Brain Research, 93 (1975) 1-13 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
1
Research Reports
THE SMOOTH ENDOPLASMIC RETICULUM: STRUCTURE AND ROLE IN THE RENEWAL OF AXONAL MEMBRANE AND SYNAPTIC VESICLES BY FAST AXONAL TRANSPORT
B E R N A R D DROZ, A L A I N R A M B O U R G AND HERBERT L. K O E N I G
D~partement de Biologic, Commissariat t~ l'l~nergie Atomique, Centre d'l~tudes Nucldaires de Saclay, B.P. 2, 91190 Gif-sur-Yvette et Laboratoire de Cytologic, Universit~ Pierre et Marie Curie, 75005 Paris (France} (Accepted February 21st, 1975)
SUMMARY
The spatial arrangement of the smooth endoplasmic reticulum (SER) was studied in 0.5-2/~m thick sections of rat spinal and chick ciliary ganglia previously impregnated with heavy metal salts. Electron microscopy at low (105 V) or high (106 V) voltage showed the impregnated SER as a continuous system extending probably from the perikaryon to the axon terminal. Tubules of the SER, which were running in a parallel direction with the axon, were occasionally seen in close apposition with the axonal membrane. Moreover in the preterminal region, anastomosed tubules of the SER formed a subsurface 'primary network' and gave rise to a deeper 'secondary network' made of thinner tubules; synaptic vesicles bulging at the tip of thin tubules of the SER were frequently observed. To specify the role played by the SER in the fast axonal transport, chicken ciliary ganglia were slightly compressed and radioautographed 3 h after the intracerebral injection of [3H]lysine. Quantitative analysis of the silver grain distribution indicated that labeled proteins, rapidly conveyed down the axon, piled up in regions containing an accumulation of SER profiles. On the basis of these results, it is concluded that: (1) the SER appears as a continuous intraaxonal pathway bridging the perikaryon and the axon terminal; (2) the SER conveys macromolecular components with the fast axonal transport; (3) the conveyed macromolecules, which are delivered to the axonal membrane and to the synaptic vesicles, are probably transferred by means of connections with the SER.
INTRODUCTION
The smooth endoplasmic reticulum (SER) of the axon has been suspected to convey materials from their sites of synthesis in the perikaryon to plasma membranes of axons and nerve endings and to synaptic vesicles4,6,s-l°,lzAs,ag,ze,z~,29. However we lack information on the spatial arrangement of the neuronal SER to determine whether or not 'the cisternae may represent a continuous system extending along the length of the axon '23. It is also necessary to specify how the SER may be connected with synaptic vesicles which 'appear to be a continuation of the delicate, canalicular, membranous structures belonging to the endoplasmic reticulum '22. In the first part of the present paper, a tridimensional study of the SER discloses its spatial configuration and its anatomical relationship with the axonal membrane and synaptic vesicles. The second part provides a quantitative analysis of experimental results supporting the fact that the SER is involved as vehicle in the fast transport of protein down the axon4,S,9.
MATERIAL AND METHODS
Double impregnation technique Ciliary ganglia from chicken or spinal ganglia from adult rats were fixed by immersion in or perfusion with a 2.5 ~ glutaraldehyde solution in cacodylate buffer. They were impregnated for 1 h at 37 °C in a 2.5 ~o aqueous solution of uranyl acetate and after a quick rinse in water, soaked for another hour at 37 °C in an aqueous copper and lead citrate solutional. They were postfixed overnight at 4 °C in a 1 ~ aqueous osmic acid solution, then dehydrated and embedded in Epon. The blocks were partially polymerized for a period of 15 h and 0.5-2 #m thick sections were prepared. Polymerization of the Epon was allowed to undergo completion during the next 4-5 h and thin sections could be prepared from the same blocks. Both thick and thin sections were mounted on formvar coated copper grids. Thick sections were examined either at 100 kV in a Siemens Elmiskop I using a wide condenser aperture (300/~m) and a small objective aperture (10 #m), or at 10C0 kV in a high-voltage electron microscope (C.I.T. Alcatel, C.N.R.S.-O.N.E.R.A.) using a 200-300/~m condenser aperture and a 20/~m objective aperture. Thin sections were examined at 60 or 80 kV in the Siemens Elmiskop 1. For stereoscopy, thick sections were placed on the goniometric stage in the high-voltage electron microscope or in the stereoscopic cartridge of the Elmiskop I. To decide whether or not structures were continuous, pictures of the same field were taken after tilting the specimen.
Radioautographic procedure Two-week-old chickens (60-80 g) were anesthetized with chloroform. The right orbital cavity was opened. A piece of gelfoam, previously soaked with a 5.10-3 M puromycin solution, was inserted between the eyeball and the ciliary ganglion to eventually produce a slight compression of the ciliary ganglion against the orbital wall. After 20-30 min 400/~Ci of L-[4-aH]lysine (30 Ci/mmole, C.E.A., Saclay) diluted
in 50 #1 of 0.9 % NaCI solution were injected into the cerebral aqueduct. The chickens were sacrificed by decapitation 3 h later. The squeezed right and the intact left ciliary ganglia were removed, fixed in 0.1 M phosphate buffered formaldehyde at 4 %, postfixed in osmium tetroxide, dehydrated and embedded in Epon. After polymerization for 16 h at 60 °C, 1/zm thick sections were cut, deposited on a glass slide and prepared for light microscope radioautography. The radioactivity concentrations were measured in the axons by counting the number of silver grains with an ocular grid. The retrimmed blocks were polymerized further and thin sections of the selected areas were prepared for electron microscope radioautography 7. After a 2-month exposure, the radioautographs were processed in phenidon developer and quantitatively analyzed 83, 34 (Table I). RESULTS Double impregnated sections
All intracellular membranes (rough and smooth endoplasmic reticulum, Golgi apparatus, mitochondria) were intensely stained whereas microtubules, microfilaments and the so-called neuroplasmic matrix were not visible in the perikaryon, axon and nerve ending. In the perikaryon, patches of rough endoplasmic reticulum (Nissl substance) appeared as tightly packed membranous sheets anastomosed to each other by elongated ends (Fig. 1). The axon hillock, known to be devoid of rough endoplasmic reticulum, contains membranous plates and tubules which were more loosely interconnected and became parallel to each other when entering the initial segment of TABLE I CRUDE COUNTS OF SILVER GRAINS AND HITS OVER VARIOUS ELEMENTS OF SQUEEZED AXONS INTRACEREBRAL INJECTION OF [3HILYSINE
3 h AFTER THE
The silver grains were assigned to the overlain structures included within a circle of 225 nm radius from the center of each silver grain 8a. The hits were counted by placing regularly spaced circles of 225 nm radius upon photographs of the radioactive axons and ascribed to the overlain structures 34. The ratio number of silver grains/number of hit circles reflects the radioactivity associated to the axonal elements. Number of silver grains
Myelin sheath 35 Axolemma + inner part of the myelin sheath + outer part of axoplasm 185 Vesicular and tubular profiles of the smooth endoplasmic reticulum + adjacent axoplasm 779 Mitochondria + adjacent axoplasm + adjacent smooth endoplasmic reticulum 70 Axoplasm 26 Total 1095
Number of hit circles
Ratio
258
0.14
46
4.02
132
5.90
42 373 851
0.07
1.67 1.29
Fig. I. The 1 f~m thick section of the perikaryon and the axon hillock in a spinal ganglion cell after double impregnation. The rough endoplasmic reticulum (RER) appears as flattened sheets connected to each other by elongated ends (horizontal arrows). In the axon hillock, sheets and tubes of the smooth endoplasmic reticulum (SER) are loosely anastomosed and are continuous with the sheets of the rough endoplasmic reticulum on the one hand, and with elements of the smooth endoplasmic reticulum present in the axon (Ax) on the other hand. Elongated and sometimes bifurcated mitochondria are labeled M. Part of the nucleus N is visible at upper left. The Golgi apparatus is not visualized in this figure.
Figs. 2 and 3. Proximal part (Fig. 2) and myelinated portion (Fig. 3) of postganglionic axons in the ciliary ganglion after double impregnation. Fig. 2. In the initial segment of the axon, tubes and elongated sheets of smooth endoplasmic reticulum (arrows) are running parallel to the long axis of the axon. They are interconnected by oblique tubular connection or solid plates (P). Note the presence of long intensely stained mitochondria (M). x 45,000. Fig. 3. In this myelinated axon, loosely anastomosed tubes (T) are running along the axonal axis. Plates (P) which are made up of smaller interconnected tubules are frequently encountered just beneath the axolemma. They probably correspond to the vesicular profiles indicated by horizontal arrows in Fig. 1. M, mitochondria, x 30,000.
J
the axon 2a (Fig. 2). In the axons o f both spinal and ciliary ganglia, tubules roughly parallel to the long axis of the axon were interconnected by oblique anastomosesa,12, 23 or m e m b r a n o u s sheets (Fig. 3). In the preterminal segment o f the axon, the tubules o f the endoplasmic reticulum (60-120 nm) were mainly disposed under the axolemma and anastomosed to f o r m an irregular network from which smaller elements originated (Figs. 4-6); these thin tubules (20-30 nm in diameter) were irregularly interconnected to f o r m a meshwork extending above the layer of the synaptic vesicles (Figs. 4 and 7). Tubular elements were observed to establish contact with the presynaptic m e m b r a n e (Fig. 6). Within the meshes of the thin tubular meshwork synaptic vesicles were found to bulge at the tip o f small tubules (Figs. 6 and 7); by tilting the specimen, these synaptic vesicles appeared to be in continuity with the endoplasmic reticulum.
Radioautographs of intact and compressed axons In the ciliary ganglia of the intact side, light microscope radioautographs showed a strong reaction over all the calyciform nerve endings. In contrast, the preganglionic axons displayed only a very weak reaction (2.2 ± 0.6 grains/1C0 sq. # m ) the silver grains were mainly scattered over the peripheral region of the axons. At the ultrastructural level, the rare silver grains detected over the preganglionic axons were closely related to the axolemma, vesicular profiles clustered underneath the axolemma and more or less elongated profiles of the s m o o t h endoplasmic reticulum (Fig. 8). In the squeezed ciliary ganglia, numerous nerve endings were devoid of a strong radioautographic reaction whereas 8-15 ~ of the preganglionic axons were the sites o f an intense retention of the label; the measured grain concentration was 10-80 times higher in these heavily labeled axons than in the axons o f the intact side. With the electron microscope, the heavily labeled axons appeared as full of vesicular and tubular m e m b r a n o u s profiles containing in their lumen a more or less electron-dense
Figs. 4--7. Preterminal segment and axon in the ciliary ganglion after double impregnation. Fig. 4. One pm thick section through the central part of the preterminal region of a preganglionic nerve ending. Small tubes (arrow) are seen to be interconnected and form the so-called secondary network. A wider tube (T) which is part of the more peripheral primary network is visible at upper right and gives rise to the smaller elements. The elongated mitochondria(M)are just fanning out towards the giant nerve ending, x 21,000. Fig. 5. One/~m-thick section through the peripheral part of the preterminal region of a preganglionic nerve ending, Wide tubes (T) anastomose to form the so-called primary network. In contrast, the deeper secondary network is scarcely visible (arrow). At lower right, mitochondria (M) are intermingled with synaptic vesicles (SV). At upper right, note the close relationship between mitochondria (M) and tubes of the primary network, x 26,000. Fig. 6. Oblique thin section through the giant nerve ending. Under the axolemma, wide tubes (T) of the primary network are continuous (vertical arrows) with the smaller tube (t) of the secondary network which extends in a deeper position. Synaptic vesicles (SV) are bulging at the end of tubes (horizontal arrow) originating from the secondary network. In circle, a tube of the latter seems to establish connection with the presynaptic plasma membrane (PPM). M, mitochondria, x 10,000. Fig. 7. Thin section through the central part of a giant nerve ending. At this higher magnification the connections (arrow) between synaptic vesicles and elements of the secondary network are obvious. At lower right, note the regular arrangement of synaptic vesicles around a mitochondrial profile. M, mitochondria, x 40,000.
Figs. 8-10. Electron microscope radioautographs of preganglionic axons 3 h aider the intracerebral injection of [3H]lysine. Fig. 8. In the intact axons of the control side, scattered silver grains are seen in the vicinity of profiles of the smooth endoplasmic reticulum (SER) and of the axolemmal region (Axl). The silver grains found over the axolemmal region are frequently associated with subsurface vesicular profiles (arrows) which probably correspond to the subaxolemmal plates observed in thick sections (see Figs. 6 and 1t ). :~ 20,000. Fig. 9. In the squeezed axon of the compressed side, numerous silver grains accumulate over clumps of tubular and vesicular profiles of the smooth endoplasmic reticulum (SER); the axoplasm (Axp) is practically devoid of radioactivity. ~ 25,000. Fig. 10. The cross-section of a node of Ranvier exhibits an accumulation of both silver grains and dilated profiles of the smooth endoplasmic reticulum (SER). • 32,000.
materiallS, z9 (Figs. 9 and 10). When consecutive sections were examined, the accumulation of both membranous profiles and silver grains reached its maximal intensity at and immediately above the node of Ranvier (Fig. 10). Grain counts indicate that most of the silver grains are distributed in close association with the stacks of membranous profiles and to lesser extent with the axolemmal region (Table I, Figs. 9 and 10). The poorly labeled axons which were lacking in stacked membranous profiles displayed ultrastructural features rather similar to those of the axons in the intact side, except that the number of mitochondrial profiles was slightly increased. DISCUSSION
The study of the tridimensional configuration of the axonal endoplasmic reticulum is greatly facilitated by the use of the double impregnation technique 31 which reveals not only the membrane but also the luminal content of the tubules. The convoluted network of the perikaryal endoplasmic reticulum (Fig. 1) appears to be continuous with the parallel bundle of tubules present in the axon hillock and the whole length of the axon (Figs. 2 and 3). In tangential or cross-sections of the axons, 'subaxolemmal plates' which consist of a dense network of smaller tubules and flat cisternae (Fig. 3), establish occasional contacts with the axolemmal membrane. Such connections could therefore account for the renewal of the axolemmal components by means of fast transported macromolecules6,S,9,13,19, 26. In the preterminal segment of the axons (Fig. 4) and in the calyciform nerve endings (Figs. 4-6), the tubular endoplasmic reticulum forms a subsurface 'primary network' from which thinner and shorter elements originate to give rise to the well developed 'secondary network' (Fig. 11). The development of the 'primary network' of endoplasmic reticulum in the preterminal region of the axon is probably responsible for the local slowing down of the fast axonal transport (see Fig. 11 in ref. 9). In the synaptic terminal, elements of the 'primary' and 'secondary network' may extend towards the presynaptic plasma membrane (Figs. 6 and 11), the contact of the endoplasmic reticulum with the membrane of the axon terminal, also observed in the growth cones 3, could be a preferential pathway to deliver protein and glycoprotein components 8,1°,19,24,26 to the highly specialized presynaptic vesicular grid 1. Among the population of synaptic vesicles, some of them appear to be directly appended to elements of the 'secondary network' (Fig. 5, 6, 7 and 11); even after tilting the specimen under the electron beam, no gap could be visualized between those synaptic vesicles and the thin tubules of the endoplasmic reticulum. The continuity of the endoplasmic reticulum with synaptic vesicles has already been suspected in nerve terminals ~,5,11,15,2°-g2, 25,2s-80. However our 3-dimensional approach provides stronger evidence for 'the direct participation of the endoplasmic reticulum in the formation of synaptic vesicles, so difficult to establish in the case of vertebrates '5. Synaptic vesicles budding from thin branches of the 'secondary network' of the endoplasmic reticulum visualize probably the biogenesis 22 or the recycling of synaptic vesicles16,17. When [aH]lysine is injected into the cerebral ventricle, the label is taken up by the nerve cell bodies of the Edinger-Westphal nucleus and incorporated into protein;
It)
AXON HILLOC
-
' ENDOPLASMIC
,xo
~
•
SUBAXOLE MMAL PL ATE
PRETERMINALREGION
~
PRIMARY NETWORK
MITOCHONDRIA
Fig. 11. Diagrammatic representation of the axonat smooth endoplasmic reticulum. The axonal endoplasmic reticulum appears as a continuous system of tubules extending from the perikaryon to the axon terminal. The convoluted tubules of the smooth endoplasmic reticulum enter the initial segment of the axon and run parallel to its long axis. Loosely anastomosed, they give rise to 'subaxolemmal plates' made up of smaller tubules. In the preterminal region of the axon, the large tubules of the smooth endoplasmic reticulum anastomose peripherally and form the primary network. The well developed secondary network, caged within the primary network, is composed of smaller elements originating from the wide tubules. Thin and wide tubules of the smooth endoplasmic reticulum appear occasionally to be closely apposed to the axolemmal and presynaptic membrane. It is postulated that the exchange of fast axonally transported macromolecules takes place at the contact between the subaxolemmal plate or tubules. The synaptic vesicles which are appended like blebs at the tip of thin tubular branches visualize probably their fission from or their fusion with the smooth endoplasmic reticulum. then labeled proteins are distally transported along the 10 m m long preganglionic axons towards their nerve endings located in the ciliary gangliong,lL T h u s 3 h after the intracerebral injection, only proteins conveyed with the fast axonal t r a n s p o r t at a rate of 288 ~ 40 m m / d a y have reached the t e r m i n a l part of the axon 9. I n the intact axons of the c o n t r o l ganglia, most of the fast transported label is f o u n d in the peripheral region of the axoplasm, as it has been observed in myelinated and u n m y e l i n a t e d axons4,8,9, 26. However it is difficult to ascertain whether the scarce
11 silver grains are associated with the axolemma or profiles of the smooth endoplasmic reticulum (Fig. 8). In contrast, radioautographs of progressively squeezed ganglia show that 8-15~o of the axons are heavily labeled, especially at and above the last node of Ranvier. It is presumed that the slight pressure exerted on the ganglion is more effective on the unmyelinated region of the axon than on segments somewhat protected by their myelin sheath (Fig. 10). These arrested proteins are found in close association with the jammed endoplasmic reticulum elements4,S, 9 (Figs. 9 and 10, Table I). This accumulation of radioactivity cannot result from a local synthesis of protein stimulated by the pressure since it persists even when puromycin is locally applied. Therefore the simultaneous increase of labeled macromolecules and of endoplasmic reticulum should be the consequence of a concomitant movement of these fast transported constituents. Furthermore, recent results obtained after administration of [aH]glycerol indicate that newly synthesized phospholipids are incorporated into membranes and migrate rapidly from the perikaryon into the axon14; when the nerve is transected, electron microscope radioautographs show that membranous profiles containing the labeled phospholipids accumulate in the proximal stumps of the axon 7a. Since membrane phospholipids and proteins are building blocks of the endoplasmic reticulum, these data provide supporting evidence for a rapid axonal transport of the endoplasmic reticulum components. However it is difficult to ascertain whether the membrane constituents move down the axon with and/or within the tubules of the SER. Furthermore some tubules of the endoplasmic reticulum seem also to be involved in the retrograde as well as in the orthograde transport of material from the axon terminal to the perikaryon as it is suggested from experiments using peroxidase as tracer20a, 21a, 24a,27,32. Thus to ensure a rapid shuttle of molecules between the perikaryon and the axon terminal, the neuron possesses a dynamic system of continuous canals which constitute the main intraaxonal pathway to ensure the rapid transfer of components making up the axonal membrane and synaptic vesicles. ACKNOWLEDGEMENTS The authors wish to thank Dr. Y. Clermont and P. Gambetti for their useful advices in writing the manuscript. They are especially indebted to Dr. A. Marraud who has facilitated the use of high voltage electron microscope, to Mrs. J. Boyenval who has prepared radioautographs and to Mrs. R. H~issig for her assistance.
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12 5 COUTEAUX, R., Remarks on the organization of axon terminals in relation to secretory processes at synapses. In B. CECCARELLI,F. CLEMENTIAND J. MELDOLESI(Eds.), Advances in Cytopharmacology, Vol. 2, Raven Press, New York, 1974, pp. 369--379. 6 CU~NOD, M., BOESCH,J., MARCO, P., PERmlC, M., SANDRI,C., AND SCnONBACH,J., Contributions of axoplasmic transport to synaptic structures and functions, Int. J. Neurosci., 4 (1972) 77-87. 7 DROZ, B., Autoradiography as a tool for visualizing neurons and neuronal processes. In W. M. COWAN AND M. CUt'NOD(Eds.), The Use of Axonal Transport for Studies of Neuronal Connectivity, Elsevier, Amsterdam, 1975, pp. 127-154. 7a DRoz, B., ET BOYENVAL,J., Le r6ticulum endoplasmique des axones: son r61e probable dans le transport axoplasmique des phospholipides membranaires, J. Microsc. Biol. Cell, 23 (1975) 45~6a. 8 DRoz, B., AND D1 GIAMBERARDINO,L., Transport of macromolecules in nerve cells, In B. C~CCARELLI, F. CLEMENTIAND J. MELDOLESl(Eds.), Advances in Cytopharmacology, Vol. 2, Raven Press, New York, 1974, pp. 249-256. 9 DRoz, B., KOEMG, H. L., AND DI GIAMBERARDINO,L., Axonal migration of protein and glycoprotein to nerve endings. 1. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [aH]lysine, Brain Research, 60 (1973) 93-127. 10 DROZ, B., KOENIG, H. L., ET RAMBOURG,A., Transport intracytoplasmique de macromol&:ules et r6ticulum endoplasmique lisse: cas du flux axonal rapide, J. Microsc., 20 (1974) 45a. 11 D~JR1N6, M. yON, Uber die Feinstruktur der motorischen Endplatte von h6heren Wirbeltieren, Z. Zellforsch., 81 (1967) 74-90. 12 GISIGER, V., VENKOV, L., ET GAUTRON, J., Ac6tylcholinestgrase et biosynth6se des protgines du ganglion sympathique au repos et stimul6, J. Neurochem., (1975) Submitted. 13 GRAFSTEIN,B., Axonal transport: the intracellular traffic of the neuron. In E. R. KANDEL (Ed.), The Handbook of the Nervous System, Vol. 1, Cellular Biology of Neurones, Amer. Physiol. Soc., Washington D.C., 1975, in press. 14 GRAFSTEIN,B., MIt.LER, J. A., LEDEEN,R. W., HALEY,J., AND SPECHT, S. C., Axonal transport of phospholipid in goldfish optic system, Exp. Neurol., 46 (1975) 261-281. 15 HERNANDEZ-NIcAJSE,M. L., The nervous system of ctenophores. 111. Ultrastructure of synapses, J. Neurocytol., 2 (1973) 249-263. 16 HEUSER,J. E., AND REESE,T. S., Morphology of synaptic vesicle discharge and reformation at the frog neuromuscular junction. In M. V. L. BENNETT (Ed.), Synaptic Transmission and Neuronal Interaction, Raven Press, New York, 1974, pp. 59-77. 17 HURLBUT, W. P., AND CECCARELLI, B., Transmitter release and recycling of synaptic vesicle membrane at the neuromuscular junction. In B. CECCARELLI, F. CLEMENTIAND J. MELDOLESI(Eds.), Advances in Cytopharmacology, Vol. 2, Raven Press, New York, 1974, pp. 141-154. 18 KAPELLER,K., AND MAYOR,D., An electron microscopic study of the early changes proximal to a constriction in sympathetic nerves, Proc. roy. Soc. B, 172 (1969) 39-51. 19 KOENIG, H. L., DI GIAMBERARDINO,L., AND B~NNE'rr, G., Renewal of proteins and glycoproteins of synaptic constituents by means of axonal flow, Brain Research, 62 (1973) 413-417. 20 KORNELIUSSEN,H,, Ultrastructure of normal and stimulated motor endplates. With comments on the origin and fate of synaptic vesicles, Z. Zellforsch., 130 (1972) 28-57. 20a LAVA1L,M. W., ANO LAVA~L,J. H., Retrograde intraaxonal transport of horseradish peroxidase in retinal ganglion cells of the chick, Brain Research, 85 (1975) 273-280. 21 LovAs, B., Tubular networks in the terminal endings of the visual receptor cells in the human, the monkey, the cat, and the dog, Z. Zellforsch., 121 (1971) 341-357. 21a NAUTA, H. J. W., KAISERMAN-ABRAMOF,|. R., AND LASEK, R. J., Electron microscope observations of horseradish peroxidase transport from the caudoputamen to the substantia nigra in the rat: possible involvement of the agranular reticulum, Brain Research, 85 (1975) 373-384. 22 PALAY,S. L., The morphology of synapses in the central nervous system, Exp. Cell Res., Suppl. 5 (1958) 275-293. 23 PETERS, A., PALAY, S.L., AND WEBSTER, H. DEF., The Fine Structure of the Nervous System, Harper and Row, New York, 1970. 24 RAMBOURG,A., AND LEBLOND, C. P., Electron microscope observations on the carbohydrate riche cell coat present at the surface of cells in the rat, J. Cell Biol., 32 (1967) 27-53. 24a REP~RANT, J., The orthograde transport of horseradish peroxidase in the visual system, Brain Research, 85 (1975) 307-312.
13 25 ROBERTSON,J. D., The ultrastructure of synapses. In F. O. SCHMITr (Ed.), The Neurosciences, Second Study Program, The Rockefeller University Press, New York, 1970, pp. 715-728. 26 SCHONBACI4,J., SCHONBACH,C., AND CU~NOD,M., Rapid phase of axoplasmic flow and synaptic proteins; an electron microscopical autoradiographic study, J. comp. NeuroL, 141 (1971) 485-198. 27 SOTELO, C., AND RICHE, D., The smooth endoplasmic reticulum and the retrograde and fast orthograde transport of horseradish peroxidase in the nigrostriatal-nigralloop, Z. Anat. Entwickl.Gesch., (1974) in press. 28 STELZNER,D. J., The relationship between synaptic vesicles, Golgi apparatus and smooth endoplasmic reticulum: a developmental study using the zinc iodine-osmiumtechnique, Z. Zellforsch., 120 (1971) 332-345. 29 TAXt, J., AND SOTELO,C., Cytological aspects of the axonal migration of catecholamines and of storage material, Brain Research, 62 (1973) 431-437. 30 TEICHBERG,S., AND HOLTZMAN,E., Axonal agranular reticulum and synaptic vesicles in cultured embryonic chick sympathetic neuron, J. Cell BioL, 57 (1973) 88-108. 31 THIERY,G., Coloration permettant l'6tude des coupes 6paisses, leur int6r~t en microscopie 61ectronique, J. Microsc., 17 (1973) 101a. 32 TURNER,P. R., AND HARRIS,A. B., Ultrastructure of exogenous peroxidase in cerebral cortex, Brain Research, 74 (1974) 305-326. 33 WHUR,P., HERSCOVlCS,A., AND LEaLOND,C. P., Radioautographic visualization of the incorporation of galactose-aH and mannose-aH by rat thyroid in vitro in relation to the stage of thyroglobulin synthesis, J. Cell Biol., 43 (1969) 289-311. 34 WILLIAMS,M. A., The assessment of electron microscopic autoradiographs, Advanc. Optic. Electr. Microsc., 3 (1969) 219-272.
1
Research Reports
THE SMOOTH ENDOPLASMIC RETICULUM: STRUCTURE AND ROLE IN THE RENEWAL OF AXONAL MEMBRANE AND SYNAPTIC VESICLES BY FAST AXONAL TRANSPORT
B E R N A R D DROZ, A L A I N R A M B O U R G AND HERBERT L. K O E N I G
D~partement de Biologic, Commissariat t~ l'l~nergie Atomique, Centre d'l~tudes Nucldaires de Saclay, B.P. 2, 91190 Gif-sur-Yvette et Laboratoire de Cytologic, Universit~ Pierre et Marie Curie, 75005 Paris (France} (Accepted February 21st, 1975)
SUMMARY
The spatial arrangement of the smooth endoplasmic reticulum (SER) was studied in 0.5-2/~m thick sections of rat spinal and chick ciliary ganglia previously impregnated with heavy metal salts. Electron microscopy at low (105 V) or high (106 V) voltage showed the impregnated SER as a continuous system extending probably from the perikaryon to the axon terminal. Tubules of the SER, which were running in a parallel direction with the axon, were occasionally seen in close apposition with the axonal membrane. Moreover in the preterminal region, anastomosed tubules of the SER formed a subsurface 'primary network' and gave rise to a deeper 'secondary network' made of thinner tubules; synaptic vesicles bulging at the tip of thin tubules of the SER were frequently observed. To specify the role played by the SER in the fast axonal transport, chicken ciliary ganglia were slightly compressed and radioautographed 3 h after the intracerebral injection of [3H]lysine. Quantitative analysis of the silver grain distribution indicated that labeled proteins, rapidly conveyed down the axon, piled up in regions containing an accumulation of SER profiles. On the basis of these results, it is concluded that: (1) the SER appears as a continuous intraaxonal pathway bridging the perikaryon and the axon terminal; (2) the SER conveys macromolecular components with the fast axonal transport; (3) the conveyed macromolecules, which are delivered to the axonal membrane and to the synaptic vesicles, are probably transferred by means of connections with the SER.
INTRODUCTION
The smooth endoplasmic reticulum (SER) of the axon has been suspected to convey materials from their sites of synthesis in the perikaryon to plasma membranes of axons and nerve endings and to synaptic vesicles4,6,s-l°,lzAs,ag,ze,z~,29. However we lack information on the spatial arrangement of the neuronal SER to determine whether or not 'the cisternae may represent a continuous system extending along the length of the axon '23. It is also necessary to specify how the SER may be connected with synaptic vesicles which 'appear to be a continuation of the delicate, canalicular, membranous structures belonging to the endoplasmic reticulum '22. In the first part of the present paper, a tridimensional study of the SER discloses its spatial configuration and its anatomical relationship with the axonal membrane and synaptic vesicles. The second part provides a quantitative analysis of experimental results supporting the fact that the SER is involved as vehicle in the fast transport of protein down the axon4,S,9.
MATERIAL AND METHODS
Double impregnation technique Ciliary ganglia from chicken or spinal ganglia from adult rats were fixed by immersion in or perfusion with a 2.5 ~ glutaraldehyde solution in cacodylate buffer. They were impregnated for 1 h at 37 °C in a 2.5 ~o aqueous solution of uranyl acetate and after a quick rinse in water, soaked for another hour at 37 °C in an aqueous copper and lead citrate solutional. They were postfixed overnight at 4 °C in a 1 ~ aqueous osmic acid solution, then dehydrated and embedded in Epon. The blocks were partially polymerized for a period of 15 h and 0.5-2 #m thick sections were prepared. Polymerization of the Epon was allowed to undergo completion during the next 4-5 h and thin sections could be prepared from the same blocks. Both thick and thin sections were mounted on formvar coated copper grids. Thick sections were examined either at 100 kV in a Siemens Elmiskop I using a wide condenser aperture (300/~m) and a small objective aperture (10 #m), or at 10C0 kV in a high-voltage electron microscope (C.I.T. Alcatel, C.N.R.S.-O.N.E.R.A.) using a 200-300/~m condenser aperture and a 20/~m objective aperture. Thin sections were examined at 60 or 80 kV in the Siemens Elmiskop 1. For stereoscopy, thick sections were placed on the goniometric stage in the high-voltage electron microscope or in the stereoscopic cartridge of the Elmiskop I. To decide whether or not structures were continuous, pictures of the same field were taken after tilting the specimen.
Radioautographic procedure Two-week-old chickens (60-80 g) were anesthetized with chloroform. The right orbital cavity was opened. A piece of gelfoam, previously soaked with a 5.10-3 M puromycin solution, was inserted between the eyeball and the ciliary ganglion to eventually produce a slight compression of the ciliary ganglion against the orbital wall. After 20-30 min 400/~Ci of L-[4-aH]lysine (30 Ci/mmole, C.E.A., Saclay) diluted
in 50 #1 of 0.9 % NaCI solution were injected into the cerebral aqueduct. The chickens were sacrificed by decapitation 3 h later. The squeezed right and the intact left ciliary ganglia were removed, fixed in 0.1 M phosphate buffered formaldehyde at 4 %, postfixed in osmium tetroxide, dehydrated and embedded in Epon. After polymerization for 16 h at 60 °C, 1/zm thick sections were cut, deposited on a glass slide and prepared for light microscope radioautography. The radioactivity concentrations were measured in the axons by counting the number of silver grains with an ocular grid. The retrimmed blocks were polymerized further and thin sections of the selected areas were prepared for electron microscope radioautography 7. After a 2-month exposure, the radioautographs were processed in phenidon developer and quantitatively analyzed 83, 34 (Table I). RESULTS Double impregnated sections
All intracellular membranes (rough and smooth endoplasmic reticulum, Golgi apparatus, mitochondria) were intensely stained whereas microtubules, microfilaments and the so-called neuroplasmic matrix were not visible in the perikaryon, axon and nerve ending. In the perikaryon, patches of rough endoplasmic reticulum (Nissl substance) appeared as tightly packed membranous sheets anastomosed to each other by elongated ends (Fig. 1). The axon hillock, known to be devoid of rough endoplasmic reticulum, contains membranous plates and tubules which were more loosely interconnected and became parallel to each other when entering the initial segment of TABLE I CRUDE COUNTS OF SILVER GRAINS AND HITS OVER VARIOUS ELEMENTS OF SQUEEZED AXONS INTRACEREBRAL INJECTION OF [3HILYSINE
3 h AFTER THE
The silver grains were assigned to the overlain structures included within a circle of 225 nm radius from the center of each silver grain 8a. The hits were counted by placing regularly spaced circles of 225 nm radius upon photographs of the radioactive axons and ascribed to the overlain structures 34. The ratio number of silver grains/number of hit circles reflects the radioactivity associated to the axonal elements. Number of silver grains
Myelin sheath 35 Axolemma + inner part of the myelin sheath + outer part of axoplasm 185 Vesicular and tubular profiles of the smooth endoplasmic reticulum + adjacent axoplasm 779 Mitochondria + adjacent axoplasm + adjacent smooth endoplasmic reticulum 70 Axoplasm 26 Total 1095
Number of hit circles
Ratio
258
0.14
46
4.02
132
5.90
42 373 851
0.07
1.67 1.29
Fig. I. The 1 f~m thick section of the perikaryon and the axon hillock in a spinal ganglion cell after double impregnation. The rough endoplasmic reticulum (RER) appears as flattened sheets connected to each other by elongated ends (horizontal arrows). In the axon hillock, sheets and tubes of the smooth endoplasmic reticulum (SER) are loosely anastomosed and are continuous with the sheets of the rough endoplasmic reticulum on the one hand, and with elements of the smooth endoplasmic reticulum present in the axon (Ax) on the other hand. Elongated and sometimes bifurcated mitochondria are labeled M. Part of the nucleus N is visible at upper left. The Golgi apparatus is not visualized in this figure.
Figs. 2 and 3. Proximal part (Fig. 2) and myelinated portion (Fig. 3) of postganglionic axons in the ciliary ganglion after double impregnation. Fig. 2. In the initial segment of the axon, tubes and elongated sheets of smooth endoplasmic reticulum (arrows) are running parallel to the long axis of the axon. They are interconnected by oblique tubular connection or solid plates (P). Note the presence of long intensely stained mitochondria (M). x 45,000. Fig. 3. In this myelinated axon, loosely anastomosed tubes (T) are running along the axonal axis. Plates (P) which are made up of smaller interconnected tubules are frequently encountered just beneath the axolemma. They probably correspond to the vesicular profiles indicated by horizontal arrows in Fig. 1. M, mitochondria, x 30,000.
J
the axon 2a (Fig. 2). In the axons o f both spinal and ciliary ganglia, tubules roughly parallel to the long axis of the axon were interconnected by oblique anastomosesa,12, 23 or m e m b r a n o u s sheets (Fig. 3). In the preterminal segment o f the axon, the tubules o f the endoplasmic reticulum (60-120 nm) were mainly disposed under the axolemma and anastomosed to f o r m an irregular network from which smaller elements originated (Figs. 4-6); these thin tubules (20-30 nm in diameter) were irregularly interconnected to f o r m a meshwork extending above the layer of the synaptic vesicles (Figs. 4 and 7). Tubular elements were observed to establish contact with the presynaptic m e m b r a n e (Fig. 6). Within the meshes of the thin tubular meshwork synaptic vesicles were found to bulge at the tip o f small tubules (Figs. 6 and 7); by tilting the specimen, these synaptic vesicles appeared to be in continuity with the endoplasmic reticulum.
Radioautographs of intact and compressed axons In the ciliary ganglia of the intact side, light microscope radioautographs showed a strong reaction over all the calyciform nerve endings. In contrast, the preganglionic axons displayed only a very weak reaction (2.2 ± 0.6 grains/1C0 sq. # m ) the silver grains were mainly scattered over the peripheral region of the axons. At the ultrastructural level, the rare silver grains detected over the preganglionic axons were closely related to the axolemma, vesicular profiles clustered underneath the axolemma and more or less elongated profiles of the s m o o t h endoplasmic reticulum (Fig. 8). In the squeezed ciliary ganglia, numerous nerve endings were devoid of a strong radioautographic reaction whereas 8-15 ~ of the preganglionic axons were the sites o f an intense retention of the label; the measured grain concentration was 10-80 times higher in these heavily labeled axons than in the axons o f the intact side. With the electron microscope, the heavily labeled axons appeared as full of vesicular and tubular m e m b r a n o u s profiles containing in their lumen a more or less electron-dense
Figs. 4--7. Preterminal segment and axon in the ciliary ganglion after double impregnation. Fig. 4. One pm thick section through the central part of the preterminal region of a preganglionic nerve ending. Small tubes (arrow) are seen to be interconnected and form the so-called secondary network. A wider tube (T) which is part of the more peripheral primary network is visible at upper right and gives rise to the smaller elements. The elongated mitochondria(M)are just fanning out towards the giant nerve ending, x 21,000. Fig. 5. One/~m-thick section through the peripheral part of the preterminal region of a preganglionic nerve ending, Wide tubes (T) anastomose to form the so-called primary network. In contrast, the deeper secondary network is scarcely visible (arrow). At lower right, mitochondria (M) are intermingled with synaptic vesicles (SV). At upper right, note the close relationship between mitochondria (M) and tubes of the primary network, x 26,000. Fig. 6. Oblique thin section through the giant nerve ending. Under the axolemma, wide tubes (T) of the primary network are continuous (vertical arrows) with the smaller tube (t) of the secondary network which extends in a deeper position. Synaptic vesicles (SV) are bulging at the end of tubes (horizontal arrow) originating from the secondary network. In circle, a tube of the latter seems to establish connection with the presynaptic plasma membrane (PPM). M, mitochondria, x 10,000. Fig. 7. Thin section through the central part of a giant nerve ending. At this higher magnification the connections (arrow) between synaptic vesicles and elements of the secondary network are obvious. At lower right, note the regular arrangement of synaptic vesicles around a mitochondrial profile. M, mitochondria, x 40,000.
Figs. 8-10. Electron microscope radioautographs of preganglionic axons 3 h aider the intracerebral injection of [3H]lysine. Fig. 8. In the intact axons of the control side, scattered silver grains are seen in the vicinity of profiles of the smooth endoplasmic reticulum (SER) and of the axolemmal region (Axl). The silver grains found over the axolemmal region are frequently associated with subsurface vesicular profiles (arrows) which probably correspond to the subaxolemmal plates observed in thick sections (see Figs. 6 and 1t ). :~ 20,000. Fig. 9. In the squeezed axon of the compressed side, numerous silver grains accumulate over clumps of tubular and vesicular profiles of the smooth endoplasmic reticulum (SER); the axoplasm (Axp) is practically devoid of radioactivity. ~ 25,000. Fig. 10. The cross-section of a node of Ranvier exhibits an accumulation of both silver grains and dilated profiles of the smooth endoplasmic reticulum (SER). • 32,000.
materiallS, z9 (Figs. 9 and 10). When consecutive sections were examined, the accumulation of both membranous profiles and silver grains reached its maximal intensity at and immediately above the node of Ranvier (Fig. 10). Grain counts indicate that most of the silver grains are distributed in close association with the stacks of membranous profiles and to lesser extent with the axolemmal region (Table I, Figs. 9 and 10). The poorly labeled axons which were lacking in stacked membranous profiles displayed ultrastructural features rather similar to those of the axons in the intact side, except that the number of mitochondrial profiles was slightly increased. DISCUSSION
The study of the tridimensional configuration of the axonal endoplasmic reticulum is greatly facilitated by the use of the double impregnation technique 31 which reveals not only the membrane but also the luminal content of the tubules. The convoluted network of the perikaryal endoplasmic reticulum (Fig. 1) appears to be continuous with the parallel bundle of tubules present in the axon hillock and the whole length of the axon (Figs. 2 and 3). In tangential or cross-sections of the axons, 'subaxolemmal plates' which consist of a dense network of smaller tubules and flat cisternae (Fig. 3), establish occasional contacts with the axolemmal membrane. Such connections could therefore account for the renewal of the axolemmal components by means of fast transported macromolecules6,S,9,13,19, 26. In the preterminal segment of the axons (Fig. 4) and in the calyciform nerve endings (Figs. 4-6), the tubular endoplasmic reticulum forms a subsurface 'primary network' from which thinner and shorter elements originate to give rise to the well developed 'secondary network' (Fig. 11). The development of the 'primary network' of endoplasmic reticulum in the preterminal region of the axon is probably responsible for the local slowing down of the fast axonal transport (see Fig. 11 in ref. 9). In the synaptic terminal, elements of the 'primary' and 'secondary network' may extend towards the presynaptic plasma membrane (Figs. 6 and 11), the contact of the endoplasmic reticulum with the membrane of the axon terminal, also observed in the growth cones 3, could be a preferential pathway to deliver protein and glycoprotein components 8,1°,19,24,26 to the highly specialized presynaptic vesicular grid 1. Among the population of synaptic vesicles, some of them appear to be directly appended to elements of the 'secondary network' (Fig. 5, 6, 7 and 11); even after tilting the specimen under the electron beam, no gap could be visualized between those synaptic vesicles and the thin tubules of the endoplasmic reticulum. The continuity of the endoplasmic reticulum with synaptic vesicles has already been suspected in nerve terminals ~,5,11,15,2°-g2, 25,2s-80. However our 3-dimensional approach provides stronger evidence for 'the direct participation of the endoplasmic reticulum in the formation of synaptic vesicles, so difficult to establish in the case of vertebrates '5. Synaptic vesicles budding from thin branches of the 'secondary network' of the endoplasmic reticulum visualize probably the biogenesis 22 or the recycling of synaptic vesicles16,17. When [aH]lysine is injected into the cerebral ventricle, the label is taken up by the nerve cell bodies of the Edinger-Westphal nucleus and incorporated into protein;
It)
AXON HILLOC
-
' ENDOPLASMIC
,xo
~
•
SUBAXOLE MMAL PL ATE
PRETERMINALREGION
~
PRIMARY NETWORK
MITOCHONDRIA
Fig. 11. Diagrammatic representation of the axonat smooth endoplasmic reticulum. The axonal endoplasmic reticulum appears as a continuous system of tubules extending from the perikaryon to the axon terminal. The convoluted tubules of the smooth endoplasmic reticulum enter the initial segment of the axon and run parallel to its long axis. Loosely anastomosed, they give rise to 'subaxolemmal plates' made up of smaller tubules. In the preterminal region of the axon, the large tubules of the smooth endoplasmic reticulum anastomose peripherally and form the primary network. The well developed secondary network, caged within the primary network, is composed of smaller elements originating from the wide tubules. Thin and wide tubules of the smooth endoplasmic reticulum appear occasionally to be closely apposed to the axolemmal and presynaptic membrane. It is postulated that the exchange of fast axonally transported macromolecules takes place at the contact between the subaxolemmal plate or tubules. The synaptic vesicles which are appended like blebs at the tip of thin tubular branches visualize probably their fission from or their fusion with the smooth endoplasmic reticulum. then labeled proteins are distally transported along the 10 m m long preganglionic axons towards their nerve endings located in the ciliary gangliong,lL T h u s 3 h after the intracerebral injection, only proteins conveyed with the fast axonal t r a n s p o r t at a rate of 288 ~ 40 m m / d a y have reached the t e r m i n a l part of the axon 9. I n the intact axons of the c o n t r o l ganglia, most of the fast transported label is f o u n d in the peripheral region of the axoplasm, as it has been observed in myelinated and u n m y e l i n a t e d axons4,8,9, 26. However it is difficult to ascertain whether the scarce
11 silver grains are associated with the axolemma or profiles of the smooth endoplasmic reticulum (Fig. 8). In contrast, radioautographs of progressively squeezed ganglia show that 8-15~o of the axons are heavily labeled, especially at and above the last node of Ranvier. It is presumed that the slight pressure exerted on the ganglion is more effective on the unmyelinated region of the axon than on segments somewhat protected by their myelin sheath (Fig. 10). These arrested proteins are found in close association with the jammed endoplasmic reticulum elements4,S, 9 (Figs. 9 and 10, Table I). This accumulation of radioactivity cannot result from a local synthesis of protein stimulated by the pressure since it persists even when puromycin is locally applied. Therefore the simultaneous increase of labeled macromolecules and of endoplasmic reticulum should be the consequence of a concomitant movement of these fast transported constituents. Furthermore, recent results obtained after administration of [aH]glycerol indicate that newly synthesized phospholipids are incorporated into membranes and migrate rapidly from the perikaryon into the axon14; when the nerve is transected, electron microscope radioautographs show that membranous profiles containing the labeled phospholipids accumulate in the proximal stumps of the axon 7a. Since membrane phospholipids and proteins are building blocks of the endoplasmic reticulum, these data provide supporting evidence for a rapid axonal transport of the endoplasmic reticulum components. However it is difficult to ascertain whether the membrane constituents move down the axon with and/or within the tubules of the SER. Furthermore some tubules of the endoplasmic reticulum seem also to be involved in the retrograde as well as in the orthograde transport of material from the axon terminal to the perikaryon as it is suggested from experiments using peroxidase as tracer20a, 21a, 24a,27,32. Thus to ensure a rapid shuttle of molecules between the perikaryon and the axon terminal, the neuron possesses a dynamic system of continuous canals which constitute the main intraaxonal pathway to ensure the rapid transfer of components making up the axonal membrane and synaptic vesicles. ACKNOWLEDGEMENTS The authors wish to thank Dr. Y. Clermont and P. Gambetti for their useful advices in writing the manuscript. They are especially indebted to Dr. A. Marraud who has facilitated the use of high voltage electron microscope, to Mrs. J. Boyenval who has prepared radioautographs and to Mrs. R. H~issig for her assistance.
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