Submicroscopic Morphology of the Synapse1

Submicroscopic Morphology of the Synapse’ EDUARDO D E ROBERTIS Director of the Instituto de Anatomia General y Embriologia, Facultad de Ciencias Mfdicas, Buenos Aires, Argentina Page I. Introduction . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 61 11. Morphology of the Synaptic Region ......................... . ..... 63 A. Preliminary Observations with the Electron Microscope . . . . . . . . 66 B. Ultrastructure of the Synaptic Region : General Description . . 66 C. Ultrastructure of Typical Terminal Synapses .. . . . .. . . . . . . . . .. . . 68 1. Synaptic Membrane ... ................ ................ .... 68 2. Mitochondria .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. .. . . . . . . . . . . 69 3. Synaptic Vesicles . .. . . . ... . . . . .. .... .. . . .. . . .. . . . . . . . . . ... 69 D. Submicroscopic Structure of Some Special Synapses . . . . . . . . . . . . 70 1. Invertebrate Synapses . . . . . . . .. . . . . .. .. . . . . . . . . . . . . . . . . . . .. 70 2. Ultrastructure of the Neuromuscular Junction . .. . . . . . . . . . . . . 72 3. Innervation of the Electric Organ . .. . . . . . . . . . . .. . . . . . . . . . . . . 74 4. Synapses in Sympathetic Ganglia . .... . .... . . . ... . . . .. . . . . . . 74 5. Ultrastructure of Some Peripheral Nerve Endings. . . . . . . . . . . . . 74 6. Microvesicles in Regenerating Nerves ... . . .. .. . . . .. . . . . . . . 76 111. Submicroscopic Morphology and Function of the Synapse . . . . . . . . . . . . . 76 A. Degenerative Changes of the Synapse .. . . . ... . . . .. . . . . . . . . .. ... 78 B. Physiological Changes in Synapses of the Retinal Rods and Cones 79 C. Changes of the Synapse after Nerve Stimulation . .. . . . . . . . . . . . . . 80 D. Dimensions and Physiology of Synapses .................... .. 85 E. Functional Role of Synaptic Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . 90 IV. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . ... . .. . . . ... . ..... 93 V. References . . . . . .. . . . .. . . . . .. . . . . . .. . ... . .. .. .. .. .. . . . . .. .. .. .. .. . 94

I. INTRODUCTION The concept of the synapse, or synaptic junction, although first elaborated by physiologists to explain how nerve elements may exert excitatory or inhibitory actions on other nerve cells (Sherrington, 1897), had from its very beginning a definite morphological basis. The so-called “neuron doctrine,” masterly developed by Cajal (see Cajal, 1934), established that the individual nerve cells are not in continuity but in close contact at certain points, where the functional connections may be effected. The synaptic junction may be considered as a specialized locus of contact, at which synaptic excitatory or inhibitory influences are transmitted and act on other cells (see Eccles, 1957). As synaptic regions, in a strict sense, we shall consider the special zones 1 Some of the latest part of this work was helped by the grant B-1549 of the National Institute of Neurological Diseases and Blindness.

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of contact between two neurons, between a receptor and a neuron, and the neuromuscular junction, thus embodying all the regions “anatomically differentiated and functionally specialized for the transmission of the liminal excitations from one element to the following in an irreciprocal direction” (Arvanitaki, 1942). These typical polarized synapses are the great majority in the nervous system of both vertebrates and invertebrates? At the synaptic junction the two cellular elements-one presynuptic and another postsynuptic-are intimately apposed, and both of them have specialized functions that can be differentiated from the signal reaching the junction or the all-or-nothing impulse originating in the postsynaptic element and conducted by the following axon (see Eccles, 1957; Luco and Davidovich, 1957). Modern physiological and pharmacological investigations have demonstrated that synaptic junctions have indeed electrophysiological and chemical properties which can be differentiated from the rest of the neuron (see Fatt, 1954; Feldberg, 1954). These physiological advances for many years were not paralleled by progresses in morphological and structural studies of the synapse. In his review published in 1942, Bodian stressed the importance of learning more about the structure of the synapse, pointing out that, since the classical works of Cajal, Retzius, Ehrlich, and others, little but technical refinement has been contributed to the methodology of study of synapses. The generally used silver staining techniques gave a considerable body of information about the size, shape, and position of the nerve ending on the postsynaptic element, but not about the intimate structure of the terminal or the interface between the ending and the postsynaptic surface. Although the morphological aspects revealed by the optical microscope gave little background for a satisfactory explanation of synaptic function, it was hoped that the enormous resolving capabilities of the electron microscope would provide more fundamental details of structure. In fact, within the range of resolution that can be now achieved in tissue sections, the macromolecular structures revealed are better related to the chemical morphology of molecular complexes and to the intimate physicochemical mechanisms of cell physiology. This review will be concerned with studies of the synaptic junction carried out during the last five years with the electron microscope. As these investigations are still fragmentary and concerned only with a few types of synapses, it is difficult at this point to establish any kind of 2 This definition would exclude the natural or artificially produced contacts (generally axo-axonic) which were designated ebltases by Arvanitaki and also the contacts with reciprocal transmission found in some giant axons of invertebrates which have been named quasiartificial synapses or contacts by Bullock (1953).

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generalization. The results so far obtained, however, have settled some of the controversies which in the past derived from the limited resolving power of the light microscope and the vagaries of the silver staining techniques and have established the existence of a submicroscopic component, “the synaptic vesicles” (De Robertis and Bennett, 1954, 1955), which seem to have some relationship to the physiology of the synaptic junction (see De Robertis, 1957).

11. MORPHOLOGY OF T H E SYNAPTIC REGION The light microscope shows the extreme variation in shape, position, and dimensions which occurs at synaptic contacts. In his general review published in 1934, Cajal described eleven types of synapses divided into two groups of axosomatic and axodendritic junctions, according to the point of contact between the axon and the postsynaptic element. Although not recognized by Cajal, axo-axonic synapses are also found particularly in the neighborhood of the axon hillock. These axo-axonic synapses are the most commonly observed in invertebrates in which neurons are usually monopolar. One should not exclude the possibility of dendrodendritic junctions as proposed by Estable (1953). The existence of polarized synapses between two homologous elements (axon-axon, dendritedendrite), as we shall see later, can be easily explained on the basis of the submicroscopic organization of the synaptic region. The junction may be of the terminal type, in which it is the axon terminus or ending that establishes contact with the postsynaptic surface. In this case the ending may be of different shapes and classified as bud or foot ending, if there is a widening of the terminal; club ending, if the axon is wider and there is no enlargement of the terminal; or c a l k or c u p ending, if the ending covers a large zone of the cell surface (Bodian, 1942, 1952). The complex variety of terminals found in the ventral acoustic nucleus of mammals has been described by Estable et al. (1953). The foot endings or boutons observed on the soma and dendrites of motoneurons may be considered as prototype of endings. Barr (1939) and Haggar and Barr (1950) have calculated that over a thousand endings may cover the surface of a motoneuron, and up to 38% of it may be occupied by synaptic contacts. The close packing of terminals is confirmed in isolated motoneurons (Chu, 1954), in preparations with modifications of silver staining (Armstrong et al. 1956; Rasmussen, 1957), and in lowpower electromicrographs. According to Wyckoff and Young ( 1956j , the Since . the number of end feet can be estimated as 15 to 20 per 1 0 0 ~ ~ total surface area of a motoneuron is about 10,000p2,there are not less than 2000 end feet per cell (see Fig. 1A and B ) .

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The complex morphology of other types of synaptic junction has been recently reviewed by Bodian (1952) and von Horstmann (1957). Bodian emphasized the difficulties of studying in detail the morphology of synapses in which the contact is not terminal but along the axon, such as the synapses “en passant,” the climbing fibers or spiral synapses, and the glomerular synapses of the cerebellum. In all these cases it has been suggested that the contact may be effected by means of an intervening layer of material such as the ground substance with special conductance properties, postulated by Cajal (1934), or by a layer of glial cytoplasm, as held by de Castro (1942, 1950). The concept of the gliotheca was generalized by de Castro for all types of synapses and even for the neuromuscular junction (Noel, 1950). Very few cytological details of the structure of the synaptic region may be observed with the optical microscope. In synapses of the giant Mauthner cells stained with cytological methods the presence of a synuptk membrane or synaptolemma between the terminal and the postsynaptic cytoplasm has been revealed (Bartelmez and Hoerr, 1933 ; Bodian, 1940). Since the true synaptic membrane is of submicroscopic dimensions, the synaptolemma probably corresponds to the limit between the two contacting elements. In some cases neurofibrils have been observed within the terminal. Also the presence of mitochondria1 granules has been detected preferably on the proximal side of the synaptic junction. These granules probably correspond to the so-called “neurosomes” observed by Held (1897) within the glomeruli of the cerebellum. Mitochondria are also concentrated in integrative regions of the brain, forming vast synaptic FIG. 1. Diagram showing bouton-like synaptic junctions a t different magnifications with the optical and electron microscope. (A) Illustrates a motoneuron as seen a t medium power of the optical microscope. The nucleus (N),the axon ( A ) , and the dendrites (d) are indicated. Numerous bouton-like endings make synaptic contact with the surface of the pericaryon (axosomatic junctions) and of the dendrites (axodendritic junctions). Enclosure B is magnified ten times in B. ( B ) End feet ( e ) , as seen at high magnification with the optical microscope. The afferent axons are enlarged at the endings. The presence of mitochondria is indicated. Enclosure C is magnified about six times with the electron microscope in C. (C) Diagram of an end foot as observed with the electron microscope. Mitochondria ( m ) , neuroprotofibrils (nf), and synaptic vesicles (m) are shown within the ending. Three clusters of synaptic vesicles become attached to the presynaptic membrane ( p s n t ) ; these are probably active points (ap) of the synapse. Both the psm and the subsynaptic membrane (ssm) show higher electron density. The glial membrane is shown in dotted lines (gm). Enclosure D is magnified about twenty times in D. ( D ) Diagram of the synaptic membrane as observed with high-resolution electron microscopy (see description in the text). Some synaptic vesicles (m) are seen attached to the p s m and opening into the synaptic cleft (sc)

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fields or neuropiles (Scharrer, 1945), and in the terminal axoplasm of neuromuscular junctions (Noel, 1950).

A . Preliminary Observations with the Electron Microscope The early electron-microscope observations published in 1953 settled some of the above-mentioned controversies on the morphology of the synapse. Pease (1953) pointed out that end feet come in direct contact with the surface of nerve cells. In the axo-axonic synapses of the giant fibers of the squid and of the abdominal ganglia of the crayfish, Robertson (1953) found that the distance between the synaptic membranes was of the order of 600 A. I n his work, however, the distortion introduced by the extraction of the plastic may have altered the relationship between the membranes. Estable et ul. (1953) found in synapses of the ventral acoustic ganglia of the cat and dog that the minimal distance between the pre- and postsynaptic cytoplasm was 320 A, which corresponds approximately to the thickness of a double membrane. In the synapse between retinal rods and bipolar cells, Sjostrand (1953) observed that there is an intimate contact with considerable digitation of the postsynaptic ending into the adjacent region of the rod cell. These early observations and all the recent ones indicate that at the level of the junction there is a direct contact of membrane surfaces without interposed cellular material alien to the two pre- and postsynaptic components. This invalidates the supposition that the synaptic terminal is surrounded by a glial sheath or by any kind of ground substance. Furthermore, the observation of a neat delimitation of both the preand postsynaptic cytoplasm confirms and extends to a submicroscopic level the concept of the individuality of the nerve element which is implicit in the neuron doctrine of Cajal. The reticularist hypothesis, which still has its followers, cannot be maintained, even in those regions of the central nervous system called neuropiles, where most of the elements are of submicroscopic dimensions. The reticular appearance is the result of technical artifacts, plus the limited resolving power of the optical microscope to detect those structures and their boundaries. These facts indicate that for an exact interpretation all structures below 1 to 0.5 p should be studied with the electron microscope.

B.

Ultrastructure of the Synaptic Region: General Description

In spite of the obvious differences existing between synapses of the peripheral and the central nervous system, between the axosomatic, the axodendritic, and the axo-axonic, between the different types of synaptic endings and the synapses “en passant,” and so forth, from the submicro-

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scopic point of view there are details that are common to all of them. In the diagrams of Figs. 1 and 2 some of the most common types of synapse are indicated. In all of them there is a presynaptic element which has a

FIG.2. Diagram of different types of synaptic junction. ( A ) A Synapse between a rod and a bipolar cell (see description in the text) : p , a blind projection of the presynaptic membrane (psm) ; d, dendrites of the bipolar cell ; er, endoplasmic reticulum; gm, glial membrane. The main characteristic of this junction is the invagination of the psm and penetration of the dendrite into the ending. (B) Ending of a neuromuscular junction. Several active points on the p s m are indicated. The main difference from other synapses is the folding of the ssm, forming the subsynaptic or postjunctional folds (ssf) (see description in the text). (C) Type of lateral junction between an axon ( A ) and an electroplaque of the electric organ of the eel. Synaptic vesicles are present along the axon at synaptic contacts. Continuity of neuroprotofibrils (nf) is observed. Sc represents Schwann cell (Diagram based on an electron micrograph of Luft ; see also Fig. 11).

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different relationship with the postsynaptic one. In Fig 1C the bouton ending of the axon is applied to the postsynaptic surface of a motoneuron, which is flat or may have a small depression. Figure 2C is a synapse “en passant,” as observed by Luft on the plates of electric organ and probably similar to other synapses of this type in the central nervous tissue. In Fig. 2A the postsynaptic element penetrates deeply into the presynaptic one, as in the case of the retinal rod (Sjostrand, 1953; DeRobertis, 1955a; De Robertis and Franchi, 1956) and cone synapses with the bipolar cells. A similar relationship of membranes is probably found in synapses of the stellate ganglion of Loligo (Young, 1939) and in the crayfish abdominal ganglia (Robertson, 1953) and is probably most frequent in invertebrate neuropile (De Robertis and Bennett, 1954, 1955). Figure 2B indicates the case observed in the neuromuscular junction, in which the nerve endings are deeply embedded into grooves of the postsynaptic element (Couteaux, 1947, 1955) and the postsynaptic membrane is extensively folded ( Palade, 1954 ; Reger, 1954 ; Robertson, 1956), forming the so-called subneural apparatus of Couteaux. C.

Ultrastructure of Typical Terminal Synapses

1. Synaptic Membrane. Detailed descriptions of a bouton-like synapse as found in motoneurons and endings of the ventral acoustic ganglion have been published by De Robertis (1955a, b, 1956) and by Palay (1956, 1957b). In the nerve terminal, a surface membrane, an amorphous matrix, mitochondria, synaptic vesicles, neuroprotofibrils, and a few tubules or vesicles of the endoplasmic reticulum may be found. The surface membrane, usually of 50 to 70 A thick, is continuous with that of the axon membrane and with the presynaptic membrane which comes into direct contact with the postsynaptic surface membrane to form the synaptic junction proper. Eccles (1957) has propounded the term subsynaptic to this juxtaposed region of the postsynaptic membrane (Fig. lC, D ) . The surface membrane of the terminal is usually covered by glial processes in central synapses or by the Schwann cell in peripheral synapses (indicated by a broken line in Fig. 2C). ‘At this junction, however, its presynaptic part becomes entirely free and comes into direct contact with the subsynaptic membrane. Palay (1957a, b) has described in some cases small glial processes interposed between the terminal and the postsynaptic surface, but these do not obstruct direct contact. At the junction, both the pre- and subsynaptic membranes may show differentiated regions, which appear as spots or patches of higher electron density. These regions were first described by De Robertis (1955a, b, 1956) in the acoustic ganglion and more recently by Palay (1957b), who finds them to be

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150 to 200mp in length. At these patches, which are probably active points of the synapse (see below), the synaptic vesicles make a closer contact with the presynaptic membrane (see Figs. 2C and 4 ) . According to Palay, this complex formed by a cluster of synaptic vesicles associated with an area of the synaptic membrane and the subjacent synaptic cleft may be considered a morphological subunit of the synaptic membrane. Both the presynaptic and subsynaptic membranes are about 60 A thick and are separated by an intervening space-the synaptic cleft-of about 120 to 200 A. The synaptic cleft represents the real discontinuity of cell cytoplasm at the level of the junction. The continuity of this cleft with the extracellular spaces may be traced particularly in peripheral synapses. In central ones the cleft is continuous only with narrow interstitial clefts, since open extracellular spaces are not found in the central nervous tissue. High-resolution observations in retinal synapses indicate that both the pre- and subsynaptic membranes may be even more complex (De Robertis, 1957). Two dense lateral layers and a central one of lower density have been observed within the 60-A thickness of both membranes (Fig. 1D). 2. Mitochondria. Mitochondria are frequently observed within the terminal among the synaptic vesicles, but their number varies considerably from one type of synapse to another. Thus they are very abundant in the glomeruli of the cerebellum within the expanded terminals of the mossy fibers (Palade, 1954; De Robertis, 1955a; Palay, 1956) (Fig. 3 ) . In sections of the ventral acoustic ganglia there are only a few per terminal, and in synapses between the retinal rods and bipolar cells of the rabbit there are generally no mitochondria in the neighborhood of the synapse (De Robertis, 1955a; De Robertis and Franchi, 1956) (Fig. 5 ) . The mitochondria show the typical structure with the double lamellar crests, described by Palade ( 1952), which are frequently oriented longitudinally. This variability in concentration, the location of mitochondria generally far from the membrane, and their function in the oxidative cycle make its direct intervention in synaptic transmission, as suggested by Bodian ( 1942), very improbable. 3. Synaptic Vesicles. Under the name of “synaptic vesicles” De Robertis and Bennett ( 1954) described a special vesicular component present in the synapse. I n their early report on synapses of the frog sympathetic ganglia and the neuropile of the earthworm, they described the presynaptic location and the intimate relationship of some of these vesicles with the synaptic membrane. Almost simultaneously Palade (1954) and Palay (1954) reported an agglomeration of small vesicles in the axon endings of several synapses of the central nervous system and in the neuromuscular junction. The full paper of De Robertis and Bennett

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(1955), submitted for publication in May 1954, described in greater detail the relationship of the membranes with the synaptic vesicles. It was suggested that they may flow toward the synaptic membrane, perforate it, and discharge their content into the intermembranal space, and even go across the postsynaptic membrane to be destroyed at the postsynaptic cytoplasm. It was speculated at that time that acetylcholine or other chemical synaptic mediators could be associated with the synaptic vesicles. A t least from the quantitative point of view, the synaptic vesicles represent the most important, constant, and specific component of the synaptic terminal. Being confined almost exclusively to the proximal side of the synaptic region, the synaptic vesicles are the only elements which may confer to the synaptic region the necessary asymmetry for a polarized functional activity. The amount and disposition of the synaptic vesicles vary in different synapses, but in all cases one may observe their close association with the synaptic membrane (Figs. 3, 4, and 8). The profiles of synaptic vesicles are spherical or oval in shape with a dense limiting membrane 40 to 50 A thick and a content that is slightly denser than the matrix. The long diameter varies between 200 and 650 A. So far, extensive measurements have been made only in the retinal synapses, showing histograms with a high peak between 350 to 400 A and a mean diameter of 386 A (De Robertis and Franchi, 1956) (Fig. 9 ) . In the frog sympathetic ganglia the vesiculous material is very compact and fills the extreme distal part of the terminal. I n the ventral acoustic ganglion and in the glomeruli of the cerebellum the synaptic vesicles occupy the entire terminal with a rather homogeneous distribution (Fig. 5 ) ; in the rod-bipolar cell junction they are accumulated at an enlargement or expansion of the rod cell in which the postsynaptic element digitates and penetrates very deeply (Figs. 6, 7, and 8).

D. Submicroscopic Structure of Some Special Synapses 1. Invertebrate Synapses. In invertebrates the most commonly observed synapses are of the axo-axonic type. Neurons are usually monoFIG. 3. Electron micrograph of a bouton-like ending of the olfactory bulb of the rat showing three mitochondria ( m ) and numerous synaptic vesicles (m). G corresponds to glial processes ; d, dendrite. ( ~ 8 9 , 0 0 0 . ) FIG.4. Axodendritic synapse of the olfactory bulb. The zone of contact is indicated between the arrows. Three active points ( u p ) are indicated in the synaptic membrane. At these points the synaptic cleft is wider than in the rest of the junction, and dense material is present on both sides. See the intimate relationship of synaptic vesicles with the active points. My, myelin sheath ; psm, presynaptic membrane; ssm, subsynaptic membrane ; G, glia. ( ~ 8 9 , 0 0 0 . )

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polar, and the cell body is generally apart from the synaptic junction. The lack of vessels and the scanty number of glial elements favors the constitution of vast synaptic fields or dense neuropiles, where there is a great number of synaptic contacts per volume unit (Bullock, 1952). In the neuropile of the nerve cord of the earthworm, De Robertis and Bennett (1954, 1955) observed a complex tangle of unmyelinated nerve fibers in contact with no interposed glial elements. The fibers interdigitate extensively, forming complex and ramifying profiles. Mitochondria and endoplasmic reticulum are observed, but not typical neuroprotofibrils. Scattered in the neuropile there are regions containing large concentrations of synaptic vesicles of 200 to 400 A. Specialized areas of synaptic contact were recognized in which the postsynaptic membrane invaginates into the presynaptic one. Some synaptic vesicles were closely related to the presynaptic membrane, and a few of them were found in the interspace or synaptic cleft. Furthermore, faint ghostlike vesicular objects were observed in some postsynaptic fibers. These observations were interpreted as suggestive that vesicles may move toward the presynaptic membrane, perforate it, and discharge their contents into the interspace, and some of them may even enter and be destroyed in the postsynaptic cell. In arthropod neuropile, De Robertis and Franchi (1954) made similar observations of synaptic fields with synaptic vesicles. Recently Edwards (1957a, b) described the presence of numerous mitochondria and synaptic vesicles within the axon near or at the neuromuscular junction of annelid muscle and in the flight leg and abdominal muscles of higher insects. 2. Ultrastructure of the Neuromuscular Junction. The study of the fine structure of the neuromuscular junction is of considerable interest in view of remarkable advances made by physiologists by means of the microelectrode technique (see Tiegs, 1953 ; Fatt, 1954 ; del Castillo and Katz, 1956a, b ) . The complex structure revealed by the light microscope and the important studies on the histochemical location of cholinesterases have been reviewed by Couteaux (1955). In mammalian (Palade, 1954; Reger, 1954), amphibian (Reger, 1957), and reptilian synapses (Robertson, 1954, 1956), a close relationship was found between the branches of the innervating axons and the synaptic trough or grooves formed,by the sarcolemma. No interposed glia (teloglia) could be observed between the two contacting elements (see Couteaux, 1955). The main difference between the neuromuscular junction FIG. 5. Electron micrograph of a synapse of the cerebellum of a rat. The enlarged irregularly shaped endings correspond to mossy fibers that establish several contacts with dendrites ( d ) ; ap indicates active points. (x72,OOO.)

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and other types of synapse is found in the fact that at the subsynaptic membrane the sarcolemma differentiates in a very special manner. Folds approximately 800 A thick and 0 . 7 ~ long are formed which run transversely across the elongated axons, so providing channels between the interstitial spaces and the synaptic clefts. These folds, called “postjunctional” by Robertson ( 1956), were recognized earlier with the optical microscope and named subneural apparatus by Couteaux (1947) and Couteaux and Taxi (1952). Another characteristic of the neuromuscular synapse is the fact that the cleft is apparently wider (about 500 A ) and more complex than in other synapses. Within the axon, terminal mitochondria and numerous synaptic vesicles of 200 to 600 A may be observed (Palade, 1954; Robertson, 1956; Reger, 1957) (see Fig. 10). The relationship of these synaptic vesicles with the presynaptic and subsynaptic membranes and the synaptic cleft must be studied in normal and different physiological conditions. 3. Innervation of the Electric Organ. Electroplaques of the electric organ of different families of fishes were studied by Luft (1956). The plates are supplied with numerous nerve endings on one surface. The nerve fibers make lateral contacts upon papillae of the electroplate. At the junction the axon becomes closely approximated to the plate surface and is separated by a synaptic cleft of about 500 A. Beyond the synapse the axon is covered by Schwann cell cytoplasm. Synaptic vesicles accumulate at the site of contact, but they are less numerous in other parts of the axon (see Figs. 2C and 11). Neuroprotofibrils are present within the axon. 4. Synapses in Sympathetic Ganglia. In the abdominal sympathetic ganglia of the bullfrog, synaptic junctions upon the cell body and the emerging axon were recognized by De Robertis and Bennett ( 1954, 1955). I t was found that the Schwann cell covering of the fiber does not extend over the enlarged presynaptic ending, and the direct contact is frequently made in a depression of the postsynaptic neuron. Numerous densely packed synaptic vesicles were observed at the ending near the synaptic membrane. In Fig. 12 an axon-dendritic synapse in sympathetic ganglion of the cat is shown. It is interesting that in this synapse there is an accumulation of mitochondria in the postsynaptic cytoplasm. 5. Ultrastructure of Some Peripheral Nerve Endings. Vesicles similar to those found in typical synapses have been found in terminals about FIG. 6. Electron micrograph of a rod-bipolar cell synapse (see diagram of Fig. 2A) : d indicates the dendrites penetrating into the rod spherule which is totally filled with synaptic vesicles. A lateral synapse between the spherule and a dendrite is marked with arrows ( d ) ; gc, glial cell ; gin, glial membrane. ( ~70,000.)

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the hair cells of the cochlea (Engstrom and Sjostrand, 1954; Smith, 1957) and certain hair cells of the vesticular organ of the guinea pig (Wersall, 1956). If they were contained in real primary afferent fibers, the vesicles would occur in these cases on the postsynaptic side of the junction. This would be an exception to the rule of the presynaptic location of synaptic vesicles. The possibility exists, however, that these endings belong to tips of efferent fibers ending on the receptor (Rasmussen, 1953). It has been suggested that stimulation of these efferent fibers may produce a depolarization and reduction of impedance at the receptor (Engstrom and Sjostrand, 1954). Synaptic granules (vesicles) have also been described in clublike endings in the taste buds (Engstrom and Rytzner, 1956; Trujillo-CCnoz, 1957). Endings of the splanchnic nerve in the adrenal medulla of rabbits show a large concentration of typical synaptic vesicles that can be modified under nerve stimulation (see below, De Robertis and Vaz Ferreira, 1957b). 6. Microvesicles in Regenerating Nerves. The first observation of a vesicular material in regenerating nerve fibers was made in tissue cultures of the nervous system of the chick embryo (De Robertis and Sotelo, 1952). The growing endings of the fibers showed an enlarged mass with fingerlike processes, filled with tightly packed microvesicular material. Recently Estable et al. (1957) found in the growing tips of regenerating adult nerve fibers, after severance of the sciatic nerve, the appearance of numerous densely packed microvesicles 200 to 700 A in diameter. In regenerating limbs of Amblystomu, Hay (1957) found bulbous nerve endings containing numerous synaptic vesicles and small mitochondria. According to the author, each ending applied to two or more epithelial cells and resembled a synapse.

MORPHOLOGY AND FUNCTION OF T H E SYNAPSE 111. SUBMICROSCOPIC Since a general review of the subject under this title was presented at the Symposium on Submicroscopic Organization and Function of Nerve Cells (De Robertis, 1957), only some data will be summarized and discussed here. FIG.7. Rod-bipolar cell synapse showing the penetrating dendrites ( d ) and in p a process or blind infolding of the presynaptic membrane. Numerous synaptic

vesicles become attached to this process. Gc, glial cell. ( x 114,000.)

FIG.8. Similar to Fig. 7. The relationship of the presynaptic membrane (psm) with the subsynaptic (ssm) and the synaptic cleft (sc) is shown. Synaptic vesicles attached to the psm are indicated with arrows ; em, surface membrane of the ending ; Gm, glial membrane. (x114,OOO.)

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A. Degenerative Changes of the Synapse The optical microscope has revealed that the alterations of the synaptic junction after section of the afferent axon consist in swelling and subsequent fragmentation and disintegration of the endings (Hoff, 1932 ;

x

x

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40.

20-

Rod Synapses

Cone Synapses

FIG.9. Histogram showing the distribution of sizes (in percentage) of the synaptic vesicles. Rod synapses: ( A) rabbit exposed for 4 hours to sunlight; (B) in darkness for 24 hours; (C) in darkness for 46 hours; ( D ) in darkness for 9 days. Cone synapses : ( A ) Rabbit under sunlight for 4 hours ; (B) in darkness for 9 days. (Taken from De Robertis and Franchi, 1956.) Foerster et al., 1933; Hoff and Hoff, 1934; Gibson, 1937; Glees et d., 1946). I n the central nervous system, swelling of nerve endings has been seen as early as 24 hours after section. In peripheral synapses and particularly in the neuromuscular junction, nerve transmission fails before the

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axon has ceased to conduct (Titeca, 1935; Lissak et al., 1939; Eyzaguirre et ul., 1952). Similar changes in sympathetic ganglia have been correlated with decrease in acetylcholine content (Coppee and Bacq, 1938 ; McIntosh, 1938). Electron-microscope observations in normal ventral acoustic ganglion (Fig. 13A) and after destruction of the cochlea have revealed a sequence of degenerative changes in the synaptic endings (De Robertis, 1956). These involve swelling of the matrix, agglutination and lysis of synaptic vesicles, lysis and disintegration of mitochondria, and finally detachment and breakdown of the membrane at the synaptic junction (Fig. 13B, C, D ) . The first (after 22 hours) and more marked changes are those of the synaptic vesicles, and it has been suggested that they may be related to the early physiological deterioration of synaptic transmission (De Robertis, 1956, 1957).

B. Physiological Changes in Synapses of the Retinal Rods and Cones A detailed description of the submicroscopic organization of rod and cone synapses with bipolar cells was published by De Robertis and Franchi (1956). The observations extend the finding of synaptic vesicles to synapses between two receptors and the corresponding neurons. One of the striking characteristics of these synapses, as first observed by Sjostrand (1953), is the fact that the dendrites of the bipolar cells penetrate and digitate into the enlarged terminal endings of the rod and cone cells (Figs. 2A, 6, 7, 8 ) . This intimate and complex junction appears in the section showing the very bizarre profiles of a folded synaptic membrane. The presynaptic membrane shows blind infolds projecting into the terminals around which synaptic vesicles tend to accumulate (Figs. 2A and 8 ) . In Fig. 6, in addition to the most common rod synapse, there is a lateral junction of the rod spherule with a dendrite of the bipolar cell. This type of junction has been described by Polyak (1941). I n order to search for physiological changes, rabbits were maintained in complete darkness for periods of 24 hours to 9 days. Others, after dark adaptation, were submitted to intense light stimulation. I n dark-adapted animals the most significant fact is the accumulation of a large number of synaptic vesicles around the presynaptic membrane and processes (Fig. 8 ) . After 46 hours in darkness, and particularly after 9 days, there is a definite and striking reduction in size of the synaptic vesicles, both in rod and in cone synapses (Fig. 9) (see De Robertis and Franchi, 1956). In darkadapted animals, after stimulation by intense light the opening of synaptic vesicles into the synaptic cleft is frequently observed and also the passage of some of them into the cleft and even beyond the subsynaptic membrane. In the postsynaptic cytoplasm there are ill-defined ghostlike vesicles

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and filamentous material, apparently related to the disintegration of vesicles. Illustrations of these findings are shown in Figs. 5, 6, and 7 cf De Robertis (1957). These results were interpreted as indicative of an active flow of vesicles under the stimulation by light. Eccles and Jaeger (1958) have made the interesting suggestion that the liberation of the synaptic vesicles at the retinal synapses is effected by the hyperpolarization induced in the receptor cell through the photochemical reaction. In fact, according to Svaetichin (1953, 1957), there appears to be no impulse mechanism in the cone but only an increase in polarization from -45 to about -70 mv. This effect is much smaller than the depolarization occurring in the neuromuscular junction and presumably in other synapses. According to Eccles and Jaeger, “the invaginated synaptic membranes of the rod and cones may be a device for slowing down loss by diffusion and ensuing a cumulative action of small quantities of transmitter liberated over many milliseconds by the relatively small hyperpolarization.” This could be also in line with the long delays that are involved in photochemical mechanisms.

C . Changes of the Synapse after Nerve Stimulation Since the discovery of the synaptic vesicles in 1953 (De Robertis and Bennett, 1954, 1955), attempts were made by the authors to induce visible changes in the synapse by electrical stimulation. Several of these attempts failed because of technical difficulties in preparing the material for the electron microscope, but interesting observations were made about the fixation of some peripheral synapses with osmium tetroxide. It was observed with Professor Amassian at the University of Washington in December, 1953, that complete stopping of synaptic transmission in the FIG.10. Electron micrograph of a neuromuscular junction of the intercostal muscle of the mouse. ( A ) At the bottom, miofibrils ( m f ) , showing the Z lines and other structural details. On top, large amounts of sarcoplasm containing numerous mitochondria (m) (sarcosomes) and three sarcosomic nuclei (sn). The sarcoplasm is limited by the sarcolemmata (s) and by a differentiation of the sarcolemmata a t the level of the neuromuscular junction. This differentiation consists in infoldings of the subsynaptic membrane, the so-called postjunctional folds or subsynaptic folds (ssf), and constitute as a whole the subneural apparatus of Couteaux. Above this are endings ( e ) of an afferent axon containing a few mitochondria (m) of smaller size than the sarcosomes. T o the top left is a Schwann cell covering the ending. (~10,000.) ( B ) Enclosure B of Fig. 10A seen a t higher magnification. The ending with a smooth presynaptic membrane (psm) is in contact with the subsynaptic folds (ssf). The interspace is the synaptic cleft (sc) . Within the ending, mitochondria (nc) and numerous synaptic vesicles ( s v ) are seen. T o interpret this electron micrograph see the diagram of Fig. 2B. (X40,OOO.)

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celiac ganglion of Rana catesbiamz took place in about 10 seconds (Fig. 14). In Bufo arenurum Hensel the postsynaptic response disappeared in 5 to 10 seconds (De Robertis and Luco, 1954, unpublished observations). The results, although far from permitting the study of single nerve impulses, opened the possibility of detecting changes induced by coarser charges, such as overstimulation and fatigue of the synapse. For this purpose the nerve endings at the adrenal medulla of the rabbit were found to be better suited than the sympathetic ganglia. In this material nerve supply is abundant and belongs almost entirely to the homolateral splanchnic nerve which can be easily stimulated (see Teitelbaum, 1942). The preganglionic nerve fibers are cholinergic (Feldberg et al., 1933, 1934) and innervate the chromaffin cells directly without intercalated neurons. This type of junction is generally considered to be of synaptic nature (Rosenblueth, 1950). The postsynaptic signal that can be recorded in this system is the amount of adrenaline, noradrenaline, or total catechol secreted into the adrenal vein under electrical stimulation (Rapela and Coviin, 1954) or the analysis of the histochemical and submicroscopic changes of the stimulated adrenal cells with the electron microscope (De Robertis and Vaz Ferreira, 1957a). Preliminary accounts of the findings have been published (De Robertis and Vaz Ferreira, 1957b ; De Robertis, 1957). In the normal nerve ending, synaptic vesicles and other components of the synapse are found (Fig. 15). In the postsynaptic cell the large catechol droplets, surrounded by a thin membrane, and the content of reduced osmium are observed. Prolonged electrical stimulation of the splanchnic nerve induces striking changes in the synaptic vesicles. With a stimulus of 400 supramaximal pulses per second, known to produce fatigue of the ending and diminished output of catechol (Rapela and Covian, 1954), considerable depletion of synaptic vesicles occurs together with less significant alterations of the matrix and mitochondria (Fig. 16). On the other hand, with a stimulus of 100 pulses per second, known to FIG.11. Electron micrograph of a synaptic junction a t the surface of an electroplaque of the eel. This is a lateral synapse similar to that shown in the Diagram of Fig. 20 in a section along the axon. On top the cross section of the axon covered by the Schwann cell (Sc) is shown. At the junction the axon is free and contains synaptic vesicles ( s v ) . A synaptic vesicle apparently opening into the synaptic cleft (sc) is marked with an arrow ; ssm, subsynaptic membrane ; Ep, electroplaque. Note that the synaptic cleft is rather wide in this synapse (about 450 A ) . (x44,OOO.) (Courtesy of J. Luft.) FIG.12. Axon-dendritic synapse in a sympathetic ganglion of the cat. Between arrows is the synaptic junction. The endings contain synaptic vesicles and mitochondria. Observe that the dendrite contains neuroprotofibrils along the axis and sev--q1 mitochondria near the junction. ( x45,OOO.)

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8

A

C

D

FIG.13. Diagram showing: ( A ) Some details of the submicroscopic structure of a synaptic ending in the normal acoustic ganglion. Description similar to that of Fig. 1C: st, stalk of the synaptic ending; nj, neuroprotofibrils; g, glia; SyE,synaptic ending ; sm, synaptic membrane ; Psy, postsynaptic cytoplasm. Three active points are indicated with arrows. ( B ) Twenty-two hours after destruction of the cochlea. (C and D ) After 44 hours. The sequence B-C-D corresponds to the most common and progressive degenerative changes observed in the endings (see description in the text). (Taken from De Robertis, 1956.)

FIG.14. Tracing of the postsynaptic response of the celiac ganglion of Rana catesbinno stimulated with supramaximal pulses a t a frequency of 2 per second. In the upper line the time a t which the osmium tetroxide is dropped on the ganglion is indicated. After 10 seconds practically all synapses have ceased transmission. (Experiment made by Prof. Amassian at the University of Washington in December, 1953.)

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induce near maximal output of catechol (Rapela and Covian, 1954), there is a definite increase in the number of synaptic vesicles and in the liberation of them at the synaptic cleft (Fig. 17 ; see also Fig. 10 of De Robertis, 1957). In Fig. 18 the results of measurements of synaptic vesicles per square micron of the surface of nerve endings are indicated. In the control the mean number is 82.6 vesicles per square micron. With 100 pulse; per second the mean increases to 132.7 vesicles per square micron, and with stimulation of 400 pulses per second it decreases considerably with a mean of 29.2 per square micron. These striking changes of the synaptic vesicles under electrical stimulation with different frequencies confirm the presumption that they play a physiological role in synaptic transmission, as first postulated by De Robertis and Bennett (1954, 1955). These experiments suggest that a balance exists between the formation of synaptic vesicles and release of the transmitter. The equilibrium may be altered in one sense or the other, according to the frequency of the stimulus (De Robertis and Vaz Ferreira, 1957b). D. Dimensions and Physiology of Synapses One of the most general conclusions that can be drawn from the submicroscopic analysis of synapses is that, in spite of the differences in morphology, distribution, and geometry of synaptic regions, they offer basic similarities. These are essentially: 1. The discontinuity between the cytoplasm of the two apposed elements of the synapse. 2. The direct contact of the presynaptic and subsynaptic surface membranes, separated only by an interspace of 100 to 500 A. 3. The presence of synaptic vesicles on the presynaptic side of the synapse. All these characteristics have been observed in synapses of vertebrates and invertebrates ; in peripheral and central synapses ; in terminal or lateral synapses ; in some synapses between receptors and neurons ; in the neuromuscular junction ; and in some neuroeffectors (see above). These factors suggest that an essentially analogous physiological mechanism may be involved in all synaptic junctions. Similar conclusions have recently been reached by physiologists, especially by the use of intracellular recording (see del Castillo and Katz, 1956a, b ; Eccles, 1957). The two essential types of synaptic actions, the excitatory and the inhibitory, are produced by an ionic flux across the synaptic cleft into the subsynaptic membrane. I n excitatory synapses a depolarization of the adjacent postsynaptic membrane occurs ( Fatt and Katz, 1951; Fatt, 1954; del Castillo and Katz, 1954, 1956a, b ; Eccles,

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1957), whereas in inhibitory synaptic action the ion flux leads either to hyperpolarization (Coombs et al., 1955a, b) or to antagonization of the depolarization induced by excitation (Fatt and Katz, 1953a, b ; Kuffler and Eyzaguirre, 1955) (Fig. 19B).

normal

1001s

4001 s

FIG.17. Diagram showing nerve endings of the adrenal medulla of the normal rabbit and after stimulation for 10 minutes with supramaximal pulses of 100 and 400 per second.

According to Eccles and Jaeger (1958) the functional operation of the synapse may involve the following processes : 1. The action potential causes the liberation of a transmitter substance from the presynaptic terminal into the synaptic cleft. 2. The liberated transmitter substance diffuses across the synaptic cleft to the subsynaptic membrane. FIG.15. Electron micrograph of a nerve ending of the adrenal medulla of the normal rabbit interposed between adrenal cells. The ending contains mitochondria and numerous synaptic vesicles: sm, synaptic membrane. In the adrenal cell large catechol-containing droplets ( c d ) and mitochondria are seen. ( x 57,000.) FIG.16. Electron micrograph of a nerve ending of an adrenal gland whose splanchnic nerve was stimulated a t 400 pulses per second for 10 minutes. The most significant change is the great depletion of synaptic vesicles (m) ; cd, catechol-containing droplets. ( ~ 3 3 , 0 0 0 . )

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3. The molecules of the transmitter become attached to specific sites of the subsynaptic membrane, and profound alterations of ionic permeability occur. 4. The consequent ionic flux alters the polarization of adjacent areas of the postsynaptic membrane, and thus a current flow (synaptic potential) is induced (Fig. 19A).

Control

Mean

Kx)

p u l a ~ ~ / ~ Mean

400 pulses/¶ Mean

FIG.18. Diagram showing results of measurements of synaptic vesicles per square micron of synaptic ending in control specimens and in rabbits with stimulation of the splanchnic nerve at 100 and 400 pulses per second for 10 minutes (see description in the text).

5. The effect of the transmitter substance on the subsynaptic membrane is ended by its removal by enzymatic destruction and diffusion into the interstitial spaces. When acetylcholine is the transmitter, its destruction may be partially effected by cholinesterase. “In summary we may state that the synapse is a device for applying minute amounts of a specific chemical substance to the specialized receptor area of the subsynaptic membrane, which in turn becomes highly permeable to some or all ions. The resulting ionic current through the subsynaptic membrane becomes effective by passing through the synaptic cleft and so to the remainder of the postsynaptic membrane” (Eccles and Jaeger, 1958). Eccles (1957) and Eccles and Jaeger ( 1958) have discussed the dimensional requirements of the synaptic cleft which will permit the efficient and

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rapid application of the transmitter and the relatively wide areas needed for the free flow of current. They have calculated the diffusion of a transmitter such as acetylcholine through a cleft of 200 A to occur in a few microseconds. Even with a distance of 500 A an effective distribution of acetylcholine could take place in about 10 psec. Therefore the dimensions

A

Inhibitory

I

Excitatory

FIG. 19. ( A ) Diagram of a synaptic ending similar to the type illustrated in Fig. lC, showing the lines of postsynaptic current flow when the subsynaptic membrane is influenced by the liberation of the transmitter substance (T) into the synaptic cleft. (B) Schematic representation of the functional operation of inhibitory and excitatory synapses. The resting potential is in both cases -70 mv. Under the action of the liberated inhibitory substance (Is) the potential is raised to -80 mv, and by the action of the excitatory substance (Es) it is diminished to 0 mv. The voltages driving the inhibitory and excitatory currents are thus -10 mv and +70 mv, respectively. (Taken from Eccles, 1957, and slightly modified.)

of the synaptic cleft shown by the electron microscope are of the kind needed for an efficient action of the transmitter. The areas needed for the passage of the postsynaptic currents must be relatively large-of the order of a few square microns-in view of the specific resistance offered by the cleft and the subsynaptic membrane. ( F o r an interesting discussion, see Eccles, 1957 ; Eccles and Jaeger, 1958 ; and also Palay, 1957b).

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E. Functional Role of Synaptic Vesicles An interpretation of the possible role of synaptic vesicles in the physiology of synaptic transmission should take into consideration the important findings made with microelectrodes in neuromuscular junctions. Fatt and Katz (1952) found that, in amphibian muscle, end plates are the seat of spontaneous subthreshold activity. This is manifested by miniature endplate potentials of the order of 1/100 of the synaptic potential in response to a nerve impulse (Fig. 20).

(A)

(B)

FIG.20. ( A ) Spontaneous miniature end-plate potentials recorded by intracellular electrodes at the end plate. ( B ) At a distance of 2 mm, in the same muscle fiber, the end-plate potentials are not recorded. In the lower part, taken at higher speed and lower amplification, the response to a nerve stimulus is shown. (Reproduced from Fatt and Katz, 1952.)

Different pharmacological properties of the miniature end-plate potentials led the authors to postulate that they must be due to the release of acetylcholine by the endings. Feldberg (1945) had already suggested that cholinergic nerve endings, even at rest, continually discharge small amounts of acetylcholine and replace it by chemical synthesis. The miniature end-plate potentials, however, could not be produced by simple molecular diffusion of acetylcholine, and Fatt and Katz (1952, 1953a, b) suggested that the release of the chemical mediator must be in multimolecular or quanta1 units (del Castillo and Katz, 1955, 1956a, b ; Boyd

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and Martin, 1956 ; Liley, 1956) arising from the synchronous discharge of a large number of acetylcholine ions. Since the amplitude of the spontaneous potential is only about onehundredth of the functional response to a nerve impulse, "it may be concluded that the apparatus for the release of acetylcholine at a junction is subdivided into large number of units (at least loo), each of which is able to operate independently of the rest" (Fatt, 1954). The authors believe that under the action of driving forces, such as their own thermal agitation catelect rotonus Presynaptic impulses

Excess K' ions

POST-ACTIVAT ION

\

'\ I

/

Activation depressed by Mg" excess ca+* deficiency

QUANTAS of ACH

(synaptic vesicles)

-1

Blocked by botulinum toxin

I DEPOLARIZATION of ENDPLATE (endplate potential) '

FIG.21. Diagram of the factors influencing the mechanism by which quantas of ACH (acetylcholine or synaptic vesicles) are ejected into the synaptic cleft. (Reproduced from Eccles, 1957.)

and the electric fields across the membrane, these quanta1 units of acetylcholine are suddenly discharged at localized points of the endings. The depolarization occurring at the arrival of the nerve impulse would produce a large synchronized action and thus the simultaneous discharge of many units which determine the end-plate potential. In Fig. 21 are illustrated some of the factors believed to be operative in the ejection of acetylcholine from nerve endings (Eccles, 1957). These physiological findings and theoretical considerations find extraordinary support in the submicroscopic organization of the synapse. The

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observations made on the structure and relationship of the synaptic vesicles with the membranes and their behavior in different physiological and pathological conditions (De Robertis, 1955a, b, 1956 ; De Robertis and Franchi, 1956; De Robertis and Vaz Ferreira, 1957b) are all consistent with the concept that the synaptic vesicle may represent the quuntal unit of acetylcholine postulated by Fatt, K a t z , and del Castillo (Fig. 21). In our first papers (De Robertis and Bennett, 1954, 1955), we suggested that acetylcholine and other chemical mediators could be associated with particles or vesicles of submicroscopic size. W e also postulated that the synaptic vesicles may move toward the presynaptic membrane and discharge their contents at the junction. The opening of synaptic vesicles and even their passage through the synaptic cleft and postsynaptic cytoplasm was postulated on the basis of observations in the earthworm neuropile (De Robertis and Bennett, 1954, 1955). Luft (1956) observed the opening of vesicles into the synaptic clefts of the electric organ; this process was most evident in the retinal synapses of dark-adapted animals after intense illumination (De Robertis, 1957). It seems possible that acetylcholine or other chemical mediators may be synthesized at the ending and segregated into packets surrounded by a membrane. The synaptic vesicles may then flow toward a position adjacent to the synaptic membrane. These points of attachment of the vesicles with the presynaptic membrane will constitute the active spots of the synapse observed by De Robertis (1955a, b) and Palay (195713) (Figs. 1C and 4). (For a biophysical consideration of these active spots see del Castillo and Katz, 1956a). One may postulate that in the resting condition single vesicles may spontaneously and randomly burst and discharge their content at localized spots of the junction, originating the miniature end-plate potentials of Fatt and Katz (1952). If the resting condition is prolonged, as in the case of dark-adapted animals, an accumulation of vesicles at the presynaptic membrane would occur (De Robertis and Franchi, 1956). When a propagated electrical disturbance in the form of an action potential reaches the presynaptic membrane, including a depolarization or a hyperpolarization of the nerve terminal, many vesicles will synchronously open at the synaptic interspace and liberate their contents of acetylcholine or other transmitter substances (Fig, 21). This process may in some cases, such as in the retinal synapses, involve the passage and rapid destruction of the vesicles into the postsynaptic cytoplasm. These concepts of flow and discharge of the synaptic vesicles are in agreement with those postulated by Bennett (1956) of membrane vesiculation as a mechanism for active transport and ion pumping. They also involve a dynamic structure for the membrane, with the possibility of local

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breakdown and restoration during synaptic transmission. This dynamic structural concept of the synapse is in agreement with physiological experiments suggesting that the chemical transmitter short-circuits the synaptic membrane (Fatt and Katz, 1953a, b ) . This results in a reduction of the resistance as well as the potential of the membrane and would be indicative of a large increase of permeability to all ions (del Castillo and Katz, 1954). According to Palay (1957b) the synaptic junction must be considered dynamic not only in its physiology but in its morphology as well. “After all, this is not a soldered junction of two hot wires, but a living system. . . . The processes of nerve cells may well be in constant play, flowing and shifting in position and in shape, as they do in tissue culture preparations. The contact points may shift from one position to another by gliding over the postsynaptic surface. At least, we may easily imagine a dynamic “scintillation” of the clustered synaptic vesicles, discharging now at one point, now at another. Such speculations are not fantastic, but are merely extensions of current knowledge concerning the dynamic life of the cell” (Palay, 1957b). IV.

SUMMARY

The electron-microscope study of synaptic regions has revealed a highly differentiated and specific submicroscopic organization, which seems to be specially fitted to carry out the transmission of the nerve impulse. In spite of differences in morphology, distribution, and geometry, synaptic regions have the following basic similarities : 1. A definite discontinuity between the cytoplasm of the two apposed cellular elements of the synapse, confirming that the individuality of the neuron applies to the finest submicroscopic expansions. 2. A direct contact of the presynaptic and subsynaptic surface membranes separated only by an interspace of 100 to 500 A, indicating that at the junction no other cellular material alien to the two synaptic elements is interposed. 3. The presence of a special microvesicular material-the synaptic vesicles (De Robertis and Bennett, 1954)-on the presynaptic side of the synapse. These structural similarities suggest that an essentially analogous physiological mechanism may be involved in all synaptic junctions. The intimate relationship of the synaptic vesicles with the junction ; their early lysis in degeneration of the synapse ; the fact that under physiological stimulation the actual flow of vesicles into the synaptic cleft and even into the near postsynaptic cytoplasm may be observed in some synapses ; the changes in size that can be found by disuse of the junction ; and finally the intense modifications of the number of synaptic vesicles

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after nerve stiniulation-all are indicative of the direct intervention of this submicroscopic component in synaptic transmission. Furthermore the presence of clusters of synaptic vesicles in contact with certain zones of the presynaptic membrane is probably an indication that there may be active “scintillating” points in the functional operation of the synapse which is in agreement with recent electrophysiological studies. The most appealing possibility is that synaptic vesicles may represent quanta1 units of a chemical transmitter, such as acetylcholine, as has been postulated by physiologists. The spontaneous discharge of single synaptic vesicles may give rise to the miniature end-plate potentials recorded by microelectrodes in the neuromuscular junction (Fatt and Katz, 1952). When a propagated electrical disturbance in the form of a nerve impulse reaches the junction, inducing a depolarization (excitatory synapses) or a hyperpolarization (inhibitory synapses), many synaptic vesicles may liberate the transmitter and determine a large end-plate potential, which in turn gives rise to the depolarization of the postsynaptic element. It seems possible that acetylcholine or other chemical mediators may be synthesized at the ending, segregated into packets by a limiting membrane, and then flow toward a position adjacent to the synaptic membrane ready for instantaneous discharge at the arrival of the nerve impulse. These concepts of flow and discharge of synaptic vesicles are in agreement with similar mechanisms observed in the synthesis and excretion of other neurohormones such as adrenaline and noradrenaline (De Robertis and Vas Ferreira, 1957a). They involve a dynamic structure for the membrane, with the possibility of local breakdown and restoration during synaptic transmission. These morphological and physiological correlations at a submicroscopic level should be continued by a closer collaboration of physiologists working with microelectrodes and electron microscopists. Furthermore they should be integrated with the study of the patterns of chemical and enzymatic organization which are operative at the different synapses.

V. REFERENCES Armstrong, J., Richardson, K. C., and Young, J. Z. (1956) Stain Technol. 31,263. Arvanitaki, A. (1942) 1. Neurophysiol. 5, 108. Barr, L. M. (1939) I . Anat. 74, 1. Bartelmez, G. W., and Hoerr, N. L. (1933) J . Comp. Neurol. 67,401. Bennett, H. S. (1956) J . Biophys. Biochem. Cytol. 2, Suppl. 99. Bodian, D. (1940) J . Comp. Ncurol. 73, 323. Bodian, D. (1942) Physiol. Revs. 22, 146. Bodian, D. (1952) Cold Spring Harbor Symposia Quant. Biol.17,1. Boyd, I. A., and Martin, A. R. (1956) I . Physiol. (London) 13a,61. Bullock, T. H. (1952) Cold Spring Harbor Symposia Quant. Biol.17,267. Bullock, T.H. (1953) J . Comp. Neurol. 28, 1.

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Cajal, S. R. y (1934) Trab. inst. Cajal invest. biol. (Madrid) 24, 1. Castillo, J. del, and Katz, B. (1954) J. Physiol. (London) 126,546. Castillo, J. del, and Katz, B. (1955) J. Physiol. (London) 128,3%. Castillo, J. del, and Katz, B. (1956a) Prog. Biophys. and Biophys. Chew. 6, 121. Castillo, J. del, and Katz, B. (195613) J. Physiol. (London) 132,630. Castro, F. de (1942) Trab. inst. Caial invest. biol. (Madrid) 34,217. Castro, F. de (1950) Verhandl. deut. Ges. Pathol. 34 Tgg. Chu, L. W. (1954) J. Comp. Xeurol. 100,381. Coombs, J. S., Eccles, J. C., and Fatt, P. (1955a) J. Physiol. (London) 130, 374. Coornbs, J. S., Eccles, J. C., and Fatt, P. (1955b) J. Physiol. (London) l30,326. CoppCe, G., and Bacq, Z. M. (1938) Arch. intern. physiol. 47, 312. Couteaux, R. (1947) Rev. can. biol. 6, 563. Couteaux, R. (1955) Intern. Rev. Cytol. 4, 335. Couteaux, R., and Taxi, J. (1952) Arch. anat. microscop. morphol. exptl. 41, 352. De Robertis, E. (1955a) Ac fa Neurol. Latinoam. 1, 1. De Robertis, E. (1955b) Anat. Record 121,284. De Robertis, E. (1956) J. Biophys. Biochem. Cytol. 2, 503. De Robertis, E. (1957) “Submicroscopic Morphology and Function of the Synapse.” Exptl. Cell Research Suppl. 6, 347 (1958). De Robertis, E., and Bennett, H. S. (1954) Federation Proc. l3,35. De Robertis, E., and Bennett, H. S. (1955) J. Biophys. Biochem. Cytol. 1, 47. De Robertis, E., and Franchi, C. M. (1954) J. Appl. Phys. 26, 1162. De Robertis, E., and Franchi, C. M. (1956) J. Biophys. Biochem. Cytol. 2, 307. De Robertis, E., and Sotelo, J. R. (1952) Exptl. Cell Research 3, 433. De Robertis, E., and Vaz Ferreira, A. (1957a) Exptl. Cell Research 12, 568. De Robertis, E., and Vaz Ferreira, A. (1957b) J . Biophgs. Biochem. Cytol. 3, 611. Eccles, J. C. (1957) “Physiology of Nerve Cells.” Johns Hopkins Press, Baltimore, Maryland. Eccles, J. C., and Jaeger, J. C. (1958) Proc. Roy. SOC.Bl48, 38. Edwards, G. A. (1957a) Anat. Record 528, 542. Edwards, G. A. (1957b) Anat. Record 128, 543. Engstrom, H., and Rytzner, C. (1956) Ann. Otol. Rhinol. & Laryngol. 65,361. Engstrom, H., and Sjostrand, F. S. (1954) Acta @to-laryngol. 44, 490. Estable, C. (1953) “Symposium on the Synapses, Montevideo” in press. Estable, C., Reissig, M., and De Robertis, E. (1953) J. Appl. Phys. 24, 1421. Estable, C., Acosta-Ferreira, W., and Sotelo, J. R. (1957) 2. Zellforsch. U. mikroskop. Anat. 46, 387. Eyzaguirre, C., Espindola, J., and Luco, J. (1952) A cfa Physiol. Latinoam. 2, 213. Fatt, P. (1954) Physiol. Revs. 34, 674. Fatt, P., and Katz, B. (1951) J. Physiol. (London) 116, 320. Fatt, P., and Katz, B. (1952) J. Physiol. (London) 117, 109. Fatt, P., and Katz, B. (1953a) Acta Physiol. S c a d . aS, 117. Fatt, P., and Katz, B. (1953b) J. Physiol. (London) 121, 374. Feldberg, W. (1945) Physiol. Revs. 26, 596. Feldberg, W. (1954) Pharmacol. Revs. 6, 85. Feldberg, W., and Minz, B. (1933) Arch. ges. Physiol. PfEiiger’s a83, 657. Feldberg, W., Minz, B., and Tsudrnizura, H. (1934) J. Physiol. (London) 80, 15; 81, 286. Foerster, O., Gagel, O., and Sheenan, D. (1933) 2. Anat. u. Ent.wicklungsgeschichte 101, 553.

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EDUARDO DE ROBERTIS

Gibson, N. C. (1937) Arch. Neurol. and Psychiat. 38,1145. Glees, P., Meyer, A., and Meyer, M. (1946) J. Anat. 80, 101. Haggar, R. A., and Barr, M. L. (1950) 1. Comp. Neurol. 93, 17. Hay, E. D. (1957) Anat. Record 128,562. Held, H. (1897) Arch. Anat. u. Physiol. Anut. Abt., Suppl. 273. Hoff, E. C. (1932) Proc. Roy. SOC.B111, 175. Hoff, E. C., and Hoff, H. E. (1934) Brain 67, 175. Horstmann, E. von (1957) Deut. med. Wochschr. 82, 731. Kuffler, S. W., and Eyzaguirre, C. (1955) J. Gen. Physiol. 39, 155. Liley, A. W. (1956) J. Physiol. (London) 132,650. Lissak, K.,Dempsey, E. W., and Rosenblueth, A. (1939) A m . 1. Physiol. 128,45. Luco, J. V., and Davidovich, A. (1956) Rev. medicinu (Argentina) 16, 295. Luft, J. (1956) J. Biophys. Biochem. Cytol. Suppl. 2,229. McIntosh, F.C. (1938) Arch. intern. Physiol. 47,312. Noel, R. (1950) Biol. mid. (Paris) 39,319. Palade, G. E. (1952) 1. Ezptl. Med. 95,285. Palade, G. E. (1954) Anat. Record 118,335. Palay, S.L. (1954) Anat. Record 118,336. Palay, S. L. (1956) J. Biophys. Biochem. Cytol. 2,193. Palay, S. L. (1957a) Progr. in Neurobiol. II. Ultrastructure and Cellular Chem. Neural Tissues, p. 31. New York, Hoeber. Palay, S. L. (195%) “The Morphology of Synapses in the Central Nervous System.” Exptl. Cell Research Suppl. 6,275 (1958). Pease, D. C. (1953) Anat. Record 115,359. Polyak, S.L. (1941) “The Retina.” Univ. Chicago Press, Chicago, Illinois. Rapela, C. E., and Coviin, M. R. (1954) Rev. soc. arg. biol. SO, 157. Kasmussen, G. L. (1953) J. Comp. Neurol. 99, 61. Rasmussen, G. L. (1957) “New Research Techniques of Neuroanatomy,” p. 27. C. C Thomas, Springfield, Illinois. Reger, J. F. (1954) Anat. Record 118,344. Reger, J. F. (1957) Exptl. Cell Research 12,662. Robertson, D. (1953) Proc. SOC.Exptl. Biol. Med. 82,219. Robertson, D. (1954) Federation Proc. 13, 119. Robertson, D. (1956) 3. Biophys. Biochem. Cytol. 2, 381. Rosenblueth, A. (1950) “The Transmission of Nerve Impulses.” Wiley, New York. Scharrer, E. (1945) J. Comp. Neurol. 83,237. Sherrington, C. S. (1897) The central nervous system. In Sir Michael Foster’s “A Textbook of Physiology,” 7th ed. Macmillan, London. Sjostrand, F. S. (1953) 1. Appl. Phys. 24, 1422. Smith, C. A. (1957) Anat. Record 127,483. Svaetichin G. (1953) Acta Physiol. Scand. 29,Suppl. 106,565. Svaetichin, G. (1957) Acta Physiol. Scand. 99, Suppl. 154, 17. Teitelbaum, H. A. (1942) Quart. Rev. Biol. 17, 135. Tiegs, 0. W. (1953) Physiol. Revs. 33,90. Titeca, J. (1935) Arch. intern. physiol. 41, 1. Trujillo-CCnoz, 0. (1957) 2. Zellforsch. u. mikroskop. Anat. 46, 272. Wersall, J. (1956) Acta Oto-Laryngol. Suppl. No. ls, 1. Wyckoff, R. W. G., and Young, J. Z. (1956) Proc. Roy. Soc. B144,440. Young, J. Z. (1939) Phil. Trans. Roy. SOC.B229, 465.