fraction was separated and diluted with physiological saline solution and centrifuged at 100,000 × g for 30 min. The pellet was resuspended in cold physiological saline solution for assays. Incubation with colloidal iron. Ferric oxide solutions were prepared at pH 1.8 and 4.0 according to the method of Gasic et al. 9 to obtain positively (Fe(+)) and negatively (Fe(--)) charged colloidal iron solutions, respectively. For incubation with colloidal * Part of this work was reported to the VI Annual Meeting of the American Society for Neurochemistry, March 10-14, 1975, Trans. Amer. Soe. Neuroehem., 6 (1975) 119.
215 iron, the synaptosome fraction was fixed with 2 . 5 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at 4 °C for 45 min 22. After brief washing with the same buffer the synaptosomes were incubated in the colloidal iron solutions for 30-45 min at room temperature. The synaptosomes were rinsed in 1 2 ~ glacial acetic acid and subsequently in bidistilled and deionized water before postfixation in phosphate buffered1 ~ osmium tetroxide for 1 h at 4 °C (see ref. 18). Some samples were washed briefly in 2 M sodium chloride solution before glutaraldehyde fixation and further incubation with the iron solutions, in order to avoid artifactual staining of material adsorbed to the synaptosome surface. Methylation. The synaptosome fraction was fixed in buffered-2.5 ~ glutaraldehyde and briefly washed in 50 ~ ethanol and absolute ethanol. Methylation was carried out by treating the samples with 0.1 N HC1 in absolute methanol for 24 h at 60 °C (see ref. 13) before incubation with the F e ( + ) solution and postfixation with osmium tetroxide. Deamination. The synaptosome fraction was fixed in buffered-2.5~ glutaraldehyde and subjected to a deamination procedure 14 before the incubation with the Fe(--) solution and postfixation with osmium tetroxide. In some experiments the samples were incubated with purified CI. perfringens neuraminidase (5/,g/mg synaptosome protein) for 60 rain at 37 °C (see below), washed twice in physiological saline solution and fixed with glutaraldehyde before the incubation with the Fe(--) solution or before the deamination process. Neuraminidase treatment. Type V Clostridium perfringens neuraminidase (Nacetyl neuraminate glycohydrolase; EC 3.2.1.18) was purified according to the method described by Bernacki and BosmannL The enzyme activity was 0.7 units/mg (one unit = 1 #mole of sialyllactose hydrolyzed/min at p H 5.0, at 37 °C.) The synaptosome preparation was washed in physiological saline solution and incubated with neuraminidase (5/~g/mg synaptosome protein) at pH 7.0 at 37 °C for 60 min. The samples were washed in cold 0.32 M sucrose and centrifuged at 7,000 x g for 15 min. The supernatant was used for measuring released sialic acid 28 and the pellet was washed with physiological saline solution before fixation with buffered-glutaraldehyde and incubation with the F e ( + ) solution. Postfixation was carried out with bufferedosmium tetroxide. Synaptosome samples incubated with boiled neuraminidase were used as controls. Sialic acid in the synaptosomalfraction. Total sialic acid was determined in the synaptosomal fraction following acid hydrolysis with 0.1 N sulfuric acid at 80 °C for 5 h. Sialic acid standards were treated similarly to correct for destruction of free sialic acid. Electron microscopy. After postfixation with osmium tetroxide the samples in all experiments were dehydrated and embedded in Epon-812 (see ref. 16). Thin sections in the silver color rangO 9 were collected in copper grids for observation without staining in a Philips EM-200 or a Zeiss EM-10 electron microscopes. Analytical determinations. Protein was determined by the method of Lowry et al. 15, whereas sialic acid was measured by the spectrophotometric method described by Warren 28.
216
Fig. 1. Incubation with the Fe(÷) solution, a: fine electron-dense deposits (arrow) were homogeneously distributed at synaptosome surface after incubation with the Fe(+) solution. Unstained section ( x 40,000). b: no deposits were observed at the synaptosome surface when the samples were treated with methanolic HC1 solution (methylation procedure) before incubation with the Fe(+) solution. m, intraterminal mitochondria; v, synaptic vesicles. Unstained section (x 40,000). c: no deposits were observed at s2cnaptosomesurface (short arrow) when the samples were incubated with purified C1. perfringens neuraminidase before fixation and incubation with the Fe(+) solution. Long arrow, synapticcontact area. Unstained section ( x 30,000). d: electron-densedeposits (arrows) appear homogeneously distributed at synaptosome surface after incubation with boiled purified Cl. perfringens neuraminidase prior to fixation and incubation with the Fe(÷) solution, m, intraterminal mitochondria; v, synaptic vesicles. Unstained section (x 40,000).
When the samples were incubated with the F e ( + ) solution, homogeneously distributed electron-dense deposits were observed along the entire synaptosomal surface (Fig. la) corresponding to exposed electronegative charges. N o particular differences in deposit distribution were noted between the synaptic contact area and the rest of synaptosomal membrane. When the synaptosomal fraction was washed with 2 M NaC1 solution before incubation with the F e ( + ) solution, the synaptosomes appeared shrunken and fine electron-dense deposits were homogeneously distributed at their surface, ruling out adsorption of extraneous charged material to the synaptosome surface during the preparation procedure. Treatment of the synaptosomes with methanolic HC1 solution before incubation with the F e ( + ) solution prevented the appearance of surface deposits at the synaptosome membrane (Fig. lb). When the synaptosomes were incubated with purified Cl. perfringens neuraminidase, nearly 20 ~ of total synaptosomal sialic acid was released (Table I). No electron-dense deposits on synaptosome surface were observed when the neuraminidase treated synapto-
217
Fig. 2. Incubation with the Fe(--) solution, a: non-homogeneously distributed pattern of electrondense deposits (arrows) was observed at synaptosome surface when the samples were incubated with the Fe(--) solution, m, intraterminal mitochondria; v, synaptic vesicles. Unstained section ( >." 40,000). b: no electron-dense deposits were observed at synaptosome surface (arrows) when the synaptosomes were subjected to a deamination procedure before incubation with the Fe(--) solution. Unstained section ( x 40,000).
218 TABLE I Sialie aeM in mouse brain synaptosomes and sialie aeM released by treatment with purified CI. perfi4ngens neuraminidase Data represent means ~ SEM for the number of experiments shown in parentheses. After severe acid hydrolysis
Release by treatment with purified neuraminidase (5 l~g/mg *) Boiled
nmoles/mg synaptosome protein
4.3 ± 0.008 (61)
0.924 ± 0.075 (6)
0.005 ± 0.002 (6)
* Synaptosome protein.
somes were incubated with Fe(+) solution (Fig. lc). A negligible amount of sialic acid was released when the synaptosomes were incubated with boiled purified neuraminidase (Table I), and electron-dense deposits were observed at the surface of these synaptosomes after incubating them with the Fe(+) solution (Fig. ld). The synaptosomes incubated with the Fe(--) solution after glutaraldehyde fixation, disclosed a non-homogeneously distributed pattern of electron-dense deposits at their surface (Fig. 2a). No changes in this labeling pattern were observed when neuraminidase treatment was carried out before incubation with the Fe(--) solution. No deposits were observed at synaptosome surface when deamination procedure was performed before incubation with the Fe(--) solution (Fig. 2b). The same negative results were obtained when the incubation of the synaptosome preparation with the Fe(--) solution was performed, after incubation with purified Cl. perfringens neuraminidase and deamination. In the present work the distribution pattern of electrical charges at the synaptosome membrane is demonstrated using high resolution cytochemical techniques. The staining method with colloidal iron at various pH as a cytochemical staining for electron microscopy appears to be in part based upon electrostatic interactions of charged groups of tissue elements with colloidal iron 2,9,17. Thus, when the colloid is negatively charged, labeling of the peripheral positively charged groups is observed. On the other hand, when positively charged colloidal iron is employed, the peripheral groups contributing to the net negative surface charge are stained. Based upon the results obtained with the Fe(+) solution after methylation and after incubation with purified neuraminidase we can conclude that the labeled electronegative charges at the synaptosome surface most likely correspond to those Conferred by the carboxyl group of sialic acid. This is further supported by the fact that in the sialidase experiments, sialic acid was demonstrated in the supernatant after centrifuging the enzymeincubated samples. The enzyme-released sialic acid corresponded to nearly 2 0 ~ of the total synaptosome sialic acid after severe acid hydrolysis. On the other hand, no sialic acid was released, while electron-dense deposits were depicted at the synaptosome surface, when incubation with boiled neuraminidase was carried out. This is in keeping with what Bosmann and Carlson have obtained after incubating the synapto-
219 somes w i t h purified n e u r a m i n i d a s e (release o f a p p r o x i m a t e l y 19 ~ o f the t o t a l synapt o s o m e sialic acid) for their e x p e r i m e n t s o f e l e c t r o p h o r e t i c m o b i l i t y o f s y n a p t o s o m e s 3. Both, gangliosidic a n d p r o t e i n - b o u n d sialic acid have b e e n c o n s i d e r e d i m p o r t a n t e l e m e n t s very likely involved in t r a n s m i s s i o n processes12, 24. T h e results o f the experim e n t s with the F e ( - - ) s o l u t i o n suggest t h a t the labeled surface positive charges are p r o b a b l y c o n f e r r e d b y e x p o s e d a m i n o groups. W h e t h e r the staining p a t t e r n w i t h the F e ( - - ) s o l u t i o n reflects the scarcity o f positively c h a r g e d g r o u p s at the s y n a p t o s o m e surface coat, c a n n o t be positively a s s e r t e d f r o m the p r e s e n t work. H o w e v e r , o t h e r a u t h o r s have o b s e r v e d a similar i r r e g u l a r d i s t r i b u t i o n p a t t e r n o f positive surface charges in o t h e r cells i n c u b a t e d with the F e ( - - ) s o l u t i o n 4. A n alternative possibility m a y be t h a t free c a r b o x y l a n d o t h e r negatively c h a r g e d groups, d e t e r m i n i n g the net negative charge o f the s y n a p t o s o m e surface, p r e v e n t the staining o f the basic groups b y p r e d o m i n a n t e l e c t r o s t a t i c r e p u l s i o n o f the negatively c h a r g e d i r o n particles. H o w e v e r , n e u r a m i n i d a s e t r e a t m e n t before i n c u b a t i o n with the F e ( - - ) s o l u t i o n d i d n o t change the labeling p a t t e r n o f the s y n a p t o s o m e surface in the present w o r k . The a u t h o r s wish to express their g r a t i t u d e to D r . R i c a r d o T a p i a for his valuable c o m m e n t s a n d careful revision o f the m a n u s c r i p t .
1 Bernacki, R. J. and Bosmann, H.B., Red cell hydrolases. Proteinase activities in human erythrocyle plasma membranes, J. Membrane Biol., 7 (1972) 1-14. 2 Blanquet, P. R. and Loiez, A., Colloidal iron used at pH's lower than 1 as electron stain for surface proteins, J. Histochem. Cytochem., 22 (1974) 368-377. 3 Bosmann, H. B. and Carlson, W., Identification of sialic acid at the nerve ending periphery and electrophoretic mobility of isolated synaptosomes, Exp. Cell Res., 72 (1972) 436-440. 4 Brandes, D., Sato, T., Ueda, H and R undell, J. O., Effect of vitamin A alcohol on the surface coat and charges of L1210 leukemic cells, Cancer Res., 34 (1974) 2151-2158. 5 Brunngraber, E. G., Dekirmenjian, H. and Brown, B. D., The distribution of protein bound Nacetyl neuraminic acid in subcellular fractions of the rat brain, Biochem. J., 103 (1967) 73-78. 6 Clausen, J. and Rosenkast, P., Isolation of acid mucopolysaccharides of human brain, J. Neurochem., 9 (1962) 392-398. 7 De Robertis, E., PeUegrino de Iraldi, A., Rodr[guez de Lores Arnaiz,G. and Salganicoff, L., Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase, J. Neuroehem., 9 (1962) 23-35. 8 Dicesare, J. L. and Rapport, M. M., Availability to neuraminidase of gangliosides and sialoglycoproteins in neuronal membranes, J. Neurochem., 20 (1973) 1781-1783. 9 Gasic, G. J., Berwich, L. and Sorrentino, M., Positive and negative colloidal iron as cell surface electron stains, Lab. Invest., 18 (1968) 63-71. 10 Gombos, G., Morgan, I., Breckenridge, W. C., Breckenridge, J. E. et Vincedon, G., Isolement et structure biochimique de la membrane synaptosomale. C.R. Soc. Biol. (Paris), 165 (1971) 499-5O6. 11 Jones, B. M., A unifying hypothesis of cell adhesion, Nature (Lond.), 212 (1966) 362-365. 12 Lehninger, A. L., The neuronal membrane, Proc. nat. Acad. Sci. (Wash.), 60 (1958) 1069--1080. 13 Lillie, R. D., Acetylation and nitrosation of tissue amines in histochemistry, J. Histochem., 6 (1958) 352 362. 14 Lillie, R. D., Histopathologic Technic and Practical Histochemistry, Blakiston, Division of McGraw-Hill, New York, N.Y., 1954, pp. 162 and 357. 15 Lowry, O., Rosebrough, N. J., Farr, A. L.and Randall, R. J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 16 Luft, J. H., Improvements in epoxy resin embedding methods, J. biophys, biochem. CytoL, 9 (1961) 409-414.
220 17 Martinez-Palomo, A., The surface coats of animal cells, bzt. Rev. Cytol., 29 (1970) 29-75. 18 Palade, G. E., A study of fixation for electron microscopy, J. exp. Med., 95 (1952) 285-297. 19 Peachy, L. D., Thin sections. I. A study of section thickness and physical distortion produced during microtomy, J. biophys, biochem. Cytol., 4 (1958) 233-242. 20 P6rez de la Mora, M., Feria-Velasco, A. and Tapia, R., Pyridoxal phosphate and glutamate decarboxylase in subcellular particles of mouse brain and their relationship to convulsions, J. Neurochem., 20 (1973) 1575-1587. 21 Roseman, S., Complex carbohydrates and intercellular adhesion. In A. A. Moscona (Ed.), The Cell Surface in Development. Chap. 13, John Wiley, New York, N.Y., 1974, pp. 255-271. 22 Sabatini, D. D., Bensch, K. and Barrnett, R. J., Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation, J. Cell Biol., 17 (1963) 19-58. 23 Svennerholm, L., Chromatographic separation of human brain gangliosides, J. Neurochem., 10 (1963) 613-623. 24 Tauc, L. and Hinzen, D. H., Neuraminidase: its effect on synaptic transmission, Brain Research, 80 (1974) 340-344. 25 Vos, J., Kuriyama, K. and Roberts, E., Distribution of acid mucopolysaccharides in subcellular fractions of mouse brain, Brain Research, 12 (1969) 172-179. 26 Vos, J., Kuriyama, K. and Roberts, E., Electrophoretic mobilities of brain subcellular particles and binding of ~-aminobutyric acid, acetylcholine, norepinephrine and 5-hydroxytriptamine, Brain Research, 9 (1968) 224-230. 27 Wallach, D. F. H. and Kamat, V. B., The contribution of sialic acid to the surface charge of fragments of plasma membrane and endoplasrnic reticulum, J. Cell Biol., 30 (1966) 660-663. 28 Warren, L., The thiobarbituric acid assay of sialic acid, J. biol. Chem., 234 (1959) 1971-1975
(Contents continued) Alteration of the hypoxanthine level in cerebrospinal fluid as an indicator of tissue hypoxia by O. D. SAUGSTAD,H. SCHRADERAND A. O. AASEN(Oslo, Norway) . . . . . . . . . . . . . . Descending spinal sympathetic p~it3away utilized by somoto-sympathetic reflex and carotid chemoreflex ...... by P. SZULCZYK(Warsaw, Poland) . . . . . . . . . . . . . . . . . . . . . . . . . . . . p-Chlorophenylalanine hyperthermia in a warm environment: reversal with 5-hydroxytryptophan by M. J. CRONIN (Los Angeles, Calif., U.S.A.) . . . . . . . . . . . . . . . . . . . . . . . Inhibitors of protein synthesis and memory: dissociation of amnesic effects and effects on adrenal steroidogenesis by L. R. SQUIRE,S. ST. JOHN AND H. P. DAVIS(San Diego, Calif., U.S.A.) . . . . . . . . . . . . Structural changes in vitro of isolated nerve endings. I. Effect of cations by F. HAj6S AND A. CSILLAG(Budapest, Hungary) . . . . . . . . . . . . . . . . . . . . . Labeling of electrical surface changes at synaptosome membrane: an electron cytochemical and biochemical study by A. FERIA-VELASCO,S. SANCHEZ-DE-LA-PE~AAND V. MAGDALENO(Mexico, D.F., Mexico) . . . .
188 190 194
200 207 214
Brain Research, 112 (1976),214-220 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Labeling of electrical surface charges at synaptosome membrane: an electron cytochemical and biochemical study*
ALFREDO FERIA-VELASCO, SALVADOR S/~NCHEZ-DE-LA-PEiqA and VICTOR MAGDALENO Section of Neurobiology, Departamento de Investigacidn en Medicina Experimental, Instituto Mexieano del Seguro Social, Mdxieo, D.F. (Mexico)
(Accepted April 28th, 1976)
The glycocalix plays an important role in cell physiology, particularly related to cell aggregation zl and contact recognition 11. Glycoproteins are essential components of glycocalix in most of the cells, including the neurons, whose membranes are rich in sialoglycoproteins and gangliosides~, 1°. In the central nervous system, the neuronal membrane, its glycocalix and the elements present in the extracellular space act together in the regulation of the ionic transport involved in membrane excitation and recovery12. Electrophoretic mobility of brain synaptosomes depends upon their negative surface charges 26, conferred either by sialic acid residues, present in sialoglycoproteins and gangliosidesS,2aY, or by acid mucopolysaccharides present in the synaptosome membrane6, 25. Because of the importance of the synaptic plasma membrane and of sialoglycoproteins and sialogangliosides at nerve cell surface coat, the present communication deals with the cytochemical identification of electrical charges at synaptosome surface by means of electron cytochemical and correlative biochemical techniques. Brains of adult albino mice were obtained quickly after decapitation and pooled in a chilled homogenizer for subcellular fractionation. A 10 ~ (w/v) homogenate in 0.32 M sucrose was prepared in a Teflon-glass homogenizer under gentle conditions. The primary fractions (nuclei, mitochondria, microsomes and supernatant) were obtained by following the procedure of De Robertis et al. 7 and the mitochondrial fraction was subfractionated in a discontinuous sucrose density gradient 2°. The synaptosomal fraction was separated and diluted with physiological saline solution and centrifuged at 100,000 × g for 30 min. The pellet was resuspended in cold physiological saline solution for assays. Incubation with colloidal iron. Ferric oxide solutions were prepared at pH 1.8 and 4.0 according to the method of Gasic et al. 9 to obtain positively (Fe(+)) and negatively (Fe(--)) charged colloidal iron solutions, respectively. For incubation with colloidal * Part of this work was reported to the VI Annual Meeting of the American Society for Neurochemistry, March 10-14, 1975, Trans. Amer. Soe. Neuroehem., 6 (1975) 119.
215 iron, the synaptosome fraction was fixed with 2 . 5 ~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at 4 °C for 45 min 22. After brief washing with the same buffer the synaptosomes were incubated in the colloidal iron solutions for 30-45 min at room temperature. The synaptosomes were rinsed in 1 2 ~ glacial acetic acid and subsequently in bidistilled and deionized water before postfixation in phosphate buffered1 ~ osmium tetroxide for 1 h at 4 °C (see ref. 18). Some samples were washed briefly in 2 M sodium chloride solution before glutaraldehyde fixation and further incubation with the iron solutions, in order to avoid artifactual staining of material adsorbed to the synaptosome surface. Methylation. The synaptosome fraction was fixed in buffered-2.5 ~ glutaraldehyde and briefly washed in 50 ~ ethanol and absolute ethanol. Methylation was carried out by treating the samples with 0.1 N HC1 in absolute methanol for 24 h at 60 °C (see ref. 13) before incubation with the F e ( + ) solution and postfixation with osmium tetroxide. Deamination. The synaptosome fraction was fixed in buffered-2.5~ glutaraldehyde and subjected to a deamination procedure 14 before the incubation with the Fe(--) solution and postfixation with osmium tetroxide. In some experiments the samples were incubated with purified CI. perfringens neuraminidase (5/,g/mg synaptosome protein) for 60 rain at 37 °C (see below), washed twice in physiological saline solution and fixed with glutaraldehyde before the incubation with the Fe(--) solution or before the deamination process. Neuraminidase treatment. Type V Clostridium perfringens neuraminidase (Nacetyl neuraminate glycohydrolase; EC 3.2.1.18) was purified according to the method described by Bernacki and BosmannL The enzyme activity was 0.7 units/mg (one unit = 1 #mole of sialyllactose hydrolyzed/min at p H 5.0, at 37 °C.) The synaptosome preparation was washed in physiological saline solution and incubated with neuraminidase (5/~g/mg synaptosome protein) at pH 7.0 at 37 °C for 60 min. The samples were washed in cold 0.32 M sucrose and centrifuged at 7,000 x g for 15 min. The supernatant was used for measuring released sialic acid 28 and the pellet was washed with physiological saline solution before fixation with buffered-glutaraldehyde and incubation with the F e ( + ) solution. Postfixation was carried out with bufferedosmium tetroxide. Synaptosome samples incubated with boiled neuraminidase were used as controls. Sialic acid in the synaptosomalfraction. Total sialic acid was determined in the synaptosomal fraction following acid hydrolysis with 0.1 N sulfuric acid at 80 °C for 5 h. Sialic acid standards were treated similarly to correct for destruction of free sialic acid. Electron microscopy. After postfixation with osmium tetroxide the samples in all experiments were dehydrated and embedded in Epon-812 (see ref. 16). Thin sections in the silver color rangO 9 were collected in copper grids for observation without staining in a Philips EM-200 or a Zeiss EM-10 electron microscopes. Analytical determinations. Protein was determined by the method of Lowry et al. 15, whereas sialic acid was measured by the spectrophotometric method described by Warren 28.
216
Fig. 1. Incubation with the Fe(÷) solution, a: fine electron-dense deposits (arrow) were homogeneously distributed at synaptosome surface after incubation with the Fe(+) solution. Unstained section ( x 40,000). b: no deposits were observed at the synaptosome surface when the samples were treated with methanolic HC1 solution (methylation procedure) before incubation with the Fe(+) solution. m, intraterminal mitochondria; v, synaptic vesicles. Unstained section (x 40,000). c: no deposits were observed at s2cnaptosomesurface (short arrow) when the samples were incubated with purified C1. perfringens neuraminidase before fixation and incubation with the Fe(+) solution. Long arrow, synapticcontact area. Unstained section ( x 30,000). d: electron-densedeposits (arrows) appear homogeneously distributed at synaptosome surface after incubation with boiled purified Cl. perfringens neuraminidase prior to fixation and incubation with the Fe(÷) solution, m, intraterminal mitochondria; v, synaptic vesicles. Unstained section (x 40,000).
When the samples were incubated with the F e ( + ) solution, homogeneously distributed electron-dense deposits were observed along the entire synaptosomal surface (Fig. la) corresponding to exposed electronegative charges. N o particular differences in deposit distribution were noted between the synaptic contact area and the rest of synaptosomal membrane. When the synaptosomal fraction was washed with 2 M NaC1 solution before incubation with the F e ( + ) solution, the synaptosomes appeared shrunken and fine electron-dense deposits were homogeneously distributed at their surface, ruling out adsorption of extraneous charged material to the synaptosome surface during the preparation procedure. Treatment of the synaptosomes with methanolic HC1 solution before incubation with the F e ( + ) solution prevented the appearance of surface deposits at the synaptosome membrane (Fig. lb). When the synaptosomes were incubated with purified Cl. perfringens neuraminidase, nearly 20 ~ of total synaptosomal sialic acid was released (Table I). No electron-dense deposits on synaptosome surface were observed when the neuraminidase treated synapto-
217
Fig. 2. Incubation with the Fe(--) solution, a: non-homogeneously distributed pattern of electrondense deposits (arrows) was observed at synaptosome surface when the samples were incubated with the Fe(--) solution, m, intraterminal mitochondria; v, synaptic vesicles. Unstained section ( >." 40,000). b: no electron-dense deposits were observed at synaptosome surface (arrows) when the synaptosomes were subjected to a deamination procedure before incubation with the Fe(--) solution. Unstained section ( x 40,000).
218 TABLE I Sialie aeM in mouse brain synaptosomes and sialie aeM released by treatment with purified CI. perfi4ngens neuraminidase Data represent means ~ SEM for the number of experiments shown in parentheses. After severe acid hydrolysis
Release by treatment with purified neuraminidase (5 l~g/mg *) Boiled
nmoles/mg synaptosome protein
4.3 ± 0.008 (61)
0.924 ± 0.075 (6)
0.005 ± 0.002 (6)
* Synaptosome protein.
somes were incubated with Fe(+) solution (Fig. lc). A negligible amount of sialic acid was released when the synaptosomes were incubated with boiled purified neuraminidase (Table I), and electron-dense deposits were observed at the surface of these synaptosomes after incubating them with the Fe(+) solution (Fig. ld). The synaptosomes incubated with the Fe(--) solution after glutaraldehyde fixation, disclosed a non-homogeneously distributed pattern of electron-dense deposits at their surface (Fig. 2a). No changes in this labeling pattern were observed when neuraminidase treatment was carried out before incubation with the Fe(--) solution. No deposits were observed at synaptosome surface when deamination procedure was performed before incubation with the Fe(--) solution (Fig. 2b). The same negative results were obtained when the incubation of the synaptosome preparation with the Fe(--) solution was performed, after incubation with purified Cl. perfringens neuraminidase and deamination. In the present work the distribution pattern of electrical charges at the synaptosome membrane is demonstrated using high resolution cytochemical techniques. The staining method with colloidal iron at various pH as a cytochemical staining for electron microscopy appears to be in part based upon electrostatic interactions of charged groups of tissue elements with colloidal iron 2,9,17. Thus, when the colloid is negatively charged, labeling of the peripheral positively charged groups is observed. On the other hand, when positively charged colloidal iron is employed, the peripheral groups contributing to the net negative surface charge are stained. Based upon the results obtained with the Fe(+) solution after methylation and after incubation with purified neuraminidase we can conclude that the labeled electronegative charges at the synaptosome surface most likely correspond to those Conferred by the carboxyl group of sialic acid. This is further supported by the fact that in the sialidase experiments, sialic acid was demonstrated in the supernatant after centrifuging the enzymeincubated samples. The enzyme-released sialic acid corresponded to nearly 2 0 ~ of the total synaptosome sialic acid after severe acid hydrolysis. On the other hand, no sialic acid was released, while electron-dense deposits were depicted at the synaptosome surface, when incubation with boiled neuraminidase was carried out. This is in keeping with what Bosmann and Carlson have obtained after incubating the synapto-
219 somes w i t h purified n e u r a m i n i d a s e (release o f a p p r o x i m a t e l y 19 ~ o f the t o t a l synapt o s o m e sialic acid) for their e x p e r i m e n t s o f e l e c t r o p h o r e t i c m o b i l i t y o f s y n a p t o s o m e s 3. Both, gangliosidic a n d p r o t e i n - b o u n d sialic acid have b e e n c o n s i d e r e d i m p o r t a n t e l e m e n t s very likely involved in t r a n s m i s s i o n processes12, 24. T h e results o f the experim e n t s with the F e ( - - ) s o l u t i o n suggest t h a t the labeled surface positive charges are p r o b a b l y c o n f e r r e d b y e x p o s e d a m i n o groups. W h e t h e r the staining p a t t e r n w i t h the F e ( - - ) s o l u t i o n reflects the scarcity o f positively c h a r g e d g r o u p s at the s y n a p t o s o m e surface coat, c a n n o t be positively a s s e r t e d f r o m the p r e s e n t work. H o w e v e r , o t h e r a u t h o r s have o b s e r v e d a similar i r r e g u l a r d i s t r i b u t i o n p a t t e r n o f positive surface charges in o t h e r cells i n c u b a t e d with the F e ( - - ) s o l u t i o n 4. A n alternative possibility m a y be t h a t free c a r b o x y l a n d o t h e r negatively c h a r g e d groups, d e t e r m i n i n g the net negative charge o f the s y n a p t o s o m e surface, p r e v e n t the staining o f the basic groups b y p r e d o m i n a n t e l e c t r o s t a t i c r e p u l s i o n o f the negatively c h a r g e d i r o n particles. H o w e v e r , n e u r a m i n i d a s e t r e a t m e n t before i n c u b a t i o n with the F e ( - - ) s o l u t i o n d i d n o t change the labeling p a t t e r n o f the s y n a p t o s o m e surface in the present w o r k . The a u t h o r s wish to express their g r a t i t u d e to D r . R i c a r d o T a p i a for his valuable c o m m e n t s a n d careful revision o f the m a n u s c r i p t .
1 Bernacki, R. J. and Bosmann, H.B., Red cell hydrolases. Proteinase activities in human erythrocyle plasma membranes, J. Membrane Biol., 7 (1972) 1-14. 2 Blanquet, P. R. and Loiez, A., Colloidal iron used at pH's lower than 1 as electron stain for surface proteins, J. Histochem. Cytochem., 22 (1974) 368-377. 3 Bosmann, H. B. and Carlson, W., Identification of sialic acid at the nerve ending periphery and electrophoretic mobility of isolated synaptosomes, Exp. Cell Res., 72 (1972) 436-440. 4 Brandes, D., Sato, T., Ueda, H and R undell, J. O., Effect of vitamin A alcohol on the surface coat and charges of L1210 leukemic cells, Cancer Res., 34 (1974) 2151-2158. 5 Brunngraber, E. G., Dekirmenjian, H. and Brown, B. D., The distribution of protein bound Nacetyl neuraminic acid in subcellular fractions of the rat brain, Biochem. J., 103 (1967) 73-78. 6 Clausen, J. and Rosenkast, P., Isolation of acid mucopolysaccharides of human brain, J. Neurochem., 9 (1962) 392-398. 7 De Robertis, E., PeUegrino de Iraldi, A., Rodr[guez de Lores Arnaiz,G. and Salganicoff, L., Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase, J. Neuroehem., 9 (1962) 23-35. 8 Dicesare, J. L. and Rapport, M. M., Availability to neuraminidase of gangliosides and sialoglycoproteins in neuronal membranes, J. Neurochem., 20 (1973) 1781-1783. 9 Gasic, G. J., Berwich, L. and Sorrentino, M., Positive and negative colloidal iron as cell surface electron stains, Lab. Invest., 18 (1968) 63-71. 10 Gombos, G., Morgan, I., Breckenridge, W. C., Breckenridge, J. E. et Vincedon, G., Isolement et structure biochimique de la membrane synaptosomale. C.R. Soc. Biol. (Paris), 165 (1971) 499-5O6. 11 Jones, B. M., A unifying hypothesis of cell adhesion, Nature (Lond.), 212 (1966) 362-365. 12 Lehninger, A. L., The neuronal membrane, Proc. nat. Acad. Sci. (Wash.), 60 (1958) 1069--1080. 13 Lillie, R. D., Acetylation and nitrosation of tissue amines in histochemistry, J. Histochem., 6 (1958) 352 362. 14 Lillie, R. D., Histopathologic Technic and Practical Histochemistry, Blakiston, Division of McGraw-Hill, New York, N.Y., 1954, pp. 162 and 357. 15 Lowry, O., Rosebrough, N. J., Farr, A. L.and Randall, R. J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 16 Luft, J. H., Improvements in epoxy resin embedding methods, J. biophys, biochem. CytoL, 9 (1961) 409-414.
220 17 Martinez-Palomo, A., The surface coats of animal cells, bzt. Rev. Cytol., 29 (1970) 29-75. 18 Palade, G. E., A study of fixation for electron microscopy, J. exp. Med., 95 (1952) 285-297. 19 Peachy, L. D., Thin sections. I. A study of section thickness and physical distortion produced during microtomy, J. biophys, biochem. Cytol., 4 (1958) 233-242. 20 P6rez de la Mora, M., Feria-Velasco, A. and Tapia, R., Pyridoxal phosphate and glutamate decarboxylase in subcellular particles of mouse brain and their relationship to convulsions, J. Neurochem., 20 (1973) 1575-1587. 21 Roseman, S., Complex carbohydrates and intercellular adhesion. In A. A. Moscona (Ed.), The Cell Surface in Development. Chap. 13, John Wiley, New York, N.Y., 1974, pp. 255-271. 22 Sabatini, D. D., Bensch, K. and Barrnett, R. J., Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation, J. Cell Biol., 17 (1963) 19-58. 23 Svennerholm, L., Chromatographic separation of human brain gangliosides, J. Neurochem., 10 (1963) 613-623. 24 Tauc, L. and Hinzen, D. H., Neuraminidase: its effect on synaptic transmission, Brain Research, 80 (1974) 340-344. 25 Vos, J., Kuriyama, K. and Roberts, E., Distribution of acid mucopolysaccharides in subcellular fractions of mouse brain, Brain Research, 12 (1969) 172-179. 26 Vos, J., Kuriyama, K. and Roberts, E., Electrophoretic mobilities of brain subcellular particles and binding of ~-aminobutyric acid, acetylcholine, norepinephrine and 5-hydroxytriptamine, Brain Research, 9 (1968) 224-230. 27 Wallach, D. F. H. and Kamat, V. B., The contribution of sialic acid to the surface charge of fragments of plasma membrane and endoplasrnic reticulum, J. Cell Biol., 30 (1966) 660-663. 28 Warren, L., The thiobarbituric acid assay of sialic acid, J. biol. Chem., 234 (1959) 1971-1975
(Contents continued) Alteration of the hypoxanthine level in cerebrospinal fluid as an indicator of tissue hypoxia by O. D. SAUGSTAD,H. SCHRADERAND A. O. AASEN(Oslo, Norway) . . . . . . . . . . . . . . Descending spinal sympathetic p~it3away utilized by somoto-sympathetic reflex and carotid chemoreflex ...... by P. SZULCZYK(Warsaw, Poland) . . . . . . . . . . . . . . . . . . . . . . . . . . . . p-Chlorophenylalanine hyperthermia in a warm environment: reversal with 5-hydroxytryptophan by M. J. CRONIN (Los Angeles, Calif., U.S.A.) . . . . . . . . . . . . . . . . . . . . . . . Inhibitors of protein synthesis and memory: dissociation of amnesic effects and effects on adrenal steroidogenesis by L. R. SQUIRE,S. ST. JOHN AND H. P. DAVIS(San Diego, Calif., U.S.A.) . . . . . . . . . . . . Structural changes in vitro of isolated nerve endings. I. Effect of cations by F. HAj6S AND A. CSILLAG(Budapest, Hungary) . . . . . . . . . . . . . . . . . . . . . Labeling of electrical surface changes at synaptosome membrane: an electron cytochemical and biochemical study by A. FERIA-VELASCO,S. SANCHEZ-DE-LA-PE~AAND V. MAGDALENO(Mexico, D.F., Mexico) . . . .
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