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Electron microscopic study of the giant nerve fiber of the giant squid Dosidicus gigas
© 1969 by Academic Press, Inc.
J. ULTRASTRUCTURE RESEARCH 26,
501-514 (1969)
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Electron Microscopic Study of the Giant Nerve Fiber of the Giont Squid Dosidicus gigas GLORIA M . VILLEGAS
Centro de Bioflsica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 1827, Caracas, Venezuela Received July 12, 1968 The present work deals with the ultrastructure of the giant nerve fiber of the giant squid Dosidieusgigas. The axons studied were 80(01200 ~ in diameter. The axolemma (membrane of the axon proper) shows a three-laminated pattern alternated with zones made up of repeated globular units. Local thickenings attached to the axolemma internal leaflet are also observed. The axoplasm shows a peculiar structure formed by clusters of vesicles, 600-1000/~ in diameter, and clusters of granules of about 400/~ in diameter. Both components form elongated massive bodies along the axon. Two types of axonal mitochondria were observed: one type rounded, over 0.6 # in diameter, found only near the axolemma; and the other, elongated, about 0.4 ~ in width by 3 # in length, scattered all over the axoplasm. The Schwann layer, 1.5-6 # thick, formed by a single row of Schwann cells, is crossed by channels. These channels are more tortuous than in the tropical species. The Schwann layer is covered outside by the basal lamina made up by a thread-like dense material, intermingled with connective tissue fibrils. Following this zone is the orderly arranged endoneurium, formed by alternated layers of cells and fibrils. Some endoneurial cells bear pigment-like granules. Blood vessels are found alongside the endoneurium. The largest axons known to the present are those of the giant squid Dosidicus gigas found in the H u m b o l d t Stream of the Pacific Ocean. The giant axons of this species are usually over 1 m m in diameter. The results already obtained by the methods of perfusion (2, 3, 15) and dialysis (4) of the squid giant axon, as well as the promising value of these experimental methods, make the axons of D. gigas of greatest interest. Electron microscopic studies of the squid giant nerve fibers have been made in the species Loligo pealii (10), Doryteuthis plei (27, 28), Sepioteuthis sepioidea (31), and Loligo forbesi (3). The present work deals with the fine structure of the giant axon of D. gigas and the Schwann cell and connective tissue sheaths surrounding it. Preliminary observations on the ultrastructure of this giant nerve fiber have been already published (30).
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Seven giant nerve fibers of the first stellar nerve of the squid D. gigas were used. The animals were obtained in the coastal waters of Peru and killed immediately by decapitation; the mantles were immersed in ice-cold seawater. Within 1.5-4 hours, the nerve fibers were isolated from the mantle in the laboratory, both their ends were tied with threads, and the fibers were tested for nerve impulse conduction and then immediately immersed in an ice-cold fixative solution of 1 To OsO~ in artificial seawater, buffered with Veronal acetate at p H 8-8.1. The fixation time was 2-8 hours. The osmolarity of the fixative was 1050 mOsm/kg of water, as determined by a freezing point osmometer. After fixation the nerve fibers were washed in artificial seawater, dehydrated in a series of acetone, and embedded in Epon. Fine sections were obtained on an LKB ultramicrotome equipped with a diamond knife, placed on Formvar-carbon-coated copper grids, double stained with uranyl acetate (32) and lead citrate (20), and examined in a Siemens Elmiskop 1A electron microscope at an accelerating voltage of 80 kV. The resolution of the microscope as measured point-to-point in a film of evaporated platinum was 4.3 A. Some longitudinal sections were mounted on disks with a single center hole. RESULTS E a c h giant nerve fiber shows a general structural o r g a n i z a t i o n similar to the one a l r e a d y described in o t h e r squid species (27-29, 31). The axon a p p e a r s s u r r o u n d e d by a single r o w o f Schwann cells, a b a s a l l a m i n a or b a s e m e n t m e m b r a n e , a n d a layer o f connective tissue (Fig. 1). The diameters o f the axons used m e a s u r e d 800-1200/z.
Axofl The a x o p l a s m is f o r m e d b y filaments 50-100 A thick, g r o u p e d in bundles m a i n l y oriented parallel to the axon m a j o r axis (Figs. 2 a n d 6). I n l o n g i t u d i n a l sections, m o s t o f the filaments seem to have a d o t t e d a p p e a r a n c e (Fig. 6). A m o n g the bundles o f filaments, n u m e r o u s m e m b r a n o u s profiles a n d m i t o c h o n d r i a are observed. The a x o p l a s m m e m b r a n o u s profiles consist o f vesicles, 200-600 A in diameter, with a m e d i u m - d e n s e h o m o g e n e o u s content. I n l o n g i t u d i n a l sections (Figs. 2 a n d 6), the vesicles a p p e a r a r r a n g e d in rows which m i g h t c o r r e s p o n d to t u b u l a r structures artifactually d i s r u p t e d into rows o f vesicles (22). Occasionally, tubule segments a n d diFIG. 1. Electron micrograph of part of the giant nerve fiber showing its structural organization: the axon (A) is covered by the Schwann layer formed by the Schwann cells (SC). A basal lamina (BM) and a layer of connective tissue (CT) cover the outer aspect of the Schwann layer. A bundle of tightlypacked fibrils (TF) separates both zones. The Schwann cell cytoplasm is filled with paired membranous profiles running along tortuous trajects which form the Schwann cell channels (Ch). Filaments (arrow), vesicles (v) and cisterns (C) are also observed in the cytoplasm. FTG. 2. Longitudinal section of a giant nerve fiber showing a massive body in the axoplasm. The vesicular (V) and the granular (G) components of the body are distinguished. The axoplasm also contains mitochondria (m), rows of endoplasmic reticulum profiles (ER), and numerous filaments longitudinally oriented.
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lated cisterns are observed (Fig. 8). The membranous profiles represent the axonal endoplasmic reticulum together with a specialized form, the massive body, to be described below. The axonal mitochondria are abundant and show two different types. One roundshaped, 0.6 # or more in diameter, with irregular cristae (Fig. 7). This type is found mainly in the most peripherally situated region of the axoplasm. The second type corresponds to elongated mitochondria, 0.2 to 0.4/z thick and up to 3/~ long with their major axis oriented longitudinally (Fig. 6). Mitochondria of this type are scattered throughout the axoplasm. The concentration of mitochondria in the axoplasm is about 40 units per 100 #2 in the periphery and decreases to about 30 per I00 #3 in regions 30-60 # deeper. Another structure, which was constantly found in the axoplasm of all the D. gigas axons studied, are the massive bodies (Figs. 2 and 3), formed by two components: vesicles and granules. The vesicles, 600-1000/~ thick are filled with a medium-dense and homogeneous material (Fig. 4). These vesicles are grouped by hundreds. The small ones appear closely packed, while the large vesicles, usually found at the periphery of the body, are separated by large interspaces. In longitudinal sections, the vesicles also appear as rounded profiles (Fig. 2). The other component, situated close to the group of vesicles, is a dense material configurated as irregular granules about 400 A in diameter, clustered in large masses (Fig. 5). Several mitochondria were observed bordering the body and also embedded in it (Figs. 2 and 3). In longitudinal sections, a massive body was followed up to 27 # without discontinuity. It is possible that they extend for larger distances along the axon, since they have been observed at variable intervals along the same line. The axon plasma membrane or axolemma showed, in the seven nerves examined, a thickness of 80-90 ~ (84_+ 0.7, mean _+SE). This membrane exhibits, as described in other squid species (30), the usual three-laminar continuous pattern, also regions where globular-repeating components seem to be distinguished (Fig. 9a). Local thickenings, attached to the internal or axoplasmic side of the membrane, described before in the axons of the tropical squids (30), were observed (Fig. 9b).
Fro, 3. Cross section of the giant nerve fiber showing a massive body with its vesicular (V) and granular (G) components. Mitochondria (m) are observed close to the body. FIG. 4. High magnification electron micrograph of the vesicular component of the massive body. A membrane showing the usual three-laminar pattern surrounds each vesicle. Elongated profiles, such as the ones seen toward the right (E), are occasionally observed. Fro. 5. High magnification electron micrograph of the granular component of the massive body. Clusters of opaque granules, ~ 400 A in diameter, with small clear areas scattered among them are observed.
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Schwann layer The Schwann layer is formed by a single row of Schwann cells closely apposed around the axon and with a space 50-105 A across, separating the Schwann cell plasma membrane from the axolemma. Each Schwann cell is shaped like a rectangular sheet estimated to measure, about 45/~ in width by about 60/~ in length. The thickness of the cell, as measured in longitudinal and cross sections, varies from about 6 # in the nuclear region to 1.5/~. The cell surface is very irregular and many processes, belonging to the same cell or to the neighboring cells, apper closely apposed or interdigitated. The nucleus, also sheet-like in shape, contains a fine-granular material with the larger granules scattered in the central region. A rounded nucleolus was often observed. The characteristic channels crossing the Schwann cell layer (10, 28, 29), are observed primarily away from the perinuclear region (Figs. 1 and 10). The channels in the D. gigas are more tortuous than those of the other squid species studied. In longitudinal sections, mounted in single-hole disks, it was possible to follow the Schwann cell layer for distances up to 360/~ and to distinguish two different patterns of channels. One pattern, situated about every 60 ~, corresponds to winding pathways that reverse their direction several times and may be as large as 8 times the thickness of the Schwann cell layer at the same region (Figs. I and 10). In the majority of the cases, this type of channels is observed ending in the axon-Schwann cell space. The second channel pattern observed corresponds to less tortuous pathways. This type is also present in the nuclear region, toward the external aspect of the cell. Finally the Schwann cell cytoplasm in the D. gigas, contains filaments 90-120 ~t thick, which are grouped in small or large bundles placed among the channels (Figs. 1 and 10). In longitudinal sections, a rather thick bundle of those filaments is seen along the cell internal aspect (Fig. 10). The Schwann cell plasma membrane measured 75-80 A. in thickness (77+0.2, mean ± SE). It invaginates to form the channel walls and is deeply indented at the cell outer surface by the so-called basement membrane or basal lamina. The indentaF~G. 6. Longitudinal section of the axoplasm situated about 40/x from the axolemma. Elongated mitochondria (m), 0.1-0.2 p thick, are observed as well as rows of vesicular and tubular profiles of the smooth endoplasmic reticulum (ER). FIG. 7. Longitudinal section of the peripheral region of the axoplasm showing the round-shaped mitochondria (m), about 0.6 ~ in diameter. FIG. 8. Branching endoplasmic reticulum cistern (ER) in the axoplasm of the giant nerve fiber. Mitochondria (m) are observed in the vicinity. FIG. 9. Two regions of the same electron micrograph show the boundary between the axon (A) and the Schwann cell (SC). In (a), the axolemma shows a trilaminated continuous pattern and also regions where globular repeating units seem to be distinguished (arrows). One of the thickenings (arrow) attached to the internal zone of the axolemma is observed in (b).
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tions of the basal lamina may be observed as islets in the Schwann cell cytoplasm. Some of these islets occupy a large part of the Schwann layer thickness, especially in the perinuclear region where channels are scarce (Fig. 1 I).
Connective tissue The endoneurial connective tissue occupies a zone 6-10 # thick placed between the outer surface of the Schwann cell sheath and the bundles of small nerve fibers. A blood vessel usually appears at the outer aspect of the endoneurial zone, between this and the small nerve fibers. The connective tissue presents two arrangements. The innermost region (Fig. 1), up to 2 # in thickness, is devoid of cells and made up of fibrils about 200-300 A thick with an axial periodicity. These fibrils are loosely arranged and also tightly packed in a bundle placed at a distance of about 0.8 # from the Schwann sheath surface (Fig. 1). Close to the Schwann cell surface, the fibrils are intermingled with a dense, thread-like material which forms the basal lamina (Figs. 1 and 11). The rest of the endoneurial region is occupied by the endoneurial cells which appear arranged in layers separated by spaces filled with fibrils. This arrangement gives to this part of the endoneurium a laminated appearance (Fig. 12). The endoneurial cell is elongated and has a nucleus about 10/~ long and 0.4/~ thick. The cytoplasm, reduced to a thin rim around the nucleus, extends away from the nuclear region, ending in fine, delicate processes. In the cytoplasm the organeIles are concentrated in the perikaryon and dense bodies, up to 1.5/~ long, are commonly observed. These dense bodies are surrounded by a membrane, 100 • thick, and show in general, a homogeneous content of variable opacity (Fig. 13). These bodies resemble pigment granules.
Blood vessels The blood vessels placed alongside the giant nerve fiber, show the same location and features observed in the tropical squid species (unpublished observations). The vessel wall is formed by two rows of cells separated by an undulating space, 0.2-0.3 # across, filled with a fine-granulous, opaque material (Figs. 14 and 15). The cells of
FIG. 10. Longitudinal section through the Schwann layer showing the channels (Ch) and the bundles of gliofibrils (GF)in the Schwann cell cytoplasm. A, axon; BM, basal lamina. FIO. 11. Electron micrograph of the Schwann layer showing the deep indentations of the basal lamina (BM) into the Schwann cell (SC). This zone is close to the perinuclear region and usually is devoid of highly cortorted channels. Note the fibrous aspect of the basal lamina ground material, as well as the connective fibrils intermingled with it. FI6. 12. Electron micrograph of a cross section through the endoneurium of the giant fiber, showing the alternated orderly arrangement of cells (EC) and fibrils (F). The fibrous layer close to the Schwann cell basal lamina is seen at the lower border.
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the outer row are larger t h a n those o f the inner row a n d their c y t o p l a s m s show a b u n d a n t microvesicles a n d bundles o f filaments a b o u t 240 A thick (Fig. 14). The adjacent cell lateral b o r d e r s are i n t e r d i g i t a t e d a n d b o t h p l a s m a m e m b r a n e s are closely a p p o s e d , with an i n t e r p o s e d n a r r o w gap filled with a highly o p a q u e m a t e r i a l (Fig. 15). The winding, dense trajects o f these intercellular spaces f o r m peculiar p a t t e r n s (Fig. 14). A basal lamina, 125-300 ~ thick, lines the external surfaces o f the cells o f the outer r o w (Fig. 15). This basal l a m i n a is less o p a q u e t h a n the m a t e r i a l filling the intercellular spaces. Outside the b l o o d vessel, the bundles o f small nerve fibers are found.
DISCUSSION The results herein presented show the m o r p h o l o g i c a l features a n d structural org a n i z a t i o n o f the cellular a n d noncellular elements in the giant nerve fiber o f the squid D. gigas. The axons o f our specimens showed an average d i a m e t e r o f a b o u t 1000 # (8001200 #). However, the thickness o f the a x o l e m m a , a b o u t 85 .& across, reveals, a c c o r d ing to previous observations (unpublished data), t h a t the specimens examined p o s • sibly belong to y o u n g individuals o f the species. The a x o l e m m a o f the D. gigas giant axon presented the t h r e e - l a m i n a r c o n t i n u o u s p a t t e r n a l t e r n a t e d with zones where spherical repeating-units, as those shown b y Sj6strand in c y t o p l a s m i c m e m b r a n e s (25), were observed. M o r e o v e r , local thickenings o f the internal leaflet o f the m e m b r a n e were also present. A l l these u l t r a s t r u c t u r a l details have been observed in the a x o l e m m a s o f the squids D. plei a n d S. sepioidea (30). Therefore, it seems t h a t the m o l e c u l a r o r g a n i z a t i o n o f the excitable m e m b r a n e in the three species o f squid is essentially the same. This finding is i m p o r t a n t to c o m p a r e the results o f biochemical a n d biophysical experiments carried o u t in axons o f different species.
FIG. 13. Longitudinal section through the endoneurium of the giant nerve fiber showing a cell containing three dense pigment-like granules (PG) in a row. Note the different densities presented by the opaque substance of the granules. FIG. 14. Low magnification electron micrograph of a blood vessel lining the outer aspect of the endoneurium. Surrounding the lumen (L) of the vessel, the endothelial cells (EN) appear as thin and irregular cytoplasmic portions, separated from the outer cellular layer (OC) by a wide space filled with a dense substance (DS). The cytoplasm of the cells of the outer layer contains filaments (f) and microvesicles (v). The lateral borders of these cells are closely apposed along winding trajects (J). F~G. 15. High magnification electron micrograph, showing part of one junction (J) between the cells of the outer layer of the vessel wall. Both plasma membranes are separated by a narrow space cemented with a very opaque material. The traject of the intercellular space is highly contorted due to the interdigitation of the adjacent cells. At the upper part, the outer border of the cell, lined by a thin basal lamina (BM) is observed, and at the lower part, the fine-granular substance (DS) filling the space between both cellular layers is shown.
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The local thickenings observed at the internal leaflet of the axolemma are considered to represent specialized areas engaged in the normal functioning of the axon (30). These thickenings already described in the giant axons of the tropical species of squid (30), exist also in L. forbesi as observed in the plate 3 of the paper by Baker et al. (3). In the D. gigas, the local thickenings, although present, are only an occasional finding. Thus, the question remains whether they are diminished by the in vitro conditions, or are scarce as compared to the D. plei axons. The axoplasmatic filaments may be considered as well preserved. Suggestions of early degeneration (26) seem unlikely since the filaments do not appear fragmented and they also exhibit the normal arrangement in bundles parallel to the axon major axis. Moreover, it should be noticed that special attention was devoted to preserve the living conditions of the D. gigas nerve fibers after decapitation of the animals. The axons remained continuous with their origen neuron or neurons sited at the stellate ganglion and they were excitable just before fixation. In the axons studied, the tubuli of the endoplasmic reticulum are mostly fragmented and appear as rows of vesicles, filled with a medium dense material. According to Rosenbluth (22), it is probable that tubular structures existing in the living animal be artifactually disrupted into vesicles by the action of the fixative. Moreover, some tubular profiles were observed among the rows of vesicles. In the case of the massive bodies cross and longitudinal sections demonstrated the vesicular nature of their constituents, although, at the periphery of the massive body some elongated profiles, similar to the ones described for the endoplasmic reticulum, were observed. Due to the existence of artifactually disrupted reticulum in the same material, the possibility that part of the vesicular component could be bundles of tubuli may not be completely discarded. Clusters of vesicles as those forming the massive bodies have been reported in nerve, muscular and sensory cells by several authors (6, 7, 11, 13, 21, 23, 24). However, the vesicles corresponded mostly to cross sections of tubular structures present in the cytoplasm. Andres (1) also has distinguished two types of tubular structures in the axoplasm of the fibers of the rat cerebellum. Both types seem to correspond to specialized forms of the axonal agranular endoplasmic reticulum, and their function, according to the same author, is to store a product that could be released to the intercellular space for neurogfial use. In the snail Helix pomatia, the works of R6hlich and T/Sr/Sk (21), Schwalbach et al. (24), and Eakin and Brandenburger (7) have demonstrated the existence of clusters of vesicles that completely filled the perikaryon of the visual cell. These structures are referred to as "biocrystal" (7, 21) or as a "sponge-like structure" (24), and considered related to the process of photoreception. This structure present in the visual cells of
GIANT NERVE FIBER OF GIANT SQUID
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H. pomatia morphologically resemble the vesicular component of the massive bodies. However, no large masses of granular material appear associated with the vesicles in the visual cell of H. pomatia. Some granules interpreted as glycogen granules, have been observed among the vesicles of the biocrystalline structure by Eakin and Brandenburger (7). The nature of the granular material in D. gigas axons is unknown at present. However, morphologically, this material exhibits, in cross and longitudinal sections, a granular appearance and size which resembles the glycogen granules as shown by Revel et al. (19). The vesicular component may be considered smooth endoplasmic reticulum. The smooth endoplasmic reticulum has been associated in the liver with the glycogen metabolism (17) and especially with the glycogenolysis (16). The mitochondria located close to the granular component of the massive body may be related to the utilization of glucose liberated from glycogen. The Schwann cells in D. gigas are thicker than the Schwann cells of other species (10, 28), and they have about the same thickness of the Schwann cell of S. sepioidea, whose dimensions, 1.5-5 # have made it suitable for impalement with microelectrodes (31). However, because the basal lamina is a fibrous structure, and the reinforcement of its adjacent fibrous layer is by the thin sheath of tightly packed fibrils, the impalement of the Schwann cell of this species with fine microelectrodes could be a rather difficult technique. The differences between the Schwann sheath of the D. gigas and the other species studied are the increase of membranous profiles for the highly contorted pathways of the channels, and the bundles of gliofibrils. These gliofibrils, not observed in the Schwann cytoplasm of the other squids studied by us, have been pointed out, however, in the Schwann cells of other invertebrates (14), as well as in vertebrate unmyelinated (8) and myelinated nerve fibers (9). The fibrous appearance of the basal lamina is morphologically different from the homogeneous, amorphous aspect of the material forming the basement membrane of other squid giant fibers (10, 27). The outer zone placed outside the basal lamina presents a laminated arrangement similar in organization to the perineurium of the vertebrate nerve bundles (5). An outer continuous layer with winding, narrow intercellular clefts lines the endothelium of the blood vessels. Those clefts are filled with a highly electron dense material which appears to offer a valuable restriction to diffusion, since micelles of thorium dioxide do not appear to penetrate through it (unpublished results). In this functional aspect, the blood vessles of the squid nerve fiber resemble the capillaries of the central nervous system which are impermeable to the peroxidase as demonstrated by Lasansky (12) and Reese and Karnovsky (18).
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The author expresses her thanks to Dr. Raimundo Villegas for the critical reading of the manuscript, to Dr. Jorge Villegas and Eng. Jos6 Whittembury for the obtention of the squid nerves in Lima, Peru, and to Messrs. Jesfis Aristimufio and Freddy Paredes and Miss Arlette Dupr6 for their technical assistance. The help of Mr. J. L. Bigorra for the photographic work is also gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16.
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
ANDRES, K., Z. Zellforsch. Mikroskop. Anat. 65, 701 (1965). BAKER, P. F., HODGKIN, A. L. and SHAW, T. I., Nature 190, 885 (1961). - - - - J. Physiol. (London) 164, 330 (1962). BRINLEY, F. J., JR. and MULLrNS, L. J., J. Gen. Physiol. 50, 2303 (1967). BURKEL, W. E., Anat. Record 158, 177 (1967). DUNCAN, D. and WILLIAMS,V., Texas Rept. Biol. Med. 20, 503 (1962). EAKIN, R. M. and BRANDENBURGER,J. L., J. Ultrastruct. Res. 18, 391 (1967). ELFVlN, L.-G., Y. Ultrastruct. Res. 5, 51 (1961). - - - - ibid. 5, 374 (1961). GEREN, B. B. and SCHMITT, F. O., Proc. Natl. Acad. Sci. U.S. 40, 863 (1954). LAMPERT, P., BLUMBERG, J. M. and PENTSCHEW, A., J. Neuropathol. Exptl. Neurol. 23, 60 (1964). LASANSKY,A., J. Cell Biol. 34, 617 (1967). MORALES, R. and DUNCAN, D., J. Ultrastruct. Res. 15, 480 (1966). NAKAHIMA,Y., g. Zellforsch. 54, 262 (1961). OIKAWA, T., SPYROPOULOS,C. S., TASAKI, I. and TEORELL, T., Acta Physiol. Scand. 52, 195 (1961). PETER, V. B., DEMmTZER, H. M., KELLY, G. W. and BARUCH, E., Proc. 5th lntern. Congr. Electron Microscopy, Philadelphia 1962, Vol. 2, TT-7. Academic Press, New York, 1962. PORTER, K. R. and BRUNL C., Cancer Res. 19, 997 (1959). REESE, T. S. and KARNOVSKY, M., J. CellBioL 34, 207 (1967). REVEL, J. P., NAVOLITANO,L. and FAWCETT, D. W., J. Biophys. Biochem. Cytol. 8, 575 (1960). REYNOLDS, E. S., J. Cell Biol. 17, 208 (1963). ROHLICH, P. and T6R6K, L. J., Z. Zellforsch. Mikroskop. Anat. 60, 348 (1963). ROSENBLUTH,J., J. Cell Biol. 16, 143 (1963). SCHLOTE, F. W., Z. Zellforsch. Mikroskop. Anat. 60, 325 (1963). SCHWALBACH,G., LICKFELD,K. G. and HAHN, M., Protoplasma 56, 242 (1963). SJ0STRAND, F. S., J. Ultrastruct. Res. 9, 340 (1963). V~AL, J. D., J. Biophys. Biochem. Cytol. 4, 551 (1958). VILLEGAS,G. M., Acta Cient. Venezolana, Suppl. l, 11 (1963). VmLEGAS, G. M. and VILLEGAS,R., J. Ultrastruct. Res. 3, 362 (1960). -ibid. 8, 197 (1963). -J. Gen. PhysioL 51, 44s (1968). VILLEGAS,R., VILLEGAS,L., GIMENEZ,M. and VILLEGAS,G. M., J. Gen. Physiol. 46, 1047 (1963). WATSON, M., J. Biophys. Biochem. Cytol. 4, 475 (1958).
J. ULTRASTRUCTURE RESEARCH 26,
501-514 (1969)
501
Electron Microscopic Study of the Giant Nerve Fiber of the Giont Squid Dosidicus gigas GLORIA M . VILLEGAS
Centro de Bioflsica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 1827, Caracas, Venezuela Received July 12, 1968 The present work deals with the ultrastructure of the giant nerve fiber of the giant squid Dosidieusgigas. The axons studied were 80(01200 ~ in diameter. The axolemma (membrane of the axon proper) shows a three-laminated pattern alternated with zones made up of repeated globular units. Local thickenings attached to the axolemma internal leaflet are also observed. The axoplasm shows a peculiar structure formed by clusters of vesicles, 600-1000/~ in diameter, and clusters of granules of about 400/~ in diameter. Both components form elongated massive bodies along the axon. Two types of axonal mitochondria were observed: one type rounded, over 0.6 # in diameter, found only near the axolemma; and the other, elongated, about 0.4 ~ in width by 3 # in length, scattered all over the axoplasm. The Schwann layer, 1.5-6 # thick, formed by a single row of Schwann cells, is crossed by channels. These channels are more tortuous than in the tropical species. The Schwann layer is covered outside by the basal lamina made up by a thread-like dense material, intermingled with connective tissue fibrils. Following this zone is the orderly arranged endoneurium, formed by alternated layers of cells and fibrils. Some endoneurial cells bear pigment-like granules. Blood vessels are found alongside the endoneurium. The largest axons known to the present are those of the giant squid Dosidicus gigas found in the H u m b o l d t Stream of the Pacific Ocean. The giant axons of this species are usually over 1 m m in diameter. The results already obtained by the methods of perfusion (2, 3, 15) and dialysis (4) of the squid giant axon, as well as the promising value of these experimental methods, make the axons of D. gigas of greatest interest. Electron microscopic studies of the squid giant nerve fibers have been made in the species Loligo pealii (10), Doryteuthis plei (27, 28), Sepioteuthis sepioidea (31), and Loligo forbesi (3). The present work deals with the fine structure of the giant axon of D. gigas and the Schwann cell and connective tissue sheaths surrounding it. Preliminary observations on the ultrastructure of this giant nerve fiber have been already published (30).
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VILLEGAS M A T E R I A L A N D METHODS
Seven giant nerve fibers of the first stellar nerve of the squid D. gigas were used. The animals were obtained in the coastal waters of Peru and killed immediately by decapitation; the mantles were immersed in ice-cold seawater. Within 1.5-4 hours, the nerve fibers were isolated from the mantle in the laboratory, both their ends were tied with threads, and the fibers were tested for nerve impulse conduction and then immediately immersed in an ice-cold fixative solution of 1 To OsO~ in artificial seawater, buffered with Veronal acetate at p H 8-8.1. The fixation time was 2-8 hours. The osmolarity of the fixative was 1050 mOsm/kg of water, as determined by a freezing point osmometer. After fixation the nerve fibers were washed in artificial seawater, dehydrated in a series of acetone, and embedded in Epon. Fine sections were obtained on an LKB ultramicrotome equipped with a diamond knife, placed on Formvar-carbon-coated copper grids, double stained with uranyl acetate (32) and lead citrate (20), and examined in a Siemens Elmiskop 1A electron microscope at an accelerating voltage of 80 kV. The resolution of the microscope as measured point-to-point in a film of evaporated platinum was 4.3 A. Some longitudinal sections were mounted on disks with a single center hole. RESULTS E a c h giant nerve fiber shows a general structural o r g a n i z a t i o n similar to the one a l r e a d y described in o t h e r squid species (27-29, 31). The axon a p p e a r s s u r r o u n d e d by a single r o w o f Schwann cells, a b a s a l l a m i n a or b a s e m e n t m e m b r a n e , a n d a layer o f connective tissue (Fig. 1). The diameters o f the axons used m e a s u r e d 800-1200/z.
Axofl The a x o p l a s m is f o r m e d b y filaments 50-100 A thick, g r o u p e d in bundles m a i n l y oriented parallel to the axon m a j o r axis (Figs. 2 a n d 6). I n l o n g i t u d i n a l sections, m o s t o f the filaments seem to have a d o t t e d a p p e a r a n c e (Fig. 6). A m o n g the bundles o f filaments, n u m e r o u s m e m b r a n o u s profiles a n d m i t o c h o n d r i a are observed. The a x o p l a s m m e m b r a n o u s profiles consist o f vesicles, 200-600 A in diameter, with a m e d i u m - d e n s e h o m o g e n e o u s content. I n l o n g i t u d i n a l sections (Figs. 2 a n d 6), the vesicles a p p e a r a r r a n g e d in rows which m i g h t c o r r e s p o n d to t u b u l a r structures artifactually d i s r u p t e d into rows o f vesicles (22). Occasionally, tubule segments a n d diFIG. 1. Electron micrograph of part of the giant nerve fiber showing its structural organization: the axon (A) is covered by the Schwann layer formed by the Schwann cells (SC). A basal lamina (BM) and a layer of connective tissue (CT) cover the outer aspect of the Schwann layer. A bundle of tightlypacked fibrils (TF) separates both zones. The Schwann cell cytoplasm is filled with paired membranous profiles running along tortuous trajects which form the Schwann cell channels (Ch). Filaments (arrow), vesicles (v) and cisterns (C) are also observed in the cytoplasm. FTG. 2. Longitudinal section of a giant nerve fiber showing a massive body in the axoplasm. The vesicular (V) and the granular (G) components of the body are distinguished. The axoplasm also contains mitochondria (m), rows of endoplasmic reticulum profiles (ER), and numerous filaments longitudinally oriented.
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lated cisterns are observed (Fig. 8). The membranous profiles represent the axonal endoplasmic reticulum together with a specialized form, the massive body, to be described below. The axonal mitochondria are abundant and show two different types. One roundshaped, 0.6 # or more in diameter, with irregular cristae (Fig. 7). This type is found mainly in the most peripherally situated region of the axoplasm. The second type corresponds to elongated mitochondria, 0.2 to 0.4/z thick and up to 3/~ long with their major axis oriented longitudinally (Fig. 6). Mitochondria of this type are scattered throughout the axoplasm. The concentration of mitochondria in the axoplasm is about 40 units per 100 #2 in the periphery and decreases to about 30 per I00 #3 in regions 30-60 # deeper. Another structure, which was constantly found in the axoplasm of all the D. gigas axons studied, are the massive bodies (Figs. 2 and 3), formed by two components: vesicles and granules. The vesicles, 600-1000/~ thick are filled with a medium-dense and homogeneous material (Fig. 4). These vesicles are grouped by hundreds. The small ones appear closely packed, while the large vesicles, usually found at the periphery of the body, are separated by large interspaces. In longitudinal sections, the vesicles also appear as rounded profiles (Fig. 2). The other component, situated close to the group of vesicles, is a dense material configurated as irregular granules about 400 A in diameter, clustered in large masses (Fig. 5). Several mitochondria were observed bordering the body and also embedded in it (Figs. 2 and 3). In longitudinal sections, a massive body was followed up to 27 # without discontinuity. It is possible that they extend for larger distances along the axon, since they have been observed at variable intervals along the same line. The axon plasma membrane or axolemma showed, in the seven nerves examined, a thickness of 80-90 ~ (84_+ 0.7, mean _+SE). This membrane exhibits, as described in other squid species (30), the usual three-laminar continuous pattern, also regions where globular-repeating components seem to be distinguished (Fig. 9a). Local thickenings, attached to the internal or axoplasmic side of the membrane, described before in the axons of the tropical squids (30), were observed (Fig. 9b).
Fro, 3. Cross section of the giant nerve fiber showing a massive body with its vesicular (V) and granular (G) components. Mitochondria (m) are observed close to the body. FIG. 4. High magnification electron micrograph of the vesicular component of the massive body. A membrane showing the usual three-laminar pattern surrounds each vesicle. Elongated profiles, such as the ones seen toward the right (E), are occasionally observed. Fro. 5. High magnification electron micrograph of the granular component of the massive body. Clusters of opaque granules, ~ 400 A in diameter, with small clear areas scattered among them are observed.
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Schwann layer The Schwann layer is formed by a single row of Schwann cells closely apposed around the axon and with a space 50-105 A across, separating the Schwann cell plasma membrane from the axolemma. Each Schwann cell is shaped like a rectangular sheet estimated to measure, about 45/~ in width by about 60/~ in length. The thickness of the cell, as measured in longitudinal and cross sections, varies from about 6 # in the nuclear region to 1.5/~. The cell surface is very irregular and many processes, belonging to the same cell or to the neighboring cells, apper closely apposed or interdigitated. The nucleus, also sheet-like in shape, contains a fine-granular material with the larger granules scattered in the central region. A rounded nucleolus was often observed. The characteristic channels crossing the Schwann cell layer (10, 28, 29), are observed primarily away from the perinuclear region (Figs. 1 and 10). The channels in the D. gigas are more tortuous than those of the other squid species studied. In longitudinal sections, mounted in single-hole disks, it was possible to follow the Schwann cell layer for distances up to 360/~ and to distinguish two different patterns of channels. One pattern, situated about every 60 ~, corresponds to winding pathways that reverse their direction several times and may be as large as 8 times the thickness of the Schwann cell layer at the same region (Figs. I and 10). In the majority of the cases, this type of channels is observed ending in the axon-Schwann cell space. The second channel pattern observed corresponds to less tortuous pathways. This type is also present in the nuclear region, toward the external aspect of the cell. Finally the Schwann cell cytoplasm in the D. gigas, contains filaments 90-120 ~t thick, which are grouped in small or large bundles placed among the channels (Figs. 1 and 10). In longitudinal sections, a rather thick bundle of those filaments is seen along the cell internal aspect (Fig. 10). The Schwann cell plasma membrane measured 75-80 A. in thickness (77+0.2, mean ± SE). It invaginates to form the channel walls and is deeply indented at the cell outer surface by the so-called basement membrane or basal lamina. The indentaF~G. 6. Longitudinal section of the axoplasm situated about 40/x from the axolemma. Elongated mitochondria (m), 0.1-0.2 p thick, are observed as well as rows of vesicular and tubular profiles of the smooth endoplasmic reticulum (ER). FIG. 7. Longitudinal section of the peripheral region of the axoplasm showing the round-shaped mitochondria (m), about 0.6 ~ in diameter. FIG. 8. Branching endoplasmic reticulum cistern (ER) in the axoplasm of the giant nerve fiber. Mitochondria (m) are observed in the vicinity. FIG. 9. Two regions of the same electron micrograph show the boundary between the axon (A) and the Schwann cell (SC). In (a), the axolemma shows a trilaminated continuous pattern and also regions where globular repeating units seem to be distinguished (arrows). One of the thickenings (arrow) attached to the internal zone of the axolemma is observed in (b).
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tions of the basal lamina may be observed as islets in the Schwann cell cytoplasm. Some of these islets occupy a large part of the Schwann layer thickness, especially in the perinuclear region where channels are scarce (Fig. 1 I).
Connective tissue The endoneurial connective tissue occupies a zone 6-10 # thick placed between the outer surface of the Schwann cell sheath and the bundles of small nerve fibers. A blood vessel usually appears at the outer aspect of the endoneurial zone, between this and the small nerve fibers. The connective tissue presents two arrangements. The innermost region (Fig. 1), up to 2 # in thickness, is devoid of cells and made up of fibrils about 200-300 A thick with an axial periodicity. These fibrils are loosely arranged and also tightly packed in a bundle placed at a distance of about 0.8 # from the Schwann sheath surface (Fig. 1). Close to the Schwann cell surface, the fibrils are intermingled with a dense, thread-like material which forms the basal lamina (Figs. 1 and 11). The rest of the endoneurial region is occupied by the endoneurial cells which appear arranged in layers separated by spaces filled with fibrils. This arrangement gives to this part of the endoneurium a laminated appearance (Fig. 12). The endoneurial cell is elongated and has a nucleus about 10/~ long and 0.4/~ thick. The cytoplasm, reduced to a thin rim around the nucleus, extends away from the nuclear region, ending in fine, delicate processes. In the cytoplasm the organeIles are concentrated in the perikaryon and dense bodies, up to 1.5/~ long, are commonly observed. These dense bodies are surrounded by a membrane, 100 • thick, and show in general, a homogeneous content of variable opacity (Fig. 13). These bodies resemble pigment granules.
Blood vessels The blood vessels placed alongside the giant nerve fiber, show the same location and features observed in the tropical squid species (unpublished observations). The vessel wall is formed by two rows of cells separated by an undulating space, 0.2-0.3 # across, filled with a fine-granulous, opaque material (Figs. 14 and 15). The cells of
FIG. 10. Longitudinal section through the Schwann layer showing the channels (Ch) and the bundles of gliofibrils (GF)in the Schwann cell cytoplasm. A, axon; BM, basal lamina. FIO. 11. Electron micrograph of the Schwann layer showing the deep indentations of the basal lamina (BM) into the Schwann cell (SC). This zone is close to the perinuclear region and usually is devoid of highly cortorted channels. Note the fibrous aspect of the basal lamina ground material, as well as the connective fibrils intermingled with it. FI6. 12. Electron micrograph of a cross section through the endoneurium of the giant fiber, showing the alternated orderly arrangement of cells (EC) and fibrils (F). The fibrous layer close to the Schwann cell basal lamina is seen at the lower border.
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the outer row are larger t h a n those o f the inner row a n d their c y t o p l a s m s show a b u n d a n t microvesicles a n d bundles o f filaments a b o u t 240 A thick (Fig. 14). The adjacent cell lateral b o r d e r s are i n t e r d i g i t a t e d a n d b o t h p l a s m a m e m b r a n e s are closely a p p o s e d , with an i n t e r p o s e d n a r r o w gap filled with a highly o p a q u e m a t e r i a l (Fig. 15). The winding, dense trajects o f these intercellular spaces f o r m peculiar p a t t e r n s (Fig. 14). A basal lamina, 125-300 ~ thick, lines the external surfaces o f the cells o f the outer r o w (Fig. 15). This basal l a m i n a is less o p a q u e t h a n the m a t e r i a l filling the intercellular spaces. Outside the b l o o d vessel, the bundles o f small nerve fibers are found.
DISCUSSION The results herein presented show the m o r p h o l o g i c a l features a n d structural org a n i z a t i o n o f the cellular a n d noncellular elements in the giant nerve fiber o f the squid D. gigas. The axons o f our specimens showed an average d i a m e t e r o f a b o u t 1000 # (8001200 #). However, the thickness o f the a x o l e m m a , a b o u t 85 .& across, reveals, a c c o r d ing to previous observations (unpublished data), t h a t the specimens examined p o s • sibly belong to y o u n g individuals o f the species. The a x o l e m m a o f the D. gigas giant axon presented the t h r e e - l a m i n a r c o n t i n u o u s p a t t e r n a l t e r n a t e d with zones where spherical repeating-units, as those shown b y Sj6strand in c y t o p l a s m i c m e m b r a n e s (25), were observed. M o r e o v e r , local thickenings o f the internal leaflet o f the m e m b r a n e were also present. A l l these u l t r a s t r u c t u r a l details have been observed in the a x o l e m m a s o f the squids D. plei a n d S. sepioidea (30). Therefore, it seems t h a t the m o l e c u l a r o r g a n i z a t i o n o f the excitable m e m b r a n e in the three species o f squid is essentially the same. This finding is i m p o r t a n t to c o m p a r e the results o f biochemical a n d biophysical experiments carried o u t in axons o f different species.
FIG. 13. Longitudinal section through the endoneurium of the giant nerve fiber showing a cell containing three dense pigment-like granules (PG) in a row. Note the different densities presented by the opaque substance of the granules. FIG. 14. Low magnification electron micrograph of a blood vessel lining the outer aspect of the endoneurium. Surrounding the lumen (L) of the vessel, the endothelial cells (EN) appear as thin and irregular cytoplasmic portions, separated from the outer cellular layer (OC) by a wide space filled with a dense substance (DS). The cytoplasm of the cells of the outer layer contains filaments (f) and microvesicles (v). The lateral borders of these cells are closely apposed along winding trajects (J). F~G. 15. High magnification electron micrograph, showing part of one junction (J) between the cells of the outer layer of the vessel wall. Both plasma membranes are separated by a narrow space cemented with a very opaque material. The traject of the intercellular space is highly contorted due to the interdigitation of the adjacent cells. At the upper part, the outer border of the cell, lined by a thin basal lamina (BM) is observed, and at the lower part, the fine-granular substance (DS) filling the space between both cellular layers is shown.
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The local thickenings observed at the internal leaflet of the axolemma are considered to represent specialized areas engaged in the normal functioning of the axon (30). These thickenings already described in the giant axons of the tropical species of squid (30), exist also in L. forbesi as observed in the plate 3 of the paper by Baker et al. (3). In the D. gigas, the local thickenings, although present, are only an occasional finding. Thus, the question remains whether they are diminished by the in vitro conditions, or are scarce as compared to the D. plei axons. The axoplasmatic filaments may be considered as well preserved. Suggestions of early degeneration (26) seem unlikely since the filaments do not appear fragmented and they also exhibit the normal arrangement in bundles parallel to the axon major axis. Moreover, it should be noticed that special attention was devoted to preserve the living conditions of the D. gigas nerve fibers after decapitation of the animals. The axons remained continuous with their origen neuron or neurons sited at the stellate ganglion and they were excitable just before fixation. In the axons studied, the tubuli of the endoplasmic reticulum are mostly fragmented and appear as rows of vesicles, filled with a medium dense material. According to Rosenbluth (22), it is probable that tubular structures existing in the living animal be artifactually disrupted into vesicles by the action of the fixative. Moreover, some tubular profiles were observed among the rows of vesicles. In the case of the massive bodies cross and longitudinal sections demonstrated the vesicular nature of their constituents, although, at the periphery of the massive body some elongated profiles, similar to the ones described for the endoplasmic reticulum, were observed. Due to the existence of artifactually disrupted reticulum in the same material, the possibility that part of the vesicular component could be bundles of tubuli may not be completely discarded. Clusters of vesicles as those forming the massive bodies have been reported in nerve, muscular and sensory cells by several authors (6, 7, 11, 13, 21, 23, 24). However, the vesicles corresponded mostly to cross sections of tubular structures present in the cytoplasm. Andres (1) also has distinguished two types of tubular structures in the axoplasm of the fibers of the rat cerebellum. Both types seem to correspond to specialized forms of the axonal agranular endoplasmic reticulum, and their function, according to the same author, is to store a product that could be released to the intercellular space for neurogfial use. In the snail Helix pomatia, the works of R6hlich and T/Sr/Sk (21), Schwalbach et al. (24), and Eakin and Brandenburger (7) have demonstrated the existence of clusters of vesicles that completely filled the perikaryon of the visual cell. These structures are referred to as "biocrystal" (7, 21) or as a "sponge-like structure" (24), and considered related to the process of photoreception. This structure present in the visual cells of
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H. pomatia morphologically resemble the vesicular component of the massive bodies. However, no large masses of granular material appear associated with the vesicles in the visual cell of H. pomatia. Some granules interpreted as glycogen granules, have been observed among the vesicles of the biocrystalline structure by Eakin and Brandenburger (7). The nature of the granular material in D. gigas axons is unknown at present. However, morphologically, this material exhibits, in cross and longitudinal sections, a granular appearance and size which resembles the glycogen granules as shown by Revel et al. (19). The vesicular component may be considered smooth endoplasmic reticulum. The smooth endoplasmic reticulum has been associated in the liver with the glycogen metabolism (17) and especially with the glycogenolysis (16). The mitochondria located close to the granular component of the massive body may be related to the utilization of glucose liberated from glycogen. The Schwann cells in D. gigas are thicker than the Schwann cells of other species (10, 28), and they have about the same thickness of the Schwann cell of S. sepioidea, whose dimensions, 1.5-5 # have made it suitable for impalement with microelectrodes (31). However, because the basal lamina is a fibrous structure, and the reinforcement of its adjacent fibrous layer is by the thin sheath of tightly packed fibrils, the impalement of the Schwann cell of this species with fine microelectrodes could be a rather difficult technique. The differences between the Schwann sheath of the D. gigas and the other species studied are the increase of membranous profiles for the highly contorted pathways of the channels, and the bundles of gliofibrils. These gliofibrils, not observed in the Schwann cytoplasm of the other squids studied by us, have been pointed out, however, in the Schwann cells of other invertebrates (14), as well as in vertebrate unmyelinated (8) and myelinated nerve fibers (9). The fibrous appearance of the basal lamina is morphologically different from the homogeneous, amorphous aspect of the material forming the basement membrane of other squid giant fibers (10, 27). The outer zone placed outside the basal lamina presents a laminated arrangement similar in organization to the perineurium of the vertebrate nerve bundles (5). An outer continuous layer with winding, narrow intercellular clefts lines the endothelium of the blood vessels. Those clefts are filled with a highly electron dense material which appears to offer a valuable restriction to diffusion, since micelles of thorium dioxide do not appear to penetrate through it (unpublished results). In this functional aspect, the blood vessles of the squid nerve fiber resemble the capillaries of the central nervous system which are impermeable to the peroxidase as demonstrated by Lasansky (12) and Reese and Karnovsky (18).
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The author expresses her thanks to Dr. Raimundo Villegas for the critical reading of the manuscript, to Dr. Jorge Villegas and Eng. Jos6 Whittembury for the obtention of the squid nerves in Lima, Peru, and to Messrs. Jesfis Aristimufio and Freddy Paredes and Miss Arlette Dupr6 for their technical assistance. The help of Mr. J. L. Bigorra for the photographic work is also gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16.
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
ANDRES, K., Z. Zellforsch. Mikroskop. Anat. 65, 701 (1965). BAKER, P. F., HODGKIN, A. L. and SHAW, T. I., Nature 190, 885 (1961). - - - - J. Physiol. (London) 164, 330 (1962). BRINLEY, F. J., JR. and MULLrNS, L. J., J. Gen. Physiol. 50, 2303 (1967). BURKEL, W. E., Anat. Record 158, 177 (1967). DUNCAN, D. and WILLIAMS,V., Texas Rept. Biol. Med. 20, 503 (1962). EAKIN, R. M. and BRANDENBURGER,J. L., J. Ultrastruct. Res. 18, 391 (1967). ELFVlN, L.-G., Y. Ultrastruct. Res. 5, 51 (1961). - - - - ibid. 5, 374 (1961). GEREN, B. B. and SCHMITT, F. O., Proc. Natl. Acad. Sci. U.S. 40, 863 (1954). LAMPERT, P., BLUMBERG, J. M. and PENTSCHEW, A., J. Neuropathol. Exptl. Neurol. 23, 60 (1964). LASANSKY,A., J. Cell Biol. 34, 617 (1967). MORALES, R. and DUNCAN, D., J. Ultrastruct. Res. 15, 480 (1966). NAKAHIMA,Y., g. Zellforsch. 54, 262 (1961). OIKAWA, T., SPYROPOULOS,C. S., TASAKI, I. and TEORELL, T., Acta Physiol. Scand. 52, 195 (1961). PETER, V. B., DEMmTZER, H. M., KELLY, G. W. and BARUCH, E., Proc. 5th lntern. Congr. Electron Microscopy, Philadelphia 1962, Vol. 2, TT-7. Academic Press, New York, 1962. PORTER, K. R. and BRUNL C., Cancer Res. 19, 997 (1959). REESE, T. S. and KARNOVSKY, M., J. CellBioL 34, 207 (1967). REVEL, J. P., NAVOLITANO,L. and FAWCETT, D. W., J. Biophys. Biochem. Cytol. 8, 575 (1960). REYNOLDS, E. S., J. Cell Biol. 17, 208 (1963). ROHLICH, P. and T6R6K, L. J., Z. Zellforsch. Mikroskop. Anat. 60, 348 (1963). ROSENBLUTH,J., J. Cell Biol. 16, 143 (1963). SCHLOTE, F. W., Z. Zellforsch. Mikroskop. Anat. 60, 325 (1963). SCHWALBACH,G., LICKFELD,K. G. and HAHN, M., Protoplasma 56, 242 (1963). SJ0STRAND, F. S., J. Ultrastruct. Res. 9, 340 (1963). V~AL, J. D., J. Biophys. Biochem. Cytol. 4, 551 (1958). VILLEGAS,G. M., Acta Cient. Venezolana, Suppl. l, 11 (1963). VmLEGAS, G. M. and VILLEGAS,R., J. Ultrastruct. Res. 3, 362 (1960). -ibid. 8, 197 (1963). -J. Gen. PhysioL 51, 44s (1968). VILLEGAS,R., VILLEGAS,L., GIMENEZ,M. and VILLEGAS,G. M., J. Gen. Physiol. 46, 1047 (1963). WATSON, M., J. Biophys. Biochem. Cytol. 4, 475 (1958).