Journal of Neurocytology 4, 697-7 I2 (I975)
Synaptic membrane structure in electric organ JACK
Torpedo
ROSENBLUTH
Departments of Physiology and Rehabilitation Medicine, New York University School of Medicine New York, New York 10016, U.S.A.
Received 15 July I975; accepted 28 July I975
Summary The innervated and noninnervated membranes of Torpedo electrocytes have been examined by electron microscopy of thin-sectioned and freeze-fractured specimens. The ventral innervated membrane is ~-~i2o A thick and is characterized by an unusually broad outer dense lamina (~-,6o A) in which a granular substructure can be resolved. The granules are "~7o A in diameter and are spaced irregularly. The same membrane specialization was noted in a previous study of amphibian myoneural junctions, and it was proposed then that the granular elements represent ACh receptor molecules. The morphologically equivalent structures presumably have the same significance in the Torpedo electric organ. However, in this case the specialized membrane covers the entire innervated surface, leading to the conclusion that high concentrations of receptors occur normally in extrajunctional as well as postjunctional regions of the innervated membrane of the electrocyte. In replicas of freeze-fractured specimens, the A face of this membrane is covered with large particles having the same distribution and approximate concentration as the granules visible in thin sections, indicating that the granules visible at the outer surface of the membrane extend at least into the hydrophobic middle layer of the membrane. The cytoplasmic surface of this membrane has an amorphous coating into which 'decorated' cytoplasmic filaments insert. Synaptic vesicle and axon terminal membranes also contain granules visible in thin sections but with a much sparser distribution. These probably correspond to the intramembranous particles seen in freeze-fractured specimens. Vesicles are occasionally attached to the axolemma by thin linear strands.
Introduction Electron micrographs of myoneural junctions in both vertebrates (Rosenbluth, 1973 a, I974) and invertebrates (Rosenbluth, i972, I973b, c) reveal a distinctive structural specialization of the postjunctional membrane. I n thin-sectioned specimens this membrane is considerably thicker than other membranes, and at high magnification a granular substructure can be resolved in its outermost lamina. On the basis of the size, location and approximate con9 1975 Chapman and Hall Ltd. Printed in Great Britain
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centration o f these granules, it was proposed that they represent the transmitter receptors. Freeze-fracture studies (Heuser, Reese and Landis, 1974) reveal high concentrations of i n t r a m e m b r a n o u s particles having roughly the same distribution and concentration as the superficially located granules seen in thin-sectioned specimens. I t is therefore likely that the elements observed b y these two different methods represent different aspects of the same structure. Daniels and Vogel (1975) have recently shown that ~-bungarotoxin binding sites also have the same distribution at myoneural junctions and there seems little doubt, therefore, that the granule-bearing m e m b r a n e is indeed the receptive m e m b r a n e . T h e present investigation consists of a study o f the equivalent m e m b r a n e s in the Torpedo electric organ, which contains extensive junctional areas along the ventral surface of the electrocytes. Thin-sectioned and freeze-fractured specimens demonstrate the same structural specializations identified previously at myoneural junctions. H o w e v e r the distribution o f these elements in the electric organ is significantly broader and leads to the conclusion that in this tissue, unlike muscle, acetylcholine receptors are normally found in high concent_ration in extrajunctional regions as well as in those regions directly under nerve endings.
Materials and methods Slices of the electric organ were fixed immediately after removal from the living animal which had been anesthetized with Tricaine. The fixatives used were 2% glutaraldehyde in o. z M phosphate buffer or 4% glutaraldehyde in o.xi M cacodylate buffer. After approximately I 8 9 -- 3 h some samples of the fixed tissues were washed in sea water, postfixed in 1% osmiumtetroxide in the same buffer for zI - 3 h, and then dehydrated and embedded in Araldite. Other fixed samples were rinsed in sea water and then dehydrated and embedded directly without postfixation, and the remaining tissue was stored in the glutaraldehyde. Thin sections were cut on a Porter-Blum MT-2 microtome. In most cases the sections used exhibited a silver interference colour, but in some instances very thin (colourless) sections were examined and in others relatively thick (red) sections were studied. The sections were stained by the permanganate-uranyl acetate method described previously (Rosenbluth, I972 ) which permits visualization of fine details of membranes even in very thin sections, but which has the disadvantage of often leaving fine filamentous deposits in the background. All sections were examined with a Philips 3oo electron microscope at 60 kV. For freeze-fracture studies, glutaraldehyde fixed specimens were passed through graded concentrations of glycerol from 5 % to 2 0 - 3 0 0 . They were then frozen in Freon 22 cooled byliquid nitrogen and fractured and shadowed in a Balzer's freeze-fracture apparatus equipped with an electron gun. The tissue was digested with several changes of Clorox; the replicas were then rinsed and mounted on i5 o mesh carbon coated grids and examined.
Results
Thin-sectioned specimens T h e Torpedo electric organ
consists o f stacks o f plate-like electrocytes (Fig. I), the ventral face of which is innervated and the dorsal face uninnervated (for review see Bennett, 1971). T h e innervated face can be identified b y the presence of nerve endings apposed to the cell m e m b r a n e and is characterized also b y the presence of rather sparse tubular invaginations o f the postjunctional m e m b r a n e which are ~-~ 0.3 ~m in diameter (Fig. 2). T h e spacing o f
Fig. i. Survey view of Torpedo electrocyte. Portions of two cells are shown separated by a connective tissue space. The dorsal noninnervated face (D) exhibits innumerable narrow infoldings, while the ventral innervated face (V) shows much less infolding and is indented by several nerve endings. • 6000. nerve endings varies from place to place. In some regions the axon terminals are almost contiguous (cf. Sheridan, I965) while in others they are more widely spaced, and stretches up to 2- 3 ~m long are devoid of endings (cf. Luft, 1956). In contrast to the innervated face, the noninnervated face exhibits an extensive plexus of narrow diameter infoldings (~--~o.I ~m diameter) which penetrate deep into the cytoplasm and occur over the entire surface of this face. A prominent basement membrane coats both faces of the cell. Within the cytoplasm, the nucleus can occasionally be identified, but other organelles are sparse consisting primarily ofmitochondria and a distinctive population of disordered filaments measuring ~-~ 12o A in diameter (Fig. 3). These are rough in appearance and sometimes have projections spaced at ~ 3-4oo • intervals extending from them. They bear a resemblance to the 'decorated' filaments described by Huxley (1963). Many of these filaments insert into the amorphous coating on the cytoplasmic surface of the ventral innervated membrane (Fig. 3), and a small number insert into the thinner coating on the dorsal noninnervated membrane. At high magnification the plasma membrane of the innervated face is seen as a threelayered structure having an overall width of ~-~ I2o A (Fig. 5). The thin (~-~25-3 o A) inner dense lamina (Robertson, I959) is usually continuous, but occasionally appears beaded (Fig. 9). It is coated on its cytoplasmic surface with an amorphous dense material. The lucent middle lamina, also measuring ~-~ 25-3 o A in width, overlies the inner dense lamina. The outer dense lamina of the membrane is the most distinctive one (Figs. 7-13). It is ~-- 60 A in width, which is much thicker than in most other cell membranes, and it frequently has a
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granular or interrupted appearance. The 'granules' visible in the outer dense lamina are 65-7o A in diameter, and in every respect resemble those described previously in the receptive membrane at amphibian motor endplates (Rosenbluth, 1974). The granular substructure is often unclear, presumably because of superimposition of randomly arrayed granules within the thickness of the section, but the characteristic thickening of the outer dense lamina is always apparent. The granules appear not to form lattices like those found at earthworm junctions (Rosenbluth, 1972) inasmuch as no extensive, regularly spaced rows of granules have been seen as would be expected in randomly oriented sections through a semicrystalline array. Short stretches of regularly spaced granules can be found, however (Figs. 7-9). The plasma membrane is covered by a basement membrane which is located ~ IOO A from the cell and which follows its invaginations faithfully with the exception that it does not extend into the narrowest branches of the invaginations arising from the noninnervated surface (Figs. 14 and 15)" A reticular pattern is sometimes visible in the basement membrane reminiscent of that in frog endplate basement membrane (Rosenbluth, 1974), and filamentous projections interconnecting the plasma membrane and basement membrane are occasionally, though not usually, visible. The specialized plasma membrane and superimposed basement membrane described cover the entire innervated face of the electrocyte including the regions directly under nerve endings (Fig. 5) and the extrajunctional regions between nerve endings (Fig. 6). Only rarely can a focal non-specialized region be found at which the plasma membrane is reduced to the more typical width of ~ 60 A (Figs. 12 and 13). The specialized membrane also extends into the tubular infoldings and can be identified in the deepest recesses of these invaginations. In marked contrast to the postjunctional membrane, the prejunctional axon terminal and synaptic vesicles are covered by limiting membranes having no such specialization of the outer dense lamina (Fig. 3). In en face views, however, the synaptic vesicle membranes exhibit a reticular pattern (Fig. 4) and, in addition, dense granules ( ~ 70 A) can be seen in the synaptic vesicle membrane (Figs. 4 and 5) (cf. Heuser and Reese, 1973, Politoffet a!., 1974) and are visible also in the axolemma (Figs. 4, 5 and 13). These probably correspond to the particles visible in replicas of the same membranes after freeze-fracturing. Linear strands
Fig. 2. Innervated face of electrocyte. Its plasma membrane (P) appears very dense in both postjunctional and extrajunctional regions. N, nerve ending; S, Schwarm cell; B, basement membrane. X 2 I 000.
Fig. 3. Innervated membrane at higher magnification. The electrocyte plasma membrane (P) is trilaminar and has a dense amorphous coating on its cytoplasmic surface. Filaments (F) bearing periodic projections abound in the cytoplasm and several of them insert into this coating. The axolemma (A) does not appear trilaminar but has an amorphous cytoplasmic density running parallel to it in one region (-~). I, infolding of electrocyte membrane; M, mitochondrion. • 70000. Fig. 4. Detail of synaptic vesicles. A reticular pattern appears in the vesicle membrane where it is cut tangentially. Dense granules are visible in the normally cut synaptic vesicle membrane and in the axolemma below (~'s). Several granules of this size are also seen superimposed on tangentially cut vesicle membranes along with two larger granules. However in oblique views it is not possible to tell precisely where the granules are with respect to the membrane. • ~52ooo.
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often extend from the synaptic vesicle membrane into the surrounding axoplasm (Figs. Io and II) and occasionally stretch between a synaptic vesicle and the prejunctional (axonal) membrane (Fig. 5). The noninnervated surface of the electrocyte (Fig. I4) has a limiting membrane which is frequently trilaminar in appearance, but it is only ~ 60-65 A in thickness and has an outer dense lamina only ~ 25/k wide. A granular substructure can occasionally be seen here (Fig. z5), but this lamina is consistently thinner than the outer dense lamina of the innervated membrane. In specimens that have not been postfixed with osmium tetroxide, differences among the various limiting membranes in the tissue are in some cases more pronounced and in others less pronounced. While most limiting membranes including those of axons, Schwann cells, nuclei, mitochondria, and synaptic vesicles are barely preserved in specimens of this kind, the innervated membrane of the electrocyte (Figs. z6 and I7) still exhibits a prominent trilaminar structure and dense cytoplasmic coating. The fact that the entire innervated face is covered with specialized membrane is particularly clear in such specimens since in contrast to ordinary membranes, which are virtually invisible, that of the innervated face stands out vividly along its entire length except for a rare gap. This technique also preserves a trilaminar structure in the noninnervated electrocyte membrane, which is specialized for quite a different function. However, the ventral and dorsal electrocyte membranes can be distinguished in preparations of this kind on the basis of the cytoplasmic coating which is distinctly heavier on the innervated membrane.
Freeze-fracture images At low magnification is is possible to distinguish both innervated and noninnervated surfaces of the same electrocyte (Fig. z8). The fracture plane does not usually pass through nerve endings as well, and therefore the identification of the two surfaces rests upon different patterns of infoldings in the membranes. The most distinctive feature of the freeze-fracture image is the occurrence of particles measuring ~ I 15 A in diameter in the A face of both the innervated (Fig. 2I) and noninnervated (Fig. 24) surfaces. The particles extend over the whole area of the membrane exposed and therefore appear not to be confined to circum-
Fig. 5. Higher magnification view of synaptic membranes. The postjunctional electrocyte membrane (P) is distinctly trilaminar in contrast to the axoIemma (A). Granules are visible in the synaptic vesicle membrane (double -~) and axolemma (-~). T h e vesicle at the extreme left is attached to the granule in the axolemma by a linear strand. B, basement membrane. • 20oooo. Fig. 6. Extrajunctional electrocyte membrane of innervated face. The dimensions and substructure of this membrane (P) are the same as those of the postjunctional membrane in Fig. 5. B, basement membrane. • 200000. Figs. 7 - I I . Details of ventral electrocyte membrane. Figs. 7-9. The outer dense lamina is thick and contains regularly spaced granules. The inner dense lamina is thin and continuous but with 'beading' evident in some places (-~'s). • 286ooo. Figs. Io a n d x I. The spacing of the granules in the outer dense lamina of the electrocyte membrane is irregular. +'s indicate linear strands attached to synaptic vesicle membranes. Fig. Io, • 292000; Fig. I I , • 23Iooo.
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scribed patches. The concentration of A face particles is ~ 5 • IO3 per ~m ~, but they usually do not exhibit any regular array. In some regions they are closely spaced, but they are usually spaced more widely and irregularly. Particles extend into the invaginations of the innervated face as far as the invaginations can be followed (Fig. 2o). The B face of the innervated membrane also exhibits scattered large particles (Fig. 22) but their concentration is only I-2ooo per ~m ~. Since the large particles in both the A and B faces are irregularly arranged, it is not possible to de termine whether they are fragments of the same elements or are separate elements that adhere by chance to one or the other fracture face. In addition to these large particles, the B face also exhibits arrays which look more square than hexagonal and which have a repeating period of 6o-7o A (Fig. 22). On the noninnervated surface, A face particles can also be followed into the depths of the invaginations; however B faces exhibit few particles and no square arrays (Fig. 25). In some instances (Fig. 2I) it has been possible to detect a central depression in the particles of both innervated and noninnervated faces (cf. Allen et al., 1974). However, this is not seen consistently. Fractures along nerve terminal membranes reveal the presence of large particles in both the A face (Fig. 19) and B face of the axolemma. However the A face particles occur in much lower concentration than in the electrocyte membrane. Occasional pits, presumably signifying exocytosis, occur in the A face (Fig. 19), but thus far no bands of particles adjacent to release sites, like those occurring in amphibian myoneural junctions (Heuser et al., 1974) , have been detected in the Torpedo electric organ. Fractures through the terminal axoplasm occasionally reveal omega-shaped profiles along the axolemma as described by Nickel and Potter (I97O), and views of either the A or B face of the synaptic vesicles are also commonly seen. The vesicle membrane frequently exhibits one or more large particles in either the A or B face (Fig. I9) (cf. Moor et al., 1969; Heuser et al., I974) which occur with about the same frequency as the granular densities seen in synaptic vesicle membranes in thin sections (Figs. 4 and 5) and probably correspond to them. Similarly, the granules visible in thin sections of the axolemma (Figs. 4, 5 and I3) probably correspond to the particles seen in the freeze-fractured axolemma. Discussion
This study shows that the innervated face of Torpedo electrocytes exhibits a membrane specialization identical to that seen previously in the receptive membrane of amphibian motor endplates and similar to those found at invertebrate myoneural junctions. The one Figs. ~z a n d I3. A short length of unspecialized membrane ( @ appears in the innervated surface of the electrocyte. A, axolemma; *, dense granules in axolemma. Fig. I2, • 200000; Fig. 13, • 252000. Figs. I4 a n d I5. Noninnervated face of electrocyte. The plasma membrane is invaginated. Basement membrane (B) extends into the broader branches of the invagination, but not into the narrowest ones. The plasma membrane (P) appears trilaminar in some places but is only ~65 A thick. At -~, granules are visible in the outer dense lamina. The overall thickness of the membrane here is ~65 A. Fig. I4, x I97OOO;Fig. 15, • 238 ooo. Figs. x6 and I7. Unosmicated specimens, innervated face. The trilaminar structure of the plasma membrane is distinct. Fig i6, • T63OOO;Fig. I7, • I56ooo.
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important difference is that the specialized membrane in Torpedo electric organ covers the extrajunctional as well as postjunctional portions of the innervated face. In previous studies of negatively stained membrane fragments, Cartaud et al. (1973) reported granules in the receptor bearing membrane which were 8o-9o A in diameter and could be resolved into 3-6 subunits. Nickel and Potter (1973) also found such subunits and in addition were able to distinguish the two faces of the cell based on the finding of'stalked particles' in the ATPase rich membrane fragments, presumably from the noninnervated face, in contrast to doughnut-shaped particles in the receptor rich membrane fragments, presumably from the innervated face. The negative staining method has the disadvantage that it is carried out on isolated fragments, and therefore it is not possible to determine whether these were originally postjunctional or extrajunctional in location. The freeze-fracture method has the same limitation inasmuch as the fracture plane does not usually pass through both pre- and postjunctional membranes in the same region, and again it is not possible to be sure whether a given region of the innervated face of the electrocyte is postjunctional or extrajunctional in such preparations. Moreoever in this organ the intramembranous particles from the noninnervated face (presumably representing ATPase) are indistinguishable from those in the innervated face (presumably representing receptors), and therefore particle-rich membrane cannot be equated with receptor-rich membrane. Only in the thin-sectioned specimens can postjunctional and extrajunctional regions be identified unambiguously, and therefore only on the basis of examining appropriately fixed and stained preparations of this kind can it be concluded that the specialized membrane covers the entire innervated face induding the extrajunctionat portions. The arguments previously offered for equating the granules visible in the outer dense lamina of the postjunctional membrane with acetylcholine receptors apply to the Torpedo electric organ as well as to the motor endplates examined originally (Rosenbluth, 1972, 1974). The specialized membrane described appears to represent regions in which the receptors are highly concentrated and constitute a significant proportion of the intrinsic structure of the membrane. It is undoubtedly their presence that allows the basic membrane architecture to be preserved even after lipid extraction in specimens whose membranes have not been stabilized by osmium tetroxide. The presence of ATPase in high concentration in the noninnervated face also seems to have the same stabilizing effect. The two faces are, however, clearly distinguishable in osmicated specimens, where the characteristic marked thickening
Fig. 18. Freeze-fracture replica of Torpedo electrocyte showing the innervated ventral membrane (V)
and the noninnervated dorsal membrane (D). The two membranes can be distinguished readily by the pattern of infoldings. • 190o0 Fig. x9. Axon terminal. The fracture plane passes through axoplasm at the left revealing A and B faces of synaptic vesicles. Two of these ( ~ 's) have singte large particles in the A face and one (white arrowhead) has large particles in the B face. At the right, fracture plane passes through the A face of the axolemma (A) revealing scattered large particles and one pit (-~). • 57ooo. Fig. 2o. Innervated surface of electrocyte showing several finger-like invaginations. In one of these the fracture plane passes from the concave (A) face to the convex (B) face. The A face of the plasma membrane at the bottom is covered with large particles. • 28000.
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of the outer dense lamina of the innervated membrane is unmistakable. Evidently the receptor molecules project from the innervated membrane in contrast to the ATPase molecules which are apparently buried in the noninnervated membrane. In addition the receptor protein, all of whose subunits are glycoprotein (Raftery, 1976), may have a greater affinity for osmium tetroxide. The occurrence of this specialization in extrajunctional regions of the Torpedo electrocyte raises an interesting question as to what governs the distribution of receptors. In vertebrate skeletal muscle, high concentrations of receptors occur under normal conditions only in the endplate regions and are probably not even present in the depths of the junctional folds (Rosenbluth, 1974). Only after denervation do the receptors appear in higher concentrations in extrajunctional regions, but this 'spread' of receptors can be reduced by periodic direct stimulation of the muscle cells (Lomo and Rosenthal, 1972). Torpedo presumably uses its electric organ intermittently. However, the cells do not generate action potentials (Bennett, 1971 ) and it may be this characteristic that underlies the broad distribution of receptors in the electrocytes. Denervation produces no increase in these receptors (Clementi et al., x975), an observation which is consistent with the conclusion reported here that in the normal Torpedo electric organ the entire innervated surface already contains receptors in high concentration. However it does not account for the apparent failure of receptors to spread to the noninnervated face of the denervated electrocyte. The significance of the cytoplasmic filaments in the electrocytes is unclear. They are presumably composed of the muscle proteins which have been shown to occur in this organ (Kaminer and Szonyi, 1973). Since the cells are derived from muscle, the filaments may merely represent vestigial structures consisting of actin filaments to which other muscle proteins and/or non-muscle proteins have become attached (perhaps during fixation) and having no contractile function. Since some of these filaments insert into the amorphous dense layer coating the inner surface of the plasma membrane, a possible alternative interpretation is that they are in some way involved in the movement of receptor molecules, ATPase, and other proteins to the plasma membrane and may thus play a role in the turnover of these membrane constituents. In summary this study makes the following points: (1) The innervated face of Torpedo electrocytes exhibits a conspicous membrane specialization consisting of a trilaminar membrane whose outer dense lamina is markedly thickened due to the present in it of ~-~ 7o A granules facing the junctional cleft. These granules probably extend into the hydrophobic middle layer of the membrane where large
Fig. 2i, Detail of Fig. 20. Innumerable randomly arranged intramembranous particles are visible in the A face of the innervated membrane. They are irregular in shape and range in size from N zoo to -~ I5o/~. Their concentration is ~ 5 • IO~per y.m2. Some appear to be arranged in short chains and at the extreme right the particles exhibit a semblance of an ordered array. Central pits are visible in a number of particles. • 136ooo. Fig. 22. B face of innervated electrocyte membrane. Intramembranous particles are present but in much lower concentration than that seen in the A face. Square arrays are visible at ~+. • 7oooo.
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numbers of particles can be seen in freeze-fractured specimens and may also extend to the cytoplasmic surface of the membrane which is covered with an amorphous dense material. The granular elements, which are probably ACh receptors, appear to form an intrinsic component of the membrane but project from its outer surface further than other intrinsic membrane elements. (2) Based on the distribution of the specialized membrane, it is concluded that high concentrations of receptors occur in the extrajunctional as well as postjunctional portions of the innervated surface of the electrocyte. (3) In thin-sectioned specimens, granules are visible in both the synaptic vesicle membranes and the axolemma having the same distribution as the intramembranous particles visible in freeze-fractured preparations of the same membranes and probably corresponding to them. Thin linear strands are attached to the outer surface of synaptic vesicles and sometimes interconnect them with the axolemma. (4) The electrocytes contain large numbers of 'decorated' filaments which insert into the amorphous coating on the cytoplasmic surface of the plasma membrane. Acknowledgement This work was supported by grants NS-o7495 and NS-o933I from the National Institutes of Health. References ALLEN, T., BAERWALD, T., POTTER, L. a n d NICKEL, E. (1974) Particles in t h e dorsal (non-innervated)
and ventral (innervated) membranes of the electroplaques of Narcine. Journal of Cell Biology 63, 6a. BENNETT, M. V. L. (1971) Electric Organs in Fish Physiology (edited by HOAR, W. S. and RANDALL, D. J.), pp. 347-491. New York: Academic Press. CARTAUD, E. L., BENEDETTI, L., COHEN, J. B., MEUNIER, J.-C. a n d CHANGEUX, J.-P. (1973) Presence of a lattice structure in membrane fragments rich in nicotinic receptor protein from the electric organ of Torpedo marmorata. Federation of European Biochemical Societies Letters 33, lO9-13. CLEMENTI, F., CONTI-TRONCONI, B., PELUCHETTI, D. and MORGOTTI, M. (1975) Effect of denervation on the organization of the postsynaptic membrane of the electric organ of Torpedo marmorata. Brain Research 90, 133-8. DANIELS, M. P. and VOGEL Z. (1975) Immunoperoxidase staining of ,-bungarotoxin binding sites in muscle endplates shows distribution of acetylcholine receptors. Nature 254, 339-41. HEIYSER, J. E. and REESE, T. S. (I973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. Journal of Cell Biology 57, 314-44.
Fig. 23. Freeze-fracture replica of noninnervated electrocyte membrane. Numerous invaginations extend into the cytoplasm. The mouths of these can be seen as circular elevations in the B face of the plasma membrane (B). • 28ooo. Fig. 24. A face of noninnervated electrocyte membrane. Innumerable particles are visible and the mouths of the invaginations in this face are seen as depressions. • 87000. Fig. zS. Invaginations of noninnervated electrocyte membrane. Concave A faces are covered with particles while convex B faces are virtually free of particles. • 58000.
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HEUSER, J. E., REESE, T. S. and LANDIS, D. M. D. (1974) Functional changes in frog neuromuscular junctions studied with freeze-fracture. Journal of Neurocytology 3, lO9-31. HUXLEY, H. E. (1963) Electron microscopic studies on the structure of natural and synthetic protein filaments from striated muscle. Journal of Molecular Biology 7, 281-3o8. KAMINER, B. and SZONYI, E. (1973) 'Muscle' proteins in the electric organ of the Torpedo. Biological Bulletin 145, 441-2. L OM O, T. and R OSEN T I-IAL, J. (I 972) Control of ACh sensitivity by muscle activity in the rat. Journal of Physiology 221, 493-513. LUFT, J. H. (1958) The fine structure of electric tissue. Experimental Cell Research, Supplement 5, 168-82. MOOR, H., PFENNINGER, K. and AKERT, K. (1969) Synaptic vesicles in electron micrographs of freezeetched nerve terminals. Science 164, 14o5-7 . NICKEL, E. and POTTER, L. T. (1970) Synaptic vesicles in freeze-etched electric tissue of Torpedo. Brain Research 23, 95-1oo. NICKEL, E. and POTTER, L. T. (1973) Ultrastructure of isolated membranes of Torpedo electric tissue. Brain Research 57, 5o8-17. POLITOFF, A. L., ROSE, S. and PAPPAS, G. D. (1974) The calcium binding sites of synaptic vesicles of the frog sartorius neuromuscular junction. Journal of Cell Biology 61, 818-23. RAFTERY, M. (1976) The Synapse I n XL Cold Spring Harbor Symposium. in press. ROBERTSON, J. D. (1959) The ultrastructure of cell membranes and their derivatives. Biochemical Society Symposium 16, 3-43. ROSENBLUTtt, J. (1972) Myoneural junctions of two ultrastructurally distinct types in earthworm body wall muscle. Journal of Cell Biology 54, 566-79ROSENBLUTH, J. (I973a) Membrane specializations at myoneural junctions. Anatomical Record 175, 428. ROSENBLUTtt, J. (I973b) Membrane specialization at an insect myoneural junction. Journal of Cell Biology 59, 143-9. ROSENBLUTH, J. (I973C) Postjunctional membrane specialization at cholinergic myoneural junctions in the leech. Journal of Comparative Neurology I5I, 399-4o6. ROSENBLUTH, J. (1974) Substructure of amphibian motor end plate. Evidence for a granular component projecting from the outer surface of the receptive membrane. Journal of Cell Biology 62, 755-66. s HER I DAN, M. N. (1965) T h e fine structure of the electric organ of Torpedo marmorata. Journal of Cell Biology 24, 129-41.
Synaptic membrane structure in electric organ JACK
Torpedo
ROSENBLUTH
Departments of Physiology and Rehabilitation Medicine, New York University School of Medicine New York, New York 10016, U.S.A.
Received 15 July I975; accepted 28 July I975
Summary The innervated and noninnervated membranes of Torpedo electrocytes have been examined by electron microscopy of thin-sectioned and freeze-fractured specimens. The ventral innervated membrane is ~-~i2o A thick and is characterized by an unusually broad outer dense lamina (~-,6o A) in which a granular substructure can be resolved. The granules are "~7o A in diameter and are spaced irregularly. The same membrane specialization was noted in a previous study of amphibian myoneural junctions, and it was proposed then that the granular elements represent ACh receptor molecules. The morphologically equivalent structures presumably have the same significance in the Torpedo electric organ. However, in this case the specialized membrane covers the entire innervated surface, leading to the conclusion that high concentrations of receptors occur normally in extrajunctional as well as postjunctional regions of the innervated membrane of the electrocyte. In replicas of freeze-fractured specimens, the A face of this membrane is covered with large particles having the same distribution and approximate concentration as the granules visible in thin sections, indicating that the granules visible at the outer surface of the membrane extend at least into the hydrophobic middle layer of the membrane. The cytoplasmic surface of this membrane has an amorphous coating into which 'decorated' cytoplasmic filaments insert. Synaptic vesicle and axon terminal membranes also contain granules visible in thin sections but with a much sparser distribution. These probably correspond to the intramembranous particles seen in freeze-fractured specimens. Vesicles are occasionally attached to the axolemma by thin linear strands.
Introduction Electron micrographs of myoneural junctions in both vertebrates (Rosenbluth, 1973 a, I974) and invertebrates (Rosenbluth, i972, I973b, c) reveal a distinctive structural specialization of the postjunctional membrane. I n thin-sectioned specimens this membrane is considerably thicker than other membranes, and at high magnification a granular substructure can be resolved in its outermost lamina. On the basis of the size, location and approximate con9 1975 Chapman and Hall Ltd. Printed in Great Britain
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centration o f these granules, it was proposed that they represent the transmitter receptors. Freeze-fracture studies (Heuser, Reese and Landis, 1974) reveal high concentrations of i n t r a m e m b r a n o u s particles having roughly the same distribution and concentration as the superficially located granules seen in thin-sectioned specimens. I t is therefore likely that the elements observed b y these two different methods represent different aspects of the same structure. Daniels and Vogel (1975) have recently shown that ~-bungarotoxin binding sites also have the same distribution at myoneural junctions and there seems little doubt, therefore, that the granule-bearing m e m b r a n e is indeed the receptive m e m b r a n e . T h e present investigation consists of a study o f the equivalent m e m b r a n e s in the Torpedo electric organ, which contains extensive junctional areas along the ventral surface of the electrocytes. Thin-sectioned and freeze-fractured specimens demonstrate the same structural specializations identified previously at myoneural junctions. H o w e v e r the distribution o f these elements in the electric organ is significantly broader and leads to the conclusion that in this tissue, unlike muscle, acetylcholine receptors are normally found in high concent_ration in extrajunctional regions as well as in those regions directly under nerve endings.
Materials and methods Slices of the electric organ were fixed immediately after removal from the living animal which had been anesthetized with Tricaine. The fixatives used were 2% glutaraldehyde in o. z M phosphate buffer or 4% glutaraldehyde in o.xi M cacodylate buffer. After approximately I 8 9 -- 3 h some samples of the fixed tissues were washed in sea water, postfixed in 1% osmiumtetroxide in the same buffer for zI - 3 h, and then dehydrated and embedded in Araldite. Other fixed samples were rinsed in sea water and then dehydrated and embedded directly without postfixation, and the remaining tissue was stored in the glutaraldehyde. Thin sections were cut on a Porter-Blum MT-2 microtome. In most cases the sections used exhibited a silver interference colour, but in some instances very thin (colourless) sections were examined and in others relatively thick (red) sections were studied. The sections were stained by the permanganate-uranyl acetate method described previously (Rosenbluth, I972 ) which permits visualization of fine details of membranes even in very thin sections, but which has the disadvantage of often leaving fine filamentous deposits in the background. All sections were examined with a Philips 3oo electron microscope at 60 kV. For freeze-fracture studies, glutaraldehyde fixed specimens were passed through graded concentrations of glycerol from 5 % to 2 0 - 3 0 0 . They were then frozen in Freon 22 cooled byliquid nitrogen and fractured and shadowed in a Balzer's freeze-fracture apparatus equipped with an electron gun. The tissue was digested with several changes of Clorox; the replicas were then rinsed and mounted on i5 o mesh carbon coated grids and examined.
Results
Thin-sectioned specimens T h e Torpedo electric organ
consists o f stacks o f plate-like electrocytes (Fig. I), the ventral face of which is innervated and the dorsal face uninnervated (for review see Bennett, 1971). T h e innervated face can be identified b y the presence of nerve endings apposed to the cell m e m b r a n e and is characterized also b y the presence of rather sparse tubular invaginations o f the postjunctional m e m b r a n e which are ~-~ 0.3 ~m in diameter (Fig. 2). T h e spacing o f
Fig. i. Survey view of Torpedo electrocyte. Portions of two cells are shown separated by a connective tissue space. The dorsal noninnervated face (D) exhibits innumerable narrow infoldings, while the ventral innervated face (V) shows much less infolding and is indented by several nerve endings. • 6000. nerve endings varies from place to place. In some regions the axon terminals are almost contiguous (cf. Sheridan, I965) while in others they are more widely spaced, and stretches up to 2- 3 ~m long are devoid of endings (cf. Luft, 1956). In contrast to the innervated face, the noninnervated face exhibits an extensive plexus of narrow diameter infoldings (~--~o.I ~m diameter) which penetrate deep into the cytoplasm and occur over the entire surface of this face. A prominent basement membrane coats both faces of the cell. Within the cytoplasm, the nucleus can occasionally be identified, but other organelles are sparse consisting primarily ofmitochondria and a distinctive population of disordered filaments measuring ~-~ 12o A in diameter (Fig. 3). These are rough in appearance and sometimes have projections spaced at ~ 3-4oo • intervals extending from them. They bear a resemblance to the 'decorated' filaments described by Huxley (1963). Many of these filaments insert into the amorphous coating on the cytoplasmic surface of the ventral innervated membrane (Fig. 3), and a small number insert into the thinner coating on the dorsal noninnervated membrane. At high magnification the plasma membrane of the innervated face is seen as a threelayered structure having an overall width of ~-~ I2o A (Fig. 5). The thin (~-~25-3 o A) inner dense lamina (Robertson, I959) is usually continuous, but occasionally appears beaded (Fig. 9). It is coated on its cytoplasmic surface with an amorphous dense material. The lucent middle lamina, also measuring ~-~ 25-3 o A in width, overlies the inner dense lamina. The outer dense lamina of the membrane is the most distinctive one (Figs. 7-13). It is ~-- 60 A in width, which is much thicker than in most other cell membranes, and it frequently has a
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granular or interrupted appearance. The 'granules' visible in the outer dense lamina are 65-7o A in diameter, and in every respect resemble those described previously in the receptive membrane at amphibian motor endplates (Rosenbluth, 1974). The granular substructure is often unclear, presumably because of superimposition of randomly arrayed granules within the thickness of the section, but the characteristic thickening of the outer dense lamina is always apparent. The granules appear not to form lattices like those found at earthworm junctions (Rosenbluth, 1972) inasmuch as no extensive, regularly spaced rows of granules have been seen as would be expected in randomly oriented sections through a semicrystalline array. Short stretches of regularly spaced granules can be found, however (Figs. 7-9). The plasma membrane is covered by a basement membrane which is located ~ IOO A from the cell and which follows its invaginations faithfully with the exception that it does not extend into the narrowest branches of the invaginations arising from the noninnervated surface (Figs. 14 and 15)" A reticular pattern is sometimes visible in the basement membrane reminiscent of that in frog endplate basement membrane (Rosenbluth, 1974), and filamentous projections interconnecting the plasma membrane and basement membrane are occasionally, though not usually, visible. The specialized plasma membrane and superimposed basement membrane described cover the entire innervated face of the electrocyte including the regions directly under nerve endings (Fig. 5) and the extrajunctional regions between nerve endings (Fig. 6). Only rarely can a focal non-specialized region be found at which the plasma membrane is reduced to the more typical width of ~ 60 A (Figs. 12 and 13). The specialized membrane also extends into the tubular infoldings and can be identified in the deepest recesses of these invaginations. In marked contrast to the postjunctional membrane, the prejunctional axon terminal and synaptic vesicles are covered by limiting membranes having no such specialization of the outer dense lamina (Fig. 3). In en face views, however, the synaptic vesicle membranes exhibit a reticular pattern (Fig. 4) and, in addition, dense granules ( ~ 70 A) can be seen in the synaptic vesicle membrane (Figs. 4 and 5) (cf. Heuser and Reese, 1973, Politoffet a!., 1974) and are visible also in the axolemma (Figs. 4, 5 and 13). These probably correspond to the particles visible in replicas of the same membranes after freeze-fracturing. Linear strands
Fig. 2. Innervated face of electrocyte. Its plasma membrane (P) appears very dense in both postjunctional and extrajunctional regions. N, nerve ending; S, Schwarm cell; B, basement membrane. X 2 I 000.
Fig. 3. Innervated membrane at higher magnification. The electrocyte plasma membrane (P) is trilaminar and has a dense amorphous coating on its cytoplasmic surface. Filaments (F) bearing periodic projections abound in the cytoplasm and several of them insert into this coating. The axolemma (A) does not appear trilaminar but has an amorphous cytoplasmic density running parallel to it in one region (-~). I, infolding of electrocyte membrane; M, mitochondrion. • 70000. Fig. 4. Detail of synaptic vesicles. A reticular pattern appears in the vesicle membrane where it is cut tangentially. Dense granules are visible in the normally cut synaptic vesicle membrane and in the axolemma below (~'s). Several granules of this size are also seen superimposed on tangentially cut vesicle membranes along with two larger granules. However in oblique views it is not possible to tell precisely where the granules are with respect to the membrane. • ~52ooo.
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often extend from the synaptic vesicle membrane into the surrounding axoplasm (Figs. Io and II) and occasionally stretch between a synaptic vesicle and the prejunctional (axonal) membrane (Fig. 5). The noninnervated surface of the electrocyte (Fig. I4) has a limiting membrane which is frequently trilaminar in appearance, but it is only ~ 60-65 A in thickness and has an outer dense lamina only ~ 25/k wide. A granular substructure can occasionally be seen here (Fig. z5), but this lamina is consistently thinner than the outer dense lamina of the innervated membrane. In specimens that have not been postfixed with osmium tetroxide, differences among the various limiting membranes in the tissue are in some cases more pronounced and in others less pronounced. While most limiting membranes including those of axons, Schwann cells, nuclei, mitochondria, and synaptic vesicles are barely preserved in specimens of this kind, the innervated membrane of the electrocyte (Figs. z6 and I7) still exhibits a prominent trilaminar structure and dense cytoplasmic coating. The fact that the entire innervated face is covered with specialized membrane is particularly clear in such specimens since in contrast to ordinary membranes, which are virtually invisible, that of the innervated face stands out vividly along its entire length except for a rare gap. This technique also preserves a trilaminar structure in the noninnervated electrocyte membrane, which is specialized for quite a different function. However, the ventral and dorsal electrocyte membranes can be distinguished in preparations of this kind on the basis of the cytoplasmic coating which is distinctly heavier on the innervated membrane.
Freeze-fracture images At low magnification is is possible to distinguish both innervated and noninnervated surfaces of the same electrocyte (Fig. z8). The fracture plane does not usually pass through nerve endings as well, and therefore the identification of the two surfaces rests upon different patterns of infoldings in the membranes. The most distinctive feature of the freeze-fracture image is the occurrence of particles measuring ~ I 15 A in diameter in the A face of both the innervated (Fig. 2I) and noninnervated (Fig. 24) surfaces. The particles extend over the whole area of the membrane exposed and therefore appear not to be confined to circum-
Fig. 5. Higher magnification view of synaptic membranes. The postjunctional electrocyte membrane (P) is distinctly trilaminar in contrast to the axoIemma (A). Granules are visible in the synaptic vesicle membrane (double -~) and axolemma (-~). T h e vesicle at the extreme left is attached to the granule in the axolemma by a linear strand. B, basement membrane. • 20oooo. Fig. 6. Extrajunctional electrocyte membrane of innervated face. The dimensions and substructure of this membrane (P) are the same as those of the postjunctional membrane in Fig. 5. B, basement membrane. • 200000. Figs. 7 - I I . Details of ventral electrocyte membrane. Figs. 7-9. The outer dense lamina is thick and contains regularly spaced granules. The inner dense lamina is thin and continuous but with 'beading' evident in some places (-~'s). • 286ooo. Figs. Io a n d x I. The spacing of the granules in the outer dense lamina of the electrocyte membrane is irregular. +'s indicate linear strands attached to synaptic vesicle membranes. Fig. Io, • 292000; Fig. I I , • 23Iooo.
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scribed patches. The concentration of A face particles is ~ 5 • IO3 per ~m ~, but they usually do not exhibit any regular array. In some regions they are closely spaced, but they are usually spaced more widely and irregularly. Particles extend into the invaginations of the innervated face as far as the invaginations can be followed (Fig. 2o). The B face of the innervated membrane also exhibits scattered large particles (Fig. 22) but their concentration is only I-2ooo per ~m ~. Since the large particles in both the A and B faces are irregularly arranged, it is not possible to de termine whether they are fragments of the same elements or are separate elements that adhere by chance to one or the other fracture face. In addition to these large particles, the B face also exhibits arrays which look more square than hexagonal and which have a repeating period of 6o-7o A (Fig. 22). On the noninnervated surface, A face particles can also be followed into the depths of the invaginations; however B faces exhibit few particles and no square arrays (Fig. 25). In some instances (Fig. 2I) it has been possible to detect a central depression in the particles of both innervated and noninnervated faces (cf. Allen et al., 1974). However, this is not seen consistently. Fractures along nerve terminal membranes reveal the presence of large particles in both the A face (Fig. 19) and B face of the axolemma. However the A face particles occur in much lower concentration than in the electrocyte membrane. Occasional pits, presumably signifying exocytosis, occur in the A face (Fig. 19), but thus far no bands of particles adjacent to release sites, like those occurring in amphibian myoneural junctions (Heuser et al., 1974) , have been detected in the Torpedo electric organ. Fractures through the terminal axoplasm occasionally reveal omega-shaped profiles along the axolemma as described by Nickel and Potter (I97O), and views of either the A or B face of the synaptic vesicles are also commonly seen. The vesicle membrane frequently exhibits one or more large particles in either the A or B face (Fig. I9) (cf. Moor et al., 1969; Heuser et al., I974) which occur with about the same frequency as the granular densities seen in synaptic vesicle membranes in thin sections (Figs. 4 and 5) and probably correspond to them. Similarly, the granules visible in thin sections of the axolemma (Figs. 4, 5 and I3) probably correspond to the particles seen in the freeze-fractured axolemma. Discussion
This study shows that the innervated face of Torpedo electrocytes exhibits a membrane specialization identical to that seen previously in the receptive membrane of amphibian motor endplates and similar to those found at invertebrate myoneural junctions. The one Figs. ~z a n d I3. A short length of unspecialized membrane ( @ appears in the innervated surface of the electrocyte. A, axolemma; *, dense granules in axolemma. Fig. I2, • 200000; Fig. 13, • 252000. Figs. I4 a n d I5. Noninnervated face of electrocyte. The plasma membrane is invaginated. Basement membrane (B) extends into the broader branches of the invagination, but not into the narrowest ones. The plasma membrane (P) appears trilaminar in some places but is only ~65 A thick. At -~, granules are visible in the outer dense lamina. The overall thickness of the membrane here is ~65 A. Fig. I4, x I97OOO;Fig. 15, • 238 ooo. Figs. x6 and I7. Unosmicated specimens, innervated face. The trilaminar structure of the plasma membrane is distinct. Fig i6, • T63OOO;Fig. I7, • I56ooo.
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important difference is that the specialized membrane in Torpedo electric organ covers the extrajunctional as well as postjunctional portions of the innervated face. In previous studies of negatively stained membrane fragments, Cartaud et al. (1973) reported granules in the receptor bearing membrane which were 8o-9o A in diameter and could be resolved into 3-6 subunits. Nickel and Potter (1973) also found such subunits and in addition were able to distinguish the two faces of the cell based on the finding of'stalked particles' in the ATPase rich membrane fragments, presumably from the noninnervated face, in contrast to doughnut-shaped particles in the receptor rich membrane fragments, presumably from the innervated face. The negative staining method has the disadvantage that it is carried out on isolated fragments, and therefore it is not possible to determine whether these were originally postjunctional or extrajunctional in location. The freeze-fracture method has the same limitation inasmuch as the fracture plane does not usually pass through both pre- and postjunctional membranes in the same region, and again it is not possible to be sure whether a given region of the innervated face of the electrocyte is postjunctional or extrajunctional in such preparations. Moreoever in this organ the intramembranous particles from the noninnervated face (presumably representing ATPase) are indistinguishable from those in the innervated face (presumably representing receptors), and therefore particle-rich membrane cannot be equated with receptor-rich membrane. Only in the thin-sectioned specimens can postjunctional and extrajunctional regions be identified unambiguously, and therefore only on the basis of examining appropriately fixed and stained preparations of this kind can it be concluded that the specialized membrane covers the entire innervated face induding the extrajunctionat portions. The arguments previously offered for equating the granules visible in the outer dense lamina of the postjunctional membrane with acetylcholine receptors apply to the Torpedo electric organ as well as to the motor endplates examined originally (Rosenbluth, 1972, 1974). The specialized membrane described appears to represent regions in which the receptors are highly concentrated and constitute a significant proportion of the intrinsic structure of the membrane. It is undoubtedly their presence that allows the basic membrane architecture to be preserved even after lipid extraction in specimens whose membranes have not been stabilized by osmium tetroxide. The presence of ATPase in high concentration in the noninnervated face also seems to have the same stabilizing effect. The two faces are, however, clearly distinguishable in osmicated specimens, where the characteristic marked thickening
Fig. 18. Freeze-fracture replica of Torpedo electrocyte showing the innervated ventral membrane (V)
and the noninnervated dorsal membrane (D). The two membranes can be distinguished readily by the pattern of infoldings. • 190o0 Fig. x9. Axon terminal. The fracture plane passes through axoplasm at the left revealing A and B faces of synaptic vesicles. Two of these ( ~ 's) have singte large particles in the A face and one (white arrowhead) has large particles in the B face. At the right, fracture plane passes through the A face of the axolemma (A) revealing scattered large particles and one pit (-~). • 57ooo. Fig. 2o. Innervated surface of electrocyte showing several finger-like invaginations. In one of these the fracture plane passes from the concave (A) face to the convex (B) face. The A face of the plasma membrane at the bottom is covered with large particles. • 28000.
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of the outer dense lamina of the innervated membrane is unmistakable. Evidently the receptor molecules project from the innervated membrane in contrast to the ATPase molecules which are apparently buried in the noninnervated membrane. In addition the receptor protein, all of whose subunits are glycoprotein (Raftery, 1976), may have a greater affinity for osmium tetroxide. The occurrence of this specialization in extrajunctional regions of the Torpedo electrocyte raises an interesting question as to what governs the distribution of receptors. In vertebrate skeletal muscle, high concentrations of receptors occur under normal conditions only in the endplate regions and are probably not even present in the depths of the junctional folds (Rosenbluth, 1974). Only after denervation do the receptors appear in higher concentrations in extrajunctional regions, but this 'spread' of receptors can be reduced by periodic direct stimulation of the muscle cells (Lomo and Rosenthal, 1972). Torpedo presumably uses its electric organ intermittently. However, the cells do not generate action potentials (Bennett, 1971 ) and it may be this characteristic that underlies the broad distribution of receptors in the electrocytes. Denervation produces no increase in these receptors (Clementi et al., x975), an observation which is consistent with the conclusion reported here that in the normal Torpedo electric organ the entire innervated surface already contains receptors in high concentration. However it does not account for the apparent failure of receptors to spread to the noninnervated face of the denervated electrocyte. The significance of the cytoplasmic filaments in the electrocytes is unclear. They are presumably composed of the muscle proteins which have been shown to occur in this organ (Kaminer and Szonyi, 1973). Since the cells are derived from muscle, the filaments may merely represent vestigial structures consisting of actin filaments to which other muscle proteins and/or non-muscle proteins have become attached (perhaps during fixation) and having no contractile function. Since some of these filaments insert into the amorphous dense layer coating the inner surface of the plasma membrane, a possible alternative interpretation is that they are in some way involved in the movement of receptor molecules, ATPase, and other proteins to the plasma membrane and may thus play a role in the turnover of these membrane constituents. In summary this study makes the following points: (1) The innervated face of Torpedo electrocytes exhibits a conspicous membrane specialization consisting of a trilaminar membrane whose outer dense lamina is markedly thickened due to the present in it of ~-~ 7o A granules facing the junctional cleft. These granules probably extend into the hydrophobic middle layer of the membrane where large
Fig. 2i, Detail of Fig. 20. Innumerable randomly arranged intramembranous particles are visible in the A face of the innervated membrane. They are irregular in shape and range in size from N zoo to -~ I5o/~. Their concentration is ~ 5 • IO~per y.m2. Some appear to be arranged in short chains and at the extreme right the particles exhibit a semblance of an ordered array. Central pits are visible in a number of particles. • 136ooo. Fig. 22. B face of innervated electrocyte membrane. Intramembranous particles are present but in much lower concentration than that seen in the A face. Square arrays are visible at ~+. • 7oooo.
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numbers of particles can be seen in freeze-fractured specimens and may also extend to the cytoplasmic surface of the membrane which is covered with an amorphous dense material. The granular elements, which are probably ACh receptors, appear to form an intrinsic component of the membrane but project from its outer surface further than other intrinsic membrane elements. (2) Based on the distribution of the specialized membrane, it is concluded that high concentrations of receptors occur in the extrajunctional as well as postjunctional portions of the innervated surface of the electrocyte. (3) In thin-sectioned specimens, granules are visible in both the synaptic vesicle membranes and the axolemma having the same distribution as the intramembranous particles visible in freeze-fractured preparations of the same membranes and probably corresponding to them. Thin linear strands are attached to the outer surface of synaptic vesicles and sometimes interconnect them with the axolemma. (4) The electrocytes contain large numbers of 'decorated' filaments which insert into the amorphous coating on the cytoplasmic surface of the plasma membrane. Acknowledgement This work was supported by grants NS-o7495 and NS-o933I from the National Institutes of Health. References ALLEN, T., BAERWALD, T., POTTER, L. a n d NICKEL, E. (1974) Particles in t h e dorsal (non-innervated)
and ventral (innervated) membranes of the electroplaques of Narcine. Journal of Cell Biology 63, 6a. BENNETT, M. V. L. (1971) Electric Organs in Fish Physiology (edited by HOAR, W. S. and RANDALL, D. J.), pp. 347-491. New York: Academic Press. CARTAUD, E. L., BENEDETTI, L., COHEN, J. B., MEUNIER, J.-C. a n d CHANGEUX, J.-P. (1973) Presence of a lattice structure in membrane fragments rich in nicotinic receptor protein from the electric organ of Torpedo marmorata. Federation of European Biochemical Societies Letters 33, lO9-13. CLEMENTI, F., CONTI-TRONCONI, B., PELUCHETTI, D. and MORGOTTI, M. (1975) Effect of denervation on the organization of the postsynaptic membrane of the electric organ of Torpedo marmorata. Brain Research 90, 133-8. DANIELS, M. P. and VOGEL Z. (1975) Immunoperoxidase staining of ,-bungarotoxin binding sites in muscle endplates shows distribution of acetylcholine receptors. Nature 254, 339-41. HEIYSER, J. E. and REESE, T. S. (I973) Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. Journal of Cell Biology 57, 314-44.
Fig. 23. Freeze-fracture replica of noninnervated electrocyte membrane. Numerous invaginations extend into the cytoplasm. The mouths of these can be seen as circular elevations in the B face of the plasma membrane (B). • 28ooo. Fig. 24. A face of noninnervated electrocyte membrane. Innumerable particles are visible and the mouths of the invaginations in this face are seen as depressions. • 87000. Fig. zS. Invaginations of noninnervated electrocyte membrane. Concave A faces are covered with particles while convex B faces are virtually free of particles. • 58000.
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HEUSER, J. E., REESE, T. S. and LANDIS, D. M. D. (1974) Functional changes in frog neuromuscular junctions studied with freeze-fracture. Journal of Neurocytology 3, lO9-31. HUXLEY, H. E. (1963) Electron microscopic studies on the structure of natural and synthetic protein filaments from striated muscle. Journal of Molecular Biology 7, 281-3o8. KAMINER, B. and SZONYI, E. (1973) 'Muscle' proteins in the electric organ of the Torpedo. Biological Bulletin 145, 441-2. L OM O, T. and R OSEN T I-IAL, J. (I 972) Control of ACh sensitivity by muscle activity in the rat. Journal of Physiology 221, 493-513. LUFT, J. H. (1958) The fine structure of electric tissue. Experimental Cell Research, Supplement 5, 168-82. MOOR, H., PFENNINGER, K. and AKERT, K. (1969) Synaptic vesicles in electron micrographs of freezeetched nerve terminals. Science 164, 14o5-7 . NICKEL, E. and POTTER, L. T. (1970) Synaptic vesicles in freeze-etched electric tissue of Torpedo. Brain Research 23, 95-1oo. NICKEL, E. and POTTER, L. T. (1973) Ultrastructure of isolated membranes of Torpedo electric tissue. Brain Research 57, 5o8-17. POLITOFF, A. L., ROSE, S. and PAPPAS, G. D. (1974) The calcium binding sites of synaptic vesicles of the frog sartorius neuromuscular junction. Journal of Cell Biology 61, 818-23. RAFTERY, M. (1976) The Synapse I n XL Cold Spring Harbor Symposium. in press. ROBERTSON, J. D. (1959) The ultrastructure of cell membranes and their derivatives. Biochemical Society Symposium 16, 3-43. ROSENBLUTtt, J. (1972) Myoneural junctions of two ultrastructurally distinct types in earthworm body wall muscle. Journal of Cell Biology 54, 566-79ROSENBLUTH, J. (I973a) Membrane specializations at myoneural junctions. Anatomical Record 175, 428. ROSENBLUTtt, J. (I973b) Membrane specialization at an insect myoneural junction. Journal of Cell Biology 59, 143-9. ROSENBLUTH, J. (I973C) Postjunctional membrane specialization at cholinergic myoneural junctions in the leech. Journal of Comparative Neurology I5I, 399-4o6. ROSENBLUTH, J. (1974) Substructure of amphibian motor end plate. Evidence for a granular component projecting from the outer surface of the receptive membrane. Journal of Cell Biology 62, 755-66. s HER I DAN, M. N. (1965) T h e fine structure of the electric organ of Torpedo marmorata. Journal of Cell Biology 24, 129-41.
