J. Cell Sci.

13, 237-255 (1973)

Printed in Great Britain

237

HISTOCHEMICAL AND BIOCHEMICAL

DEMONSTRATION OF SIALIC ACID AND
SULPHATE IN VESICLES AND MEMBRANES
ISOLATED FROM NERVE ENDINGS OF
RAT BRAIN
RUTH MARX, ELKE GRAF AND W. WESEMANN
Department of Biochemistry, Philipps-University, 355 Marburg, Lahnberge, Germany

SUMMARY
The reaction of colloidal iron hydroxide (CIH) with acidic groups was applied for an ultrastructural study of the distribution of sulphuric acid monoesters and sialic acid in synaptic
vesicles and external nerve ending membranes isolated from rat brain. At p H 1-7 C I H was
precipitated as electron-dense granules with a uniform size of 67 nm specifically labelling the
carboxyl group of sialic acid and the sulphate group of monoesters of sulphuric acid. T h e
differentiation of these 2 groups was achieved by treatment with neuraminidase and methylation followed by saponification. After preincubation with neuraminidase, which released 90
100 % of the sialic acid from the membranes of the synaptic vesicles and the nerve endings, the
electron-dense deposits marked the reaction sites of sulphate with CIH. The sulphate groups
which were present at a concentration of 2-3 and 2-2 /jmol/mg protein for the synaptic vesicle
and nerve ending membrane preparations, respectively, were rendered soluble as methyl
monosulphate by trans-esterification with acid/methanol and quantitatively removed from the
structures. By this treatment membrane-bound sialic acid was blocked as sialic acid methyl
ester and partly split off by acid hydrolysis. About 55 % of the sialic acid found in the nerve
ending membranes remained attached to the structure as compared with about 35 % of the
sialic acid of the vesicles. The acid-resistant proportion of the sialic acid could be localized with
CIH after saponification of the esterified preparations. The method described allows the
electron-microscopical demonstration of acid-resistant, neuraminidase-sensitive sialic acid in
synaptic structures and the discrimination from sulphated mucopolysaccharides.

INTRODUCTION

Substances containing sialic acid are now considered as common constituents of

membranes of mammalian cells fulfilling structural as well as dynamic functions
(Martinez-Palomo, 1970). In the central nervous system sialoglycolipids and sialoglycoproteins appear to be of significance for synaptic transmission as it was shown
that these substances restore the electrical excitability of inactivated brain tissue
(Mcllwain, 1963) and possess binding capacity towards transmitter molecules
(Wesemann, Henkel & Marx, 1971) and cations such as Na+, K+, and Ca2+ (Derry &
Wolfe, 1967; Spence & Wolfe, 1967).
It is well established by biochemical methods that, besides gangliosides, a large
portion of the glycoproteins found in brain is associated with subcellular structures of
the nerve endings (Brunngraber, Dekirmenjian & Brown, 1967). Distribution studies

238

R. Marx, E. Graf and W. Wesemann

suggest that, compared with other neuronal membranes, nerve endings contain a
higher percentage of glycoproteins relative to that of gangliosides (Dekirmenjian,
Brunngraber, Johnston & Larramendi, 1969). Application of histochemical methods
using ruthenium red and the periodic acid-Schiff test at the ultrastructural level
reveals that carbohydrates are especially present at the synaptic cleft (Rambourg &
Leblond, 1967; BondarefF, 1967a, b). In the present study a further characterization
and differentiation of the carbohydrate-rich cell coat of synaptosomal fractions was
achieved by marking the sulphate and/or sialic acid carboxyl groups of glycolipids,
glycoproteins, and acid mucopolysaccharides with colloidal iron hydroxide (CIH).
Since the 5-hydroxytryptamine (serotonin) receptor of the synaptic vesicles (SV)
and of the nerve ending membranes (external synaptosome membranes, ESM) is very
likely a structure-bound sialoglycolipid or sialoglycoprotein (Wesemann et al. 1971),
the demonstration of sialic acid at the ultrastructural level can be used to localize
possible sites of reaction between 5-hydroxytryptamine and the membranes.
The staining procedure with CIH is derived from Hale's method for demonstrating
acidic mucopolysaccharides in animal tissues (Hale, 1946) and was adapted to electron
microscopy (Gasic & Berwick, 1963; Wetzel, Wetzel & Spicer, 1966; Benedetti &
Emmelot, 1967; Wesemann et al. 1971). A procedure for the specific identification of
sulphuric acid monoesters and sialic acid as well as the differentiation of these 2 acids
are presented here together with the biochemical data on sialic acid and the distribution of sulphate in synaptic structures.
MATERIALS AND METHODS

Isolation
Synaptic vesicles and nerve ending membranes were isolated from rat brain (males, strain
Wistar II, 180200 g) by differential and sucrose-density gradient centrifugation (Wesemann
et al. 1971). Vesicles obtained from the 0'2 and 0 3 M sucrose layer and membranes enriched
in the i-o and 12 M sucrose layer of the gradient were used throughout the experiments.

Analysis
The fractions obtained were analysed for 5-hydroxytryptamine fluorimetrically (Snyder,
Axelrod & Zweig, 1965). Sialic acid was assayed with thiobarbituric acid (Warren, 1959) after
1 h hydrolysis with 0 1 N H.SOj at 80 C with jV-acetyl-neuramin-lactose (Sigma Type I) as
reference or after treatment with purified Vibrio cholerae neuraminidase (mucopolysaccharide
A^-acetylneuraminyl-hydrolase, E.C. 3 . 2 . 1 . 18), activity 500 U./ml, from Behringwerke,
Marburg, Germany (Wesemann et al. 1971). In the latter case, iV-acetylneuraminic acid
(m.p. 181-183 C; [a]ff = 32 ; o-i % H 2 O) prepared according to Wesemann & Zilliken
(1966) was used as a standard in the thiobarbituric acid assay. Sulphate was assayed turbidimetrically after 8 h hydrolysis with 6 N formic acid at 100 C (Dodgson, 1961). Protein was
measured according to Lowry, Rosebrough, Fair & Randall (1951).

Esterification and saponification

The preparations of SV and ESM were once washed with methanol and centrifuged at
145000 gu^/30 rain before esterification. The pellets were resuspended in 40 ml acidified
methanol (01 N HC1 in methanol) and heated at 60 C for 18 h (Wetzel et al. 1966). The
esterified fractions were centrifuged and the pellets obtained were treated with CIH either
directly or after saponification with alkaline ethanol ( 1 % KOH in 8 0 % ethanol, room
temperature, 25 min) followed by recentrifugation (Spicer & Lillie, 1959).

Sialic acid and sulphate in nerve endings

239

Colloidal iron hydroxide

The colloidal iron hydroxide solution was prepared according to Muller (1955) and stored at
room temperature for 1-3 weeks before use. The staining solution (Hale's stain) was obtained
by diluting the dialysed CIH with water and adjusting it to pH 17 with glacial acetic acid
(Benedetti & Emmelot, 1967). To eliminate coarse aggregates, the solution was finally centrifuged under the same conditions as were the isolates during the treatments. For the staining
procedure, the fractions were washed twice in 5 % acetic acid, incubated in freshly prepared
staining solution of CIH for 1 h at room temperature and washed 3 times in 5 % acetic acid.
Controls were incubated for the same time at pH 1-7 in a solution in which CIH was replaced
by an equivalent amount of water. For washing and incubation the pellets were resuspended,
and every step was followed by centrifugation at 145000 gmnl/3o min.
Electron microscopy
The pellets were prefixed in 3 % glutaraldehyde in 01 M phosphate buffer, pH 7-2, at 4 C
for 30 min and washed in several changes of the same buffer overnight. After staining with
CIH the final pellets were postfixed in osmium tetroxide in the same buffer at 4 C for 1 h,
dehydrated in a graded series of ethanols and embedded in Epon.
In the experiments with neuraminidase, the enzyme treatment was performed prior to
fixation in glutaraldehyde. In the case of methylation the material was usually fixed in glutaraldehyde before the blocking treatment. In some series of experiments,fixationin glutaraldehyde
was completely omitted. Immediately after isolation controls werefixedin 1 % osmium tetroxide
in veronal acetate buffer for 1 h in the cold, with or without prefixation in glutaraldehyde.
Sections were cut on a Reichert ultramicrotome 0mU2, stained with uranyl acetate for 1 h
or with lead citrate for 20 min, and examined with a Siemens Elmiskop 101.
RESULTS
Morphology

The electron micrographs in Figs. 1-4 show 2 vesicle and 2 membrane fractions
isolated from the nerve endings of total rat brain. These 4 fractions have the highest
serotonin content and the highest serotonin-binding capacity of all brain fractions
tested so far (Wesemann, 1969; Wesemann et al. 1971).
The synaptic vesicles (SV) isolated from the 0-2 M sucrose layer of the gradient
(Fig. 1) exhibit various diameters ranging from about 50 to 130 nm with maximum
incidence at about 75 nm (Wesemann, 1969). These vesicles are bound by a single
membrane which, at higher magnification, appears triple-layered showing the typical
unit membrane structure with 2 opaque lines separated by an electron-lucent layer
(Fig. 5). Some of the vesicles seem to have an electron-dense core. The triple-layered
structure was also seen in vesicles still enclosed in isolated synaptosomes.
In the 0-3 M sucrose layer vesicles of more varying sizes were found (Fig. 2).
In the membrane fractions, the external synaptosome membranes (ESM) are enriched. The membranes obtained from the i-o M sucrose layer are demonstrated in
Fig. 3. The large vesicular structures with their round or more irregular shapes represent the membranes; they also reveal a triple-layered structure at higher magnification
(Fig. 6).
In the i-2-M sucrose layer, besides membranes, some myelin-like structures and
free mitochondria are present as well as synaptosomes which contain small vesicles
and mitochondria (Fig. 4).

240

R. Marx, E. Graf and W. Wesemann

Histochemistry and biochemistry

In the histochemical and biochemical experiments all 4 fractions were examined.
From the morphological point of view, the results are very similar within the 2 SV
fractions and within the 2 ESM fractions, so that the electron micrographs shown
here may be restricted to the 0-2 M and i-o M fractions respectively, without any loss
of information. In the comparative biochemical analysis for sialic acid and sulphate
the 2 SV fractions on the one side and the 2 ESM fractions on the other side were
combined.
Colloidal iron hydroxide. After treatment with CIH electron-dense granules of iron
hydroxide are deposited on the membranes of the SV and ESM fractions. These
granules have a uniform size of about 6-7 nm and are arranged along the membranes.
In the SV, the deposits are located around the circumference (Figs. 7, 8); in the
ESM, the granules are distributed more irregularly with focal accumulations (Figs. 9,
10). Strings of single iron granules along a membrane as shown in Fig. 10 are rarely
seen. Obliquely cut membranes are seldom seen, since their shapes are irregular or
smaller than the thickness of the sections. In most cases a picture of dense particles
superimposed from different planes is obtained. In some areas the deposits are
arranged in a hexagonal pattern.
The treatment with CIH has some unfavourable effects. The vesicles and the
membranes do not maintain their original shapes, but are deformed and may adhere
to one another; furthermore the contrast in the electron micrographs is diminished.
The best results are found with material completely unfixed before treatment with
CIH. In preparations prefixed in glutaraldehyde, the granular deposits appear denser
and less regular.
Differentiation between sialic acid and monosulphate. CIH reacts with acidic groups
forming a precipitate of electron-dense material. At the low pH of 1-7 where the
staining procedure is carried out only sialic acid and monosulphates give a reaction
with CIH. The electron-dense granules in the micrographs are considered as marking
the reaction sites of the carboxyl group of sialic acid and the sulphate group of monoesters of sulphuric acid with CIH. Both groups are present in the SV and ESM membrane material as shown by the following experiments. To ensure the specificity of
the staining, blocking reactions and enzyme treatments combined with biochemical
analyses are employed.
Methylation. Esterification by acidic methanol is used to block the anionic groups.
After this treatment the reactivity of the SV and ESM preparations towards CIH is
almost totally lost (Figs, n , 12). Only random deposits at the marginal zones are
visible, which may be due to contamination with substances solubilized during
methylation and not eliminated during the washing procedure. The morphological
integrity of the structures is damaged, especially in the SV. The destructive effect of
methylation is less serious in unfixed material.
The 2 groups that are to be discriminated are affected by the methylation procedure in different ways. The carboxyl group of sialic acid becomes esterified and
should remain attached to the membrane structures. By analysis, between 35 and

Sialic acid and sulphate in nerve endings

241

Table 1. Release of sialic acid (N-acetylneuraminic acid) from nerve ending membranes
and vesicles after successive esterification, saponification and neuraminidase treatment
Total and neuraminidase-sensitive sialic acid was measured after 1 h hydrolysis
with o-i N H,SO4 at 80 C using the thiobarbituric acid assay of Warren (1959). The
same method was applied to analyse sialic acid released after incubation with Vibrio
cholerae neuraminidase, 500 U./ml (1 ml enzyme/i-1-1-4 m g protein) at 37 C and
pH 5-5 (0-05 M Na-acetate, 0-9 % NaCl, o-i % CaCl2) for 14 h. The amount of sialic
acid released by the various successive treatments is expressed as percentage of the
total sialic acid content before esterification. The figures in parentheses give the
percentage of sialic acid found after acid hydrolysis of the esterified and saponified
preparations. Mean values of 4 experiments with the standard deviations are stated.
Ultrastructure

Esterification
supernatant, %

Synaptic vesicles
Nerve ending membranes

6713
44 1

Saponification
supernatant, %
3i
8 05

Neuraminidase Recovery,
supernatant, %
%
15 1(17 2)
292(29i)

87
81

Table 2. Content of sulphate in nerve ending membranes and vesicles before and
after esterification
Total sulphate was measured turbidimetrically after 8 h hydrolysis with 6 N formic
acid at 100 C. After trans-esterification with methanol, sulphate was assayed in the
supernatant following hydrolysis of the soluble methyl sulphate with 2 N HC1 at
100 C for s h. Mean values with standard deviations are stated. The number of
experiments was 16 before and 4 after esterification. Recovery of sulphate after
esterification and hydrolysis of the supernatant is given in parentheses.
Sulphate, /tmol/mg protein
After esterification
Ultrastructure
Synaptic vesicles
Nerve ending membranes

Total content

Supernatant

Residue

2-3 0-2
2-2 0-2

2-1 0-1(92%)
I-9 O-2(86%)

0
O

55 % of the sialic acid is found associated with the membrane material, the rest is
removed and found in the methanol supernatant after centrifugation (Table 1). The
sulphate groups are split off by trans-esterification as soluble methyl sulphate. From
the total sulphate content of 2-3 and 2-2 /tmol/mg protein nothing is recovered from
the pellets of the SV and ESM fractions after methylation and centrifugation (Table 2).
In order to hydrolyse the methyl sulphate, the clear supernatants are evaporated in
vacuo and heated in 2 N HC1 at 100 C for 5 h. The sulphate content in the supernatant of the SV preparations corresponds to 2-1 /imol/mg protein of the desulphated
SV pellet or 92 % of the theoretical value. The corresponding yield for the ESM is
1-9 /fmol/mg protein or 86 %.
Saponification. The reactivity towards CIH is partially restored by alkaline hydrolysis of the sialic acid methyl ester (Figs. 13, 14), since free carboxyl groups of
sialic acid still ketosidically bound to the membranes are available for the reaction
l6

CEL

13

242

R. Marx, E. Graf and W. Wesemann

Table 3. Analysis of static acid [N-acetylneuraminic acid) in subcellular fractions

from rat brain
Total and neuraminidase-sensitive iV-acetylneuraminic acid was measured as
described in the legend to Table 1. Mean values are stated, standard deviations,
with numbers of experiments in parentheses.
nmol iV-acetylneuraminic acid/mg protein

Ultrastructure
Synaptic vesicles 0 2 M sucrose
Synaptic vesicles 0-3 M sucrose
Nerve ending membranes 1 -o M sucrose
Nerve ending membranes 1-2 M sucrose

Hydrolysis with
o-i N H.SO4
A

,
22-2 i-o (10)
2i'i2'4(io)
i4'53'2(7)
III2-7(6)

Incubation with
neuraminidase
*
20-6 3 5 (10)
2i-i2s(io)
14-414-1(7)
8-2i'3(6)

93 %
100%
99%
93%

with CIH. More electron-dense granules are present in the ESM than in the SV
fraction. This corresponds well with the analytical findings (Table 1) that only 52%
of the total sialic acid of the ESM is released by esterification and saponification and
found in the supernatants (columns 2 and 3 of Table 1) as compared with 70% for
the SV.
Neuraminidase. To ensure the specificity of the reaction with CIH, SV and ESM
fractions are treated with neuraminidase either directly (a) or after esterification and
saponification (b).
(a) The enzyme releases 93-100% of the total amount of sialic acid when freshly
prepared fractions are incubated (Table 3). The number of iron granules is reduced
(Figs. 17-20). The difference in CIH staining before and after enzyme treatment is
obvious when series of pictures are compared. They are not significant when single
micrographs are examined. The loss of reactive sites seems to be higher in the ESM
fractions. The preparations exhibit the same distribution pattern of electron-dense
deposits before and after enzyme incubation.
(b) To confirm that sialic acid is the only CIH-reactive group of the desulphated
preparations they are incubated with neuraminidase after esterification and subsequent
saponification. The total amount of sialic acid left after this pretreatment is assayed
after acid hydrolysis with o-i N H 2 S O J . Table 1 shows that sialic acid remaining after
methylation and saponification is almost completely removed by incubation with
neuraminidase. Only minor amounts - 2 % - are found in the vesicle-containing
residue of the enzyme treatment. The low recoveries of 87 and 8 1 % , respectively,
are probably due to degradation of sialic acid by acid or alkali. The analytical data
agree well with the morphological finding that the number of the electron-dense
granules is decreased (Figs. 15, 16). The few iron deposits still present are randomly
distributed and seem to be unspecific. In contrast to this, the numerous iron deposits
visible in electron micrographs of freshly prepared fractions which were directly
incubated with the enzyme (Figs. 17-20) are ascribed to the sulphate groups.

Static acid and sulphate in nerve endings

243

DISCUSSION

It was previously shown that sialic acid and monoesters of sulphuric acid are
localized in synaptic structures (Wesemann et at. 1971). According to our results, the
reaction of CIH at pH 17 is restricted on the carboxyl group of sialic acid (pK = 2-6)
and the sulphate group of monoesters of sulphuric acid (pK = 1-9) while the dissociation of other possible membrane components like phosphate with a pK2 of 7-2
is not sufficient to give a reaction with CIH. Omission of the prussian-blue reaction
(Hale, 1946) and ultracentrifugation of the CIH solution prior to the staining procedure prevents the formation of coarser particles and aggregates described in the
literature (Wetzel et al. 1966; Benedetti & Emmelot, 1967; Halaris & Jatzkewitz,
1969). Thus fine electron-dense granules with a uniform size of 6-7 nm are deposited
on the membranes of SV and ESM marking the reaction sites of the acidic groups
with CIH. Histochemistry and biochemical analyses are in accordance with regard to
the determination of sulphuric acid monoesters. The number of iron granules observed on electron micrographs of SV and ESM is diminished when the staining
procedure is applied after esterification and saponification. By this treatment structurebound sulphate is released by means of trans-esterification and found in the methanol
supernatant. The almost quantitative removal of sulphate by acid/methanol agrees
well with the findings of Kantor & Schubert (1957), who showed in a detailed
analytical study that isolated chondroitin sulphate can be desulphated with methanol
without serious destruction of the carbohydrate moiety. Since the second CIHreactive substance of the membranes - the sialic acid - can be removed enzymically
the sulphate-containing structures can be localized on the membranes of SV and
ESM after incubation with neuraminidase. The histochemical and chemical identification of sulphate in the synaptic membranes is in accordance with data published
by Vos, Kuriyama & Roberts (1969) and Kuriyama & Okada (1971). These authors
described the occurrence of sulphated acid mucopolysaccharides, predominantly
chondroitin sulphate, in subcellular fractions of nerve endings from mouse brain
with a remarkable higher concentration in the synaptic vesicles than in the whole
synaptosome. Though preincubation with neuraminidase and staining with CIH at
pH 17 is a suitable procedure for the specific demonstration of sulphuric acid monoesters in synaptic membranes, it cannot be excluded that during the isolation procedure an association of the membranes with acid mucopolysaccharides from some
other source, as for instance from the intercellular space, causes an artifact (Vos
et al. 1969; Martinez-Palomo, 1970).
Benedetti & Emmelot (1967) described a method for the specific staining of sialic
acid in plasma membranes of rat liver with CIH. The specificity of the method
employed was proved by the complete loss of CIH-reactive sites after incubation
with neuraminidase which released about 70 % of the total amount of the sialic acid.
Applying the CIH staining to subcellular fractions isolated from guinea-pig brain,
Halaris & Jatzkewitz (1969) found an intense reaction with the membranes of SV and
nerve endings. In contrast to the results obtained with liver plasma membranes,
incubation with neuraminidase failed to remove the CIH-reactive sites. According to
16-2

244

R- Marx, E. Graf and W. Wesemann

our results the positive reaction of CIH at pH 1-7 after the complete removal of sialic
acid is due to the sulphate content of the nerve ending structures. To confirm that,
besides sulphate, sialic acid is the only CIH-reactive group of the desulphated
preparation, sialic acid is split off by incubation with neuraminidase which releases
almost 100% of the total. The analytical findings that after esterification, saponification, and incubation with neuraminidase neither sulphate nor sialic acid could be
detected in the membrane preparations are in agreement with the histochemical result
that after these various treatments no specific precipitate of CIH could be demonstrated. Contrasting with liver plasma membranes, only after esterification and
saponification can sialic acid-containing substances be localized with CIH in nerve
ending membranes and vesicles. Since the ketosidic linkage of sialic acid can be split
by acid hydrolysis (Svennerholm, 1956; Zilliken & O'Brien, i960), it is not surprising
that after esterification a high percentage of the total sialic acid is found in the supernatant. As some neuraminosidic linkages are hardly affected by dilute acid (Karkas &
Chargaff, 1964; Gibbons, 1963), it cannot be excluded that only sialic-acid-containing
compounds more resistant to the acid treatment are localized by CIH after esterification and saponification. The different amounts of sialic acid released from SV and
ESM during the treatment with methanol may reflect a difference in composition or
accessibility of the sialic-acid-containing components of the 2 structures. This
assumption would be in favour of results first described by Whittaker (1966) that in
the ESM sialic acid is incorporated into glycolipids, like gangliosides, and into glycoproteins; in the SV, however, gangliosides are absent (Whittaker, 1966; Wiegandt,
1967; Wesemann, 1969). Since sialic acid and gangliosides are incorporated in nerveending membranes (Barondes, 1968; Halaris & Jatzkewitz, 1969) the ultrastructural
Iocali2ation in synaptic membranes fits well into the concept that sialic acid-containing
substances form an integral part of the membrane and have a dynamic function at the
process of neurotransmission.
The authors are grateful to Professor P. Emmelot, Amsterdam, and Professor W. Vogell,
Konstanz, for valuable discussions. They also wish to thank Miss A. Behle-Schalk and Miss R.
Henkel for excellent technical assistance.
Professor H. G. Schwick, Behringwerke, Marburg, kindly provided purified Vibrio cholerae
neuraminidase.
This work has been supported by grants from the Deutsche Forschungsgemeinschaft.

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(Received 10 October 1972)

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R. Marx, E. Graf and W. Wesemann

Figs. 1-4. Synaptic structures, isolated from nen'e endings of rat brain by sucrose
density-gradient centrifugation. Double fixation in glutaraldehyde and osmium
tetroxide, lead citrate staining, x 50000.
Fig. 1. Synaptic vesicles isolated from 0-2 M sucrose.
Fig. 2. Synaptic vesicles isolated from 0-3 M sucrose.
Fig. 3. Nerve ending membranes (external synaptosome membranes) isolated from
1 -o M sucrose.
Fig. 4. Nerve ending membranes isolated from 1-2 M sucrose.

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R. Marx, E. Graf and W. Wesemann

Figs, 5, 6. Isolated synapdc structures. Doublefixation,lead citrate staining, x 250000.

Fig. 5. Synaptic vesicles (0-2 M sucrose), showing the triple-layered structure cf the
surrounding membranes.
Fig. 6. Nerve ending membranes (i-o M sucrose).

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Figs. 7-10. Reaction of synaptic structures with colloidal iron hydroxide. Electrondense granules are deposited on the structures. No prefixation. Fixation in osmium
tetroxide, post-staining with uranyl acetate.
Fig. 7. Synaptic vesicles (o-2 M sucrose), x 80000.
Fig. 8. Synaptic vesicles (02 M sucrose), x 250000.
Fig. 9. Nerve ending membranes (10 M sucrose), x 80000.
Fig. 10. Nerve ending membranes (10 M sucrose). Individual granules of colloidal
iron hydroxide with diameters of 6-7 nm are shown to be arranged along the membranes (arrow), x 250000.

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Fig. 11. Synaptic vesicles (0-2 M sucrose) stained with CIH after treatment with
acid/methanol. The electron-dense granules are practically absent. No prefixation.
Fixation in osmium tetroxide, post-staining with uranyl acetate, x 250000.
Fig. 12. Nerve ending membranes (I-OM sucrose) treated and stained as described
for Fig. 11. Only unspecific precipitates of CIH can be observed, x 250000.
Fig. 13. Synaptic vesicles (0-2 M sucrose) after esterification with acid/methanol and
alkaline saponificarion. The reactivity towards CIH is partly restored. Fixation as for
Fig. 11. x 250000.
Fig. 14. Nerve ending membranes (1 -o M sucrose) stained with CIH after esterification
and saponification. The restored reaction sites show focal accumulation of electrondense granules. Fixation as for Fig. 11. x 250000.
Fig. 15. Isolated synaptic vesicles (0-2 M sucrose) stained with CIH after incubation
with neuraminidase following methylation and saponification. The number of
electron-dense granules is significantly reduced. Fixation as for Fig. 11. x 250000.
Fig. 16. Nerve ending membranes (i-o M sucrose) stained with CIH after pretreatment
as described for Fig. 15. The reactivity towards CIH is almost totally lost, x 250000.

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& Marx, E. Graf and W. Wesemann

Figs. 17-20. Reaction of synaptic structures with colloidal iron hydroxide after
treatment with neuraminidase. The number of CIH granules is diminished. No
prefixation. Fixation in osmium tetroxide, post-staining with uranyl acetate.
Fig. 17. Synaptic vesicles (02 M sucrose), x 80000.
Fig. 18. Synaptic vesicles, x 250000.
Fig. 19. Nen'e ending membranes (i-o M sucrose), x 80000.
Fig. 20. Nerve ending membranes, x 250000.

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