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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). 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(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