Download on the effect of motor nerve degeneration on the fine

J. Cell Set. 3, 529-538 (1968)
Printed in Great Britain
529
ON THE EFFECT OF MOTOR NERVE
DEGENERATION ON THE FINE-STRUCTURAL
LOCALIZATION OF ESTERASES IN THE
MAMMALIAN MOTOR END-PLATE
B. CSILLIK AND ELIZABETH KNYIHAR
Department of Anatomy, University Medical School, Szeged, Hungary
SUMMARY
Electron-microscopic localizations of acetylcholinesterase and non-specific esterases have
been studied in the motor end-plates of the rat diaphragm, using a modified Koelle acetylthiocholine technique for acetylcholinesterase, the Holt technique for non-specific esterases (substrates: indoxyl acetate and indoxyl butyrate) and the Crevier-Belanger technique for thiolacetic acid esterases. Acetylcholinesterase is located in the pre- and post-synaptic membranes
and in the junctional folds built up by the latter. Indoxyl acetate esterase shows a homogeneous
localization in primary and secondary synaptic clefts. Indoxyl butyrate esterase is located in
the 'middle dense layer' of the synaptolemma. Thiolacetic acid esterase is predominantly
post-synaptic, accompanied by a slighter pre-synaptic activity.
Eleven days after motor nerve transection, the synaptic gutter is occupied by a large number
of collagenous fibres, and only small granular fragments of the degenerated nerve fibres remain.
These fragments are devoid of acetyl- and non-specific esterases, but exert a thiolacetic acid
esterase activity, probably demonstrating proteolytic enzymes. The post-synaptic membrane
exhibits a slightly reduced enzyme activity without any major alteration in the fine-structural
localization of acetyl- and non-specific esterases.
INTRODUCTION
Light-microscopic histochemical observations of the early 1950s revealed the unique
survival of synaptic esterases in motor end-plates after nerve degeneration. Studies of
Couteaux (1951), Kupfer (1951), Portugalov & Yakovlev (1951), Coers (1953), Savay
& Csillik (1954), Snell & Mclntyre (1956), Schwarzacher (1957), etc., established
marked species and regional differences with regard to the period of survival; yet, as
a general rule, it could be stated that Couteaux's 'subneural apparatus' and the
associated esterolytic enzyme(s) are virtuaUy independent of the innervating motor
nerve fibre, since it is only several weeks or rather several months after nerve deprivation that the subneural apparatus itself undergoes structural and enzyme-histochemical degradation.
Electron-microscopic studies of Robertson (1954), Reger (1954) and Palade & Palay
(1954) revealed the intricate system of junctional folds of the muscle plasma membrane
that build up the subneural apparatus. According to electron histocheniical studies (see
Discussion) acetylcholinesterase (AChE) and other esterases are associated with the
junctional folds. The fine-structural relation of these enzymes to the various com-
530
B. Csillik and E. Knyiliar
ponents of the junctional membrane complex could, however, hardly be established,
since these studies were performed on different muscles, using not only different
substrates for enzyme reactions but employing also different fixation and incubation
parameters, etc. Our first aim was, therefore, to demonstrate the electron histochemical
localizations of the junctional enzymes in motor end-plates of the very same muscle, in
order to decide the functional importance of the different layers of the synaptolemma.
Closely related to this first aim was the second aim of this study: we sought degenerative alterations of the neuromuscular junction in electron histochemical specimens.
A relatively brief degenerative period (i i days) was chosen for this purpose, when the
motor axons already undergo degeneration, whereas the light-microscopic appearance
of the subneural apparatus is virtually unchanged (Csillik, 1967).
MATERIAL AND METHODS
Investigations were performed on the diaphragms of albino rats. After decapitation,
abdomen and thorax were dissected. The left hemidiaphragm was exposed and a
sagittal strip of 3 mm width, containing the intramuscular course of the phrenic
nerve, was excised. Samples were pre-fixed in 10% formalin buffered with sodium
cacodylate (O-IM, pH 7-6) for 3 h at 4 °C.
Frozen sections 50 /t thick were then prepared and incubated at room temperature
in one of the following solutions:
A, for acetylcholinesterase (AChE): acetylthiocholine iodide, 7 mg; sodium acetate
(O-IM), 2-2 ml; acetic acid (O-IM), 0-3 ml; copper glycinate, o-i ml; lead nitrate
(o-1 %), o-1 ml, or uranyl acetate (o-1 %), o-1 ml. After incubation, the sections were
rinsed in distilled water (5 sec) and post-treated in a neutralized yellow ammonium
sulphide solution (2 min).
B, C, for non-specific esterases: citrate buffer (O-IM), 5 ml; distilled water, 8-5 ml;
hexazotized pararosaniline, 0-3 ml; indoxyl acetate (B) or indoxyl butyrate (C),
10 mM, 1-5 ml.
D,/or thiolacetic acid hydrolases: thiolacetic acid, o-i ml; sodium barbiturate (5 %),
io-o ml; lead nitrate (o-i %), o-i ml.
After this treatment the samples were post-fixed in Millonig's osmium tetroxide
(1 h), rinsed in Millonig's phosphate buffer (1 h), dehydrated in graded alcohols and
embedded in Durcupan (Fluka). Thin sections were obtained on an LKB Ultrotome
and inspected and photographed on Tesla 242 and 413 electron microscopes.
End-products. Reactions A and D yield metallic sulphides (copper, lead and uranyl)
exhibiting a high electron density. The end-products of reactions B and C are identical:
indoxyl coupled by hexazotized pararosaniline yields a coloured azo dye. Though
this compound is electron lucent, secondary reaction with osmic acid results in a fine,
non-granular, electron-dense end-product, as shown by Holt & Hicks (1966). The
reactions are summarized in Fig. 1.
Electron histochemistry of the motor end-plate
.CHj.CH^S.ICO.CH,
(Pb, U)
531
O-j-CO—CH,
(hexaio)
O-j-CO—CHj.CHj.CH3
(hexazo)
Fig. 1. The reactions employed to detect the different esterases.
RESULTS
Normal motor end-plates
In diaphragms with an intact innervation apparatus, light-microscopic enzyme histochemistry reveals the characteristic form and structure of the subneural apparatus.
Though the individual apparatus may vary considerably both in size and shape, the
general plan is the same: the enzyme staining outlines the synaptic gutter; that is, the
reaction appears to be confined to the post-synaptic membrane. In this respect, there
is no major difference between the enzyme localizations obtained by the four different chromogenic substrates, though indoxyl butyrate appears to stain also the axoplasm to a certain extent. The results of the light-microscopic staining techniques
are summarized in Fig. 2.
In striking contrast to the uniformity of the light-microscopic enzyme localizations,
electron microscopy reveals considerable differences in the elaborate enzyme pattern
of the motor end-plate.
Acetylcholinesterase. The activity of this enzyme is clearly confined to pre- and
post-synaptic membranes (Fig. 3). In a virtually transverse cross-section of the motor
terminal, the semicircular profile of the axon is surrounded by the geometrically
regular muscle plasma membrane. Both axolemma and muscle plasma membrane
(sarcolemma) exert AChE activity of the same intensity. Also the junctional folds,
emerging from the latter and intruding into the muscle cytoplasm, exhibit the same
degree of AChE activity. Due to a slight over-incubation, also the synaptic cleft may
contain more or less of the electron-dense precipitate. However, in those parts of the
34
Cell. Sci. 3
532
B. Csillik and E. Knyihdr
section where the reaction is weaker, the membrane-bound character of the enzyme
activity is clearly visible.
Even more striking is the membrane-bound localization of the AChE activity in
specimens fixed for a brief time only and treated according to the uranyl thiocholine
technique (Fig. 4). In such sections, due to the briefness of the fixation, the ultrastructural preservation is poor; brief fixation renders possible, however, a very brief
incubation, too, sufficient to 'stain' the starting-points of the enzyme reaction only.
These 'starting-points' (spots of the uranyl thiocholine precipitate) are presumably
identical with or located close to the active centres of AChE molecules. Their diameter
is in the range of 80-120 A; the interspaces measure 300-400 A. The number, size
and density of the 'spots' is just the same in both pre- and post-synaptic membranes,
as well as in the membranes of the junctional folds.
Non-specific indoxyl acetate esterase. In striking contrast to AChE, this enzyme
exhibits a diffuse localization in the synaptic cleft, without any tendency to be concentrated either in pre- or post-synaptic membranes (Fig. 5). In some cases, the
'middle dense layer' of the synaptolemma exerts a slightly stronger reaction than
other constituents of the synaptic gap.
Non-specific indoxyl butyrate esterase. The main localization of this enzyme, like
that of the preceding one, is in the synaptic cleft. More obviously, however, the
activity of this enzyme is concentrated in the ' middle dense layer' of the synaptolemma
(Fig. 6). In addition, a more or less intense enzyme staining can be observed within
the terminal axoplasm; this outlines mitochondria and synaptic vesicles as blank
(non-reacting) units.
Thiolacetic acid esterase. The bulk of this enzyme is located on the post-synaptic
membrane, in the form of relatively large (300—500 A) strongly electron-dense globular
particles. Smaller particles were observed at the pre-synaptic membrane, in a considerably smaller number. Scattered particles were found within the terminal axoplasm, attached to or located within synaptic vesicles (Fig. 7). A number of reaction
product particles was present also in the muscle fibres themselves, most probably
corresponding to the 'M-band enzyme' described by Barrnett & Palade (1959).
Denervated end-plates
Light microscopically, no major alteration of the subneural apparatus can be observed in this period, as described by us several years ago (Savay & Csillik, 1956).
The only alteration that can be seen is a slight 'hypersegmentation', i.e. dislocation,
of the enzyme-active gutters. Correspondingly, electron-microscopic enzyme localization of the post-synaptic membrane does not show any conspicuous changes either,
the main alterations being confined to pre-synaptic structures.
AcetylchoUnesterase. The post-synaptic system of junctional folds is virtually intact;
the intensity of the enzyme raction is slightly decreased, however. Due to axonal
degeneration, the pre-synaptic membrane (axolemma) disappears, so no pre-synaptic
AChE activity can be observed.
Within the synaptic gutter, occupied originally by the telodendrial motor axon,
Electron histochemistry of the motor end-plate
533
granules of the degenerated nerve fibres and many collagenous fibres can be observed
(Figs. 8, 9).
Non-specific indoxyl acetate esterase. The electron-dense end-product fills secondary
synaptic clefts in the same way as under normal conditions. Though in a slightly
reduced amount, precipitate can be found also in the primary synaptic cleft (Fig. 10).
Non-specific indoxyl butyrate esterase. Enzyme reaction product can be seen in both
primary and secondary synaptic clefts. Staining of the 'middle dense layer' of the
synaptolemma, characteristic of normal neuromuscular junctions, is reduced. No activity can be seen in the axoplasm of the terminal nerve fibre (Fig. 11).
Thiolacetic acid esterase. The heavy electron-dense reaction product (lead sulphide)
is present in a virtually unchanged localization on the post-synaptic membrane. Activity of the pre-synaptic membrane is virtually abolished. Granular fragments of the
degenerated axons, easily recognizable among abundant collagenous fibres within the
synaptic gutter, are surrounded by reaction-product particles; some of the fragments
contain lysosome-like bodies which also exert an enzyme reaction (Figs. 12-14).
DISCUSSION
According to the pioneering studies of Robertson (1956) the fundamental plan of
the vertebrate motor nerve ending consists of a synaptic gutter, surrounded by junctional folds; the terminal arborization of the motor axon lies within this gutter.
Essentially, the same structural organization can be observed in the extrafusal motor
end-plates of all mammalian species, including those in the diaphragm of the rat.
The relation of the ultrastructural organization to the light-microscopic structure of
the motor nerve ending has been discussed in detail recently (Csillik, 1967).
Light-microscopic histochemistry disclosed the association of acetylcholinesterase
and other less-specific esterases with the 'subneural apparatus' of Couteaux (1947)
years before the fine structure of the end-plate could have been unequivocally established. The Koelle & Friedenwald (1949) technique—in the hands of Couteaux (1951),
Coers (1953) and Gerebtzoff (1953)—as well as the indoxylacetate (Holt, 1952), the
naphthylacetate (Ravin, Zacks & Seligman, 1953; Csillik & Savay, 1953) and the thiolacetic acid techniques (Crevier & Be'langer, 1955; SaVay & Csillik, 1959) clearly
showed the localization of eserine-sensitive esterases in the non-nervous structures of
the motor end-plate, as well as the survival of esterase activity after nerve deprivation
(for a summary of such experiments, see Csillik, 1967). For the electron-histochemical
localization of end-plate esterases, various substrates and experimental parameters were
employed in various muscles, so that a clear-cut comparison of these studies is extremely
difficult. In 1959, Lehrer & Ornstein demonstrated an eserine- anddiisopropylalkylfluorophosphate-sensitive esterase in the end-plates of the mouse intercostal muscle,
using a-naphthylacetate substrate for block (in toto) incubation; the reaction product,
naphthol-hexazo red, was found in the primary and secondary synaptic clefts, as well
as in the gaps between axons and teloglial (Schwann) cells. Apart from their historical
importance, these experiments have shown for the first time that an electron-lucid
end-product of a histochemical reaction (consisting of elements exclusively of a low
534
B. CsiUik and E. Knyihdr
atomic weight: C, 0, N, H) gains sufficient electron density after osmication, probably
by an intermolecular interaction between hydrophilic moieties of the hexazo dye and
osmium ions.
Zacks & Blumberg (1961) introduced thiolacetic acid for end-plate electron histochemistry (block incubation); the reaction product yielded a very coarse precipitate,
filling both primary and secondary synaptic clefts. It has been noted, furthermore,
that central parts of the tissue blocks showed only a very slight enzyme reaction, if
any, in striking contrast to the heavy reactions observed in the superficial regions of
the samples.
Barrnett (1962) and his co-workers considerably improved the thiolacetic acid
reaction, by subjecting tissue blocks to a mild osmium tetroxide fixation prior to
enzyme ' staining'. Ultrastructural preservation became comparable to normal electronmicroscopic patterns and also the reaction product became considerably finer. Yet,
osmium pre-fixation introduced a haphazard element, unknown in its effect on enzyme
kinetics.
For the first time, Couteaux (1963) used the original Koelle thiocholine technique
for the electron-microscopic localization of AChE in motor end-plates and obtained a
fair enzyme staining at low-power level. Lewis & Shute (1964) considerably improved
the thiocholine techniques, obtaining good ultrastructural preservation and delicate
enzyme staining. In their specimens the precipitate of the AChE reaction appeared
in a membrane-bound form, exerting a medium electron density. Karnovsky (1964)
obtained a heavy electron-dense precipitate by means of his Fe-Cu-thiocholine technique ; yet when this was applied to motor end-plates the reaction product appeared
in quite a coarse form.
Based upon these experiments, we tried to introduce various heavy metals in order
to enhance the electron density of the enzyme reaction. Lead alone or lead and copper
together were found the most suitable for such purposes (Jo 6, Savay & Csillik, 1965;
Csillik, J06, Kasa & Savay, 1966; Kasa & Csillik, 1966), In special cases, when very
brief incubation periods were employed or when a very faint enzyme reaction had to
be demonstrated, combinations with uranyl ions were found useful.
In a recent publication on esterase electron histochemistry Holt & Hicks (1966) recommended the use of indoxyl acetate. In contrast to the light-microscopic esterase reaction
(Holt, 1954) that yields a crystalline diindigo, an azo-dye formation between indoxyl
and hexazotized pararosaniline was employed in the electron-microscopic procedure.
Thus, instead of the rough oxydized diindigo granules a very fine, non-granular
precipitate was obtained, with a high degree of osmiophilia. However, Holt did not
study motor end-plates electron microscopically.
In this present study, experiments were performed on the very same muscle (rat
diaphragm). In order to obtain comparable results, care was taken to minimize differences in fixation, tissue thickness, etc. All the above staining reactions were performed on formalin-fixed thick frozen sections. Though freezing in general is regarded
as a rough destructive agent, we did not encounter any serious ultrastructural disorganization in our speciments, provided the microtome knives used for the production
of frozen sections were freshly polished. (Even microscopic irregularities of the knife
Electron histochemistry of the motor end-plate
535
edge may produce imponderable structural alterations of the section surface, extending
in most cases also into the interior of the section.) The use of frozen sections makes it
possible to observe and trim end-plates under the light microscope. Formalin-fixed
frozen sections are easily permeable to the substrates and other components of the
histochemical reactions, in contrast to intact muscle fibres, which are virtually impermeable to positively charged molecules (e.g. acetylthiocholine (Ulbrecht & Kruckenberg, 1965)).
The main structural characteristic of the specific junctional enzyme, AChE, is its
definite membrane-bound appearance. Biochemical studies long ago revealed the
presence of AChE in coarse biological 'membranes', e.g. in the axolemmal sheath of
the Loligo giant nerve fibres (Boell & Nachmansohn, 1940). Experiments performed
by means of differential centrifugation (Karlin, 1965) proved that AChE is actually
bound to membrane structures, even in the microstructural sense of the word. The
first electron-histochemical studies on AChE (Lehrer & Ornstein, 1959; Zacks &
Blumberg, 1961) raised doubts about the membrane-bound character of this enzyme,
but, it appears, mainly because of the non-specificity of the substrates used in these
early experiments. Electron-histochemical reactions performed using the specific substrate (acetylthiocholine) and specific inhibitor (BW284C51 (Kasa & Csillik, 1966))
prove unequivocally that the activity of AChE 19 confined to both junctional membranes. The same localization has been obtained by the gold-thiocholine technique
(Davis & Koelle, 1965, 1967). The slight reaction observed here and there within the
synaptic cleft is, in all probability, due to diffusion or over-incubation.
The fact that both pre- and post-synaptic membranes exert the same intensity of
AChE disproves the earlier view that in the neuromuscular junction AChE activity
is located in the post-synaptic membrane—a widely adopted opinion based upon
light-microscopic studies. Undoubtedly the activity of the post-synaptic membrane
is much higher than that of the pre-synaptic one; this uneven distribution of the
enzyme activity is due, however, only to the structural plan of the junction, the surface
area of the post-synaptic membrane (junctional folds) being 10-20 times larger than
that of the pre-synaptic membrane.
While AChE is definitely a membrane-bound enzyme, the two non-specific esterases, splitting acetyl- and butyryl-indoxyl, are located within the layers of the intersynaptic gap. Acetate esterase appears to fill out the synaptic gap, whereas butyryl
esterase exhibits a clear-cut localization in the middle dense layer of the synaptic
membrane complex. Since the end-products of both enzyme reactions are identical,
the micro-topographically different localizations of these two enzyme reactions cannot
be ascribed to preferential bindings of reagents and/or end-products to special structures in the junctional tissue.
The role of the ' middle dense layer' is enigmatic since Robertson's first notion
(1956) of this slightly electron-opaque structure. It has been tentatively identified as
a 'PAS-positive ground substance' (Zacks & Blumberg, 1961). According to our
studies, the function of the middle dense layer is related to the hydrolysis of 'nonspecific' substrates, differing from acetylcholine; and the question emerges whether
such compounds may take part also in the physiological events of impulse trans-
536
B. Csillik and E. Knyihdr
mission. On the other hand, indoxyl butyrate esterase appears to be located also
within the pre-synaptic axoplasm. Such a localization is in full agreement with the
recent light-microscopic observation of Eranko & Teravainen (1967).
Finally, the localization of the thiolacetic acid splitting enzyme(s)—as seen in our
formalin-fixed specimens—appears to be predominantly post-synaptic. Since a slight
post-synaptic activity remains after eserine treatment too, one is inclined to believe
that this activity is, at least partly, due to proteolytic enzymes. The same holds good
also for the reaction seen in the terminal axoplasm (probably representing activity of
synaptic vesicles), the latter being observed also by Barrnett in his osmium-prefixed
specimens. The membrane reaction in osmium-prefixed specimens is smooth and
delicate, in contrast to the coarse granular precipitate in our formalin-prefixed
samples. We are inclined to believe, however, that the very vital organization of the
enzyme activity is more reliably reflected by aldehyde-fixed than by osmium-fixed
specimens, although the electron-microscopic patterns may be more attractive in the
latter case than in the former.
Therefore, the neuromuscular membrane complex ('synaptolemma') seems to consist of several layers of esterolytic (and proteolytic) enzyme proteins. The fundamental
plan of this enzyme stratification is summarized in Fig. 2. It is evident from this
pattern that non-specific esterases are virtually sandwiched in between two layers of
AChE. It is tempting to speculate on the possible role of these cascade-connected
enzymes, the physiological substrates of which are unknown, except for AChE. One
cannot escape the idea that chemical events resulting in the hberation and enzymic
degradation of acetylcholine take place in both pre- and post-synaptic membranes
(as proposed years ago by Nachmansohn, 1955), whereas the enzymes present in the
synaptic gap (especially the butyryl esterase of the 'middle dense layer') play some
part in the processes that link pre-synaptic events to post-synaptic ones. Such an
enzyme organization appears to be a general phenomenon, not restricted to motor
end-plates. Preliminary experiments in the cerebellar cortex prove that AChE is
located in pre- and post-synaptic membranes of mossy fibre/granule cell dendrite
junctions, whereas indoxyl esterase is located within the narrow synaptic gap.
In full accordance with light-microscopic histochemical studies, electron histochemistry reveals the survival of junctional esterases after motor nerve degeneration.
Specific AChE activity of the pre-synaptic membrane, however, disappears due to
the degradation of the axon. Non-specific esterases remain virtually intact, especially
those located within the secondary synaptic gaps. Some activity remains in the
primary gap, too, proving that non-specific esterases are attached to the external
surfaces of the junctional membranes. Most striking is the behaviour of thiolacetic
esterases. Their survival in the post-synaptic membrane could be anticipated already
on the basis of the survival of the membrane itself. Strangely enough, however, a
quite strong enzyme activity appears on the surfaces of the granular remnants of the
degenerating axon also; and since such structures do not show up in the indoxyl
esterase patterns, one has to conclude that this staining is due to the activity of proteolytic enzymes (cathepsin C).
The fate of the synaptic gutter can easily be envisaged on the basis of the above
Electron histochemistry of the motor end-plate
537
patterns. The fragments of the degenerating axon undergo proteolysis and, at the
same time, the space left behind is filled by collagenous fibres that grow into the
abandoned gutter. It appears, however, that these collagenous fibres do not hinder
regenerating axons in finding their way into the abandoned gutters, at least in early
states after degeneration (Csillik & Savay, 1958).
Authors are grateful to Dr G. Kalman for his help in the chemistry of the histochemical
experiments. Fig. 3 has been obtained in the course of other, unpublished, experiments, carried
out in collaboration with Dr F. J06. We are deeply indebted to Dr H. Hamori (Department of
Anatomy, Budapest) for his help in using the Tesla 413 electron microscope.
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SAVAY, G. & CSILLIK, B. (1959). BeitrSge zur Methodik der histochemischen CholinesteraseReaktion. Acta histochem. 6, 307-312.
SCHWARZACHER, H. G. (1957). Der histochemisch nachweisbare Cholinesterasegehalt in Muskelendplatten nach Durchschneidung des motorischen Nerven. Acta anat. 31, 507-521.
SNELL, R. C. & MCINTYRE, N . (1956). Changes in the histochemical appearances of cholinesterase at the motor end plate following denervation. Br. J. exp. Path. 37, 44-48.
ULBRECHT, G. & KRUCKENBERG, P. (1965). Acetylcholinesterase in the sarcoplasmic reticulum
of skeletal muscle. Nature, Lond. 206, 305-306.
ZACKS, S. I. & BLUMBERG, J. M. (1961). Observations on the fine structure and cytochemistry
of mouse and human intercostal neuromuscular junctions. J. biophys. biochem. Cytol. 10,
517-528.
(Received 4 December 1967)
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D
Fig. 2. Light-microscopic appearance of the subneural apparatus in rat diaphragm.
Four motor endings are depicted from specimens treated according to various histochemical procedures (A, acetyl thiocholine; B, indoxyl acetate; c, indoxyl butyrate;
D, thiolacetic acid). All of these reactions 'stain' the junctional membrane complex,
x 800.
B. CSILLIK AND E. KNYIHAR
(Facing p. 538)
Fig. 3. Electron-microscopic localization of acetylcholinesterase. The axon appears in
a virtually transverse section. The nearly geometrically regular semicircular profile of
the motor terminal contains ghosts of synaptic vesicles (sv) and mitochondria (m).
Enzyme reaction is confined to pre- and post-synaptic membranes and to the elongated
junctional folds (Jf) of the latter. A slight activity can be seen in the synaptic cleft;
yet the main bulk of this enzyme is definitely membrane-bound. Two fundamental
nuclei (Jn) and several fundamental mitochondria (fm) are present in the postjunctional sarcoplasm. Cu-Pb—Thiocholine technique.
Fig. 4. Electron-microscopic appearance of acetylcholinesterase, after brief fixation
and brief incubation, using the uranyl thiocholine technique. Axoplasmatic structure
(a) is deteriorated, yet the granular appearance of the reaction product on pre- and
post-synaptic membranes and on the membranes of the junctional folds (Jf) is distinct.
The outlines of two fundamental nuclei (fn) appear in the section.
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Fig. 5. Electron-microscopic localization of non-specific esterase (indoxyl acetate
substrate). The axon containing synaptic vesicles (sv) is surrounded by the intricate
system of junctional folds (jf); enzyme reaction fills the primary and the secondary
(arrow) synaptic clefts. Two fundamental nuclei (Jn) and several fundamental mitochondria (jm) are present in the post-junctional sarcoplasm. No enzyme reaction can
be observed in the myofibrils (my) or in the muscle mitochondria (mm).
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B. CSILLIK AND E. KNYIHAR
Fig. 6. Electron-microscopic localization of non-specific esterase (indoxyl butyrate
substrate). A slight enzyme reaction can be seen within the terminal axoplasm (a),
surrounding synaptic vesicles (sv) and mitochondria (m). The bulk of the reaction is
confined, however, to the ' middle dense layer' of the synaptolemma, both in the
primary and secondary synaptic clefts (arrows and double arrows, respectively). The
junctional folds (jf) intrude into the post-junctional sarcoplasm, containing several
mitochondria (Jm), ribosomes (r) and tubular structures, probably terminal elongations of the sarcotubular system.
Fig. 7. Electron-microscopic localization of thiolacetic acid esterase. In the profile of
the terminal axon, reaction product is confined to fine granular units (synaptic
vesicles, sv), and to the pre-synaptic membrane (pre). A heavy electron-dense deposit,
consisting of larger granular units, occurs on the post-synaptic membrane (post), most
conspicuously in the junctional folds (jf). Note the presence of a slight reaction in the
M-band (arrow).
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Fig. 8. Electron-microscopic localization of acetylcholinesterase in a denervated endplate. Enzyme activity, though slightly decreased, is present in the post-synaptic membrane (post), especially conspicuous in the junctional folds (Jf). Pre-synaptic activity
has disappeared. The synaptic gutter is filled by collagenous fibres (coll). Note the
accumulation of ribosomes, attached to the post-synaptic membrane (arrows, r).
Uranylthiocholine technique.
Fig. 9. Electron-microscopic localization of acetylcholinesterase in a denervated endplate. Enzyme reaction is present in the post-synaptic membrane (post); no activity can
be seen at the surfaces of the granular remnants of the degenerated axon (arrows).
Uranyl-thiocholine technique.
Fig. 10. Indoxyl acetate esterase activity of a denervated end-plate. Enzyme activity
is still persistent in the junctional folds (if), and slight activity can be observed also in
the former primary synaptic cleft (arrows). The synaptic gutter is filled by collagenousfibres (coll). A small portion of the myofilamentous apparatus (my) can be seen.
Fig. 11. Indoxyl butyrate esterase activity of a denervated end-plate. The activity of
the middle dense layer is considerably decreased (mdl). The gutter contains small
granular fragments of the axon (deg) and a filamentous material.
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Fig. 12. Thiolacetic acid esterase activity of a denervated end-plate. The intricate
system of the junctional folds (jf) exerts a strong, granular enzyme activity; the synaptic gutter contains collagenous fibres (coll) and two fragments of the degenerated axon
(deg). (Jm, fundamental mitochondria.)
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Fig. 13. Thiolacetic acid esterase activity of a denervated end-plate. Enzyme activity
of the junctional folds (Jf) is virtually intact; the synaptic gutter is filled by collagenous
fibres (coll). Small fragments of the degenerated axon (marked by arrows) exert a slight
enzyme activity on the surfaces. (Jm, mitochondria in the post-junctional sarcoplasm.)
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B. CSILLIK AND E. KNYIHAR
Fig. 14. Thiolacetic acid esterase activity of a denervated end-plate. Enzyme activity
of the junctional folds (jf) is virtually intact. In the synaptic gutter, collagenous fibres
(coll) replaced the axon; two granular fragments of the degenerated nervefibre,however,
are still visible (deg 1, deg 2). In deg 1, mitochondria (m) and lysosomes (ly) are discernible, and several enzyme-active granules (arrows) are also present. Two vacuoles
(vac) appear in the degenerating fragment, probably in their way of disruption of the
degenerating fragment into smaller parts. In deg 2 only enzyme-active particles
(arrows) can be seen. (Jm, fundamental mitochondria; fn, part of a fundamental
nucleus.)
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