ELECTRON STEREOSCOPY AS A MEANS OF CLASSIFYING SYNAPTIC VESICLES

Transcription

1 J. Cell Sci. 8, ( Printed in Great Britain ELECTRON STEREOSCOPY AS A MEANS OF CLASSIFYING SYNAPTIC VESICLES MARION E. DENNISON Department of Anatomy, University College London, Gower Street, London, W.C. 1, England SUMMARY In addition to the well known ' round' synaptic vesicles, 2 distinct types of ' flat' vesicle have been found by electron stereoscopy of aldehyde-fixed central nervous tissue. In the goldfish spinal cord presynaptic terminals are found which contain cylindrical vesicles, and others which contain disk-shaped vesicles. In the rat olfactory bulb no cylindrical vesicles are found; all the flat vesicles there appear to be disk-shaped. INTRODUCTION Uchizono (1965) identified 2 types of presynaptic nerve terminals in the cerebellar cortex of the cat after perfusion fixation with a formalin solution. One contained spherical, the other ellipsoidal vesicles. He claimed that the synapses on the soma of the Purkinje cells contained exclusively (sic) ellipsoidal vesicles and, since electrophysiological evidence indicated that the soma of the Purkinje cell was innervated by inhibitory synapses, he concluded that the synapses containing ellipsoidal vesicles were inhibitory and called them I-type. The synapses containing spherical vesicles he called E-type, assuming them to be excitatory. This would then provide a simple means of distinguishing between excitatory and inhibitory synapses on morphological grounds. Ellipsoidal vesicles had been observed before this (Pellegrino de Iraldi, Duggan & De Robertis, 1963; Robertson, Bodenheimer & Stage, 1963), but no special significance had been attached to them, and their appearance had not been connected with aldehyde fixation. A little later, Walberg (1965) found 'cylindrical' vesicles in synapses of the inferior olive of the cat brain, and Hirata (1966) found 'cylindrical' synaptic vesicles at many sites in the CNS in both cats and rats. These 2 authors did not attribute any functional significance to these 'cylindrical' vesicles but rather regarded them as an interesting artifact offixation. Initial fixation with osmium tetroxide, the most usual method for electron microscopy at that time and widely regarded as producing the 'true' ultrastructural appearance, showed only the round vesicles, even at synapses which when fixed with aldehyde appeared to be 'ellipsoidal' or 'cylindrical'. Walberg (1968), therefore, maintained that the 'elongated' or 'flat' vesicles were merely the result of aldehyde fixation and without functional significance, while Uchizono (1968) provided more evidence that they were related to inhibitory synapses. His argument was further strengthened by Fukami (1969) who demonstrated 'flat' synaptic vesicles

2 526 M. E. Dennison after osmium tetroxide fixation without initial fixation with aldehyde in a proportion of the synapses in the spinal cord of frog and snake. 'Flat' vesicles have now been demonstrated by many workers in various synapses in several species, and it has been suggested that there may be more than 2 types of (agranular) synaptic vesicle (Larramendi, Fickenscher & Lemkey-Johnston, 1967; Gray, 1969a; Bodian, 1970; Price & Powell, 1970). It was therefore decided to compare the shapes of round and flat vesicles more closely by electron stereoscopy, using the tilting methods described by Gray & Willis (1968). Two areas were chosen for study: the goldfish spinal cord (Gray, 19696) and olfactory bulb of the rat (Price, 1969). METHODS Goldfish (Carassius aurattu L.) were anaesthetized with MS-222 (Sandoz) and perfused through the heart with about 5 ml of phosphate-buffered (ph 74) aldehyde mixture (formaldehyde 4%, glutaraldehyde 025 %) at room temperature. The spinal cord was then exposed and flooded with more of the same fixative. Transverse cuts were made in the cord approximately 1 mm apart and the pieces were removed and placed in fresh fixative for about 15 min. After a brief rinse in phosphate buffer (Millonig, 1962) the pieces were postfixed in 1 % osmium tetroxide in phosphate buffer for 2 h. Other goldfish were killed by decapitation and the cord fixed by immersion only, the rest of the procedure being the same as for the perfused fish. Some material was block-stained with 1 % uranyl acetate for 16 h at the 70 % ethanol stage of dehydration; some was impregnated with zinc iodide-osmium tetroxide (ZIO) according to Kawana, Akert & Sandri (1969) after the initial aldehyde fixation and the remainder was processed in the usual way without further treatment. Ether-anaesthetized albino rats ( g) were perfused with either a phosphate-buffered (ph 74) aldehyde mixture (formaldehyde 4 %, glutaraldehyde 1 %) or similarly buffered 4 % glutaraldehyde, at room temperature for about 10 min. The olfactory bulbs were removed, cut into thin slices and after a brief rinse in phosphate buffer were either post-fixed in 1 % osmium tetroxide in phosphate buffer for 2 h or impregnated with ZIO for 16 h. All the material was dehydrated in ethanol and transferred to Araldite via epoxypropane (Pease, 1964). Silver or silver-gold sections were cut on a Cambridge ultramicrotome. Some of the block-stained material and the ZIO impregnated material was stained on the grid with 04% lead citrate in 01 N NaOH; the unstained material was grid-stained with either saturated uranyl acetate in 70% ethanol or 04% lead citrate in o-i N NaOH or with both stains in succession. The stained sections were examined in a Philips EM 300 electron microscope fitted with a goniometer stage. Large numbers of synapses were examined, both axo-somatic and axo-dendritic. Presynaptic terminals containing 'flat' or round vesicles were seen in all the variously treated material. In the rat, most attention was paid to the reciprocal synapses which have 'flat' vesicles in the gemmules of the granule cells and round vesicles in the dendrites of the mitral cell which they contact (Price, 1969). Many synapses were photographed with the section horizontal and then at a number of tilt positions on both sides of horizontal. These sets of tilted pictures are arranged in the figures so that the apparent change in shape of the vesicles can be followed through from one angle of specimen tilt to another. The degree of tilt is stated in the caption to each figure. The direction of tilt is up and down the page, so that if a stereoviewer is available adjacent pairs can be viewed with the page turned sideways to produce a stereoscopic image. Some people also find it possible to fuse a stereopair by first crossing the eyes and then slowly uncrossing them (Gray & Willis, 1968).

3 Stereoscopy of synaptic vesicles 527 OBSERVATIONS AND DISCUSSION Most of the vesicles seen are lying enclosed within the thickness of the section, which is of the order of nm thick (Peachy, 1958). As the whole depth of the section is in focus one is seeing complete vesicles rather than optical sections of them. The vesicles which have been cut through so that only parts are enclosed within the thickness of the section do not provide sufficient electron-scattering material aligned parallel to the beam to produce a distinct image on the photographic plate and can CATEGORY 45 TILT 90 TILT Spherical 90 (a) Cylindrical 90 o 90 Disk-shaped Fig. 1. Diagrams of expected appearances of round and 'flat' vesicles when tilted through various angles (see text for details).

4 528 M. E. Dermison be seen only as faint outlines which disappear altogether at some angles of tilt. So we are considering here projected images of whole vesicles, and by tilting the section through various angles in the electron beam we can study different projections of the vesicle. The change in appearance expected to occur in different shaped vesicles is shown diagramatically in Fig. i. For a full discussion of the problems of electron stereoscopy see Gray & Willis (1968). Spherical vesicles (Fig. 1, category 1) will always show a round profile in whatever direction and however far they are tilted. It is therefore reasonable to assume that a synaptic ending which shows only round profiles contains only spherical or round vesicles. However, if a synaptic ending shows a mixture of elongated and round profiles it cannot be assumed that it contains a mixture of elongated and round vesicles. If the elongated vesicles are cylindrical (Fig. 1, category 2 (a)), they will show a mixture of profiles ranging from a full length cylinder to a small circle of the same diameter as the cylinder. If the section is tilted in the same direction as an elongated vesicle is pointing, and that vesicle can be shown to change shape to a small circle then it is a cylinder. To confirm this, tilting the section at right angles to the axis of the elongated profile would show no change in shape, as it is the equivalent of rolling the vesicle from side to side. A variant of this shape is the curved cylinder (category 2(6)). When interpreting this sort of shape it should be kept in mind that one is looking through things rather than at them with the electron microscope, that is, one is seeing a projection rather than a reflection (surface view) of a given structure. This means that one can see the curved ends of such a vesicle through the main body and the change in shape with tilting will be as shown in Fig. 1, category 2(6). A synaptic ending which shows a mixture of elongated, ellipsoidal and large round profiles could be manifesting the different views of disk-shaped vesicles (Fig. 1, category 3). If by tilting the section at right angles to the direction in which an elongated profile is lying, it is possible to change that profile through an ellipse to a large but faint (since the thickness of scattering material has been reduced, see Fig. 1) round outline of the same diameter as the length of the elongated profile, then it is a disk. This is confirmed by tilting in the direction at right angles and producing no change of shape, as one would still be seeing the disk edgeways. Now turning to the observations made on the actual vesicles it was found that the stains used and the methods of application did not significantly affect the appearance of the vesicles. It has been shown that stains applied to the surface of Araldite blocks will penetrate for at least 100 nm (R. A. Willis, personal communication). Since this is approximately the upper limit of the section thickness, it can be assumed that the stain even when applied on the grid penetrates the whole depth of the section and effects due only to surface staining can be discounted. This was confirmed by stereoviewing. The ZIO mixture impregnated both round and 'flat' vesicles, as stated by Kawana et al. (1969). First, vesicles of the round type were examined by tilting the section. As expected they showed a round profile from every angle, thus confirming that they were spherical. Then the synaptic endings containing ' flat' vesicles were examined to decide into which of the other categories described in Fig. 1 they best fitted. In synapses of the goldfish

5 Stereoscopy of synaptic vesicles 529 spinal cord (Fig. 2) many of the synapses which could be broadly classified as containing 'flat' vesicles were found to contain vesicles which were mostly in category 2. The vesicle arrowed on the right-hand side is lying at right angles to the direction of tilt. In all 5 views it is seen as an elongated profile which shows that it must be a cylinder (category 2(0)). In Fig. 2 A it is confused with another vesicle but with a stereo viewer one can see that it is lying above the other vesicle. This figure also shows an example of a curved cylinder (category z(b), arrowed on the left). Fig. 2A-D show an elongated double structure but tilting another 24 (Fig. 2E) reveals a dense round profile with a fainter curve to one side, the appearance to be expected from seeing a curved vesicle end on. Fig. 3 shows examples of disk-shaped vesicles (category 3) in the goldfish spinal cord. All the arrowed vesicles show a broadening to an ellipsoidal or roundish shape as the section is tilted. The vesicle in the bottom left-hand corner is most clearly elongated in Fig. 3 A and the other 2 are 'flat' in Fig. 3E. It should be noted that the vesicles are most clearly seen when the profile is at its narrowest; this is because the disk is being viewed from the edge and thus presents the maximum amount of material aligned with the electron beam. It is not possible to fit all the profiles seen into a definite category but even if all the vesicles in one synaptic terminal do not fit into a single category the majority of them do. Since the ZIO technique appears to fill the vesicles with an electron-dense precipitate, this provides a solid object to tilt, instead of the semi-transparent vesicles produced by conventional staining. Kawana et al. (1969) decided that the ZIO-positive 'flat' vesicles that they found in the spinal cord of the rat were 'coin-like disks' and indeed Fig. 4 shows that this type of vesicle is also present in the goldfish spinal cord. The arrowed vesicles show a change from elongated to circular profiles as the section is tilted, suggesting disk-shaped vesicles. However, some of the cylindrical vesicles which have been described above in the goldfish spinal cord were also found to be ZIO-positive. The 2 vesicles arrowed in Fig. 5 lying at right angles to the direction of tilt remain as elongated profiles through 60 of tilt, an appearance which is not compatible with a disk shape. In the rat olfactory bulb all the 'flat' vesicles examined proved to be disk-shaped. Fig. 6 shows part of one of the reciprocal synapses from the outer plexiform layer of the olfactory bulb. On the far left-hand side of Fig. 6 A there is an elongated vesicle lying at about 45 0 to the direction of tilt. As the section is tilted this vesicle becomes broader and less distinct. A cylindrical vesicle could not show this broadening effect. The 2 vesicles seen clearly as 'flat' in Fig. 6E and which are lying at about 65 0 to the direction of tilt show this effect more clearly. The marked vesicle on the right of the pictures remains more or less 'flat' throughout, as it is lying almost in the direction of tilt. Fig. 7 shows the 'flat' vesicles of the reciprocal synapse impregnated with ZIO. Two vesicles are indicated which show a change from an elongated to a round form as the section is tilted. Some profiles are present which are clearly spherical and some which are difficult to interpret but there is none which could be interpreted as a cylinder. These vesicles of the reciprocal synapses are of the order of nm in diameter, but there are other disk-shaped vesicles present in other synapses of the 34 CEL 8

6 530 M. E. Dennison olfactory bulb which are also ZIO-positive but smaller (40-50 nm). A synaptic terminal containing vesicles of this type is shown in Fig. 9. The recent work of Price & Powell (1970) also shows there to be 2 separate populations of flattened vesicles in the olfactory bulb of rat: 'a larger one found within the granule cells and a smaller one in axon terminals which form symmetrical synapses upon the granule cells'. These differences are based on a statistical analysis of the average mean diameter of the vesicles. This method of measuring the size of the vesicle blurrs the distinction between the 2 populations. If the vesicles are considered to be disk-shaped the longer diameter of the flattened vesicle is the only one that need be taken into account, as the shorter diameter depends on the orientation of the vesicle within the section. The ZIO mixture impregnates spherical (not illustrated here), cylindrical and diskshaped vesicles, which confirms recent opinion that the ZIO reaction is not specific for any one class of vesicle (Kawana et at. 1969; Matus, 1970). However, it was interesting to note that the spherical vesicles of the reciprocal synapse (i.e. the vesicles of the mitral cell) were very resistant to ZIO. In some animals they did not stain at all and in others the stained vesicles were very few and far between and then rarely at the synapse itself but more usually scattered in the dendrite. This may be compared with the situation in Loligo vulgaris (Martin, Barlow & Miralto, 1969) where the vesicles of the first-order giant axon are ZIO-negative and the vesicles in the afferent terminals are nearly all ZIO-positive. In both goldfish and rat the synaptic thickenings associated with 'flat' vesicles were of the symmetrical type (Colonnier, 1968). The main conclusion to be drawn from this study is that there are at least 2 different types of 'flat' vesicle present in the goldfish spinal cord, whereas in the rat olfactory bulb there appears to be only one. In the goldfish spinal cord both cylindrical and disk-shaped 'flat' vesicles were seen. In the rat olfactory bulb the 'flat' vesicles were all disk-shaped. There are other differences in synaptic terminals containing the 2 types of 'flat' vesicles (Fig. 8). Cylindrical vesicles tend to be tightly packed, with several mitochondria in the terminal. Disk-shaped vesicles, on the other hand, are found with few mitochondria and are much more widely separated in the terminal. Fig. 8 shows 3 different synaptic terminals from goldfish spinal cord, containing cylindrical synaptic vesicles (c), disk-shaped vesicles (d) and spherical vesicles (s). The disk-shaped vesicles are probably the origin of reports of ellipsoidal vesicles in the literature, since the ellipitical shape is the most frequently seen profile in a synapse containing disk-shaped vesicles, although the most noticeable profile in this type of synapse is the elongated shape of a disk seen from the side (i.e. edge on). This is because in this orientation the maximum amount of electron-dense material is aligned with the beam (see Fig. 1). The cylindrical vesicles, which I have so far observed only in the goldfish, also present a variety of profiles in one synapse, but the range extends from the fully elongated vesicle through a series of profiles decreasing in length but remaining the same width to a small very dense circle. It is therefore possible to classify synapses as containing either cylindrical or disk-shaped vesicles

7 Stereoscopy of synoptic vesicles 531 without tilting the specimen, but bearing in mind the information obtained from such a procedure. A synapse showing cylindrical vesicles is shown in Gray (19696). Fig. 2 of his paper clearly shows a mixture of small dense rings and elongated profiles. Another example is seen in a paper by Sotelo (1969) on the cerebellar cortex of the frog where there is a picture of a Golgi axon terminal containing flattened vesicles which appear to be cylindrical, which raises the interesting possibility that cylindrical vesicles are characteristic of synapses of lower vertebrates. The relationship between 'flat' vesicles and inhibitory transmitters is neither verified nor disproved by this study, but if vesicle shape is related to the transmitter that it presumably contains then it does increase the possibility of being able to correlate different transmitters that are being recognized in the CNS, with morphological differences observable among vesicles. Bodian (1970) has found 5 types of synaptic vesicles in the monkey, 2 of which are 'flat'. He separates them on the basis of their response to post-fixation washing with sucrose-containing cacodylate buffer. One type is flat without the buffer wash and becomes slightly flatter after it. The other appears flat only after the buffer wash. This effect would appear to be due to the sucrose since he states that storage in cacodylate-buffered fixative did not have the same flattening effect. However, it could be due to the absence of fixative rather than the presence of sucrose. Since the material in the present study was buffered after initial fixation with Millonig's (1962) buffer which contains sucrose it is possible that the 2 types of 'flat' vesicle seen in the goldfish spinal cord correspond to Bodian's 2 types of flat vesicle found in the monkey. However, it would be extremely rash to transfer findings of this nature from one class of vertebrate to another without making a more direct comparison. It has been suggested that Gray's (1959) type II or Colonnier's (1968) symmetrical synapses are inhibitory (see Gray, 1969). In the goldfish both the synapses containing cylindrical and those containing disk-shaped vesicles were of the symmetrical type. Assuming that differences in vesicle shape do reflect differences in transmitter, this would suggest the existence of 2 inhibitory transmitters in the goldfish spinal cord. The author wishes to express her gratitude to Professor E. G. Gray for his constant encouragement and advice and to Professor J. Z. Young, F.R.S., for use of facilities. Skilful technical assistance with the perfusions was rendered by Miss Eva Franke and the text-figure was drawn by Mrs J. Astfiev. This work was supported by a grant from the Medical Research Council to Professor Gray. REFERENCES BODIAN, D. (1970). An electron microscopic characterization of classes of synaptic vesicles by means of controlled aldehyde fixation. J. Cell Biol. 44, COLONNIER, M. (1968). Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study. Brain Res. 9, FUKAMI, Y. (1969). Two types of synaptic bulb in snake and frog spinal cord, the effect of fixation. Brain Res. 14, GRAY, E. G. (1959)- Axo-somatic and axodendritic synapses of the cerebral cortex: an electron microscope study. J. Anat. 93, GRAY, E. G. (1969a). Electron microscopy of excitatory and inhibitory synapses: a brief review. In Progress in Brain Research, vol. 31 (ed. K. Akert & P. G. Waser), pp Amsterdam: Elsevier. 34-2

8 532 M. E. Dennison GRAY, E. G. (19696). Round and flat synaptic vesicles in the fish central nervous system. In The Structure and Function of Nervous Tissue, vol. 3 (ed. S. H. Barondes), pp New York: Academic Press. GRAY, E. G. & WILLIS, R. A. (1968). Problems of electron stereoscopy of biological tissue. J. Cell Sd. 3, HIRATA, Y. (1966). Occurrence of cylindrical synaptic vesicles in the central nervous system perfused with buffered formalin solution prior to OsO 4 -fixation. Archvm histol. jap. 26, 269. KAWANA, E., AKERT, K. & SANDRI, C. (1969). Zinc iodide-osmium tetroxide impregnation of nerve terminals in the spinal cord. Brain Res. 16, LARRAMENDI, L. M. H., FICKENSCHER, L. & LEMKEY-JOHNSTON, N. (1967). Synaptic vesicles of inhibitory and excitatory terminals in the cerebellum. Science, N. Y. 156, MARTIN, R., BARLOW, J. & MIRALTO, A. (1969). Application of the zinc iodide-osmium tetroxide impregnation of synaptic vesicles in cephalopod nerves. Brain Res. 15, MATUS, A. I. (1970). Ultrastructure of the superior cervical ganglion fixed with zinc iodide and osmium tetroxide. Brain Res. 17, MILLONIG, G. (1962). Further observations on phosphate buffer for osmium solutions. In Vth Int. Congr. Electron Microsc, vol. 2 (ed. S. S. Breese), p. P8. New York and London: Academic Press. PEACHY, L. D. (1958). Section thickness and compression. In IVth Int. Congr. Electron Microsc. vol. 2 (ed. W. Bargmann), pp Berlin: Springer Verlag. PEASE, D. C. (1964). In Histological Techniques for Electron Microscopy, pp New York and London: Academic Press. PELLEGRINO DE IRALDI, A., DUGGAN, H. F. & DE ROBERTIS, E. (1963). Adrenergic synaptic vesicles in the anterior hypothalamus of the rat. Anat. Rec. 145, PRICE, J. L. (1969). The synaptic vesicles of the reciprocal synapse of the olfactory bulb. Brain Res. 11, PRICE, J. L. & POWELL, T. P. S. (1970). Synaptology of the granule cells of the olfactory bulb. J. Cell Sd. 7, 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, SOTELLO, C. (1969). Ultrastructural aspects of the cerebellar cortex of the frog. In Neurobiology of Cerebellar Evolution and Developvwnt (ed. R. Llinas), pp Chicago: American Medical Association. UCHIZONO, K. (1965). Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat. Nature, Lond. 207, UCHIZONO, K. (1968). Morphological background of excitation and inhibition at synapses. J. Electron Microsc. 17, WALBERG, F. (1965). A special type of synaptic vesicles in boutons in the inferior olive. J. Ultrastruct. Res. 12, 237. WALBERG, F. (1968). Morphological correlates of postsynaptic inhibitory processes. In Structure and Function of Inhibitory Neuronal Mechanisms (ed. C. von Euler, S. Skoglund & U. S6derberg), pp Oxford: Pergamon Press. (Received 21 August 1970) Fig. 2. Cylindrical synaptic vesicles from goldfish spinal cord fixed by immersion, stained with lead citrate and tilted through angles: A, +39 ;B, + 15 ; C, O ; D, 15 0 ; E, 39. x Fig. 3. Disk-shaped synaptic vesicles from goldfish spinal cord fixed by perfusion, stained with lead citrate and tilted through angles: A, +36 ;B, +I5 ;C,O ;D, 15 0 ; E, x

9 Stereoscopy of synapttc vesicles 55J B m. /.:* D?t r V'-'

10 534 M. E. Dennison Fig. 4. Disk-shaped synaptic vesicles from goldfish spinal cord fixed by immersion, impregnated with ZIO and tilted through angles: A, +30 ; B, + 15 ; C, O ; D, 15 ; E, x Fig. 5. Cylindrical synaptic vesicles from goldfish spinal cord fixed by immersion, impregnated with ZIO and tilted through angles: A, ; B, + 15 ; C, O ; D, 15 0 ; E, x

11 Stereoscopy of synaptic vesicles 535 B J i *.',«'

12 1536 M. E. Dennison Fig. 6. Disk-shaped synaptic vesicles from reciprocal synapse of rat olfactory bulb fixed by perfusion with aldehyde mixture, stained with lead citrate and tilted through angles: A, +30 ; B, +I5 ;C,O ;D, 15 0 ; E, x Fig. 7. Disk-shaped synaptic vesicles from reciprocal synapse of rat olfactory bulb fixed by perfusion with glutaraldehyde, impregnated with ZIO and tilted through angles: A, + I5 ;B,O ;C, 15 0 ; D, 30 0 ; E, 45. x

13 Stereoscopy of synaptic vesicles 537 A' B t

14 538 M. E. Dennison Fig. 8. Three different types of synaptic terminals containing cylindrical (c), diskshaped (d) and spherical (s) vesicles, from goldfish spinal cord fixed by perfusion and stained with lead citrate. The membrane thickenings of c and d are not shown on the micrograph. Fig. 9. Synaptic terminal from outer plexiform layer of rat olfactory bulb containing small disk-shaped vesicles, fixed by perfusion with glutaraldehyde and impregnated with ZIO. x

15 Stereoscopy of synaptic vesicles 539

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