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Synaptic morphology and cytoplasmic densities: Rapid post-mortem effects
TISSUE & CELL 1974 6 (4) 777-788 Published by Longman Group Ltd. Printed in Great Britain
ARYEH ROUTTENBERG
SYNAPTIC MORPHOLOGY CYTOPLASMIC DENSITIES: POST-MORTEM EFFECTS
and SALLY TARRANT
AND RAPID
ABSTRACT. In the study of nervous tissue, the shift from the living state to the postmortem condition affects the ultrastructure of the neuron in ways incompletely understood. We have observed rapid post-mortem changes associated with the synaptic region in the molecular layer of the rat dentate gyrus. These changes are evident in tissue taken from anesthetized animals in which perfusion was beguri while the heart was beating but breathing had stopped. Such alterations were rarely in evidence if perfusion was started while the animal was breathing, either with or without artificial means. The following morphological alterations were observed: (a) an increase in spherical electron densities seen near the synapse as well as in the perinuclear cytoplasm; (b) a variation in the amount of density attached to the thickening of postsynaptic membrane; (c) a change in the curvature of the synaptic cleft. Because of these rapid alterations, caution is recommended in the interpretation of the in viva morphology of the synapse and associated densities.
microscopy, two methods of primary fixation were used: immersion in aldehyde and intracardiac aldehyde perfusion. For immersion fixation of anesthetized animals, subjects were decapitated and the brain was rapidly dissected from the cranial cavity. Frontal sections 3-4 mm thick were made with a clean razor blade and placed in lo-20 times their volume of cold (5°C) dilute fixative recommended by Karnovsky (1965) and Peters (1970). 90-120 set elapsed from the time of decapitation to immersion of the frontal sections in fixative. After 24 hr in the dilute aldehyde, the tissue was placed in a concentrated fixative (Karnovsky, 1965 ; Peters, 1970) at 5°C for 1 hr. For perfusion fixation, animals were anesthetized with 1 ml of 7% chloral hydrate (0.07 mg) or 40 mg/kg sodium pentobarbital. The dilute fixative, identical to that used for immersion, was perfused through the left ventricle for 7 min with a cut made in the right auricle by a tissue forceps providing escape for the blood and perfusate. This was immediately followed by perfusion for 3 min with the concentrated solution. Both dilute and concentrated fixatives were maintained at
Introduction
SEVERALdescriptions exist of post-mortem changes in central nervous system fine structure (Karlsson and Schultz, 1966; Larramendi and Wolosewick, 1971; Palacios and Larramendi, 1973). These reports indicate that alterations in synaptic morphology may occur 5 min post-mortem. Because we observed changes in morphology in rapidly dissected, immersion-fixed material we thought it possible that post-mortem effects more rapid than 5 min might occur in perfusion-fixed tissue. The present report describes such rapid alterations both in cytoplasmic dense material and in synaptic morphology. Materials and Methods
Subjects were adult male and female albino rats, 60-90 days of age, obtained from Holtzman Research, Madison, Wisconsin. In order to prepare brain tissue for electron Northwestern
University,
Evanston,
Illinois.
Received 21 February 1974. Revised 22 April 1974. 777
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37” prior to perfusion. The flow rate of the fixative was 40 ml/min; the brain was removed O-60 min following perfusion and then immersed in fixative. We have subsequently observed tissue fixed following artificial respiration with 95% 02/S% COZ, prior to perfusion, a flow rate of 100 ml/min, and removal of the brain 24 hr following perfusion. Although the tissue fixation is better, it does not depart radically from the control material presented in this experiment. In order to evaluate quantitatively the possibility of post-mortem effects on synaptic fine structure, perfusions were begun with dilute fixative at various times following the administration of an overdose of anesthetic: (1) at the onset of anesthesia, animal breathing and heart beating-control condition; (2) 3-5 min following onset of anesthesia, breathing stopped and heart beating-O min condition; (3) breathing stopped and heart stopped-l min condition; (4) breathing and heart stopped for 5 min-5 min condition; (5) breathing and heart stopped for 10 min10 min condition.
After aldehyde fixation, both immersion and perfusion materials were treated identically. Small tissue cubes, approximately 0.5 by 3 mm, were removed from the hippocampus, immersed in a 5°C concentrated aldehyde for 1 hr, washed in 0.2 M sodium cacodylate buffer and post-fixed for 1 hr in 2 % osmium tetroxide. Following dehydration in a graded ethanol and propylene series, they were embedded in Araldite 502. On the basis of light microscopic observations of 1 p sections stained with Mallory’s methylene blue, fine markings were made with a needle directly on the plastic block so that the region of the molecular layer of the dentate gyrus could be accurately and reliably blocked for thin sectioning. Because we wished to study the same region in all animals, we arbitrarily focused our attention on the dorsal lip of the dentate gyrus in the supragranular portion of the molecular layer. Gray sections were picked up on uncoated, 400 mesh copper grids and double stained, first with 2.5% uranyl acetate in 50% ethyl alcohol for 15 min and then with lead citrate at high pH for 5 min. Serial sections were prepared following the recommendations of Anderson and Brenner ( 1971). Four electron micrographs of neuropil and four of perinuclear cytoplasm were taken as
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a montage of perfused material from each of the five conditions (control, 0, 1, 5 and 10 min). The areas photographed were of a clean, unfolded section covering a single grid hole of a 400 mesh grid with an initial magnification of 10,000 later enlarged 2.6 x photographically. A total area of 5846 p-‘” was then analyzed for each of the conditions. Although material was observed from several animals at each condition, only one animal was selected for the quantitative study on the basis of best fixation in that condition. Thus, counts of synaptic curvature and postsynaptic thickness were based on 71, 49, 52. 57, 26 synapses in the control, 0, 1, 5 and IO min conditions. Measurements were taken of the spherical densities on the four micrographs used to make the montage. Densities were identified by their size, dark gray shade, lack of a membranous surround. Synapses meeting the criteria of Gray (1966) were evaluated for curvature by using a transparency with 9 steps where I was concave, 5 straight, 9 convex and the remaining numbers represented gradations between these points (see Fig. 16 for a concave synapse, Fig. 19 for an example of a convex synapse). The width of the thickening of the post-synaptic membrane was measured from the 26,000 x prints using an 0.1 mm rule under a dissecting microscope at a 10-30x magnification. A one-way analysis of variance was used to evaluate statistically both the overall effects of post-mortem condition and comparisons between any two conditions.
In tissue fixed by immersion, observation of the perinuclear cytoplasm of the granule cell (Fig. 1) indicated the presence of spherical 100-300 rnp diameter electron densities, which were frequently associated with the outer aspect of the rough endoplasmic reticulum membrane. Observation of dendritic cytoplasm (Fig. 2) and subsynaptic regions (Fig. 3) in tissue fixed by immersion revealed the presence of spherical densities with a similar alveolate appearance to those observed in the cell body cytoplasm (Fig. I ). These densities were attached to smooth membranous saccules of endoplasmic reticulum in the dendrite (Fig. 2) and were seen in close proximity to synaptic regions (Fig. 3). Serial sections revealed that these densi-
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ties were invariably apposed or attached to membrane, particularly endoplasmic reticulum (Fig. 4ac), outer nuclear envelope or subsynaptic regions. It cannot be stated, however, that the densities in the region of the post-synaptic membrane are, in fact, similar in composition to those observed in dendrite or perinuclear cytoplasm. It can be stated, however, that the densities seen in all three regions were not the result of a staining artifact since the spherical densities could be observed in unstained material as well as in material stained either with lead citrate or with uranyl acetate. When material obtained following perfusion fixation in the control condition was observed, however, a considerably reduced number of spherical densities in perinuclear cytoplasm (Fig. 5), dendritic and subsynaptic regions (Fig. 10) was apparent. Because of the abundance of electron dense spherical profiles seen in immersion fixation tissue and their paucity in perfused material, it seemed possible that their presence was related to the 90-120 set delay in the immersion fixation procedure plus any additional time taken for the fixative to diffuse through the 334 mm thick tissue sections. To evaluate this suspected change in number of densities as a function of postmortem time, perfusion fixed material of control and perfusion fixed post-mortem tissue (see Methods) was studied. It can be seen that in the micrographs of post-mortem perinuclear cytoplasm from the 0 min (Fig. 6), 1 min (Fig. 7), 5 min (Fig. 8) and 10 min (Fig. 9) conditions, that electron densities similar to those observed with immersion fixation were frequently observed. The nematosome (Grillo, 1970) was observed in the control condition as well as in the four postmortem categories. Clumping of nuclear chromatin in the post-mortem conditions was obvious (Figs. 6-9). In neuropil of perfused post-mortem material (Figs. 11-14) a similar pattern to that seen in immersion material was observed. Few densities were present in the control condition (Fig. lo), but an increasing number of densities was seen in all four post-mortem times (Figs.
11-14) in dendritic cytoplasm. Several favorable sections revealed spherical densities in myelinated axons. A count of the number of densities appear-
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Table 1. Effects of post-mortem manipulation on number of densities, synaptic curvature and post-synaptic thickness in control, 0, 1, 5 and 10 min animals
PostCondition (min) Control 0 1 5 10
Number of densities (&-SD.)
Synaptic curvature (+ SD.)
synaptic thickness ( f S.D.)
4.2k2.06 27.7+6.84 26.Oz3.16 27.Ok1.91 35.Ok5.44
3.9k1.32 4.9+1.25 5.2z1.27 4.5k1.25 6.8k1.20
0.47kO.16 0.75+0.18 1X)070.35 0.81+0.20 0.46kO.15
ing at each post-mortem time is shown in Table 1, which is based on an analysis of dentate gyrus molecular layer neuropil perfusion-fixed material. It can be seen that there is a sharp rise in the number of spherical electron densities as a function of the postmortem manipulation. This is statistically significant for the analysis across all conditions (P
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Figs. l-4. Spherical electron densities following immersion fixation in a glutaraldehyde-formaldehyde fixative. The example shown here is from rat hippocampal dentate gyrus, molecular layer. Fig. 1. Arrows cell. x 36,000.
indicate
densities
Fig. 2. Densities are observed
in the perinuclear
cytoplasm
in the apical dendrite.
x 30,000.
Fig. 3. Density seen near post-synaptic
membrane.
of dentate
granule
x 36,000.
Fig. 4. Density associated with post-synaptic region (a-c are adjacent serial sections). Note the absence of densities along the inner dendrite membrane except at the synaptic region. x 20,000. Figs. 5-9. material.
Dentate
gyrus
granule
Fig. 5. Control condition. Note clumping (upper right hand corner). Fig. 6. 0 min condition. X 34,000. Fig. 7.
I min condition.
cell perinuclear
absence of spherical x 38,000.
Note spherical Note spherical
Fig. 8. 5 mln condition.
densities
(arrows)
lack of nuclear
and nuclear
clumping.
x 32,000.
x 32,000.
Note densities (arrows).
Figs. 10-14. Dentate gyrus molecular
of perfusion-tixed
densities,
densities (arrows).
Note densities (arrows).
Fig. 9. 10 min condition.
cytoplasm
x 34,000.
layer neuropil
of perfusion-fixed
material
Fig. IO. Control condition. Note dispersion of synaptic vesicles and absence spherical densities of the type seen in Figs. 2 and 3. x 36,000.
of
Fig. 1 I. 0 min condition. Note aggregation of synaptic vesicles (arrowhead), convex synapse (large arrow), and increased number of densities (small arrows). x 27,000. Fig. 12. I min condition. Note the size of the densities (small arrow), curvature (large arrow). x 20,000.
and synaptic
Fig. 13. 5 min condition. Note convex synapse (large arrow) and the spherical (arrow) in the dendritic cytoplasm. x 38,000.
density
Fig. 14. IO min condition. Note the presence of a convex profile of synaptic curvature (large arrow) and the aggregation of pre-synaptic vesicles (arrowheads). x 40,000. Figs. 15-20. Synaptic Fig. 15. Control
junctions
of perfused
dentate
gyrus molecular
condition
showing
a slightly concave
Fig. 16. Control condition apparatus. x 96,000.
showing
a concave
Fig. density. process of cleft
synapse.
dendritic
layer neuropil.
x 102,000.
spine synapse.
Note spine
17. 0 min condition. Note slightly convex synapse with thick post-synaptic Note spherical electron densities in subsynaptic cytoplasm and in an adjacent (arrowsj. Note wavy shape of pre-synaptic membrane and possible clumping material. x 78,000.
Fig. 18. I min condition. Example of convex synaptic clumping of pre-synaptic vesicles (arrowhead). x 72,000. Fig. 19. 5 min condition. arrows). x 67,000.
Several
examples
of convex
Fig. 20. IO min condition. Examples of clumping and further convexity of synapse. x 84,000.
curvature
(rating
synaptic
of pre-synaptic
of 8) and
curvature
(large
vesicles (arrowhead)
__
--.-
POST-MORTEM
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were bulging slightly into the post-synaptic process. An example of a concave synapse with a rating of 3 is shown in Fig. 16. An analysis of the curvature of synaptic junctions of molecular layer neuropil control material indicated a mean curvature of 3.9. No attempt was made to classify synaptic complexes by Type l-Type 2 (Gray, 1959) or the F-S system (Uchizono, 1965). In the perfusion tixed post-mortem material, the concavity of the synapse was diminished at all four time periods. Although the mean bowing of the Omin synaptic junction (Fig. 18) was 4.97, which is virtually straight, this difference was statistically significant in comparison with the 3.9 mean bowing seen in the control material. Close inspection of the neuropil shown in Figs. 12-14, the I,5 and 10 min condition, indicates that in many of the synapses, the postsynaptic process bulges into the pre-synaptic terminal, hereinafter referred to as a convex synapse. Particularly clear examples of convex synapses are shown in Figs. 19 and 20. Inspection of the post-synaptic membrane gives the impression that the bowing is occurring in the region of the density, since there is often a bend and a straightening of the post-synaptic membrane itself adjacent to the thickening. An overall analysis of curvature as a function of the post-mortem manipulation was statistically significant (PC 0.001) indicating that with increasing post-mortem time, synaptic curvature becomes increasingly more convex. This result is shown in Table 1. We wish to emphasize that the synaptic curvature is undergoing a rapid alteration, but that this effect is not quite so obvious as the increase in spherical electron densities. Because Larramendi and Wolosewick (1971) noted increased post-synaptic thickening 5 min and longer post-mortem, we measured the thickness of the post-synaptic specialization in our material. Table 1 shows that there is, in fact, an increased thickening in the 0, 1 and 5 min conditions, as compared to the control condition (P
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10 min condition (P > 0.20). A dissolution of the increasing post-synaptic density was noted by Palacios and Larramendi (1973) to occur 24 hr post-mortem. We wish to emphasize that there is a statistically significant difference between the control and the 0 min condition (PC 0.01) indicating a rapid change in another aspect of synaptic morphology, the thickening associated with the post-synaptic membrane. Discussion These results indicate that discernible changes occur in synaptic morphology as the animal enters the post-mortem condition. Thus, changes occur in the morphology of the synapse if perfusion is begun after breathing has stopped but while the heart is still beating. Rapid chemical alterations in brain have been documented following death (Passonneau and Lowry, 1971; Kakiuchi and Rail, 1972), but there is no direct evidence relating the morphological alterations presented here to specific chemical events. The rapidity of the change requires emphasis since it seems likely that interesting and commonly accepted morphological features of the synapse may be related, at times, to events associated with the transition from the living state to the dead state. This conclusion is strengthened by the report of Larramendi and co-workers (Larramendi and Wolosewick, 1971; Palacios and Larramendi, 1973), who to our knowledge were the first investigators to note post-mortem effects on synaptic morphology. Following a post-mortem period of 5 min they observed an increase in the post-synaptic thickening in Type 1 synapses on cerebellar Purkyne cells. Palacios and Larramendi (1973) noted that within the first post-mortem hour, cerebellar material showed synaptic bowing similar to that observed here. The spherical densities observed in our post-mortem material may represent the aggregation of material in the cell cytoplasm. The impression gained by observation of our post-mortem material is of a cytoplasmic matrix (‘cytonet’) which aggregates or precipitates, increasing aggregation with increasing post-mortem time, and attaching to certain membranes: the outer nuclear envelope, endoplasmic reticulum and the post-synaptic specialization. This is similar
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to the theory of the coated vesicle artifact proposed by Gray (1973). If the densities represent aggregation of existing cytoplasmic material, then a parallel process can be observed in the nucleus where clumping of chromatin in post-mortem material is obvious (Figs. 6-8). Perhaps not unrelated to these considerations is the aggregation of synaptic vesicles which is particularly pronounced in the 5 and 10 min post-mortem material (Figs. 19, 20). The spherical densities observed in the cell cytoplasm and dendritic trees and the increased thickness of the post-synaptic density in the present study have been described by others as part of the in vivo morphology in other brain regions. In particular, LeBeux (1972) has described these aggregates attached to endoplasmic reticulum in substantia nigra cell cytoplasm, Streit et al. (1972) have described such densities near post-synaptic membranes, and Grill0 (1970), LeBeux et al. (1971) and Sotelo and Llinas (1972) have described these aggregates associated with the post-synaptic membrane. The latter authors described the density as a ‘dendritic differentiation with an alveolate aspect’ (1972; Figs. 4, 16). On the basis of the present observations it may be necessary to view these descriptions of synaptic morphology with some caution even when perfusion fixation was used, since ‘local post-mortem effects’ may account for these morphological events. A possible explanation for this may be based on the suggestion by Fitzgerald et al. (1972), who have observed that ‘the process of fixation is spread over a period of seconds due to perfusion barriers: therefore fixation is not an instantaneous process’ (p. 224). It may be recalled in this context, then, that in our control material (see Table 1) a few densities were noted. Whether these represent ‘local post-mortem effects’ or in vivo morphology cannot be stated, though the present results caution against the latter alternative. LeBeaux et al. (1971) and Grill0 (1970) have described the presence of a threadlike body, the nematosome. This structure was present in both control and post-mortem material. Although the nematosome has been linked by LeBeaux et al. (1971) with the dense aggregates seen in the cell body and adjacent to the subsynaptic membrane, the fact that they respond differently to the post-
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mortem manipulation suggests that they may not be of the same origin. The concave to straight appearance of control synaptic material contrasts with the less concave and, in the 5 and 10 min postmortem condition, the convex appearance of the synaptic curvature. It is interesting that in electron micrographs of highly purified synaptosome preparations (Davis and Bloom, 1970; Cotman and Taylor, 1972) the curvature of the synaptic membranes is convex. In addition, the density of the post-synaptic thickening in synaptosome preparations is often considerably greater than one observes in well-fixed material. It seems reasonable, therefore, to raise the possibility that certain findings of synaptosome chemistry may be based on prior post-mortem chemical events. Streit et al. (1972) have correctly pointed out that the majority of fine structural descriptions of the synapse are derived from anesthetized preparations. They demonstrated that aldehyde fixation of unanesthetized rats leads to an altered synaptic morphology. This morphology, however, is similar to that observed in our post-mortem material and raises the possibility that their methods of fixation, which were incompletely described, were causing post-mortem changes. This view is compatible with the fact that these authors also observed an increased number of densities, which is a consistent feature of our post-mortem material. It is possible, however, that some metabolic feature 01 the awake condition and of the transition between the alive and dead condition is similar. It would, therefore, be unwise at this juncture to conclude that their results were obtained as a result of post-mortem effects, though this possibility cannot now be dismissed. Although a major point of this paper relates to post-mortem artifacts in the morphology of the synapse, it may be a mistake to ignore the possibilities which the post-mortem manipulation might provide in studying the synaptic complex. The rapidity of the change in synaptic morphology and in the appearance of the electron densities might suggest some particularly unique function of the molecules associated with the synapse. Are the morphological alterations a consequence of an exaggeration of a more evanescent event, such as ionic
POST-MORTEM
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permeability changes, which can be seen only rarely in ‘alive’ fine structural material ? It is interesting that Schwartz et al. (1970) were unable to find any discernible alterations in hippocampal fine structure even after several hours of continuous epileptiform activity. The present results suggest, however, that alterations in fine structure can occur quite rapidly, and recommend study of relatively short-term manipulations. There are several problems inherent in such an experiment which require consideration in assessing the significance of the present findings. First, it is conceivable that the differences observed were related to differences in the quality of perfusion fixation, and not effects of post-mortem time. Thus, post-mortem induced alterations in vasculature patency might cause alterations in the distribution of fixative leading to alterations in the preservation of synaptic morphology. Although this remains a possibility, it should be recalled that we first observed these effects in immersion fixed material, hence alterations in vasculature patency may not be a significant contributing factor. Second, it is possible that, in the extreme case, the fixation we obtained was, in fact, an immersion fixation, and that little fixative reached the brain during the initial perfusion fixation. Although we do not have independent evidence using, for example, a dye marker to assess the extent of perfusion, the tissue was, indeed, fixed by the perfusion itself since the blood vessels were clear and the brain tissue was hard to the touch. Third, it may be objected that our observations were from a heterogeneous synaptic population hence our system is not a well-defined one. Although we counted only synapses which were asymmetrical, pre-synaptic vesicles clearly visible and post-synaptic specialization in evidence, it is likely that we were studying several different input systems to the hippocampus. The number, however, would be restricted
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since we studied the internal lamina of the molecular layer which is predominated by choiinergic afferents from the septal area (Raisman et al., 1965). It may be, then, that our sample is somewhat restricted. Other synaptic terminals in other loci will have to be studied to determine the generality of the post-mortem effect reported here. Although we have pointed out potential answers to these objections, it should be emphasized that the inherent problem in these studies is that post-mortem alterations observed may be related to fixation alteration, brought about by differing times after death. Use of different methods of fixation, both chemical and physical, will be of value, but it will be difficult to prove that the altered morphology occurred either before or after the initial fixation. Although varying degrees of fixative spread represents a potential factor, changes in the number of densities, synaptic curvature and postsynaptic thickening occurred increasingly over time, whether as a result of immediate fixation following beginning of perfusion (if such is the case, then our 0, 1, 5 and 10 min times are absolute) or a fixation delay, due to loss of vasculature patency added to our post-mortem times. In either case these can be called post-mortem times, and the relative measurements can then be considered significant. Acknowledgements Initial studies were performed at the Department of Anatomy. The first author wishes to thank Dr William Bondareff for his generosity in providing use of his laboratory facilities. Microscopy was also carried out in the Department of Biological Sciences. Gratitude is expressed to Mrs P. Larramendi for her capable assistance. Dr L. Larramendi provided helpful discussion of our findings as well as demonstrating his own electron micrographs. Supported by the Alfred P. Sloan Foundation.
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References
ANDERSON, R. G. W. and BRENNER, R. M. 1971. Accurate placement of ultrathin sections on grids: control by sol-gel phases of a gelatin flotation fluid. Stain Tech&., 46, I-6. COTMAN, C. W. and TAYLOR, D. 1972. Isolation and structural studies on synaptic complexes from rat brain. J. Cell Biol., 55, 696-712. DAVIS, G. and BLOOM, F. E. 1970. Proteins of synaptic junctional complexes. J. Cell Biol.. 47, 46A. FITZGERALD, T., BLOODGOOD,R. and MCINTOSH, J. R. 1972. The effect of fixation on the wave propagation of the protozoan axostyle. Tissue & Cell, 4 (2), 219-225. GRAY, E. G. 1959. Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study. J. Anat., 93, 420433. GRAY, E. G. 1966. Problems of interpreting the fine structure of vertebrate and invertebrate synapses. Irrt. Rev. Gen. Exp. Zool., 2, 139-170. GRAY, E. G. 1972. Are the coats of coated vesicles artifacts? J. Newocytol., 1 (4), 363-382. GRILLO, M. A. 1970. Cytoplasmic inclusions resembling nucleoli in sympathetic neurons of adult rats. ./. Cell Biol., 45, 100-l 17. KAKIUCHI, S. and RALL, T. W. 1968. Studies on adenosine 3’,5’-phosphate in rabbit cerebral cortex. Biochem. Pharmnc.,
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KARLSSON, U. and SCHULTZ, R. L. 1966. Fixation of the central nervous system for electron microscopy by aldehyde perfusion. J. Ultrastruct. Res., 14, 47-63. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol., 27, 137A. LARRAMENDI,L. M. H. and WOLOSEWICK,I. 1971. Post-mortem effects on cerebellar synapses: an electron microscopic study. Anat. Rec., 169, 364. LEBEUX, Y. J. 1972. An ultrastructural study of a cytoplasmic filamentous body termed nematosome, in the neurons of the rat and cat substantia nigra. The association of nematosomes with the other cytoplasmic organelles in the neuron. Z. Zellforsrh. mikrosk. Anat., 133, 298-326. LEBEUX, Y. J., LANGELIER, P. and POIRIER, L. J. 1971. Further ultrastructural data on the cytoplasmic nucleolus resembling bodies or nematosomes. Their relationship with the subsynaptic web and a cytoplasmic filamentous network. Z. Zellfororsch. mikrosk. Anat., 118, 147-l 55. PALACIOS, E. and LARRAMENDI,L. M. H. 1973. Post-mortem effects on synapses: a spinodendritic net. AN
PAPPAS, G. D. and PURPURA, D. P. (eds.) 1972. Strrtct~r and Function of Synapses. Raven Press, Nem York. PASSONNEALJ, J. V. and LOWRY, 0. H. 1971. Metabolite flux in single neurons during ischemia and anesthesia. In Recent Advances in Quantitative Histochemistr_v and Cytochemistry (eds. U. C. Dubach and U. Schmidt). Huber, Berne. PETERS, A. 1970. The fixation of central nervous tissue and the analysis of electron micrographs of the neuropil, with special reference to cerebral cortex. In Contemporary Research Methods in Newwanatomy (eds. W. J. H. Nauta and S. 0. E. Ebbesson), pp. 56-76. Springer-Verlag, New York. RAISMAN,G., COWAN, W. M. and POWELL, T. P. S. 1965. The extrinsic afferent. commissural and association fibers of the hippocampus. Brain, 88, 963-976. SCHWARTZ, I. R., BROGGI, G. and PAPPAS, G. D. 1970. Fine structure of cat hippocampus during sustamed seizure. Brain Res., 18, 179-180. SOTELO, C. and LLINAS, R. 1972. Specialized membrane junctions between neurons in the vertebrate cercbellar cortex. J. CeN Biol., 53, 271-289. STREW, P., AKERT, K., SANDRI, C., LIVINGSTON, R. B. and MOOR, H. 1972. Dynamic ultrastructure of presynaptic membranes at nerve terminals in the spinal cord of rats. Anesthetized and unanesthetized preparations compared. Brain Res., 48, 1 l-26. UCHIZONO, J. 1965. Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat. Nature, Lotrd., 207, 642-643.
ARYEH ROUTTENBERG
SYNAPTIC MORPHOLOGY CYTOPLASMIC DENSITIES: POST-MORTEM EFFECTS
and SALLY TARRANT
AND RAPID
ABSTRACT. In the study of nervous tissue, the shift from the living state to the postmortem condition affects the ultrastructure of the neuron in ways incompletely understood. We have observed rapid post-mortem changes associated with the synaptic region in the molecular layer of the rat dentate gyrus. These changes are evident in tissue taken from anesthetized animals in which perfusion was beguri while the heart was beating but breathing had stopped. Such alterations were rarely in evidence if perfusion was started while the animal was breathing, either with or without artificial means. The following morphological alterations were observed: (a) an increase in spherical electron densities seen near the synapse as well as in the perinuclear cytoplasm; (b) a variation in the amount of density attached to the thickening of postsynaptic membrane; (c) a change in the curvature of the synaptic cleft. Because of these rapid alterations, caution is recommended in the interpretation of the in viva morphology of the synapse and associated densities.
microscopy, two methods of primary fixation were used: immersion in aldehyde and intracardiac aldehyde perfusion. For immersion fixation of anesthetized animals, subjects were decapitated and the brain was rapidly dissected from the cranial cavity. Frontal sections 3-4 mm thick were made with a clean razor blade and placed in lo-20 times their volume of cold (5°C) dilute fixative recommended by Karnovsky (1965) and Peters (1970). 90-120 set elapsed from the time of decapitation to immersion of the frontal sections in fixative. After 24 hr in the dilute aldehyde, the tissue was placed in a concentrated fixative (Karnovsky, 1965 ; Peters, 1970) at 5°C for 1 hr. For perfusion fixation, animals were anesthetized with 1 ml of 7% chloral hydrate (0.07 mg) or 40 mg/kg sodium pentobarbital. The dilute fixative, identical to that used for immersion, was perfused through the left ventricle for 7 min with a cut made in the right auricle by a tissue forceps providing escape for the blood and perfusate. This was immediately followed by perfusion for 3 min with the concentrated solution. Both dilute and concentrated fixatives were maintained at
Introduction
SEVERALdescriptions exist of post-mortem changes in central nervous system fine structure (Karlsson and Schultz, 1966; Larramendi and Wolosewick, 1971; Palacios and Larramendi, 1973). These reports indicate that alterations in synaptic morphology may occur 5 min post-mortem. Because we observed changes in morphology in rapidly dissected, immersion-fixed material we thought it possible that post-mortem effects more rapid than 5 min might occur in perfusion-fixed tissue. The present report describes such rapid alterations both in cytoplasmic dense material and in synaptic morphology. Materials and Methods
Subjects were adult male and female albino rats, 60-90 days of age, obtained from Holtzman Research, Madison, Wisconsin. In order to prepare brain tissue for electron Northwestern
University,
Evanston,
Illinois.
Received 21 February 1974. Revised 22 April 1974. 777
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37” prior to perfusion. The flow rate of the fixative was 40 ml/min; the brain was removed O-60 min following perfusion and then immersed in fixative. We have subsequently observed tissue fixed following artificial respiration with 95% 02/S% COZ, prior to perfusion, a flow rate of 100 ml/min, and removal of the brain 24 hr following perfusion. Although the tissue fixation is better, it does not depart radically from the control material presented in this experiment. In order to evaluate quantitatively the possibility of post-mortem effects on synaptic fine structure, perfusions were begun with dilute fixative at various times following the administration of an overdose of anesthetic: (1) at the onset of anesthesia, animal breathing and heart beating-control condition; (2) 3-5 min following onset of anesthesia, breathing stopped and heart beating-O min condition; (3) breathing stopped and heart stopped-l min condition; (4) breathing and heart stopped for 5 min-5 min condition; (5) breathing and heart stopped for 10 min10 min condition.
After aldehyde fixation, both immersion and perfusion materials were treated identically. Small tissue cubes, approximately 0.5 by 3 mm, were removed from the hippocampus, immersed in a 5°C concentrated aldehyde for 1 hr, washed in 0.2 M sodium cacodylate buffer and post-fixed for 1 hr in 2 % osmium tetroxide. Following dehydration in a graded ethanol and propylene series, they were embedded in Araldite 502. On the basis of light microscopic observations of 1 p sections stained with Mallory’s methylene blue, fine markings were made with a needle directly on the plastic block so that the region of the molecular layer of the dentate gyrus could be accurately and reliably blocked for thin sectioning. Because we wished to study the same region in all animals, we arbitrarily focused our attention on the dorsal lip of the dentate gyrus in the supragranular portion of the molecular layer. Gray sections were picked up on uncoated, 400 mesh copper grids and double stained, first with 2.5% uranyl acetate in 50% ethyl alcohol for 15 min and then with lead citrate at high pH for 5 min. Serial sections were prepared following the recommendations of Anderson and Brenner ( 1971). Four electron micrographs of neuropil and four of perinuclear cytoplasm were taken as
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a montage of perfused material from each of the five conditions (control, 0, 1, 5 and 10 min). The areas photographed were of a clean, unfolded section covering a single grid hole of a 400 mesh grid with an initial magnification of 10,000 later enlarged 2.6 x photographically. A total area of 5846 p-‘” was then analyzed for each of the conditions. Although material was observed from several animals at each condition, only one animal was selected for the quantitative study on the basis of best fixation in that condition. Thus, counts of synaptic curvature and postsynaptic thickness were based on 71, 49, 52. 57, 26 synapses in the control, 0, 1, 5 and IO min conditions. Measurements were taken of the spherical densities on the four micrographs used to make the montage. Densities were identified by their size, dark gray shade, lack of a membranous surround. Synapses meeting the criteria of Gray (1966) were evaluated for curvature by using a transparency with 9 steps where I was concave, 5 straight, 9 convex and the remaining numbers represented gradations between these points (see Fig. 16 for a concave synapse, Fig. 19 for an example of a convex synapse). The width of the thickening of the post-synaptic membrane was measured from the 26,000 x prints using an 0.1 mm rule under a dissecting microscope at a 10-30x magnification. A one-way analysis of variance was used to evaluate statistically both the overall effects of post-mortem condition and comparisons between any two conditions.
In tissue fixed by immersion, observation of the perinuclear cytoplasm of the granule cell (Fig. 1) indicated the presence of spherical 100-300 rnp diameter electron densities, which were frequently associated with the outer aspect of the rough endoplasmic reticulum membrane. Observation of dendritic cytoplasm (Fig. 2) and subsynaptic regions (Fig. 3) in tissue fixed by immersion revealed the presence of spherical densities with a similar alveolate appearance to those observed in the cell body cytoplasm (Fig. I ). These densities were attached to smooth membranous saccules of endoplasmic reticulum in the dendrite (Fig. 2) and were seen in close proximity to synaptic regions (Fig. 3). Serial sections revealed that these densi-
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ties were invariably apposed or attached to membrane, particularly endoplasmic reticulum (Fig. 4ac), outer nuclear envelope or subsynaptic regions. It cannot be stated, however, that the densities in the region of the post-synaptic membrane are, in fact, similar in composition to those observed in dendrite or perinuclear cytoplasm. It can be stated, however, that the densities seen in all three regions were not the result of a staining artifact since the spherical densities could be observed in unstained material as well as in material stained either with lead citrate or with uranyl acetate. When material obtained following perfusion fixation in the control condition was observed, however, a considerably reduced number of spherical densities in perinuclear cytoplasm (Fig. 5), dendritic and subsynaptic regions (Fig. 10) was apparent. Because of the abundance of electron dense spherical profiles seen in immersion fixation tissue and their paucity in perfused material, it seemed possible that their presence was related to the 90-120 set delay in the immersion fixation procedure plus any additional time taken for the fixative to diffuse through the 334 mm thick tissue sections. To evaluate this suspected change in number of densities as a function of postmortem time, perfusion fixed material of control and perfusion fixed post-mortem tissue (see Methods) was studied. It can be seen that in the micrographs of post-mortem perinuclear cytoplasm from the 0 min (Fig. 6), 1 min (Fig. 7), 5 min (Fig. 8) and 10 min (Fig. 9) conditions, that electron densities similar to those observed with immersion fixation were frequently observed. The nematosome (Grillo, 1970) was observed in the control condition as well as in the four postmortem categories. Clumping of nuclear chromatin in the post-mortem conditions was obvious (Figs. 6-9). In neuropil of perfused post-mortem material (Figs. 11-14) a similar pattern to that seen in immersion material was observed. Few densities were present in the control condition (Fig. lo), but an increasing number of densities was seen in all four post-mortem times (Figs.
11-14) in dendritic cytoplasm. Several favorable sections revealed spherical densities in myelinated axons. A count of the number of densities appear-
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Table 1. Effects of post-mortem manipulation on number of densities, synaptic curvature and post-synaptic thickness in control, 0, 1, 5 and 10 min animals
PostCondition (min) Control 0 1 5 10
Number of densities (&-SD.)
Synaptic curvature (+ SD.)
synaptic thickness ( f S.D.)
4.2k2.06 27.7+6.84 26.Oz3.16 27.Ok1.91 35.Ok5.44
3.9k1.32 4.9+1.25 5.2z1.27 4.5k1.25 6.8k1.20
0.47kO.16 0.75+0.18 1X)070.35 0.81+0.20 0.46kO.15
ing at each post-mortem time is shown in Table 1, which is based on an analysis of dentate gyrus molecular layer neuropil perfusion-fixed material. It can be seen that there is a sharp rise in the number of spherical electron densities as a function of the postmortem manipulation. This is statistically significant for the analysis across all conditions (P
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Figs. l-4. Spherical electron densities following immersion fixation in a glutaraldehyde-formaldehyde fixative. The example shown here is from rat hippocampal dentate gyrus, molecular layer. Fig. 1. Arrows cell. x 36,000.
indicate
densities
Fig. 2. Densities are observed
in the perinuclear
cytoplasm
in the apical dendrite.
x 30,000.
Fig. 3. Density seen near post-synaptic
membrane.
of dentate
granule
x 36,000.
Fig. 4. Density associated with post-synaptic region (a-c are adjacent serial sections). Note the absence of densities along the inner dendrite membrane except at the synaptic region. x 20,000. Figs. 5-9. material.
Dentate
gyrus
granule
Fig. 5. Control condition. Note clumping (upper right hand corner). Fig. 6. 0 min condition. X 34,000. Fig. 7.
I min condition.
cell perinuclear
absence of spherical x 38,000.
Note spherical Note spherical
Fig. 8. 5 mln condition.
densities
(arrows)
lack of nuclear
and nuclear
clumping.
x 32,000.
x 32,000.
Note densities (arrows).
Figs. 10-14. Dentate gyrus molecular
of perfusion-tixed
densities,
densities (arrows).
Note densities (arrows).
Fig. 9. 10 min condition.
cytoplasm
x 34,000.
layer neuropil
of perfusion-fixed
material
Fig. IO. Control condition. Note dispersion of synaptic vesicles and absence spherical densities of the type seen in Figs. 2 and 3. x 36,000.
of
Fig. 1 I. 0 min condition. Note aggregation of synaptic vesicles (arrowhead), convex synapse (large arrow), and increased number of densities (small arrows). x 27,000. Fig. 12. I min condition. Note the size of the densities (small arrow), curvature (large arrow). x 20,000.
and synaptic
Fig. 13. 5 min condition. Note convex synapse (large arrow) and the spherical (arrow) in the dendritic cytoplasm. x 38,000.
density
Fig. 14. IO min condition. Note the presence of a convex profile of synaptic curvature (large arrow) and the aggregation of pre-synaptic vesicles (arrowheads). x 40,000. Figs. 15-20. Synaptic Fig. 15. Control
junctions
of perfused
dentate
gyrus molecular
condition
showing
a slightly concave
Fig. 16. Control condition apparatus. x 96,000.
showing
a concave
Fig. density. process of cleft
synapse.
dendritic
layer neuropil.
x 102,000.
spine synapse.
Note spine
17. 0 min condition. Note slightly convex synapse with thick post-synaptic Note spherical electron densities in subsynaptic cytoplasm and in an adjacent (arrowsj. Note wavy shape of pre-synaptic membrane and possible clumping material. x 78,000.
Fig. 18. I min condition. Example of convex synaptic clumping of pre-synaptic vesicles (arrowhead). x 72,000. Fig. 19. 5 min condition. arrows). x 67,000.
Several
examples
of convex
Fig. 20. IO min condition. Examples of clumping and further convexity of synapse. x 84,000.
curvature
(rating
synaptic
of pre-synaptic
of 8) and
curvature
(large
vesicles (arrowhead)
__
--.-
POST-MORTEM
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were bulging slightly into the post-synaptic process. An example of a concave synapse with a rating of 3 is shown in Fig. 16. An analysis of the curvature of synaptic junctions of molecular layer neuropil control material indicated a mean curvature of 3.9. No attempt was made to classify synaptic complexes by Type l-Type 2 (Gray, 1959) or the F-S system (Uchizono, 1965). In the perfusion tixed post-mortem material, the concavity of the synapse was diminished at all four time periods. Although the mean bowing of the Omin synaptic junction (Fig. 18) was 4.97, which is virtually straight, this difference was statistically significant in comparison with the 3.9 mean bowing seen in the control material. Close inspection of the neuropil shown in Figs. 12-14, the I,5 and 10 min condition, indicates that in many of the synapses, the postsynaptic process bulges into the pre-synaptic terminal, hereinafter referred to as a convex synapse. Particularly clear examples of convex synapses are shown in Figs. 19 and 20. Inspection of the post-synaptic membrane gives the impression that the bowing is occurring in the region of the density, since there is often a bend and a straightening of the post-synaptic membrane itself adjacent to the thickening. An overall analysis of curvature as a function of the post-mortem manipulation was statistically significant (PC 0.001) indicating that with increasing post-mortem time, synaptic curvature becomes increasingly more convex. This result is shown in Table 1. We wish to emphasize that the synaptic curvature is undergoing a rapid alteration, but that this effect is not quite so obvious as the increase in spherical electron densities. Because Larramendi and Wolosewick (1971) noted increased post-synaptic thickening 5 min and longer post-mortem, we measured the thickness of the post-synaptic specialization in our material. Table 1 shows that there is, in fact, an increased thickening in the 0, 1 and 5 min conditions, as compared to the control condition (P
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10 min condition (P > 0.20). A dissolution of the increasing post-synaptic density was noted by Palacios and Larramendi (1973) to occur 24 hr post-mortem. We wish to emphasize that there is a statistically significant difference between the control and the 0 min condition (PC 0.01) indicating a rapid change in another aspect of synaptic morphology, the thickening associated with the post-synaptic membrane. Discussion These results indicate that discernible changes occur in synaptic morphology as the animal enters the post-mortem condition. Thus, changes occur in the morphology of the synapse if perfusion is begun after breathing has stopped but while the heart is still beating. Rapid chemical alterations in brain have been documented following death (Passonneau and Lowry, 1971; Kakiuchi and Rail, 1972), but there is no direct evidence relating the morphological alterations presented here to specific chemical events. The rapidity of the change requires emphasis since it seems likely that interesting and commonly accepted morphological features of the synapse may be related, at times, to events associated with the transition from the living state to the dead state. This conclusion is strengthened by the report of Larramendi and co-workers (Larramendi and Wolosewick, 1971; Palacios and Larramendi, 1973), who to our knowledge were the first investigators to note post-mortem effects on synaptic morphology. Following a post-mortem period of 5 min they observed an increase in the post-synaptic thickening in Type 1 synapses on cerebellar Purkyne cells. Palacios and Larramendi (1973) noted that within the first post-mortem hour, cerebellar material showed synaptic bowing similar to that observed here. The spherical densities observed in our post-mortem material may represent the aggregation of material in the cell cytoplasm. The impression gained by observation of our post-mortem material is of a cytoplasmic matrix (‘cytonet’) which aggregates or precipitates, increasing aggregation with increasing post-mortem time, and attaching to certain membranes: the outer nuclear envelope, endoplasmic reticulum and the post-synaptic specialization. This is similar
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to the theory of the coated vesicle artifact proposed by Gray (1973). If the densities represent aggregation of existing cytoplasmic material, then a parallel process can be observed in the nucleus where clumping of chromatin in post-mortem material is obvious (Figs. 6-8). Perhaps not unrelated to these considerations is the aggregation of synaptic vesicles which is particularly pronounced in the 5 and 10 min post-mortem material (Figs. 19, 20). The spherical densities observed in the cell cytoplasm and dendritic trees and the increased thickness of the post-synaptic density in the present study have been described by others as part of the in vivo morphology in other brain regions. In particular, LeBeux (1972) has described these aggregates attached to endoplasmic reticulum in substantia nigra cell cytoplasm, Streit et al. (1972) have described such densities near post-synaptic membranes, and Grill0 (1970), LeBeux et al. (1971) and Sotelo and Llinas (1972) have described these aggregates associated with the post-synaptic membrane. The latter authors described the density as a ‘dendritic differentiation with an alveolate aspect’ (1972; Figs. 4, 16). On the basis of the present observations it may be necessary to view these descriptions of synaptic morphology with some caution even when perfusion fixation was used, since ‘local post-mortem effects’ may account for these morphological events. A possible explanation for this may be based on the suggestion by Fitzgerald et al. (1972), who have observed that ‘the process of fixation is spread over a period of seconds due to perfusion barriers: therefore fixation is not an instantaneous process’ (p. 224). It may be recalled in this context, then, that in our control material (see Table 1) a few densities were noted. Whether these represent ‘local post-mortem effects’ or in vivo morphology cannot be stated, though the present results caution against the latter alternative. LeBeaux et al. (1971) and Grill0 (1970) have described the presence of a threadlike body, the nematosome. This structure was present in both control and post-mortem material. Although the nematosome has been linked by LeBeaux et al. (1971) with the dense aggregates seen in the cell body and adjacent to the subsynaptic membrane, the fact that they respond differently to the post-
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mortem manipulation suggests that they may not be of the same origin. The concave to straight appearance of control synaptic material contrasts with the less concave and, in the 5 and 10 min postmortem condition, the convex appearance of the synaptic curvature. It is interesting that in electron micrographs of highly purified synaptosome preparations (Davis and Bloom, 1970; Cotman and Taylor, 1972) the curvature of the synaptic membranes is convex. In addition, the density of the post-synaptic thickening in synaptosome preparations is often considerably greater than one observes in well-fixed material. It seems reasonable, therefore, to raise the possibility that certain findings of synaptosome chemistry may be based on prior post-mortem chemical events. Streit et al. (1972) have correctly pointed out that the majority of fine structural descriptions of the synapse are derived from anesthetized preparations. They demonstrated that aldehyde fixation of unanesthetized rats leads to an altered synaptic morphology. This morphology, however, is similar to that observed in our post-mortem material and raises the possibility that their methods of fixation, which were incompletely described, were causing post-mortem changes. This view is compatible with the fact that these authors also observed an increased number of densities, which is a consistent feature of our post-mortem material. It is possible, however, that some metabolic feature 01 the awake condition and of the transition between the alive and dead condition is similar. It would, therefore, be unwise at this juncture to conclude that their results were obtained as a result of post-mortem effects, though this possibility cannot now be dismissed. Although a major point of this paper relates to post-mortem artifacts in the morphology of the synapse, it may be a mistake to ignore the possibilities which the post-mortem manipulation might provide in studying the synaptic complex. The rapidity of the change in synaptic morphology and in the appearance of the electron densities might suggest some particularly unique function of the molecules associated with the synapse. Are the morphological alterations a consequence of an exaggeration of a more evanescent event, such as ionic
POST-MORTEM
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SYNAPTIC
permeability changes, which can be seen only rarely in ‘alive’ fine structural material ? It is interesting that Schwartz et al. (1970) were unable to find any discernible alterations in hippocampal fine structure even after several hours of continuous epileptiform activity. The present results suggest, however, that alterations in fine structure can occur quite rapidly, and recommend study of relatively short-term manipulations. There are several problems inherent in such an experiment which require consideration in assessing the significance of the present findings. First, it is conceivable that the differences observed were related to differences in the quality of perfusion fixation, and not effects of post-mortem time. Thus, post-mortem induced alterations in vasculature patency might cause alterations in the distribution of fixative leading to alterations in the preservation of synaptic morphology. Although this remains a possibility, it should be recalled that we first observed these effects in immersion fixed material, hence alterations in vasculature patency may not be a significant contributing factor. Second, it is possible that, in the extreme case, the fixation we obtained was, in fact, an immersion fixation, and that little fixative reached the brain during the initial perfusion fixation. Although we do not have independent evidence using, for example, a dye marker to assess the extent of perfusion, the tissue was, indeed, fixed by the perfusion itself since the blood vessels were clear and the brain tissue was hard to the touch. Third, it may be objected that our observations were from a heterogeneous synaptic population hence our system is not a well-defined one. Although we counted only synapses which were asymmetrical, pre-synaptic vesicles clearly visible and post-synaptic specialization in evidence, it is likely that we were studying several different input systems to the hippocampus. The number, however, would be restricted
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since we studied the internal lamina of the molecular layer which is predominated by choiinergic afferents from the septal area (Raisman et al., 1965). It may be, then, that our sample is somewhat restricted. Other synaptic terminals in other loci will have to be studied to determine the generality of the post-mortem effect reported here. Although we have pointed out potential answers to these objections, it should be emphasized that the inherent problem in these studies is that post-mortem alterations observed may be related to fixation alteration, brought about by differing times after death. Use of different methods of fixation, both chemical and physical, will be of value, but it will be difficult to prove that the altered morphology occurred either before or after the initial fixation. Although varying degrees of fixative spread represents a potential factor, changes in the number of densities, synaptic curvature and postsynaptic thickening occurred increasingly over time, whether as a result of immediate fixation following beginning of perfusion (if such is the case, then our 0, 1, 5 and 10 min times are absolute) or a fixation delay, due to loss of vasculature patency added to our post-mortem times. In either case these can be called post-mortem times, and the relative measurements can then be considered significant. Acknowledgements Initial studies were performed at the Department of Anatomy. The first author wishes to thank Dr William Bondareff for his generosity in providing use of his laboratory facilities. Microscopy was also carried out in the Department of Biological Sciences. Gratitude is expressed to Mrs P. Larramendi for her capable assistance. Dr L. Larramendi provided helpful discussion of our findings as well as demonstrating his own electron micrographs. Supported by the Alfred P. Sloan Foundation.
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References
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