Download formalin perfusion for correlative light- and

J. Cell Sci. I, 229-238 (1966)
Printed in Great Britain
229
FORMALIN PERFUSION FOR CORRELATIVE
LIGHT- AND ELECTRON-MICROSCOPICAL
STUDIES OF THE NERVOUS SYSTEM
L. E. WESTRUM AND R. D. LUND
Department of Anatomy, University College London, Gower Street, London, W.C. 1
SUMMARY
The formalin perfusion technique of Pease has been shown to be a satisfactory fixation
method for both light and electron microscopy of the nervous system of rat and goldfish, so
allowing correlative studies to be made on tissue from the same region of one brain.
Immediate post-osmication is not necessary for good ultrastructural preservation and even
if it is delayed for 1 week, neuronal structure is adequate for all but detailed cytological work.
Unosmicated tissue stained with uranyl acetate and lead citrate showed protein-like structures
but not lipids, with membranes appearing as unstained bands. 'Dark' glial cells occurred near
the surface of the cortex and could not be eliminated by prolonged fixation in situ. ' Dark'
neurons were found rarely.
It is suggested that, with this method of fixation, any poor preservation of tissue in direct
light- and electron-microscopical correlative studies is due to the subsequent processing rather
than the initial fixation. Preliminary results on material stained by a new Golgi-Kopsch
modification and by reduced silver methods and subsequently examined with the electron
microscope suggest that such damage is not extensive.
INTRODUCTION
In studies of the normal and degenerating nervous system it is often essential that
the details found by electron microscopy should be correlated with those found by
the standard techniques of light microscopy. Such problems as, for example, deciding
what type of cell lies post-synaptically to a degenerating bouton seen under the electron
microscope, or to what extent the Nauta method may be considered to show terminal
degeneration, are typical difficulties encountered. The main obstacle to correlative
studies is that different techniques of fixation are used for light-microscope and ultrastructural studies (see, for example, Walberg, 1964), and as a result, the possibility
of differential fixation artifacts is introduced. Also, except in isolated circumstances,
it is impossible to duplicate experimental conditions exactly in any two animals.
Clearly the use of one animal fixed by one method is essential.
For the standard techniques of light microscopy of the nervous system, with the
notable exception of the Golgi methods, formalin perfusion methods are generally
used, and are considered essential for Nauta and Glees methods. Several formalin
perfusion methods exist for electron microscopy (Holt & Hicks, 1961, as used by
Walberg, 1964; Pease, 1962, 1964; Aguilar & de Robertis, 1963; Bodian & Taylor,
1963). Although these have all been developed from fixatives used for specific light
microscopy, their application to direct or indirect correlative studies in the nervous
230
L. E. Westrum and R. D. Lund
system has not been investigated. Maxwell & Kruger (1965 a) mention that the Pease
technique is suitable for Nissl and PAS staining. Guillery & Ralston (1964) found Holt &
Hicks's method at pH 7-2 suitable for the Nauta method (1957), although Cragg (1961)
has suggested that formol saline buffered at pH 6-5 is necessary for satisfactory Nauta
staining under the light microscope. Such a pH is, however, incompatible with good
ultrastructural preservation (Schultz & Karlsson, 1965). Walberg (1964) found the
Holt & Hicks procedure unsatisfactory for the Glees method. Richardson (i960) found
best preservation of Bielschowsky-stained material for light microscopy with a
buffered formalin solution, the basis of which has been adopted by Pease for his perfusion method. The principal drawback of formalin as a primary fixative for light
microscopy is the presence of dark cells, thought to be due to the slowness of fixation
as well as to pressure effects during biopsy (see Cammermeyer, 1962). It is somewhat
surprising that although similar cells have been described from electron microscopy
with standard methods of fixation (e.g. Gray, 1961 a), they have never been described
after formalin fixation by perfusion or immersion (Richardson, 1961). Does this mean
perhaps that the dark cells of electron microscopy are different from those of light
microscopy, or that a wide enough survey has not yet been made?
All the methods of fixation with formalin for electron microscopy of the nervous
system demand fairly rapid post-osmication after initial formalin fixation. Since longer
formalin fixation times are required for light-microscopical staining, post-osmication
is delayed and direct electron-microscopical study of material fixed and stained for
light microscopy would be expected to show poor preservation. However, it is not
certain to what extent the poor preservation found by Gray & Guillery (1961) with
the Bielschowsky method and Guillery & Ralston (1964) with the Nauta method is
due tofixationor to damage during staining. Direct correlation after early osmication, as
is possible with some Golgi techniques (Stell, 1964; Blackstad, 1965), circumvents these
difficulties, but such fixation is unsatisfactory for most other nervous-system stains.
The present study is intended to investigate to what extent formalin is satisfactory
as a fixative for correlative studies and to find whether any of the formalin perfusion
methods for electron microscopy are reliable, consistent and not too complex to
perform for routine fixation.
MATERIALS AND METHODS
The regions investigated in detail were the primary olfactory cortex (prepyriform
cortex) and superior colliculus of the rat, the cerebrum and optic tectum of the goldfish (Carassius auratus) and superior colliculus of the monkey.
Perfusion techniques by Aguilar & de Robertis (1963), Holt & Hicks (1961) as used
by Walberg (1964) and Pease (1962, 1964) were used, but since in the hands of the
present investigators only the last gave consistent, good preservation of ultrastructural
detail, this was used for most of the study. The fixative was prepared by adding 4 g of
paraformaldehyde (trioxymethylene, Hopkin and Williams Ltd.) to 100 ml of phosphate buffer at 60 °C; complete solution was effected by addition of 15-20 drops of
2-5 % sodium hydroxide, after which the fixative was cooled and the pH adjusted to
Fixation of nervous system
231
7*3 by addition of hydrochloric acid. For some animals the perfusate contained either
°'5 % glucose or o-oi % calcium chloride. The solution prior to perfusion was usually
at or near room temperature but in some cases was at 10-15 °C. I*1 t n e rats > anaesthetized with open ether, the perfusate was delivered by a 20 ml syringe and no. 1
needle via the left ventricle, the descending aorta being clamped and right atrium cut.
About 20 ml were delivered over 2-3 min without previous washing in most animals.
However, a few trials were carried out with larger volumes of solution given over a
longer period (40-100 ml over 5-15 min). In the monkey, under Nembutal anaesthesia, the perfusate was delivered over 15 min by gravity flow, but in the goldfish,
anaesthetized with MS 222 (Sandoz), a rapid direct perfusion was carried out. None
of the animals was artificially respired.
The brains were usually removed immediately after perfusion for electron microscopy and put into the perfusate for an additional 10-30 min, 1 h, 2 days or 1, 3, or
4 weeks at room temperature, prior to trimming and osmication. Animals perfused
with the larger volumes of fixative were allowed to remain undissected for 30 min.
In view of Cammermeyer's recommendation to reduce the effect of pressure which
causes some dark cells in light-microscope preparations (1962), two animals were
perfused with 100 ml of solution, and the brains left undissected for 4 h.
In all cases small wafers (about 0-5 mm thick) of the desired area were cut freehand
with a clean razor blade. The pieces were immersed immediately, without previous
rinsing, in 2 % osmium tetroxide, buffered at pH 7-3 with phosphate according to
Millonig (1961), and fixed for 1^-2 h at 4 °C. Dehydration was carried out in graded
solutions of acetone or ethanol. Blocks were stained in 1 % uranyl acetate or uranyl
nitrate in ethanol or acetone (Westrum, 1965) or 1 % phosphotungstic acid in ethanol
(Gray, 1959). Some blocks were not osmicated but were stored for up to 1 week in
perfusate, transferred to 1 % aqueous uranyl acetate for 2 h and dehydrated in 1 %
uranyl acetate in 50 %, 75 %, 95 % and absolute ethanol.
Araldite was used for embedding, either slowly or rapidly (see Gray, 1959; Robertson, Bodenheimer & Stage, 1963) and thin sections from a Servall ultramicrotome
were stained on copper grids in lead citrate (Reynolds, 1963; Westrum, 1965).
A Siemens Elmiskop 1 b was used at 80 kV and fitted with a 200-/1 condenser aperture
and a 30-/4 or 50-/^ objective aperture.
The material for light microscopy was left in the perfusate for 1-3 days, a few
specimens being stored for up to 1 week. The methods used thereafter on rat tissue
included Glees (1946), Nauta & Gygax (1951, 1954), Holmes (1943), Nissl (0-5%
cresyl-violet acetate) and a modification of the Golgi-Kopsch technique. The last
method is described here:
(1) Leave entire brain or pieces in perfusate for 2-3 days.
(2) Transfer small wafers (2-3 mm in thickness) to 100 ml of fresh 3-5 % potassium
dichromate.
(3) Leave at room temperature in the dark for 5-7 days.
(4) Wash in several changes of distilled water for 10-15 min.
(5) Place pieces in several changes of 0-75 % silver nitrate (freshly prepared) with
washing in distilled water between changes.
232
L. E. Westrum and R. D. Lund
(6) Leave in 0-75 % silver nitrate for 2-3 days at room temperature in a dark or
amber bottle.
(7) Wash in several changes of distilled water for £ h (this may be omitted).
(8) Dehydrate in acetone-ethanol for 2 h.
(9) Embed in celloidin and section at 100-200 fi.
Nauta-Gygax (1954), Nissl, and Golgi techniques were also used on goldfish
tissue with success.
RESULTS
Electron microscopy
The success of the fixation has been judged first by the quality of ultrastructural
preservation. The criteria of good preservation have been outlined by Palay, McGeeRussell, Gordon & Grillo (1962) and Pease (1964). In addition, special attention was
paid to the occurrence of 'dark' cells.
After rapid direct perfusion, not preceded by saline and followed by osmication
within 30 min, consistently satisfactory preservation was found throughout the tissue
examined (Fig. 1). Maximum contrast was obtained by double staining (Westrum,
1965) but unit-membrane structure was not seen in all cases. Staining with PTA for
up to 4 h was not found to be as intense with this fixation method as with immersion
techniques of fixation.
The tissue was routinely found to have uniform, uninterrupted surface, nuclear
and internal membranes, intact mitochondria and Golgi apparatus and only slightly
swollen endoplasmic reticulum as compared with immersed tissue (Fig. 2; see also
Pease, 1964). The membranes themselves, however, appeared more granular and less
crisp than when observed after immersion fixation only. Extracellular spaces were of
usual proportions as described by many authors (e.g. Palay et ah, 1962). With the
exception of the well-known tight junctions between glia (Gray, 1961 b) the membranes
did not show fusion, in contrast to the observations of Karlsson & Schultz (1965).
Irregularities of myelin (Fig. 1) were seen after uranyl staining to a greater extent than
with PTA. Neurofilaments (about 90 A in diameter) were well demonstrated (Fig. 6,
inset).
'Dark' cells were found in some preparations of the upper cortical layers of the
cerebrum (Fig. 5) but less frequently in the superior colliculus. They were characterized by dense cytoplasm and nucleus, usually intact but expanded nuclear envelope
and scalloped surface membrane. There was normally no evidence for discontinuities
or swellings of the profiles adjacent to the dark cells. Two populations were distinguished. Some occurring very infrequently in both cortex and superior colliculus
were neurons, showing subsurface cisternae and synaptic contacts on their processes.
The others, occurring more frequently and particularly in the cortex, were nonneuronal, situated in close relation either to fibre tracts or blood vessels or as satellite
cells to normal neurons. Other glial cells were found in similar locations, being the
same size with a similar nuclear pattern, high nucleocytoplasmic volume ratio, and
a considerable amount of granular reticulum. These were also found in a previous
Fixation of nervous system
233
study on the prepyriform cortex (L. E. Westrum, unpublished), but the ' dark' glial
cells were notably uncommon.
The addition of sucrose gave results little different from those described for normal
rapid perfusion, but with the addition of calcium chloride more tubules were seen in
both dendritic and axonal profiles (Fig. 3; compare Fig. 1). Longer perfusion times
gave results similar to those obtained with the shorter times. When the brain was left
undissected for 4 h after perfusion, the general standard of tissue preservation was not
as good as after rapid dissection, but the frequency of 'dark' cells in the cortex remained unaltered.
Tissue left in perfusate for 1 h before osmication showed no detectable difference
in fine structure. In that left for 1 week in perfusate there was still little difference in
the general neuronal preservation as seen in the electron microscope (Fig. 4). There
were, however, occasional interruptions in surface membranes (Fig. 4) not due to
oblique sectioning, and an apparent general reduction in intercellular space. Tight
membrane appositions were seen sometimes associated with patches of 'quintuple
layering' between profiles other than glia (Fig. 4, inset). After 4 weeks, broken membranes were more frequently seen in many structures but the general cytoplasmic
preservation was still good. The endoplasmic reticulum was more swollen than after
shorter fixation times, but synaptic structure, nuclear membranes and other details
were not much altered in comparison with tissue fixed for 1 week prior to osmication.
Glial cells showed the most severe changes with long fixation. After 1 week the
endoplasmic reticulum became swollen, giving membrane-bound sacs approaching
0-5 /i in greatest diameter in the perinuclear cytoplasm. Filaments, mitochondria
and surface membranes were normal in appearance. With fixation in perfusate prolonged for 4 weeks the glia contained large membranous 'whorls'.
The formalin-fixed unosmicated tissue, treated with uranyl acetate only, showed little
evidence of membranes. The position of membranes is marked by clear spaces bounded
by electron-dense material (Fig. 8). This is particularly evident at 'synapses' when
pre- and postsynaptic material and cleft substance is stained showing the interposed
membrane regions in negative contrast. RNP granules were heavily stained, as were the
nuclear granules. In the absence of membrane detail the general quality of preservation
was difficult to assess, but cytoplasmic organelles could be clearly recognized.
The goldfish tissue yielded exceedingly good results, with excellent fine-structural
detail. Membrane contrast was particularly enhanced by uranyl-lead double staining,
as were synaptic relationships (Figs. 6, 7).
Light microscopy
The general light-optical quality of tissue was considered superior to that fixed by
routine lithium-carbonate-neutralized formol-saline.
The Nissl-stained cells had contours comparable to those in tissue fixed according
to the method of Koenig, Groat & Windle (1945). A delicate background neuropil
reticulation was seen throughout.' Dark' cells judged by light-microscopic criteria (see
Cammermeyer, 1962) were seen in these preparations. 'Dark' neurons occurred
mainly in the neocortex, which was usually compressed in biopsy, and were seen
234
L. E. Westrum and R. D. Lund
infrequently in the superior colliculus and the prepyriform area of the cortex. 'Dark'
satellite cells of the kind shown in Fig. 5 (inset) were not uncommon in the prepyriform area and presumably provide a light-microscopic correlate of those found
in the electron microscope.
Golgi preparations (Fig. 12) were characterized by impregnation of large numbers
of cells near the surface of the block and fewer towards the centre. The individual
cells were lightly impregnated, appearing amber in colour, and showed reticulation
in the perikaryon. Dendritic contours, especially spines, were well shown, as were
some fine axons with their boutons 'en passant'. Astrocytes were also impregnated
and showed fine detail.
Degeneration, shown by the Nauta (Nauta & Gygax, 1954) method after olfactory
bulb or optic nerve lesions, was clearly demonstrated even after 48 h survival (Fig. 9).
A background granulation was also seen in all preparations. The individual background
granules were about 0-5 /* in diameter and were unevenly distributed throughout the
sections. Preliminary electron-microscopic observations on these sections suggested
a selective distribution of background silver granules in certain fine structures (see
Discussion), rather than a random precipitate as has been noted when using the
unmodified perfusion method of Koenig et al. (1945) for this technique (see, for example,
Zimmerman, Chambers & Liu, 1964). The Nauta method (1951) on frozen and
celloidin-embedded tissue of the superior colliculus gave comparable results, with a
larger number of normal fibres in the background.
The Glees method on the superior colliculus 5 days after optic nerve section showed
a large number of typical degenerating boutons on the side contralateral to the lesion
(Fig. 11) with none seen on the control side. There was no background granulation
in these preparations. Material embedded in paraffin and stained by the Holmes
method (1943) also gave satisfactory results.
DISCUSSION
The general conclusion of this study is that a uniform, reproducible, fine-structural
preservation can be seen with the electron microscope where the Pease perfusion
technique is used, and that light-microscopical preparations of excellent quality are
produced with the same method of fixation. The rapidity and ease of this fixation
recommends it as a routine technique for light- as well as electron-microscopic studies
of the nervous system. The additional advantage is that tissue may be kept in the
perfusate for several days without drastic changes in cytoplasmic detail. This allows
adequate time for fixation for light-microscopic techniques, and indicates that material
stained for light microscopy within this period is unlikely to suffer from serious
fixation artifacts.
The presence of' dark' cells in some electron microscopic preparations needs careful
consideration. They appear in material fixed by standard electron microscopic techniques, but they have not been described in material fixed by formalin perfusion,
although Cammermeyer (1962) found them unavoidable in a light-microscopic study
with such fixation. Electron-dense cells other than neurons have previously been considered as microglia (e.g. Schultz, Maynard & Pease, 1957), normal oligodendroglia
Fixation of nervous system
235
(e.g. Maxwell & Kruger, 19656) or degenerating oligodendroglia (Mugnaini, 1965).
Schultz (1964) has described oligodendroglia with RNP-rich cytoplasm, which,
although electron-dense, are different from the 'microglia' of his earlier study
(Schultz et ah, 1957) and the 'dark' glia of this study. Such cells correspond to the
paler glial cells described in this work, which satisfy the criteria for oligodendroglia.
Since the 'dark' glia of this study are basically similar, it is thought that they represent pathological oligodendroglia, as suggested by Mugnaini (1965). They are uncommon in tissue fixed by direct immersion in osmium tetroxide, which suggests that
they may be the result of a specific reaction to formalin fixation. Since the immediately
adjacent tissue to these 'dark' cells appears normal and frequently contains normal
oligodendroglia it is suggested that the 'dark' cells are a special population of oligodendroglia recognized by their reaction to formalin.
An alternative possibility is that suggested by Cammermeyer (i960), that 'dark'
glia result from traumatic force in biopsy being unevenly distributed throughout the
nervous system, causing haphazard damage to glia, which then turn 'dark'. Since the
preparation of tissue for immersion in osmium tetroxide is inevitably a more traumatic
procedure than formalin perfusion, this argument does not apply here.
Gray (1961a) has described 'dark' neurons in the cerebellum, which he found to
result from the effects of pressure during biopsy. This conclusion was partly based on
Nissl-stained preparations of slices from the same brain immersed in formol saline,
but since such fixation is bound to produce dark cells of light microscopy by itself
(Scharrer, 1938), correlation of light- and electron-microscopic findings is difficult in
this instance.
Since the fixation technique used here allowed the material to be used directly for
light or electron microscopy, it is possible to correlate closely the ultrastructural
findings with those from light microscopy. The 'dark' cells appear in the electron
microscope similar to those of light microscopy with regard to size, approximate
frequency and density of Nissl-positive material. The 'dark' neurons of the light
microscope correspond closely with those demonstrated by Cammermeyer (1962),
although the pale area he described surrounding them is not evident nor can it be
correlated with any ultrastructural feature.
For correlative work of the kind proposed in this study, it is not possible to avoid
'dark' cells by using either the suggested fixative of Cammermeyer (1962) or the
osmium perfusion method of Palay et al. (1962); and longer fixation in situ, also
suggested by Cammermeyer (1962), is contra-indicated by the poor ultrastructural
preservation. These cells are a feature which must always be anticipated.
The effectiveness of formalin as a good primary fixative and agent for blocking
post-mortem autolysis was fully confirmed by observations on the tissue left in the
perfusate for several days before osmication and processing for electron microscopy.
Even if such tissue was not optimal for electron-optical studies, the lack of major
changes in cytoplasmic preservation suggests that these components have largely been
stabilized prior to osmication (Sabatini, Bensch & Barrnett, 1963). The decrease in
intercellular space (compared with controls fixed for short times) should caution one
in using longer-fixed material for fine-structural studies. This phenomenon could be
236
L. E. Westrum and R. D. Lund
explained by slight swelling of the structures during long soaking in aqueous fixative
and the presence of the aldehyde causing the cross-linking of proteins. It is doubtful,
however, whether the light-optical preparations would be significantly affected,
especially in view of the good preservation of other elements. It is necessary, however,
to osmicate the material after formalinfixationto stain lipoprotein membranes. Treating
unosmicated, formalin-fixed tissue with uranyl acetate results in preparations mostly devoid of these structures, but accentuatesfilamentousprotein structures. The membranes
occur as clear 'negative' images, with adherent stained material. These observations
seem to confirm the suggested affinity of uranyl salts for protein sites (Westrum, 1965).
The background granulation found in sections stained by the Nauta method is
difficult to correlate with any particular feature of the tissue. It is unlikely to be a nonspecific precipitate, since the granules are not random and do show some differential
orientation, particularly around nuclei. Sections from the same preparations were
examined in the electron microscope and showed a characteristic distribution of silver
grains. Apart from degeneration granules, further smaller granules were seen in the
mitochondria of various types of cell profiles. It may be this last component which
accounts for the background granulation seen in the light microscope.
The Glees impregnation of the superior colliculus after eye removal gave a higher
density of ring-shaped or solid boutons than had previously been found using formol
saline neutralized with lithium carbonate (Lund, 1965). In addition to large rings, the
demonstration of very delicate and small rings is striking, and these have apparently
been resistant to staining in previous preparations from similar areas.
Staining by the Golgi-Kopsch method for light-optical study has been particularly
successful. Critical features seem to be the size of the tissue block, length of time in
potassium dichromate, and amount of washing before using silver. Pieces smaller than
2 mm thick are less reliably impregnated in silver, and more than 5-7 days in dichromate results in over-impregnation. Extensive washing before silver treatment results
in very light impregnation of structures and a particularly clear background. Delicate
structures such as small dendritic spines often lose their identity with long periods of
washing. It is recommended that a range of times be tried to obtain detail with least
background precipitate.
It is therefore clear that the buffered formalin used here as a primary fixative is
suitable for the major light-microscopic techniques, and it would be expected that
others requiring formalin as a primary fixative should be equally successful.
Having shown that prolonged formalin fixation, even without post-osmication, does
not result in poor ultrastructural preservation of tissue, the next step in a light- and
electron-microscopical study is to find to what extent the tissue may be damaged
during the staining procedure. Studies on material embedded in Araldite after staining
by the Golgi method described in this paper indicate that the standard of background
preservation is similar to that shown by Blackstad (1965).
The standard of preservation after subjecting the tissue to the Nauta & Gygax
technique (1954) is also satisfactory (Fig. 10) and is a considerable improvement on
the method used by Guillery & Ralston (1964), allowing further interpretation of the
fine structure of organelles than was previously possible.
Fixation of nervous system
237
The authors wish to thank DrE. G. Gray and Professor J. Z. Young, F.R.S., for much helpful
advice, Mr S. Waterman for assistance with the photographs and Mr G. Savage for assistance
with the goldfish material. Dr L. E. Westrum is supported by a U.S. Public Health Service
Fellowship, 2F2, NB20, 844-02 (National Institute of Neurological Diseases and Blindness).
REFERENCES
F. G. & ROBERTIS, E. DE (1963). A formalin-perfusion fixation method for histophysiological study of the central nervous system with the electron microscope. Neurology,
Minneap. 13, 758-771.
BLACKSTAD, T. W. (1965). Mapping of experimental axon degeneration by electron microscopy
of Golgi preparations. Z. Zellforsch. mikrosk. Anat. 67, 819-834.
BODIAN, D. & TAYLOR, N. (1963). Synapse arising at cental node of Ranvier and note on
fixation of the central nervous system. Science, N.Y. 139, 330-332.
CAMMERMEYER, J. (i960). Reappraisal of the perivascular distribution of oligodendrocytes.
Am. J. Anat. 106, 197-232.
CAMMERMEYER, J. (1962). An evaluation of the significance of the dark neurone. Ergebn. Anat.
AGUILAR,
EntwGesch. 36, 1-61.
CRAGC, B. G. (1961). The connections of the habenula in the rabbit. Expl Neurol. 3, 388-409.
GLEES, P. (1946). Terminal degeneration within the central nervous system as studied by a new
silver method. J. Neuropath, exp. Neurol. 5, 54-59.
GRAY, E. G. (1959), Axo-somatic and axo-dendritic synapses of the cerebral cortex; an electron
microscope study. J. Anat. 93, 420-433.
GRAY, E. G. (1961a). The granule cells, mossy synapses and Purkinje spine synapses of the
cerebellum; light and electron microscope observations. X Anat. 95, 345-356.
GRAY, E. G. (1961 b). Ultrastructure of synapses of the cerebral cortex and of certain specialisations of neuroglial membranes. In Electron Microscopy in Anatomy (ed. J. D. Boyd et al.),
PP- 54~73- London: Edward Arnold.
GRAY, E. G. (1963). Electron microscopy of presynaptic organelles of the spinal cord. J. Anat.
97,
101-106.
E. G. & GUILLERY, R. W. (1961). The basis for silver staining of synapses of the mammalian spinal cord: a light and electron microscope study. J. Physiol., Lond. 157, 581-588.
GUILLERY, R. W. & RALSTON, H. J. (1964). Nerve fibers and terminals: electron microscopy
after Nauta staining. Science, N.Y. 143, 1331-1332.
HOLMES, W. (1943). Silver staining of nerve axons in paraffin sections. Anat. Rec. 86, 157-187.
HOLT, E. J. & HICKS, R. M. (1961). Studies on formalin fixation for electron microscopy and
cytochemical staining purposes. .7- biophys. biochem. Cytol. 11, 31-45.
KARLSSON, U. & SCHULTZ, R. L. (1965). Fixation of the central nervous system for electron
microscopy by aldehyde perfusion. J. Ultrastruct. Res. 12, 160-186.
KOENIG, H., GROAT, R. A. & WINDLE, W. F. (1945). A physiological approach to perfusionfixation of tissues with formalin. Stain Technol. 20, 13-22.
LUND, R. D. (1965). The occipitotectal pathway of the rat. J. Anat. 100, 51-62.
MAXWELL, D. S. & KRUGER, L. (1965 a). Small blood vessels and the origin of phagocytes in
the rat cerebral cortex following heavy particle irradiation. Expl neurol. 12, 33-54.
MAXWELL, D. S. & KRUGER, L. (1965 b). Thefinestructure of astrocytes in the cerebral cortex and
their response to focal injury produced by heavy ionising particles. J. Cell Biol. 25, 141-157.
MILLONIG, G. (1961). Advantages of a phosphate buffer for osmium tetroxide solutions in
fixation. X appl. Phys. 32, 1637.
MUGNAINI, E. (1965). 'Dark cells' in electron micrographs from the central nervous system of
vertebrates. J. Ultrastruct. Res. 12, 235-236 (Bi).
NAUTA, W. J. H. (1957). In New Research Techniques in Nenro-anatomy (ed. W. F. Windle).
Springfield: Thomas.
NAUTA, W. J. H. & GYGAX, P. A. (1951). Silver impregnation of degenerating axon terminals
in the central nervous system: (1) Technic, (2) Chemical notes. Stain Technol. 26, 5-11.
NAUTA, W. J. H. & GYGAX, P. A. (1954). Silver impregnation of degenerating axons in the
central nervous system: a modified technic. Stain Technol. 29, 91-93.
GRAY,
238
L. E. Westrum and R. D. Lund
S. L., MCGEE-RUSSELL, S. M., GORDON, S. & GRILLO, M. A. (1962). Fixation of neural
tissues for electron microscopy by perfusion with solutions of osmium tetroxide. J. Cell Biol.
iz, 385-410.
PEASE, D. C. (1962). Buffered formaldehyde as a killing agent and primary fixative for electron
microscopy. Anat. Rec. 142, 342.
PEASE, D. C. (1964). Histological Techniques for Electron Microscopy, 2nd edition, pp. 50-56.
New York and London: Academic Press.
REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in
electron microscopy. J. Cell Biol. 17, 208-212.
RICHARDSON, K. G. (i960). Studies on the structure of autonomic nerves in the small intestine,
correlating the silver impregnated image in light microscopy with permanganate-fixed
ultrastructure in electron microscopy. J. Anat. 94, 457-472.
RICHARDSON, K. C. (1961). Formalin-osmium tetroxide fixation of nuclei, tracts or discrete
regions in the central nervous system for electron microscopy. Anat. Rec. 139, 333.
PALAY,
ROBERTSON, J. D., BODENHEIMER, T. S. & STAGE, D. E. (1963). The ultrastructure of Mauthner
cell synapses and nodes in goldfish brains. J. Cell Biol. 19, 159-199.
D. D., BENSCH, K. G. & BARRNETT, R. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation.
J. Cell Biol. 17, 19-58.
SCHARRER, E. (1938). On dark and light cells in the brain and in the liver. Anat. Rec. 72, 53-65.
SCHTJLTZ, R. L. (1964). Macroglial identification in electron micrographs. J. comp. Neurol. 122,
281-295.
SCHULTZ, R. L. & KARLSSON, U. (1965). Fixation of the central nervous system for electron
microscopy by aldehyde perfusion. J'. Ultrastruct. Res. 12, 187-206.
SCHULTZ, R. L., MAYNARD, E. A. & PEASE, D. C. (1957). Electron microscopy of neurons and
neuroglia of cerebral cortex and corpus callosum. Am. J. Anat. 100, 369-407.
STELL, W. K. (1964). Correlated light and electron microscope observations on Golgi preparations of goldfish retina. J. Cell Biol. 23, 89 A.
WALBERG, F. (1964). The early changes in degenerating boutons and the problem of argyrophilia. J. comp. Neurol. 122, 113-137.
WESTRUM, L. E. (1965). A combination staining technique for electron microscopy. I. Nervous
tissue. J. Microscopie 4, 275-278.
ZIMMERMAN, E. A., CHAMBERS, W. W. & Liu, C. N. (1964). An experimental study of the
anatomical organisation of the corticobulbar system in the albino rat. J. comp. Neurol. 123,
301-324.
{Received 27 November 1965)
SABATINI,
ABBREVIATIONS
a
ar
b
c
d
axon
agranular reticulum
bouton
closed contact between glia
dendrite
/
g
gf
gr
m
neurofilaments
astrocyte
glial filaments
granular reticulum
mitochondrion
n
nn
r
s
sv
nucleus
normal neuron
ribosomes
dendritic spine
synaptic vesicles
Fig. i. A survey electron micrograph of the neuropil in rat prepyriform cortex,
molecular layer. Two synapses are seen on dendritic spines and a ' bouton en passage'
forms a synapse with a larger dendritic profile (d). An axon (a) in the upper left corner
shows moderate myelin distortion, x 36000.
Fig. 2. Cytoplasmic detail within a neuron from the pyramidal layer of the prepyriform
cortex. The nucleus is above. Granular (gr) and agranular (ar) endoplasmic reticulum
and free ribosomes are seen throughout the cytoplasm, x 30000.
Fig. 3. Apposing dendrites showing neurotubules in longitudinal and cross section.
Rat prepyriform cortex, after addition of CaCl2 to the fixative. In the upper right a
mitochondrion shows dilatation of its outer membrane (x). Synaptic vesicles are
present in a bouton at the bottom of the picture for comparison with the tubules in cross
section, x 36000.
Journal of Cell Science, Vol. i, No. 2
L. E. WESTRUM AND R. D. LUND
{Facing p. 238)
Fig. 4. Detail of a neuron and some surrounding neuropil from the prepyriform
cortex of the rat. This material remained in perfusate for 1 week before osmication.
Occasional membrane discontinuities may be seen (arrow), x 30 000. Inset: higher
magnification of a similar preparation showing areas of membrane fusion (arrows)
between a bouton containing synaptic vesicles (sv) and an adjacent glial profile,
x 80000.
Fig. 5. A 'dark' cell in the pyramidal layer of the prepyriform cortex, lying close to
normal neurons (tin). There is no retraction of cytoplasm from the surrounding
neuropil, which is well preserved, x 17500. Inset: light micrograph from similar
material stained with cresyl violet showing a ' dark' satellite cell (arrow) corresponding
to that in the adjacent electron micrograph, x 1000.
Journal of Cell Science, Vol. i, No. 2
L. E. WESTRUM AND R. D. LUND
Fig. 6. A survey of the neuropil from the optic tectum of the goldfish. Small myelinated
axons (a) contain filaments (/) as does the synaptic knob at the left, x 30000. Inset:
detail of neurofilaments from a neighbouring region, x 85 000.
Fig. 7. A terminal synapsing on a dendritic profile (d) showing presynaptic dense projections (Gray, 1963). Goldfish, lateral forebrain, molecular layer, x 75 000.
Fig. 8. A synapse from the rat prepyriform cortex, treated with uranyl acetate and lead
citrate, without previous osmication. The synaptic membranes appear light and arc
outlined by pre- and postsynaptic cleft densities, x 80000.
Journal of Cell Science, Vol. i, No.
• ;.
L. E. WESTRUM AND R. D. LUND
8
Fig. 9. Nauta-Gygax preparation from rat prepyriform cortex, molecular layer 2 days
after cutting the olfactory tract, showing small and large degenerating fibres and the
background granularity (see text), x 2000.
Fig. 10. Superior colliculus of rat treated by a slightly modified Nauta-Gygax procedure and stained as usual for electron microscopy to show the standard of background preservation. The damage indicated (arrows) is probably due to freezing the
tissue for cutting. No specific silver deposition is shown in this micrograph, x 35000.
Fig. 11. Glees preparation from superior colliculus after section of optic tract (5 days'
survival), showing rings and solid boutons. x 3000.
Fig. 12. Golgi-Kopsch preparation of goldfish forebrain showing a neuron and
dendritic arborizations with delicate spines, x 850.
Journal of Cell Science, Vol. i, No. 2
•
.'
L. E. WESTRUM AND R. D. LUND