Progress in Neurobiology Vol. 39. pp. 443 to 474, 1992 Printed in Great Britain.All rights reserved

0301-0082/92/$15.00 ~ 1992PergamonPress Ltd

THE CYTOPLASMIC STRUCTURE OF THE AXON TERMINAL TAKAHIRO GOTOW Department of Anatomy, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan (Received 11 December 1991)

CONTENTS I. Introduction 2. General features of the axon terminal obtained by conventional electron microscopy 2.1. Thin .section 2.2. Freeze-fracture 3. Direct-freeze (quick-freeze) technique 3.1. Freeze-substitution 3.2. Deep etching 4. Mitochondrial domain 4. I. Microtubules 4.2. Neurofilaments 4.3. Smooth endoplasmic reticulum (smooth ER) 4.4. Large dense core vesicle (LDCV) 4.5. Filamentous strands connecting organelles with each other in the mitochondrial domain 4.6. Granular materials 43. Possible functions performed in the mitochondrial domain 5. Synaptic vesicle domain 5.1. Deep-etch overall profile of the synaptic vesicle domain 5.2. Synaptic vesicle 5.3. Synaptic vesicle membrane particles 5.4. Synaptic vesicle-associated filamentous strands 5.5. Distribution of synaptic vesicle-associated filamentous strands 5.6. Actin filament 5.7. Granular materials in the synaptic vesicle domain 5.8. Movement of the synaptic vesicles within the synaptic vesicle domain 6. Conclusions Acknowledgements References

1. INTRODUCTION The neuron is a cell unique in having a remarkably complex set of asymmetrical extensions. Usually one of these extensions is morphologically, physiologically and biochemically different from the others, and is called the axon (Matus et al., 1983; Sternberger and Sternberger, 1983; Riederer et al., 1986; Black and Baas, 1989; Goslin et al., 1990; Fletcher et al., 1991; Peters et al., 1991). This extension has a characteristic bulge at its tip and sometimes one or more on its shaft. These bulges are called axon or presynaptic terminals, and, unlike other parts of the axon, predominantly contain synaptic vesicles which are always associated with a specialized membrane region underlain with dense materials: the presynaptic active zone (Palay, 1958). The active zone is well known to be essential for the neurotransmitter release, and its structure and dynamics have been extensively examined by many authors (see Kelly, 1988; Eccles, 1990; Peters et al., 1991). Still many questions remain unanswered. How is the neurotransmitter loaded into

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the synaptic vesicles? for example; is it released by vesicle exocytosis?; and how do the vesicles move down to the active zone (Marchbanks, 1975; Tauc, 1979, 1982; Zimmermann, 1979; Ceccarelli and Hurlbut, 1980; Melega and Howard, 1984; Wickelgren et al., 1985; Dunant, 1986; Poulain et al., 1986; Van der Kloot, 1988)? Furthermore, why does the interior of small clear synaptic vesicles, called just the synaptic vesicles here, not stain immunocytochemically with antibodies against neurotransmitters even where the vesicle membrane is ruptured, whereas the interior of large dense core vesicles (LDCV) mingled with the synaptic vesicles is always stained even with the antibody to the transmitter synthesizing enzyme (Soteio et al., 1986; Gotow and Sotelo, 1987; Bourrat et al., 1989; Gotow et al., 1989). Possibly the neurotransmitter is loaded only into LDCV or synthesized by only LDCV, or possibly, if the transmitter is loaded into synaptic vesicles, it is very unstable or in very little amounts. Further, the exocytotic profile of synaptic vesicles, usually observed in the active zone in chemically-fixed tissues, is hardly detectable in tissues

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directly frozen from life (Hirokawa and Kirino, 1980; Nakajima and Reese, 1983; Ichimura and Hashimoto, 1988; Tatsuoka and Reese, 1989) or in tissue fixed with concentrated aldehydes (Gonzalez-Aguilar et al., 1988). Probably in in vivo there is no exocytotic profile of synaptic vesicles, and it is only the drastic stimulation of various artificial agents that induces fusion of the synaptic vesicles with the presynaptic membrane (Heuser and Reese, 1973; Heuser et al., 1974; Heuser et al., 1979; Ceccarelli et al., 1979a, b). This suggests that vesicle exocytosis is not necessary for neurotransmitter release in normal in rivo conditions. There are many phenomena regarding neurotransmitter release and synaptic transmission, such as these, that we do not understand. It is now generally accepted that neurotransmitter release is triggered when Ca -'~ binds to some unknown receptor within the axon terminal after the cytoplasmic Ca 2- concentration is elevated by the depolarization-dependent Ca-' ÷ influx through Ca-" channels in the axolemma (Zucker and Fogelson, 1986; Augustine et al., 1987; Smith and Augustine, 1988). The intrinsic membrane protein of the synaptic vesicle, synaptophysin or p38 (Jahn et al., 1985), which possesses a cytoplasmic Ca"'-binding site (Rehm et al., 1986), is one candidate for the unknown Ca 2" receptor. Another is the Ca: ÷-dependent kinase that phosphorylates synapsin I, a synaptic vesicle associated phosphoprotein (De Camilli et al., 1983a, b; Navone et al., 1984; De Camilli and Greengard, 1986; Schiebler et al., 1986; Sfidhof et al., 1989b) possibly linking the vesicles to other cytoskeletal components (Bahler and Greengard, 1987; Petrucci and Morrow, 1987; Horokawa et al., 1989b). If synaptic vesicles really perform exocytosis, it appears to be important for them to associate with cytoskeletal or other cytoplasmic structures, in order to reach appropriate sites and be ready for transmitter release. However, since neurotransmitter release is much faster than membrane fusion in other cells (Almers and Tse, 1990), the direct participation in it of cytoplasmic and/or cytoskeletal components is doubtful. In order to understand the dynamics of synaptic vesicle behavior, we need to examine first of all the cytoplasmic environment. In this review, I describe, primarily from our own data obtained by the modern electron microscopic technique (Heuser and Salpeter, 1979; Hirokawa, 1982; Sehnapp and Reese, 1982; Landis and Reese, 1983), the cytoplasmic organization of axon terminals in the central nervous system, to see at the macromolecular level how membranebound organelles, especially the synaptic vesicles, are distributed, and how they are associated with each other and with cytoskeletal or cytoplasmic components. 2. GENERAL FEATURES OF THE AXON TERMINAL OBTAINED BY CONVENTIONAL ELECTRON MICROSCOPY

2.1. THIN SECTION Although the main aim of this article is to describe the morphology of the axon terminal as revealed by modern electron microscopy, I describe briefly, as a helpful background, what has been shown by con-

ventional techniques, thin section and freeze-fracture (see Section 2.2.). When thin sections are cut from mammalian brain tissue routinely perfused with aldehyde mixture through the ascending aorta (chemical fixation), postfixed with OsO~, dehydrated and embedded in resin, the axon terminal image in these thin sections shows clearly the presence of two prominent kinds of membrane-bound organelle: Synaptic vesicles and mitochondria (Palay, 1958). The synaptic vesicles, which are the most characteristic organelle in the terminal and are intimately related with the presynaptic active zone (cf. Peters et al., 1991), are always detectable irrespective of the size of the terminal. The mitochondria are sometimes invisible in very small axon terminals. Since these two types of organelle tend to segregate (Peters et al., 1991) we divide the terminal axoplasm into the peripheral portion, the s y n a p t i c vesicle domain, and the central portion, the m i t o c h o n d r i a l d o m a i n (Gotow et al., 1991). The mitochondrial domain is usually almost completely surrounded by the synaptic vesicles (Fig. I), and a few synaptic vesicles are also present within it. In relatively large axon terminals, neurofilaments and microtubles can be seen in the mitochondrial domain, especially in its middle portion (Peters et al., 1991). A smooth endoplasmic reticulum (smooth ER) is also characteristic of the mitochondrial domain, distributed among mitochondria, cytoskeletal structures or both. The synaptic vesicle domain consists almost exclusively of synaptic vesicles. Large dense core vesicles (LDCV) sometimes appear, but they are rather associated with the mitochondrial domain and usually do not appear in the area close to the presynaptic active zone where synaptic vesicles are especially concentrated. This suggests that synaptic vesicles participate in the release of neurotransmitter in the active zone, while LDCV do not, a suggestion supported by the difference in molecular marker distribution (Navone et al.. 1984). 2.2. FREEZE-FRACTURE Conventional freeze-fracture provides no information about the cytoplasmic environment of synaptic vesicles in the axon terminal. With this technique membrane structure is nicely observable but cytoplasmic structure, except for organelle membranes, is not. Large intramembrane particles can be seen accumulated in the active zone of the axolemma (Fig. 2). These are believed to be ion channels for Ca 2 + influx from extracellular space into the axon terminal (Pumplin et al., 1981). If they are, entry of Ca 2÷ through these channels after depolarization of the terminal may induce immediate release of the neurotransmitter from synaptic vesicles already attached to the presynaptic membrane. In this case, synaptic vesicles not attached to the presynaptic membrane probably take no part in the transmitter release, but may be influenced by the entry of Ca 2+ in their movement in the cytoplasm or in their degree of association with the presynaptic membrane. Freeze-fracture, showing depressions on P face membrane or protrusions on E face, probably corresponding to vesicle exocytosis and/or endocytosis, provides important information concerning the relationship between synaptic vesicles and the

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presynaptic membrane (Heuser et al., 1974; CeccareUi et al., 1979a, b) and reports concerning this relationship are abundant, as are those concerning the mechanism of neurotransmitter release from synaptic vesicles (Ceccarelli and Hurlbut, 1980; Triller and Korn, 1985; Dunant, 1986), some from the quickfreeze method (Heuser et al., 1979; Heuser and Reese, 1981). However, there is no substantial evidence that the neurotransmitter is released by vesicle exocytosis. Many reports support this idea (cf. Van der Koot, 1988), while some oppose it. Some evidence that the presynaptic membrane of the active zone is different in fluidity or chemical composition from other parts of the axolemma was produced by a cytochemical technique, using the antibiotic filipin that detects membrane cholesterol (Elias et al., 1979), applied to conventional freeze-fracture method (Fig. 3). As the presynaptic membrane was not affected by the filipin (Fig. 3), we suppose that, even considering other relevant factors (cf. Gotow and Hashimoto, 1983a, b; Gotow, 1984), cholesterol is either absent or much less concentrated in the presynaptic membrane than in other membrane domains. It is probable that exocytosis for neurotransmitter release is performed only in the presynaptic membrane regions. The retrieval of synaptic vesicle membrane from axolemma after release of the neurotransmitter may also occur in this membrane domain as suggested by Ceccarelli et al. (1979). 3. DIRECT-FREEZE (QUICK-FREEZE) TECHNIQUE Chemical fixation tends to induce artifactural images of structure and organization of the cell (cf. Gray, 1975a), so that images obtained with the conventional techniques (above) are sometimes considerably distorted. The structure and organization of the cytoplasm are especially difficult to see in the chemically-fixed cell, because most cytoplasmic proteins are lost during technical processing and some are cross-linked and changed in structure by fixatives. With the recently advanced electron microscopic technique, direct freezing of the tissue from life, cytoplasmic structure is preserved much better and the image is much closer to the living condition of the cell (Hirokawa and Kirino, 1980; Schnapp and Reese, 1982; Landis and Reese, 1983; Bridgman and Reese, 1984; Bridgman et al., 1986; Bridgman, 1987). This method is called rapid freezing or quick freezing where the fresh tissue from life is frozen within 104~C/sec by slamming against pure copper block cooled by liquid helium to temperatures a few degrees above absolute zero (4K). With this freezing method, we "fix" all cell structures almost instantaneously ( < 10msec) with no formation of detectable ice crystals, thus providing good temporal resolution for macromolecule or organelle movement within cell cytoplasm. After the tissue is frozen it is further prepared for the electron microscopy by freeze-substitution for thin section or deep-etch replica. 3. I. FREEZE-SuBsTITUTION

Freeze-substitution is a method in which directlyfrozen tissue is immersed in OsO4 in acetone while the

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temperature is raised from - 8 0 ° C to 4~C. Tissue is then dehydrated and embedded, and thin sections are cut in a manner similar to the conventional technique. The axon terminal viewed in freeze-substitution thin section is fundamentally the same in profile as that viewed in conventional chemically-fixed thin section, but with some important differences. The most characteristic difference is the ambiguity of presynaptic density in the active zone (Hirokawa and Kirino, 1980; Nakajima and Reese, 1983; Landis et al., 1987; Ichimura and Hashimoto, 1988; Tatsuoka and Reese, 1989). This presynaptic density is prominent in the chemically-fixed specimen, especially that stained with phosphotungstic acid (Triller and Korn, 1985; Landis et al., 1988). Another difference is that all synaptic vesicles, whether excitatory or inhibitory, are spherical in shape but not necessarily the same size (Nakajima and Reese, 1983; Tatsuoka and Reese, 1989). Finally, cytoskeletons, especially microtubules, are better preserved and are sometimes seen extending into the synaptic vesicle domain or presynaptic active zone (Hirokawa and Kirino, 1980). Although synaptic vesicles contact the presynaptic membrane directly, curiously if the freezing is optimal, profiles of exocytosis or endocytosis (Heuser et al., 1979; Hirokawa and Kirino, 1980; Heuser and Reese, 1981; Nakajima and Reese, 1983; Ichimura and Hashimoto, 1988; Tatsuoka and Reese, 1989) are hardly observable. This suggests that exo- or endocytotic profiles frequently observed in chemicallyfixed specimens are due to stimulation by the fixative (Heuser et al., 1974) and do not occur in the normal in rivo conditions. More importantly, a close examination of the freeze-substituted samples shows that, whereas cytoplasm of the mitochondrial domain is relatively lucent, that of the synaptic vesicle domain is filled with fuzzy material (Hirokawa and Kirino, 1980; lchimura and Hashimoto, 1988; Tatsuoka and Reese, 1989). Usually this fuzzy material obscures the synaptic vesicles, which does not occur in chemically-fixed terminals. The fuzzy material in the presynaptic active zone may be different in chemical composition from that among the synaptic vesicles, since in chemically-fixed specimens the density of this zone is much greater (Landis et al., 1988; Ichimura and Hashimoto, 1988; Tatsuoka and Reese, 1989), In spite of the better structural preservation, the interior of the synaptic vesicle is still electron-lucent (Hirokawa and Kirino, 1980; Tatsuoka and Reese, 1989) as in chemically-fixed terminals. In brief, freeze-substitution reveals a difference in the cytoplasm of the synaptic vesicle domain and that of the mitochondrial domain in the axon terminal in respect to the presence of flocculent material. It is difficult to tell, however, whether this flocculent material is composed of granular materials, filamentous elements, or both, and how this material is associated with the synaptic vesicles and with the presynaptic membrane, and exactly what the difference is between the cytoplasms of the two domains. It seems important to know this difference in order to understand the functional significance of the synaptic vesicles and their contribution to the neurotransmitter release.

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I-. (itrrow 3.2. DEEP E'ICHING

Deep etching, by exposing the interior of the terminal axoplasm, reveals cytoplasmic structure more accurately than freeze-substitution. There is always the possibility, however, even when freezing conditions are appropriate, of structure collapse and; or artifactural image during either fracturing, etching or shadowing (Schnapp and Reese, 1982: Landis and Reese, 1983; Landis et al.. 1987: Golow and Hashimoto, 1988. 1989: Gotow etal.. 1991). Thus it is necessary to compare images shown by various techniques. Further, some false images may bc produced if the tissue is completely fresh and unextracted, especially when the etching is prolonged (Schnapp and Rcese, 1982). Thus it is necessary to examine with the same technique both tissue that is extracted by detergent such as saponin or Triton X-100 before freezing, and tissue which is fixed with aldehydes and washed with water immediately before freezing. Structures left by such treatments are, however. mainly the cytoskcletal components. Unfortunately. very delicate structures or ground substance predominantly composed of soluble proteins (Bridgman and Reese, 1984) may be almost completely removed. Thus, the deep-etch images from fresh unextracted tissue should be compared with images obtained by freeze-substitution where structural preservation is better. Deep-etch axon terminals directly frozen from life without any treatment show a difference in texture between the axoplasm of mitochondrial domain and the axoplasm of synaptic vesicle domains. I describe these in Sections 4 and 5.

4. MITOCHONDRIAL DOMAIN In deep-etch preparation, as in thin section, the mitochondrial domain is observed as an accumulation of mitochondria situated at the center of the axon terminal (Figs 4-6). The most prominent organelle of the axon terminal, the synaptic vesicle, numerous in other parts of the terminal, is less abundant in the mitochondrial domain. The cytoplasm of the mitochondrial domain appears to be empty in chemicallyfixed thin section (Fig. 1), and more electron-lucent than the cytoplasm of the synaptic vesicle domain in freeze-substituted thin section (Hirokawa and Kirino, 1980; Ichimura and Hashimoto, 1988; Tatsuoka and Reese, 1989), but is clearly crowded with variouslysized filamentous profiles and granular materials in deep-etch replica (Figs 4-6). 4. I. MICROTUBULES The microtubule is the most prominent filamentous component of the mitochondrial domain and one of the major cytoskeletons of the neuron (Figs 4-7A) (Hirokawa, 1991; Peters et al., 1991). Microtubules are intimately associated with mitochondria and sometimes almost completely surround them (Figs 6, 12). Some microtubules are directly attached to the wall of mitochondria (Figs 6, 12), as observed with freeze-substitution in Drosophila axons (Benshaiom and Reese, 1985), and some occasionally form bundles

at the periphery of the mitochondrial domain (Fig. 6). Microtubules sometimes extend into the synaptic vesicle domain but never as a bundle, and, unlike those observed by Gray (1975) or Bird (1976) where tissues were treated with albumin before chemical fixation, they rarely come very close to the subaxolemmal region (Fig. 4). This deep-etch image, almost the same as freeze-substitution image, may suggest that microtubules in the mitochondrial domain are relatively stable, while thosc in the synaptic vesicle domain are very unstable, always polymerizing and depolymerizing and being easily disintegrated by chemical fixation or other factors. 4.2. NI!UROFn.AMENIS

Neurofilamcnts, the most numerous cytoskeleton in the axon (Fig. 7A) (Hirokawa, 1982, 1991; Schnapp and Reese, 1982; Tsukita et al., 1982; Peters et al.. t991), appear in the mitochondrial domain, but arc more conspicuous near the axon shaft (Figs 4, 7B). Unlike microtubules, which tend to be disintegrated and become less detectable in chemically-fixed material (Gray, 1975a; Bird, 1975: Gordon-Weeks et al., 1982), neurofilaments appear to be even more detectable in chemically-fixed tissue because of the removal of surrounding cytoplasmic matrix (Hirokawa. 1982). In deep-etch preparation, neurofilaments are easy to disccrn in the axon shaft, but difficult to discern in the terminal, probably because of their random orientation and of their decoration with numerous granular materials. However. terminal filaments still have the cross-bridges (Fig. 7B) characteristic of the axonal ncurofilament. Neurofilaments tend to segregate separately to the microtubules that are more intimate with both mitochondria and synaptic vesicles. This segregation is also recognized in chemically-fixed thin sections. Immunocytochemistry shows the two larger neurofilament subunit proteins, NF-M and NF-H, restricted to the mitochondrial domain, and not extending to the synaptic vesicle domain (Figs 8, 9). Even in their unpolymerized form. that is. not incorporated into the filament, they do not exist in the synaptic vesicle domain. Compartmentalization of neurofilaments and their constituting proteins is thus very clear in the axon terminal. Further, NF-M and NF-H are known to be heavily phosphorylated in the axon but not in soma and dendrite (Fig. 8) ~,Sternberger and Sternberger, 1983; Glicksman et al., 1987). The fact that antibodies to the phosphorylated forms specifically stain the mitochondrial domain (Fig. 9) suggests that the terminal ncurofilaments are similar in chemical nature, i.e. in phosphorylation, to those of the axon shaft, and different from those of soma or dendrites. The deep-etch image of cross-bridges between the filaments here supports this suggestion. 4.3. SMOOTH ENDOPLASMIC RETICULUM (SMOOTH ER) This third organelle specifically related with the mitochondrial domain, visible also in conventional thin sections, is somewhat difficult to distinguish from the mitochondria in deep-etch preparation if only the membrane is exposed. Difference in membrane

CYTOPLASMIC STRUCTUREOF AXON TERMINALS

particle organization, however, is usually recognizable, and more importantly, compared with the cytoplasmic matrix surrounding the mitochondria that around smooth ER is more compact and filaments extending from its membrane less conspicuous (Fig. 10). A stereo view often makes clearer distinction possible (Fig. 10), that is, the smooth ER is tubular or cistern-like in shape while the mitochondria are oval or round. This organelle is considered to be one of the structural sources of synaptic vesicles (Holtzman et al., 1973; Droz et al., 1975). Indeed, in conventional thin section smooth ER occasionally extend into the synaptic vesicle domain, sometimes becoming relatively spherical, but remaining larger than synaptic vesicles. The appearance, however, is very infrequent, except perhaps in the frog central axon terminal where smooth ER often mingle with synaptic vesicles (Lieberman, 1971). The synaptic vesicle associated molecular markers, such as synapsin I and synaptophysin, do not associate with smooth ER membrane (De Camilli et al., 1983b; De Camilli and Navone, 1987), which suggests that its molecular membrane organization is different from that of synaptic vesicles, but does not rule out the possibility that smooth ER is a precursor. 4.4. LARGEDENSE CORE VESICLE(LDCV) The fourth predominant organelle of the mitochondrial domain is LDCV, 80-100 nm in diameter, which has an electron-dense core (Fig. 1). Most authors have considered LDCV to be mingled with the synaptic vesicles in the synaptic vesicle domain (see Peters et al., 1991). Closer examination of conventional thin sections (see Section 2.2) shows LDCV situated far from the presynaptic active zone, and more closely associated with the mitochondria. This is also shown by freeze-substituted thin sections. This localization within the axon terminal, along with the absence of molecular markers of synaptic vesicle associated proteins such as synapsin I and synaptophysin (De Camilli and Greengard, 1986; De Camilli and Navone, 1987) suggests that LDCV are different from synaptic vesicles in function and in contribution to the neurotransmitter release. Indeed, secretion of neuronal polypeptides, which are usually contained in LDCV (Pelletier et al., 1981; Coulter, 1988), appears to occur without the involvement of the presynaptic active zone (De Camilli and Navone, 1987). LDCV in the peptidergic neuron, for example, are known to release their contents into extracellular space from outside the active zone (Zhu et al., 1986). In deep-etch preparations, even when the dense core cannot be seen, LDCV are distinguishable from the synaptic vesicles by their larger size, ~ 100nm (Figs 4, 6 and l I). We found LDCV preferentially in the mitochondrial domain and usually linked with microtubules by thinner filamentous strands (Fig. 6). If they appear in the synaptic vesicle domain, they are here also in the same way linked with microtubules (Fig. 4). LDCV thus appear, in localization and association with microtubules, to be similar to the mitochondria, but quite different from the synaptic vesicles. This suggests that their function and movement in the terminal are different from those of the synaptic vesicles. In this article I look for differences

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in the cytoplasmic environment governing the function and movement of LDCV and of synaptic vesicles. 4.5. FILAMENTOUSSTRANDSCONNECTINGORGANELLES WITH EACH OTHER IN THE MITOCHONDRIALDOMAIN With deep-etch image, of course, we cannot distinguish between mechanical contact and functional interaction (Gotow et al., 1991). A great advantage of deep-etch technique is, however, that it reveals filamentous strands more distinctly than any other electron microscopic approach. In spite of the possibility of artifactural image through the technical limitations of freezing, fracturing, etching and shadowing (Heuser and Salpeter, 1979; Heuser and Krischner, 1980; Schnapp and Reese, 1982; Landis and Reese, 1983), if the tissues are fresh and unextracted and alive until freezing, deep etch provides a truer image of cytoplasmic and filamentous structures than any other method. The deep-etch filamentous profiles described below have been carefully examined and compared with materials extracted by detergents or chemically-fixed, and we believe that they are probably not artifacturally produced during tissue preparation (Gotow et al., 1991). In low magnification of deep-etch preparations, filamentous profiles are conspicuous in the mitochondrial domain (Figs 4-6), but higher magnification shows them more conspicuous in the synaptic vesicle domain (Fig. 12). Furthermore, filamentous structures in the mitochondrial domain, which mainly connect mitochondria with each other, are somewhat thinner and shorter than those among the synaptic vesicles (cf. quantitative analysis in Section 5.4). The filamentous strands connecting mitochondria with each other appear relatively constant in interval. They are, more conspicuous when the mitochondria are close to each other, less conspicuous when surrounded only by granular materials of the cytoplasmic matrix with no organelles, either cytoskeletal or membrane-bound, associated with them. Probably these filaments serve to keep the mitochondria from contacting each other. The strands appear to be specifically associated with the outer membrane of the mitochondria, and keep space just around the membrane for particles extruding from the cytoplasmic surface (Fig. 7B) which carry out some specific function, e.g. interaction with cytoplasmic proteins. Similar-sized filaments connect mitochondria to microtubules and to LDCV, and also microtubules to LDCV. This suggests that there are differences in chemical composition among filamentous strands in the mitochondrial domain. Strands from the microtubules are probably composed of microtubuleassociated proteins (MAPs), such as MAPIA,B, M A P I C (brain dynein), tau or kinesin (Matus et aL, 1983; Shiomura and Hirokawa, 1987; Hirokawa et al., 1988, 1989a, 1991 ; Goedert et al., 1991 ). St rands connecting the mitochondria with each other are probably composed not of MAPs but of different types of proteins that might be mitochondrial membraneassociated. Strands connecting mitochondria with other organeiles, such as LDCV and synaptic vesicles, would again be different in chemical composition from those connecting mitochondria with each other; and since synaptic vesicles and LDCV are different

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in intrinsic as well as associated membrane protein their composition and in the manner of their fusion with axolemma (exocytosis) (Navone et al., 1984; De Camilli and Navone, 1987; Matteoli et al., 1988; De Camilli and Jahn, 1990), as mentioned above, filaments connecting mitochondria with LDCV are probably different in nature from those connecting mitochondria with synaptic vesicles. There are some very thin filaments between mitochondria, less that 5-6 nm in diameter. We counted these as within the variation of mitochondrionassociated filaments, in spite of the possibility that they differ in nature from others or represent a more simplified protein structure. Filamentous strands connecting microtubules to each other in the axon shaft, where microtubules are abundant, arc thinner (Hirokawa et al., 1988) than those extending from microtubules in the terminal. The axonal strands may be predominantly composed of the axon specific MAPs mentioned above (Matus et al.. 1983: Hirokawa et al., 1988. 1991; Goedert et al.. 1991). while those of the terminal may be composed of several kinds of MAPs combined, of both MAPs and other proteins or of proteins different from MAPs. 4.6. GRANULAR MATERIALS

In addition to filamentous strands, there are some granular materials between the organelles, similar in diameter to the strands and sometimes difficult to distinguish from them with a low magnification micrograph (Figs 4--6). These granular materials are almost spherical, vary considerably in size (6-20 nm in diameter) and are distributed throughout the axoplasm of the terminal. They are less conspicuous in the mitochondrial domain than in the synaptic vesicle domain (cf. Section 5.7) (Figs 4-6), and again less conspicuous in the axon shaft where the neurofilaments and microtubules predominate and membrane-bound organelles are sparse (Fig. 7A). The terminal cytoplasmic matrix is observed as electron-lucent space between organelles when tissue is chemically-fixed. If tissue is detergent-extracted before chemical fixation, the space appears completely empty with no structural profile. However, if tissue is quick frozen from life without any treatment and observed in thin section after freeze-substitution, the space is filled with fine filamentous and granular materials (Hirokawa and Kirino, 1980; lchimura and Hashimoto, 1988; Tatsuoka and Reese, 1989). This cytoplasmic matrix appears to be very delicate and easily destroyed or extracted by chemical treatment. It does not seem to be constituted of cytoskeletons, which are usually known to be detergent-resistant, yet biochemically many cytoskeleton-associated proteins have been reported, and cytoplasmic proteins, especially the filamentous materials, may be cytoskeleton-associated. In any case, the granular materials and fine filamentous profiles revealed by the quickfreeze deep-etch technique are real structures and not artifacts produced during tissue preparation, and the relationship between them can be analyzed in deep-etch images. Characteristically the granular materials decrease abruptly in density in the immediate vicinity of the

mitochondria. This granular material-poor space, usually 15-20 nm in width, surrounds the mitochondria almost completely like a halo. In this space, in the relative absence of granular materials filamentous strands are clearly visible (Figs 4-6). The halo space around the mitochondria in freeze-substituted preparations (Benhalom and Reese, 1985) is probably due to shrinkage of this originally. However, even where, judging from the preservation of other organelles surrounding the mitochondria, the freezing condition is appropriate, closer examination with freeze-substituted thin section shows that compared with other cytoplasmic domains the layer immediately surrounding the mitochondria is likely to be relatively electronlucent and devoid of other organelles (Hirokawa and Kirino. 1980; lchimura and Hashimoto, 1988; Tatsuoka and Reese, 1989). Indeed, in deep-etch sample even where the preservation of cytoplasmic structure is appropriate, the cytoplasm surrounding the mitochondria tends to be empty of granular materials (Figs 4 and 6) (Schnapp and Reese, 1982; Landis et al., 1988; Gotow et al., 1991). Further, in this empty space filamentous profiles connecting the mitochondria and other organelles are not broken by shrinkage of mitochondria but look intact, and microtubules are the only organelles directly attached to the mitochondria. This cytoplasmic organization around the mitochondria probably reflects the actual living condition. A cytoplasmic space almost devoid of granular materials may facilitate the translocation of mitochondria along the microtubules. 4.7. POSSIBLEFUNCTIONS PERFORMED IN THE MITOCHONDRIAL DOMAIN

The association of mitochondria with microtubules has been observed in conventional thin sections of in t, it:o and in vitro axon terminals (Gordon-Weeks et al., 1982), although the chemical fixation used for preparing these samples can not preserve well the cytoplasmic structure. The association of mitochondria, or other membrane-bound organelles, with microtubules has been observed in preparations directlyfrozen from life from other parts of neuron and other kinds of cells (Benshalom and Reese, 1985; Bridgman et al., 1986; Kacher et al., 1987). Thus, the mitochondria might have a specific association with microtubules wherever they appear. The mitochondria might need the microtubules for their translocation from perikaryon to axon terminal and vice versa. LDCV formed in the perikaryon from the Golgi apparatus (Broadwell and Cataldo, 1984; De Camilli and Navone, 1987; De Camilli and Jahn, 1990), also appear in the axon (Fig. 11) (Tsukita and Ishikawa, 1980), suggesting that they, like mitochondria, may need the microtubule to move down from perikaryon to axon terminal. Both LDCV and mitochondria, when they are old or malfunctioning, are degraded, probably at the perikaryon, to remain as the secondary lysosomes there, since there is no lysosomal system in normal terminal (Broadwell and Cataldo, 1984). These organelles may thus go back to the perikaryon after operating at the terminals, and their movement may be controlled by microtubule-associated motor proteins, such as kinesin or brain dynein (Hirokawa et al., 1989a, 1990, 1991). The functions of membrane-

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bound organelles in the mitochondrial domain appear to be always regulated by the perikaryon. Smooth ER also associated with the mitochondrial and not with the synaptic vesicle domain, probably, like mitochondria and LDCV, moves from perikaryon to terminal and vice versa. Many authors have considered that smooth ER is continuous throughout the length of the axon or at least over long distances (Palay, 1958; Droz et al., 1975; Alonso and Assenmacher, 1979; Broadwell and Cataldo, 1984). Smooth ER is in most parts tubular in profile and sometimes similar in width to microtubules but easily distinguished from the latter in deep-etch preparation because of the protofilament structure in the microtubule wall. The cytoplasmic structure around the smooth ER is somewhat different from that around the mitochondria; filamentous structure connecting the smooth ER to other organelles especially to the mitochondria is not so frequent. Unlike mitochondria or LDCV, smooth ER is not associated with microtubules, but may contribute to translocation from perikaryon to the axon terminal of some materials essential to the terminal, probably related with the formation of synaptic vesicles and neurotransmitter. The mitochondrial domain, in addition to mechanically stabilizing the axon terminal, may also, through the organelles operating from the perikaryon and down the axon shaft, be necessary for metabolism and energy supply for the synaptic vesicle domain that participates in the essential neuronal function. The organelles may go down to the terminal and back to the perikaryon with less friction because of the cytoplasmic matrix with fewer granular materials and filamentous profiles than that of the synaptic vesicle domain. 5. SYNAPTIC VESICLE DOMAIN 5.1. DEEP-ETCH OVERALL PROFILE OF THE SYNAPTIC VESICLE DOMAIN

In deep etching, the synaptic vesicle domain, compared with the mitochondrial domain, is almost devoid of empty spaces, the cytoplasmic matrix around the vesicles being almost filled with granular and filamentous materials. The synaptic vesicles appear to be connected by these thin filaments and fixed in specific sites, not randomly or freely suspended in the axoplasm. Although the organelles in this domain are almost exclusively synaptic vesicles, LDCV and microtubules appear very occasionally and they are closely associated with each other (Fig. 4) as they are in the mitochondrial domain. LDCV may be different in mobility from the synaptic vesicles, and may need microtubules for their movement even within the terminal. Synaptic vesicles tend to decrease in number around the microtubules as if to leave a route around the microtubules for other organelles such as LDCV and mitochondria (Fig. 4). In cultured spinal cord neurons treated with albumin before chemical fixation, microtubules are reported to associate with synaptic vesicles as well as LDCV, and to approach very closely to the presynaptic density (Gray, 1975a; Bird, 1976). These phenomena are not observed in quick-freeze deep-etch preparation, JpN 39.' ~.--

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however, suggesting that they may be induced by high concentration of albumin. Coated vesicles also are observed mingled with the synaptic vesicles, sometimes close to the active zone (Fig. 4). They are formed from axolemma outside the active zone (Heuser and Reese, 1973; Heuser et al., 1974). Synaptic vesicles fuse only with the presynaptic membrane. Their membrane does not mix, however, with presynaptic membrane or any other part of axolemma (Valtorta et al., 1988a). Thus, although the coated vesicle is the only organelle which, like the synaptic vesicle, concentrates in the synaptic vesicle domain of the axon terminal (it appears in any part of soma or dendrite or in non-neuronal cells), it may be different in chemical composition from the synaptic vesicle. In deep-etch preparations tubular profiles of smooth ER are easily distinguishable from microtubules, but none are clearly encountered in the synaptic vesicle domain. 5.2. SYNAPTIC VESICLE

All synaptic vesicles in any axon terminal are spherical in shape. Most are 50-60 nm in diameter, and vary in size even at the same depth from the fracturing surface in the same terminal. No elongate or flattened vesicles are observed in tissues directly frozen from life (Figs 4-6), nor in freeze-substituted thin sections (Nakajima and Reese, 1983; Tatsuoka and Reese, 1989). In deep-etch preparation chemically fixed with aldehydes before freezing, however, the synaptic vesicles are irregular in shape and some are elongated (Fig. 13). Since the shape is thus alterable with chemical fixation, it is possible that in other ways also the synaptic vesicle may be more influenced by chemical fixation than by freezing, and that direct freezing thus provides a more accurate close to life image. In cross fracture the inside of synaptic vesicles look almost empty, a few granules remaining in the bottom (Fig. 5B). These granules may be a neurotransmitter or the transmitter combined with its associated proteins and they may be diluted to fill the interior of the vesicle. Such extensive dilution of the neurotransmitter might be the reason for the electron-lucent appearance of the inside of the vesicle in thin section. 5.3. SYNAPTIC VESICLE MEMBRANE PARTICLES The cytoplasmic surface of the synaptic vesicle membrane is studded with various-sized particles, 5 15 nm in diameter (Fig. 14A), which cover the whole membrane surface. Since only a few are observed on the E-face, most of these particles are evidently not transmembrane structures but from their variation in size appear to represent several kinds of membraneassociated protein. The protein most commonly considered to associate with the cytoplasmic surface of the synaptic vesicle membrane and not penetrate into the membrane itself is synapsin I (De Camilli et al., 1983b; Trimble and Scheller, 1988; De Camilli and Jahn, 1990). A single molecule of synapsin I has a tadpole-like profile with a head ~ 14 nm in diameter, and a tail ~33 nm long (Hirokawa et al., 1989b) and very thin, probably 4-5 nm. Some of the synaptic

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vesicle-membrane particles, larger ones, are similar in size to the head of synapsin I. but with no profile corresponding to the tail. The tail could be engaged in contact with the vesicle surface (Ueda and Greengard, 1977: Dc Camilli and Greengard. 1986; Trimble and Schcller, 1988), and may be hidden behind the head. lmmunoferritin particles for synapsin 1 have been located on the cytoplasmic side slightly away from the vesicle surface. Because of the size of the two IgG of the first and second antibodies, the distance of the fcrritin particles from the vesicle surface means that synapsin I antigen is located on the vesicle membrane. The head with its tail hidden behind it on the vesicle membrane might extend just a bit from the vesicle surface, and the particles protruding from the cytoplasmic surface of the synaptic vesicle membrane could be synapsin I. Also it is recently reported that the head domain of synapsin I could interact with the hydrophobic core of the lipid bilaycr of the synaptic vesicle membrane (Bcnfenati et al., 1989a,b), so synapsin I may be tightly associated with vesicle membrane and thus. rather than appearing as the filamentous profile extending from the vesicle, could appear as the membrane particle. With their considerable variation in size, however, the membrane particles may represent not only synapsin I but several other kinds of vesicle membrane-associated proteins as well. The considerably fewer transmembranc particles may be intrinsic membrane proteins, such as synaptophysin (p38), SV2, p65 or synaptobrevin/VAMP-I (Leubc et al.. 1987; Smith and Augustine, 1988; Baumert et al., 1989; Sfidhofet al., 1989a; De Camilli and Jahn. 1990). Among these intrinsic vesicle membrane proteins, synaptophysin is known to exist at the axon terminals of all neurons and to form a hcxameric channel (Thomas et al., 1988), like a connexon of gap junction particles (l_,eube et al.. 1987: Sfidhof et al., 1987). It may also form a small pore allowing neurotransmitter molecules to he released when the synaptic vesicle is attached to the presynaptic membrane (De Camilli and Jahn, 1990). The membrane particles observed in the E face of the vesicle membrane measure 9-10 nm in diameter, which includes platinum thickness 2 nm in our deep-etch preparation, and is close to the 7-8 nm range tbr synaptophysin molecules revealed by negative staining (Thomas et ul., 1988). In our materials single membrane particles at the point of contact of synaptic vesicle with presynaptic membrane are observed, probably being engaged in the transmitter release (Fig. 15). Synaptophysin forms a channel and has binding sitc for Ca-" in its collagenase-sensitive carboxy-terminal tail domain on the cytoplasmic side of the synaptic vesicle (Rchm et al., 1986), so it might allow the release of the neurotransmitter to the synaptic cleft immediately after Ca -'' enters into the axon terminal. However, unlike the connexon particles o1" gap junction (Hirokawa and Heuscr, 1982), in deep-etch replica vesicle-membrane particles either on E face or on the cytoplasmic surface show no central depression corresponding to a channel. Large membrane particles accumulated in pre- and postsynaptic membranes. which are considered to represent Ca z+ influx channels (Pumplin et al., 1981) and receptor ion channels respectively, do not show clear central depression

either (Figs 2. 3). Some of the large membrane particles in the presynaptic membrane are probably not Ca-" influx channels and could be related with fusion of the synaptic vesicles with prcsynaptic membrane and possibly combined with synaptophysin to make a channel for the neurotransmitter release. 5.4. SYNAP'IICVESICLIi-AssOCIATtiD FII.AMENFOUSSTRANDS As mentioned above, the protein most commonly associated with synaptic vesicles is the neuronspecific phosphoprotein, synapsin I. Judging from the immunoreactivity of synapsin I revealed by De Camilli et al. (1983b), showing a characteristic distribution pattern of the antibody covering the synaptic vesicles but at a slight distance from the cytoplasmic surface. the actual site of the synapsin I antigen may be only a little away from or on the cytoplasmic surface of the synaptic vesicle. That is. although we do not know which domain of synapsin I. the head or the tail. is bound by the antibody De Camilli et al. (1983b) used. synapsin I appears to associate continuously with the whole vesicular surface, and could bind to the vesicle membrane with its collagenase-sensitive tail. leaving its head extending slightly from the vesicle surface. ,,'cry much like thc schema drawn by De Camilli and Greengard (1986). Morphological profiles possibly corresponding to synapsin I are not detectable in conventional electron microscopy. They are first demonstrated with a quick-freeze and deep-etch technique (Landis et al.. 1988: Hirokawa et al., 1989). In our deep-etch images, especially from fresh unextracted materials, the synaptic vesicles are extensively linked with filamentous profiles (Figs 4 6). whose distribution is very similar to that of synapsin 1 demonstrated by immunocytochemistry (De Camilli et al.. 1983b; De Camilli and Greengard. 1986: Valtorta et al.. 1988b). Landis eta/. (1988) showed filaments up to 40 nm long including their globular tips. Measurement of thickness using their micrographs showed a thickness of 4 5 nm. less than half that of their actin filaments. Hirokawa et al. (1989) measured length at - 3 0 nm, which, as it did not include globular tips, is very close to Landis et al. (1988). Thickness in their pictures, however, is by my estimate almost equal to that of their actin filaments which they measure at - 9 nm. Hirokawa et al. (1989) also observed structures formed by synapsin I in vitro using the combination of synapsin I and synaptic vesicles or eytoskeletons such as actin filaments or microtubules. The strands they observed h7 ritro look similar to those they observed in the m viro axon tcrminal, but usually thinner and with heads clearly visible when contacting the cytoskeletons. Aggregated globular structures corresponding to synapsin I heads are also visible in strands among synaptic vesicles in their in vitro pictures, but very difficult to secm rivo. Landis et al. (1988) made no mention of the connection between synapsin I-like structure and actin filaments or microtubules, as these cytoskeletons are almost undetectable in the synaptic vesicle domain in their preparations. Our synaptic vesicle-associated strands are 42.7 nm in mean length and I 1.7 nm in mean thickness, much thicker than the measurements taken by the above

FIG. 1. All micrographs (Figs 1 17) are obtained from the adult rat cerebellar cortex unless otherwise noted. Thin section of axon terminal of a mossy fiber chemically fixed with I% paraformaldehyde and 1% glutaraldehyde for conventional electron microscopy. (A) Mitochondria accumulate in the middle portion of the axon terminal, the mitochondrial domain, synaptic vesicles predominate in the peripheral part, the synaptic ~'esicle domain. The synaptic vesicles tend not to enter even wide spaces (asterisks in A and B) between mitochondria, but tend to concentrate in the area close to the synaptic contacts (arrowheads) with the granule cell dendrite (D). Cytoskeletal elements such as microtubules (thick arrows) and neurofilaments (thin arrows) are observed in the mitochondrial domain. (B) Microtubules (thick arrows), smooth ER (long thin arrows) and LDCV (short thin arrows) in the mitochondrial domain. One LDCV is well within the synaptic vesicle domain (circle). Arrowhead, presynaptic active zone. Bar, I ~m.

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FIG. 2. Conventional freeze-fracture replica of axon terminal (AT) at axosomatic synapse. Presynaptic P face membrane (asterisks) is b u m p y and has numerous membrane particles. With this technique membrane-bound organelles are detectable in the axon terminal bul cytoplasmic and cytoskeletal slructures are not. Rat cerebral cortex. Bar, 1/am.

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FIG. 3. Conventionally freeze-fractured axon terminals (AT) showing in (A), P face of the presynaptic membrane (asterisk) and E face ofpostsynaptic membrane (E), and in (B), E face of presynaptic membrane (asterisk). These .samples were treated with antibiotic fllipin to detect the presence of cholesterol. If the membrane contains cholesterol, which is known to regulate membrane fluidity, filipin will bind with it to show protrusions or depressions, as in extrasynaptic membranes (arrows). In pre- and postsynaptic membranes, aggregated large intramembrane particles are clearly visible, but there are scarcely any protrusions or depressions, suggesting that there is less cholesterol here than in the extrasynaptic membrane. Rat cerebral cortex. Bar, 0.2 #m.

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FIG. 4. Deep-etch replica of mossy fiber terminal directly frozen from life. Mitochondria (M) accumulate in central region, synaptic vesicles are concentrated in its periphery (lower half), the synaptic vesicle domain. Microtubules (long thick arrows) and smooth ER (asterisks) are observed between mitochondria. with one microtubule (arrowhead) extending into the synaptic vesicle domain, where an LDCV is connected to it by a single thin filamentous strand (short thin arrow). Mitochondria are connected with each other by thin strands, and the space between them is relatively empty. Synaptic vesicles are frequently connected with each other by filamentous strands, and the space between them is granular and fibrous. An area crowded with filamentous profiles (long thin arrowl underlies the axolemma. Judging from their periodic structure some of these filaments may be constituted from actin. Clathrin coated vesicles (short thick arrows) are visible near this area. Neurofilament-like profiles arc observed (open arrows) but granular materials make them difficult to identify. D, granular cell dendrites. Bar, 0.5 ltm.

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FIG. 5. Deep etching. (A) Immature mossy fiber terminal from 10-day-old rat. Mitochondria (M) are not accumulated and synaptic vesicles are distributed throughout the axoplasm, but microtubules (thick arrows) and LDCV (arrowheads) appear only close to mitochondria. A single actin filament (long thin arrow) with periodic profile is observed among synaptic vesicles. Most filamentous strands (short thin arrow) among synaptic vesicles are much shorter than the actin filament and have no periodic structure, and are clearly attached to the vesicles. D, granule cell dendrite. (B) Part of an axon terminal from a three-week-old rat. Filamentous strands (thick arrows) between synaptic vesicles are thicker than those (thin arrows) between mitochondria (M). Synaptic vesicles are connected to axolemma by strands (arrowhead) similar to those between the vesicles. Granular materials are more conspicuous in the synaptic vesicle domain (left) than in the mitochondrial domain (right). Microtubules (open arrows) visible only in mitochondrial domain. Circle, a synaptic vesicle fractured at its middle, giving a rare glimpse of interior of the vesicle, small granules at the bottom. Asterisks, smooth ER. Bar, 0.5 tam. Fig. 5A is reproduced from Gotow et al. (1991).

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FIG. 6. Deep-etch axon terminal from a three-week-old rat clearly shows the two distinct axoplasmic domains: the mitochondrial (center to left) and the synaptic vesicle (right). Mitochondria (M) are connected with each other, with microtubules (long thicker arrows), with LDCV (open arrow) and with synaptic vesicles by filamentous strands (short thin arrows). Microtubules appear more frequently in the upper part of the mitochondrial domain, where smooth ER is also visible (asterisk). Synaptic vesicles in the synaptic vesicle domain are attached by filamentous strands (short thick arrows) slightly thicker than those in the mitochondrial domain. Cytoplasmic matrix between mitochondria is relatively free of granular materials, while that between synaptic vesicles is crowded with them. Bar, 0.5 ,urn. (From Gotow et al., 1991.)

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FIG. 8. Thin section of immunoperoxidase labeling of anti-NF-M, the middle-sized neurofilament subunit, in the molecular layer of rabbit cerebellum. The anti-NF-M antibody recognizes only the phosphorylated form of NF-M (data not shown), the labeling is observable in all parts of the axon (arrows) except the synaptic vesicle domains (asterisks). D, Purkinje cell dendrites. Bar, I/am.

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FIG. 9. Cryothin-section immunogold labeling of anti-NF-H, the largest neurofilament subunit, in rabbit spinal cord anterior horn. Labeling, with 5 nm colloidal gold particles, is clearly observable in the mitochondrial domain except in the area close to presynaptic active zone. Synaptic contact area is between arrowheads. The postsynaptic partner, a dendrite of anterior horn cell (D), is significantly labeled. This antibody recognizes only the phosphorylated form of NF-H, but another NF-H antibody recognizing this protein irrespective of degree of phosphorylation shows the same distribution pattern as here (data not shown). Together with the data of Fig. 8 this suggests that there are no neurofilament proteins (NF-M, NF-H) in the synaptic vesicle domain. M, mitochondria. Bar, 1 pm.

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FIG. 10. Stereo pair of a part of the mitochondrial domain of the axon terminal shown in Fig. 4. This stereo view clearly shows the difference in profile between mitochondria (M) and smooth ER (asterisks). Mitochondria are oval, while smooth ER is tubular or cistern. Smooth ER intrude into the space between the mitochondria and appear to cover them in part. Arrowheads. microtubules. Bar, 0.5 tim. FIG. 1 1. Deep-etch expanded portion of small neuronal process, either dendrite or unmyelinated axon, in a 10-day-old rat. Microtubules (arrowheads), compact in thinner parts of the process, spread out here. Mitochondrion (M) and a profile corresponding to LDCV (asterisks) are connected with microtubules by thin filamentous strands (arrows). Bar, 0.5 ~m.

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FIG. 13. Two small axon terminals and a dendrite (D) synapscd with one of them (area of synapse is between arrowheads), chemically fixed by vascular perfusion with 0.1% glutaraldehyde and 3% paraformaldehyde, quick-frozen and deep-etched. Most of the filamentous strands attached to synaptic vesicles are broken away and removed, but some are still attached (long thick arrows). Most granular materials arc also removed and the cytoplasmic matrix looks empty. Globular structures (thin arrows), different from membrane particles, extend from the synaptie vesicle surfaces. These are probably exposed bases of filamentous strands attached to the vesicles. Actin filaments are conspicuous in the dendrite (short thick arrows) but not in the axon terminals. (From Gotow et al., 1991.) Bar, 0.5 urn. FI6. 14. (A) Deep-etched synaptic vesicles. The true cytoplasmic surface of synaptic vesicles (arrows), showing various-sized particles protruding into the cytoplasm from the vesicle surface. E face membrane (arrowheads) shows very few intramembrane particles. Since no filamentous strands associated with synaptic vesicles are observed, the strands have probably been broken, and some of the particles protruding from the vesicle surface, especially large-sized ones. may represent bases of these strands. (B) This deep-etch micrograph shows a presynaptic active zone (between arrowheads) synapsed with postsynapse dendrite (D). The cytoplasmic surface of the presynaptic active zone (asterisk) is crowded with particles protruding from the membrane. Such particle accumulation is specific to the presynaptic membrane area and not found elsewhere. Filamentous strands from the synaptic vesicles appear to be linked with these particles. Some of the particles may be Ca:+ influx channels, but others may be necessary for synaptic vesicles to bind the membrane. Bar, 0.2 pro. FtG. 16. Mossy fiber axon terminal of a 10-day-old rat. Cytoplasmic surface of the axon terminal contacting the granular cell dendrite (D) is underlain characteristically with thin filaments attached to the axolemma in a lattice-like network (between arrowheads). The filaments themselves (thin arrows) are thinner than the vesicle-associated strands (long arrows). Some vesicle-associated strands appear to connect to the membrane underlying filaments (two long arrows in the left side). This area underlain with filamentous network may be a part of the active zone, although membrane particle specialization is not detectable here. Microtubules (short thick arrows) are visible in the postsynaptic partner, the granule cell dendrite. Bar, 0 . 2 ~ m . 464

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ll Biol. 102, 1510 1521. BROADWELI,, R. D. and CAI'ALI.~), A. M. (1984) The neuronal endoplasmic reticulum: its cytochemistry and contribution to the endomembrane system. It. Axons and terminals. J. comp. Neurol. 230, 231-248. CECCARELLI,B., GROHOVAZ,F. and HURLBUT,W. P. (1979a) Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca-' " -free solutions on the structure of the active zone. J. Cell Biol. 81, 163 177. CECCARELLI,B.. GROHOVAZ,F. and HURLBUT,W. P. (1979b) Freeze-fracture studies of flog neuromuscular junctions during intense release of neurotransmitter. It. Effects of electrical stimulation and high potassium. J. ('ell Biol., 81, 178 192. CFCCAREI.LI, B. and HURLBUI, W. P. (1980) Vesicle hypothesis of the release of quanta of acetylcholine. Physiol. Rev. 60, 396 441. COL'LrER, H. D. (1988) Vesicular localization of immunoreactive [MetS]enkephalin in the globus pallidus. Proc. hath. Acad. Sci. U.S.A. 85, 7028 7032. DE CAMIt.LI, P., CAMERON, R. and GREENGARD,P. (1983a) Synapsin I ( protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluoresence in frozen and plastic sections. J. ('ell Biol. 96, 1337-1354. DE CAMU.t.I, P., HARRIS, JR, S. M., Ht;rTNER. W. B. and GREENGARO. P. (1983b) Synapsin 1 (Protein I), a nerve terminal-specific phosphoprotein. 11. Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J. Cell Biol. 96, 1355 1373. DE CAMILLI. P. and GREENGARD, P. (1986) Synapsin I: a synaptic vesicle-associated neuronal phosphoprotein. Bioehern. Pharmac. 35, 4349-4357. DE CAMILLI,P. and NAVONE,F. (1987) Regulated secretory pathways of neurons and their relation to the regulated secretory pathway of endocrine cells. Ann. N. Y. Acad. Sci. 493, 461 479. DE CAMILLLP. and JAHN, R. (1990) Pathways to regulated exocytosis in neurons. A. Rev. Physiol. 52, 625-645. DRENCKHAHN, D. and K*ISER, H.-W. (1983) Evidence for the concentration of F-actin and myosin in synapses and in the plasmalemmal zone of axons. Eur. J. Cell Biol. 31, 235--240. DUNANT, Y. (1986) On the mechanism of acetylcholine release. Prog. Neurobiol. 26, 55-92.

DROZ, B., RAMBOL:RG,A. and KOENIG(1975) The smooth endoplasmic reticulum: Structure and role in the renewal of axonal membrane and synaptic vesicles by fast axonal transport. Brain Res. 193, I -.13. ECCLES, J. C. (1990) Developing concepts of the synapses J. Neurosci. I0, 3769- 378l. ELIAS. P M., FRIEND,D. S. and GOERKE,J. (1979) Membrane sterol heterogeneity. Freeze-fracture detection with saponin and filipin. J. HL~tochem. Cytoehem. 27, 1247 1260. FATH, K. R. and LASEK.R. J. (1988) Two classes of actin microfilaments are associated with the inner cytoskeleton of axons. J. Cell Biol. 107, 613-621. FLET('HER, T. L., CAMERON,P., DE CAMILLI,P. and BANKER, G. (1991) The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture. J. Neurosci. II, 1617. 1626. GLICKSMAN, M. A., SOPPET, D. and WILLARD, M. B. (1987) Posttranslational modification of neurofilament polypeptides in rabbit retina. J. Neurobiol. 18, 167 196. GOEDERT, M., CROWTHER, R. A. and GARNER,C. C. (1991) Molecular characterization of microtubule-associated proteins tau and MAP2. Trend~" Neurosci. 14, 193 199. GOEt,Z, S. E., NESILER, E. J. and GREENG.ARD, P. (1985) Phylogenetic survey of proteins related to synapsin I and biochemical analysis of four such proteins from fish brain. J. Neurochem. 45, 63 72. GONZAt.EZ-AGuIt.AR, F., RODRIGUEZ,J. A., ALZOLA, R. H. and l.uon)lo, M. ('. (1989) Synaptic vesicle relationships with the presynaptic membrane as shown by a new method of fast chemical fixation. Neuroscience 24, 9-17. GORDON-WEEKS, P. R., BURGOYNE, R. D. and GRAY, E. G. (1982) Presynaptic microtubules: organisation and assembly,disassembly. Neuroscience 7, 739 749. GOSLIN. K., SCHREYER,D. J., SKENE,J. H. P. and BANKER,G. (1990) Changes in the distribution of GAP-43 during the development of neuronal polarity. J. /Veurosci. 10, 588 602. Gorow, T. (1984) Cytochemical characteristics of astrocytic plasma membranes specialized with numerous orthogonal arrays. J. Neuro