Chapter IV Neural Processes

CHAPTER IV

NEURAL PROCESSES

A. DEVELOPING NEURAL PROCESSES

Cajal (1909) gave an early description of the growing tip of dorsal root neuroblasts and coined the term “c8ne de croissance”. Electron-microscopic studies of growing nerve processes (Bodian, 1966; Del Cerro and Snider, 1968; Tennyson, 1970; Kawana et al., 1971; Bunge, 1973) have shown more structural details in growing tips and Rees et al. (1976) concentrated on their developmental stages in synaptogenesis. Freeze-etch replicas have been obtained from organotypic cultures of rat olfactory bulb and spinal cord by Pfenninger and Bunge (1974). They found that neural growth cones may be distinguished from glial pseudopodia by the low number of membrane-associated particles within their plasmalemma. A special feature of the growing neurite is the increase in the number of particles (about 8-fold) during maturation. The bulbous growing tip is characterized by extremely slender filopodia containing microfilamentous material. The tip, itself, is filled with clusters of vesicles, vacuoles, branched membranous reticulum and lysosomes. The general situation of developing neural processes as seen in thin sections is depicted in Fig. 40. The subfornical organ of the newborn cat offers an opportunity to study interactions between growing elements of developing neuropil in vivo. The formation of the freeze-etch aspects of axonal contacts with soma and growing dendrites is shown in Fig. 41. Growth cones containing clusters of endoplasmic reticulum (Fig. 43a, c) are equipped with filopodia (Fig. 42, 43a). Growth cone filopodia, after their initial contact with the target neuron, become extensively applied to the neuronal plasmalemma. There, they form numerous punctate regions of contact in which the apposing plasma membranes are separated by only 7-10 nm. The first sign of synaptogenesis seems to consist in the undercoating of the postsynaptic site (not demonstrable in freeze-etched material). Subsequently, the form and content of the growth cone is altered by the loss of filopodia and the appearance of synaptic vesicles which gradually become clustered opposite

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Fig. 40. Developing neural processes, diagram

The interactions of filopodia extending from axonal and dendritic growth cones are represented in terms of apposition and synapse formation. Vacuoles (Vc) are possibly the result of fusion and endocytosis in filopodia (Fp). CV = coated invagination of plasma membranes; dp = dense projection; dV = dark cored vesicles; f = fine fibrillary material; G = Golgi complex; Mit = mitochondria; m = microtubules; Nc = nucleus; PO = postsynaptic density; R = ribosomes; ER = rough surfaced endoplasmic reticulum; sER = smooth surfaced endoplasmic reticulum; sV = synaptic vesicles. (from Kawana et al., Z. Zellforsch., 115: 295, Springer-Verlag, 1971) the postsynaptic density (Rees et al., 1976). The relative paucity or absence of synaptic vesicles in developing presynaptic axon terminals is shown in Fig. 31 (p. 79) and Fig. 41. A n important question regarding the morphology of early contacts is whether gap junctions are formed to allow electrical and chemical coupling of cells as is the case in other tissues (Bennett, 1973; Gilula, 1974). Fig. 43a gives a n example of a gap junction between a filopodium and a neuron. Further studies (Van Buren et al., 1977) disclosed that gap junctions appeared between growing nerve processes and target cells and might even

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Fig. 41. Formation of synaptic contacts, developing neuron Cat, 3 days, subfornical organ. Neuron with nucleus (Nc). The cytoplasm contains many rounded structures, probably largely endoplasmic reticulum. Immature axon terminals (triangles) with a few if any synaptic vesicles abut the P face of the soma. Double triangle marks the site of a growing dendrite approached by the tip of an axonal ending containing synaptic vesicles. The bases of two dendrites (arrows) are seen above. The area shown in the rectangle is enlarged in Fig. 31, p.79.

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Fig. 42. Growth cone with filopodium Cat, 5 days, subfornical organ. Filopodium (Fp) of a presumed neuron (NI) presents the P face (PF). Rounded outlines of endoplasmic reticulum lie in the cytoplasm of N' and N2. EF = E face of neuron NZ.

Fig. 43. Gap junctions in developing brain Cat, neonatal, subfornical organ. a: A growth cone with a filopodium (FP) forms a gap junction (GJ) with the apposed membrane E face (EF) of an unidentified element. V = growth cone vesicles. Inset: higher magnification of the gap junction (arrows). 6: Gap junction (GJ) between neural elements (not further identified). c: Gap junction (GJ) between a slender nerve process and a dendritic (?)growth cone. V = growth cone vesicles. (from Van Buren et al., Cell Tiss. Res., 181: 30, Springer-Verlag, 1977).

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appear on filopodia (Fig. 43a). The morphology of these junctions was similar to that described earlier but they were of small size (0.2-0.3 pm). Shoukimas and Hinds (1978) found in the early histogenesis of the mouse cerebral cortex that initially the neuron destined to migrate toward the surface of the cortex was bound to the junctional region next to the ventricle by zonulae adhaerentes, gap junctions and atypical tight junctions. A point against gap junctions being functional synaptic precursors was the finding (Crain and Peterson, 1965; Bunge et al., 1967) that the time of appearance of bioelectric activity correlated well with the first appearance of synapses in explants of fetal rat spinal cord rather than the antecedent gap junctions. Furthermore, similar gap junctions appear on filopodia of developing fibroblasts (Hasty and Hay, 1977).

B. DENDRITES Dendrites are processes of the neuronal perikarya. In contrast to axons, they contain Nissl bodies and are unmyelinated (for exception, see Pinching, 1971). Another difference between dendrites and axons is their functional polarization: the former being centripetally oriented and therefore considered to be postsynaptic or receiving elements, while the latter conduct centrifugally and constitute presynaptic elements. However, exceptions to this rule are numerous. For dendrites are known to form presynaptic bags (see review by Reese and Shepherd, 1972) in various regions of the brain, and axons may be the site of axo-axonal (Gray, 1962; Palay et al., 1968) synapses. The cytoplasm of dendrites in standard freeze-etch preparations both in cross-fractures and in longitudinal profiles (Figs. 44, 45) shows mitochondria, endoplasmic reticulum, neurotubules and occasionally microfilaments. High-pressure freezing of unfixed tissue has resulted in considerable improvement in the resolution of intracytoplasmic detail. In the rat cerebellar cortex the dendrites are seen to be densely packed with neurotubules and other organelles (Fig. 44a, b; cf. Moor et al., 1980). The surface of dendrites is studded with synapses (Figs. 45-47) which will be treated in a subsequent section (1V.D). However, it should be pointed out here, that the surface of dendrites may be specialized by small projections, called thorns, spines, appendages and crests (literature in Fig. 44. Purkinje dendrites Rat, cerebellar cortex. Unfixed tissue, frozen under high pressure. a: Cross-fracture. The dense packing of neurotubules (nt) disclosed by this method is evident. On the upper right a higher magnification shows the pits in the center of the neurotubules. Mit = mitochondria; ER = endoplasmic reticulum. b: Oblique fracture with longitudinally fractured neurotubules (nt).

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Gray, 1959; Akert et al., 1967; Jones and Powell, 1969). These formations are the sites of synaptic contacts, and their structural integrity seems to essentially depend on synaptic activity (Globus and Scheibel, 1967; Valverde, 1968). Spines may have a length of about 0.5-1.0 pm and a diameter of about 0.3-0.5 pm. Their shape compares with that of the end of a drumstick; it seems important to point out that the profile is narrowest at the exit from the dendritic branch (Figs. 48-51). Landis and Reese (1974a) have presented freeze-fracture preparations with identified dendritic shafts and spines showing their profiles in both cross-fractures and in longitudinal orientation. Spines prove to be rather fragile structures in the freeze-etch procedure; they are frequently fractured at the neck region (Fig. 48). Gray (1959) first described the spine apparatus. This structure seems to occur exclusively in mammals (Scheibel and Scheibel, 1968). It seems to be the only organelle inside the spine and consists of a series of double sacs separated from one another by dense bands, 15-20 nm wide. Unfortunately, the spine apparatus was elusive in our freeze-etch material.

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Fig. 45. Dendrite with axon terminals Rat, spinal cord. Three axon terminals ( A x ~ 'make . ~ ) contact with a dendrite (Den). The active zones on the axonal presynaptic P faces (pr PF) are outlined by arrows. Triangles indicate collections of postsynaptic particles underlying the active zones on the postsynaptic E face (POEF) of the dendrite. ER = endoplasmic reticulum; Mit = mitochondria.

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Fig. 46. Smooth dendritic shaft with synaptic contacts Rat, spinal cord. The P face of a dendrite (Den PF) exposed in longitudinal fracture is partially covered by two large axonal terminals (Axt', Axt'). They present multiple regions of vesicular attachment (arrows). A third axon terminal (Axt3) lies above and a glial process below (GE).

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Fig. 47. Smooth dendritic shaft with postsynaptic site Cat, subfornical organ. The E face of a dendrite (Den EF) presents postsynaptic aggregations of particles (arrow). The dendritic cytoplasm with endoplasmic reticulum (ER) and probable mitochondrion (Mit) to the left. An axon terminal with vesicles above (Axt).

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Fig. 48. Purkinje cell dendrite with broken spines Pigeon, Purkinje cell. The P face of the Purkinje dendrite (Den PF) presents numerous spines broken near their base (arrows). Examples at the top of the photograph show slightly more peripheral fractures. A few fragments of the E face of adjacent processes (EF) are seen. A section of dendritic cytoplasm is exposed with endoplasmic reticulum (ER) and a probable mitochondrion (Mit).

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Fig. 49. Spiny dendrites of Purkinje cells a: Cat, cerebellar cortex. The dendritic E face (Den EF) shows numerous dendritic spines (DSp) in tangential and cross-fracture. The postsynaptic P face (PO PF) and presynaptic E face (pr EF) are shown in the DSp on the left (see Inset). b: Rat, cerebellar cortex. A dendrite, seen from outside demonstrates the P face (Den PF) and numerous spines (DSp). The spine at the lower left is partially surrounded by an axon terminal (Axt PF). The P face of this terminal exhibits an active zone (arrows). This tissue was prepared without fixation and frozen under high pressure.

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Fig. 50. Axon terminal with multiple spine contacts Cat, spinal cord. Four dendritic spines form invaginating contacts with a single axon terminal (Axt) exposing presynaptic E face (pr EF) and postsynaptic P face (PO PF). One postsynaptic EF of a spine DSp2 is seen. Among the vesicles of the terminal several larger ones of uniform size (arrows) may represent dense core vesicles. Mit = mitochondrion. Fig. 51. Postsynaptic sites at dendritic spines Cat, cerebellar cortex. Particle aggregations at E and P faces of postsynaptic membranes are exposed. a: Dendritic spine viewed from “within”. The postsynaptic E face (PO EF) contains a fairly circumscribed aggregation of particles (triangles) which stands out conspicuously against the particle-poor surround. b: Dendritic spine viewed from “without”. The postsynaptic P face (PO PF) contains particle aggregation (triangles). Den = dendritic shaft with various organelles. c: Dendritic spines with synaptic sites. Above is an axo-spiny synapse seen in profile. The presynaptic (pr PF) and the postsynaptic (PO EF) are depicted. Particle aggregation marked by triangles. sV = synaptic vesicles. Below is a dendritic spine containing postsynaptic (PO PF) particle aggregation.

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114 C . AXONS

Axons consist of several segments which differ in morphology and function. In a proximo-distal direction one may differentiate between axon hillock, initial segment, main axon cylinder and terminal segment. The axon hillock consists of the slightly flared portion of the axon at its junction with the soma. Although free of Nissl substance and quite distinct in outline in large neurons it may be difficult to distinguish in smaller cells. The transition of the hillock to the initial segment is marked by the appearance both of bundles of microfilaments and of an electron-dense layer or undercoating below the axonal membrane (Palay et al., 1968). Synaptic contacts often appear at the initial segment and below them the dense layer is missing. The main axon cylinder is characterized by the presence of the myelin sheath. In unmyelinated fibers the transition between the initial segment and the hillock is marked by the loss of the electron-dense layer and the fascicular arrangement of microtubules (Palay et al., 1968). The freeze-etch characteristics of the myelin sheath including the nodes of Ranvier are discussed in Chapter V, Sect. A and B, p. 201ff. The terminalsegment is marked by the termination of the myelin sheath and the formation of boutons en passage (Fig. 54) and boutons terminaux (Fig. 55). Although propagation of impulses is the classical function of the axon, evidence recently reviewed by Weiss (1970) emphasizes its role in the transport of macromolecules between soma and synapses (CuCnod and Schonbach, 1971). While endoplasmic reticulum, microtubules and neuro-

Fig. 52. Myelinated axon, cross fracture Knifefish (Sternarchus albifrons), spinal cord. Axon (Ax). Neurofilaments in cross-fracture (nf) with mitochondria (Mit) and segments of endoplasmic reticulum (ER). Axon P face (Ax PF) and Schwann cell E face (Sch EF) are indicated. My = myelin; Mit = mitochondria.

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116 filaments may be functionally related to these transport mechanisms, it seems that the morphological correlate of electrical excitability lies primarily in the fine structure of the axolemma. Kristol et al. (1977) have examined the differential distribution of membrane-associated particles in excitable versus non-excitable segments of axonal membrane in the neurogenic electric organ of a gymnotid (Sternarchus) and demonstrated that the number of E face particles is significantly increased in the nodal as compared with the internodal axolemma. The relatively dense aggregation of E face particles in the nodal membranes in the rat is shown in Figs. 115, 120, 123. Conceivably, these particles represent ionic channels. It should be noted that central axons may be the receiving site of afferent nerve signals. Postsynaptic sites along the axon are found specifically at the initial (Peters et al., 1968) and at the terminal segment (Gray, 1962). Presynaptic sites are not only present at the axonal endings (Fig. 5 9 , but also at the nodes of Ranvier (Bodian and Taylor, 1963; Waxman, 1972, 1974). An example is given in Fig. 124 (See also Chapter V, Sect. A.4.b, p. 218). The identification of myelinated axons in freeze-etch material presents no difficulty (Babe1 et al., 1970; Orci and Perrelet, 1975; Peters et al., 1976). The axon hillock and initial segment are less readily identified in thin section material and are rarely encountered in freeze-etch replicas. Although the characteristic electron-dense undercoating of the axonal membrane in the initial segment cannot be resolved against the background of frozen glycerinated axoplasm, organelles such as mitochondria, neurofilaments, microtubules, agranular reticulum, vesicles and multivesicular bodies are readily identified (Figs. 52, 53). In recent studies (Droz et al., 1975) the presence of endoplasmic reticulum in axons has been emphasized. The tubules and sacs extend throughout the axon cylinder and even into the terminal bags. These elements can be clearly visualized in freeze-etched profiles of axons (Figs. 14, 53).

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Fig. 53. Axoplasm, longitudinal fracture

Knifefish (Sternarchus albifrons), spinal cord. Neurofilaments (nf)of an axon (Ax) show an open uniform distribution. This is in contrast to glial filaments (cf. Fig. 136, p. 271) which tend to be grouped in densely populated fascicles. ER = endoplasmic reticulum.

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Fig. 54. Terminal segment of axon: bouton en passage Rat, spinal cord. Unmyelinated axon (Ax) forming a conspicuous varicosity. Arrow marks region where cytoplasm with synaptic vesicles (sV) is exposed. Several active zones (triangles) are identified on the basis of slight indentations of the presynaptic P face (pr PF). The vesicle attachment sites are barely visible. Note that the bouton is closely apposed to an underlying dendrite (Den) seen in a cross-fracture.

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Fig. 55. Axon, terminal segment Cat, spinal cord. The terminal portion of a myelinated axon (Ax) is seen at the lower right accompanied by the final turn of the glial cell (glial loop, GL') which leaves its impression on the axon P face (Ax PF). The axon terminal (Axt) contains many vesicles and mitochondria (Mit).

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D. SYNAPSES AND SYNAPTOID JUNCTIONS 1. Central chemical synapses

The synapse as a functional element was recognized and named by Sherrington (1897). One of the major advances was made when the principle of chemical transmission was recognized by Otto Loewi in 1921 in the vagal nerve endings of the frog heart. This discovery led eventually to the general concept of chemical synapses both in the peripheral and central nervous system. Ultrastructural studies on chemical synapses began with the classical papers by Palade and Palay (1954) and by Palay (1956) and have since rapidly accumulated. This vast literature has been reviewed in a series of symposia and monographs to which the reader is referred (De Robertis, 1964; Robertson, 1965; Taxi, 1965; Gray, 1966; Gray and Guillery, 1966; Akert et al., 1969, 1972; Bloom et al., 1970; Peters et al., 1976; Sotelo, 1971; Pappas and Waxman, 1972; Pfenninger, 1973; Andres, 1975). This section may be appropriately introduced by showing the very intensive impingement of synaptic terminals upon the perikaryal surface. The model (Fig. 56) of a motoneural perikaryon in the cat spinal cord was constructed by Poritsky (1969) from serial electron-microscopic sections. Figs. 57-59 expose the enormous number of synaptic sites on the soma of a large nerve cell as seen with the freeze-etching method. Two of these figures (Figs. 57, 59) are complementary in that the soma membrane is viewed from opposite sides. The fracture plane shifts from soma to axon and vice versa at the border of the active site, thus revealing the “stickiness” which exists between pre- and postsynaptic membranes. Fig. 60 depicts the classical features of a chemical synapse between an axon terminal and a smooth dendritic process. The terminal has the shape of a knob or bag. Its profile contains synaptic vesicles and mitochondria.

a. Synaptic cleft

The synaptic cleft is clearly wider (20 nm) than the normal gap between

122 plasma membranes of neuropil. Bondareff (1967) has shown by cytochemical methods that the cleft contains a vast number of macromolecules (presumably glycoproteins). De Robertis (1964) and others have suggested that this material may be arranged in a columnar or filamentous fashion. Glial elements ensheath the axonal endfeet, yet spare the synaptic region (Fig. 60). The synaptic cleft seems to communicate freely with the general extracellular space since no occluding junctions have been found in the vicinity of synapses. The extrasynaptic region of the terminal axolemma in Fig. 60 reveals numerous stomata which are interpreted as indicating pinocytosis.

b. Synaptic vesicles The vesicles are of spheric shape and have a mean diameter of about 50 nm. In freeze-etch replicas they appear in the form of convex and concave profiles thereby exposing the E faces and the P faces, respectively. Careful examination of synaptic vesicles has shown that their freezeetched profiles are always spherical (Moor et al., 1969). No elliptoid or cylindrical elements could be found either in unfixed or in glutaraldehydetreated specimens. A small fraction of vesicles have a somewhat larger diameter (80-150 nm) and may correspond to the so-called dense core vesicles (Fig. 61a). Solitary 10-nm particles are found on the P faces of the concavities. They are particularly obvious in experimentally enlarged vesicles (Fig. 61) such as may be obtained during the initial stages of Wallerian degeneration (CuCnod et al., 1970). These particles have a striking similarity with cytochemically identified calcium binding sites which seem to occur in the form of single membrane-bound granules (Politoff et al., 1974; Akert et al., 1977).

c. Presynaptic membrane complex

The main advantage of the freeze-etch technique lies in the fact that the active zone may be studied en face. On the other hand, fine granular material adhering specifically to the presynaptic membrane (presynaptic dense projections of Gray, 1963) fails to show up in these preparations*. The presynaptic membrane may be examined from “without”, i.e. the P face or from “within” the terminal, i.e. the E face. Fig. 62 reveals presynaptic P faces of nerve terminals impinging upon motoneural soma

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Gray has emphasized the presence of microtubules in the vicinity of the presynaptic membrane in “albuminized” brain and retina1 synapses (1975, 1976). These microtubules may be associated with the presynaptic vesicular grid and with cisternae and tubules of the smooth endoplasmic reticulum (Lieberman, 1971). The functional significance of this arrangement is not clear and its freeze-etch aspects have not yet been revealed.

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Fig. 56. Motoneuron with synaptic contacts Reconstruction of a cat spinal motoneuron and its enormous coverage with synaptic boutons. (from Poritsky, J. comp. Neurol., 135: 447, Wistar Press, 1969).

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or dendrites in the rat spinal cord. The “active zone” is frequently characterized by a shallow inwards curving of the membrane. Within this region there are numerous tiny dimples representing the vesicle attachment sites. These dimples, originally named “synaptopores”, were first described by Pfenninger et al. in 1971. Their counterpart, on thepresynaptic E face (Fig. 63) consists of small protuberances many of which bear a crater-like opening at the top. It is concluded that these membrane modulations represent complementary views of the same structures: The attachment sites of vesicles at the presynaptic active zone. The underlying process is believed to be exo- and/or endocytosis, both of which may represent important phases in the release and re-uptake of transmitter molecules. The attachment of vesicles to the plasmalemma is demonstrated in Fig. 63. The “synaptopores” seem to be randomly localized within the active zone. Yet, with appropriate methods, close examination of their organization into active zones reveals that they are regularly arranged in hexagonal arrays (Gray, 1963; Pfenninger et al., 1972). This is in agreement with earlier findings based on thin-section electron-microscopic studies using special heavy metal contrast (Pfenninger et al., 1969) which led to the concept of the “presynaptic grid” (Fig. 64). The hexagonal arrangement of vesicle attachment sites is shown in Fig. 65. Three observations may be added at this point which may relate the descriptive data to more dynamic aspects of synaptic organization: (i) “Synaptopores” are far more conspicuous and numerous in synaptic regions obtained from unanesthetized animals. The same is true for the inward curving of the synaptic site (Cooke et al., 1974). Streit et al. (1972) have compared the presynaptic sites of anesthetized with those of unanesthetized animals and found that significant differences exist which readily explain the fact that endo-exocytosis is only rarely observed in the conventionally treated specimens. Examples of anesthetized synapses are given in Fig. 66; here, the synaptic sites are barely detectable. Following

Fig. 57. Synaptic coverage of perikaryal surface

Electric fish (Hypopomus artedi), medullary relay nucleus, MS 222 anesthesia. The neural soma membrane (N SM) is covered with a dense pavement of synaptic end feet (Axt PF). The view is from within the soma towards the synaptic interface. The soma membrane is best preserved at the clefts between nerve terminals. An arrow marks the cluster of particles characteristic of a postsynaptic site. Active zones at the axon terminals contain vesicle attachment sites (vas); note also that these zones may bulge inward slightly (triangles).

acoustic stimuli Gulley (1978) found an increase of vesicular attachment sites in the presynaptic' membrane of synapses in the anteroventral cochlear nucleus. The administration of 4-aminopyridine has provided another means to increase the exocytosis of synaptic vesicles. In the rat spinal cord, Tokunaga et al. (1979b) found that the presynaptic membrane of contacts in the ventral horn showed a statistically significant increase in both omega-shaped profiles in thin sections and the presynaptic membrane modulations (PMM) on the E face of the axon in freeze-etch preparations. Activation of isolated synaptosomes from the electric organ of Torpedo (More1 et al., 1980) by excess potassium also resulted in increasing the number of micropits (which they described on the P face rather than the E face). (ii) Many active zones have a round or oval shape. However, unexpected configurations of active zones exist as well. They have been described by Akert in 1973 and examples of ring-like and patch-like shapes are given in Fig. 67. The functional significance of these variations remains to be clarified. It is conceivable that the arrangement and number of vesicle attachment sites correspond to functional and/or developmental states. An additional example of the wide range of variation of active zones is given in Fig. 68. (iii) In a morphometric investigation Venzin et al. (1977) pointed out that the P face of the presynaptic active zone is further characterized by about 3 times more of the large intramembraneous particles than the surrounding P face. It was also found that the active zones in unanesthetized animals contained about double the number of 10-nm particles as compared with the active zones of anesthetized animals. Further confirmation of the relationship between particle density and activity has been gained by studies of synapses activated by 4-aminopyridine (Tokunaga et al., 1979a).

Fig. 58. Synaptic coverage of perikaryal surface

Electric fish (Hypopomus artedi), medullary relay nucleus. An extensive region of the E face of a postsynaptic membrane (POEF) of a large neuron is seen with postsynaptic intramembranous collections of particles (triangles). Through breaks in the postsynaptic E face the presynaptic P face (pr PF) of several axons is seen. The shallow grooves outline the axon terminals. Apart from the postsynaptic regions the E face has fewer particles than the P face. The areas of faint dimpling (arrows) indicate vesicular attachment sites of relatively inactive synapses.

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Fig. 59. Axosomatic synapses

Rat spinal cord after treatment with 4-amino-pyridine. The E face of 5 axonal terminals (Axt EF) with active zones showing numerous vesicular attachement sites are applied to the P face of a neuron (N PF). An axon terminal P face (Axt PF) is seen above. The nucleus of the neuron showing nuclear pores (NP) on the P face (Nc PF) is seen in the right lower corner.

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Fig. 60. Axon terminal (bouton terminal) Cat, 15 days, subfornical organ. Synaptic vesicles (sV) presenting both E face (elevations) and P face (depressions) are evident. The pinocytotic pores (P) (characteristic of immature tissue) are seen at the left. They present as dimples on the P F of Axt’ and as craters on the EF of Axt’. A glial process (GE) separates terminal Axt’ and Axt’. The asterisk marks a region of contamination. sV = synaptic vesicles; Mit = mitochondrion. (from Akert et al., In: K. Akert and P. G . Waser (Eds.), Mechanisms of Synaptic Transmission (Progr. in Brain Res., Vol. 31), Elsevier, 1969, p. 234.)

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Fig. 61. Solitary particles in vesicle membrane a: Cat, spinal cord. An axon terminal (Axt) presents both the presynaptic P face (pr PF) and a cross-fracture of the cytoplasm. In the cytoplasm are many synaptic vesicles (sV) which present either the P face or the E face. Triangles indicate solitary particles mostly on the P face of the vesicle membrane. In addition four larger vesicles (arrows) are seen. Their uniform size suggests they may be dense core vesicles rather than endoplasmic reticulum but identification is not certain. The dendrite (Den) shows the P face of a dendritic spine (DSp PF) forming an invaginated contact with the axon terminal. b: Pigeon, optic tectum. Nerve terminal of retino-tectal fiber in the stage of anterograde degeneration. The vesicles are conspicuously enlarged. The solitary particles (triangles) are clearly visible at the P face of vesicle membrane. Mit = mitochondrion: pr EF = presynaptic E face of axolemma; Axt = axon terminal. (from Akert et al., Brain Res., 25: 261, Elsevier, 1971).

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Fig. 62. Axon terminal with active zone a: Rat, spinal cord (without pentobarbital). Axon terminal (Axt) presents the presynaptic P face (pr PF) with particles and vesicular attachment sites (vas). Fracture into the cytoplasm exposes synaptic vesicles (sV). The terminal is connected with a narrow axon (Ax). b: Rat, spinal cord (without pentobarbital). P face of an axon terminal (Axt pr PF) shows vesicular attachment sites (vas). The E face of an adjacent dendrite (Den EF) is seen below. A glial process (GE) is entering the space between the axon terminal and dendrite from the left. Ax = axon.

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Fig. 63. Presynaptic membrane with attached vesicles

Rat, spinal cord. Vesicle attachment sites (vas) are shown at various fracture planes. a: Axon terminal with plasmalemma exposed from “within”. Many protuberances with crater-like openings are seen at the presynaptic E face (pr EF). Note that the bouton is partly cross-fractured at both sides (asterisks). The attachment of a synaptic vesicle to the plasmalemma is shown in profile (triangle). This situation corresponds to the “omega-forms” seen in thin sections. b: Presynaptic bag, exposing the cross-fractured cytoplasm containing synaptic vesicles (sV) as well as the presynaptic E face (pr EF) with many “synaptopores” ( = protuberances with crater-like openings). Triangle marks the site where one synaptic vesicle is in close connection with the plasmalemma. The craters mark the necks from which vesicles have been fractured artifactually during the procedure. c: Synaptic region showing the slightly curved presynaptic and postsynaptic membranes and the synaptic cleft (arrows). Two vesicles (triangles) are attached to the presynaptic membrane (pr EF). The postsynaptic P face (PO PF) is also seen. (from Streit et al., Brain Res., 48: 18, Elsevier, 1972).

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gs/; AXON

TERMINAL

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ACTIVE ZONE

Fig. 64. The presynaptic vesicular grid, diagram Three-dimensional reconstruction of presynaptic active zone. The hexagonal peak and hole pattern is demonstrated. The proportions are slightly distorted for sake of clarity. The exact dimensions of the grid are represented in the inset (upper left), SV = synaptic vesicles; dp = dense projections; dV = dense core vesicles; Mit = mitochondrion. (after Pfenninger et al., Brain Res., 12: 17, Elsevier, 1969.

Fig. 65. Hexagonally arranged vesicle attachment sites Rat, spinal cord. Unanesthetized. Two presynaptic membrane faces (pr EF) overlie the membrane of a perikaryon or large dendrite (PO PF). The vesicle attachment sites are partially arranged in hexagonal order (asterisks). The active zones are outlined by triangles. (from Pfenninger et al., J. Neurocytol., 1: 136, Chapman and Hall, 1972).

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Fig. 66. Axon terminal with active zone Rat, spinal cord. Pentobarbital (50 mg/kg i.v.) anesthesia. The typical membrane modulations of the active zone are barely visible. a: Axon terminal (Axt) exposing the cytoplasm with synaptic vesicles (sV) and the presynaptic P face (pr PF) en face. The active site (marked by triangles) is very slightly indented and contains numerous large particles. Vesicle attachment sites are not clearly visible. b: Axon terminal (Axt) exposing the presynaptic E face (pr EF) with a few vesicle attachment sites (vas) whose number is clearly reduced when compared with unanesthetized preparations (cf. Fig. 65). No bulging of the plasmalemma is seen.

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Fig. 67. Variability of active zones Rat, spinal cord (without pentobarbital). Varied forms of active zones with vesicular attachment sites. In axon terminal Axt' they form isolated irregular groups (arrows). In Axt2 the triangles outline a confluent reniform arrangement. The asterisk indicates a fingerlike protrusion of presynaptic membrane outlined by glial processes (GE). PO EF = postsynaptic E face; pr PF = presynaptic P face. (from Akert, Brain Res., 49: 515, Elsevier, 1973).

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Fig. 68. Multiple active zones in large axon terminal Rat, spinal cord. Ketalar anesthesia (Ketamine hydrochloride, 100 mg/kg i.m.) The presynaptic E face (pr EF) of an axon terminal (Axt) is seen en face with 6 regions of vesicular attachment sites bearing the characteristic elevated craters (triangles). Den = dendrite; PO PF = postsynaptic P face. @

144 d. Postsynaptic membrane complex Postsynaptic regions are encountered on soma and dendritic membranes; they are readily identified on the basis of a sharply limited aggregation of 10-nm particles (Sandri et al., 1972). This aggregation is more conspicuous on the E face which is generally particle-poor. Figure 69 shows the particle aggregation in cross-fracture in which the presynaptic site is identified (vesicle attachment sites). The extent of the particle aggregation and that of the presynaptic active site are identical. This correspondence between areas of pre- and postsynaptic membrane specializations is also demonstrated in Fig. 70 showing a fracture that shifts in the midline of a synapse from one apposed membrane to the other. It should be noted that in both aldehyde-treated and in unfixed material there is a similarly dense aggregation of particles. Yet, this aggregation does not clearly reveal a patterned structure. This finding is confirmed in synaptosomal membranes (Fig. 71), where the postsynaptic sites can be readily detected and used for the identification of the fractured material. Similar aggregates of particles are also found in the P faces of postsynaptic sites (Fig. 5 1). The question arises whether these particles have any relationship with the fibrillar undercoating (“web” of De Robertis, 1964) of the postsynaptic membrane. The coat itself is seen only rarely in cross-fractures (Fig. 72). It is noteworthy that Landis et al. (1974) were unable to find particle aggregations in postsynaptic membranes of the inhibitory granule-tomitral cell synaptic contact in the olfactory bulb. In the same preparation, however, these authors confirmed the presence of particle aggregations in postsynaptic E faces of the excitatory contacts between secondary dendrites of the mitral cell and gemmules of granule cell dendrites. A similar structural difference of postsynaptic membranes was established by Landis and Reese (1974a) between synapses identified as excitatory and inhibitory in the cerebellar cortex.

Fig. 69. Postsynaptic membrane, cross-fracture Rat, spinal Ford (without pentobarbital). An axon terminal (Axt’) demonstrates a presynaptic P face (pr PF) with vesicular attachment sites (vas). The widened interspace with fine granular material forms the synaptic cleft (Syc) (cf. Fig. 92b). The adjacent dendrite (Den) presents a narrow segment of postsynaptic E face (PO EF) below the synapse and postsynaptic particles in this membrane are seen between triangles. A second terminal AXt2 presents vesicular attachment sites and E face fragments of an adjacent cell. Mit = mitochondrion.

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146 An interesting apparent inconsistency was revealed by the report of Hanna et al. (1976) on postsynaptic membrane specializations in the cerebellar cortex of the mutant weaver mouse. Since the dendritic spines of these animals seem to lack presynaptic active zones as a counterpart, the specific relationship of postsynaptic membrane specializations to the synapse is open to question. Many rectilinear arrays of particles and gap junctions were seen in glial membranes around the dendritic spines in freeze-etch preparations of these animals. In thin sections these membranes could be identified as astrocytic processes.

e. Synaptic arrangements The neuropil of the various regions of the central nervous system is characteristically complex. There is a wide variety of synaptic arrangement between soma, dendrites and axons. Only a few examples are illustrated here: Axo-somatic synapses (Fig. 59), axo-dendritic synapses (Fig. 72) and axo-axonic contacts (Figs. 73, 74).

f. Attachment plaques (puncta adhaerentia)

Chemical synapses often appear in close association with attachment plaques which are generally considered to be anchoring devices. These junctions are usually much smaller than synapses and characterized by distinct membrane thickenings comparable to those of the postsynaptic membrane. This similarity to chemical synapses has led to considerable confusion although the chemical synapses (of all types) are strictly asymmetrical in contrast to the symmetrical construction of the puncta adhaerentia. Unequivocally identified puncta adhaerentia have not been reported in freeze-etched material. Landis and Reese (1974a) describe on the E face of cerebellar granule cell dendrite membranes small circular clusters of particles coextensive with the widened intercellular spaces. The particles seen in these spots were smaller than those at synaptic contacts. Numerous puncta adhaerentia have been identified at these sites on the granule cell dendrites. In our own freeze-etch material, we have failed to identify puncta adhaerentia.

Fig. 70. Pre- and postsynaptic membranes, en face Rat, spinal cord (without pentobarbital). A fortunate fracture broke the membrane in the middle of a synapse to expose half of the synapse on each face. The presynaptic P face (Axt pr PF) shows a region of vesicular attachment with large particles (arrows). On the postsynaptic membrane (Den PO EF) the particles form a discrete aggregation corresponding to the region occupied by the synapse (triangles) in contrast to the remainder of the E face which is relatively free of particles. An unidentified PF is seen below. Ax'-~= axons. (from Sandri et al., Brain Res., 41: 8 , Elsevier, 1972).

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Fig. 71. Synaptosorne with postsynaptic site Pigeon, optic tectum, synaptosome fraction. (This material was kindly given to us by Dr. Michel Cutnod.) Note profiles of synaptosomes; both outer and inner leaflets (EF, PF) of synaptosomal membranes are revealed. Triangles mark a typical postsynaptic E face site (PO EF) with a densely packed aggregation of particles. Note that this site is sharply delineated from the particle-poor surround.

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Fig. 72. Axo-dendritic synapses a: Rat, spinal cord (without pentobarbital). Axon and an axon terminal (Ax’, Axt’) are disclosed with two active zones with vesicular attachment sites (vas) and fragments of E face from unknown cell, possibly glia (EF). Between the Axt’ and the dendrite (Den) adjacent to the vesicular attachment sites are two areas of widening of the intercellular space representing synaptic clefts (Syc). Granular material appears in these synaptic spaces. The second axon terminal (Axt2) shows many synaptic vesicles and a small segment of P face. The E face of Axt2 shows a small line at the junction with the cytoplasm (arrow) which represents the inner lamella of the cell membrane. The inset shows an enlargement of the rectangle. Note the presynaptic P face (PF), the presynaptic E surface (ES), the postsynaptic E face (EF) and the postsynaptic P surface (PS). b: Monkey, spinal cord (pentobarbital, 40 mg/kg i.p.) A synapse between an axon terminal (Axt’) and a dendrite (Den) displays an active zone with vesicular attachment sites (triangles) on the presynaptic P face (pr PF) and widening of the synaptic cleft (Syc) which is filled with fine granular material. Below the postsynaptic membrane, the coarsely granular cytoplasm of the dendrite takes on a finer granular appearance (arrow). This may represent the postsynaptic dense material (“web of De Robertis”) seen in thin section (Gray type I junction). On the right a non-synaptic apposition with a second axon terminal (Axt2)is seen with vesicles, mitochondrion (Mit) and pr PF. The E face of an unidentified structure is seen between the two axon terminals.

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Fig. 73. Axo-axonic contacts Rat, spinal cord, substantia gelatinosa. a: An axon (Ax') makes an invaginating contact with another axon (Ax'). Both are identified as axon terminals by the presence of synaptic vesicles (sV). Quiescent active zones are outlined by triangles. Den = dendrites; Mit = mitochondria. b: An axon terminal (Axt') makes synaptic contact with an axon terminal (Axt') (arrows) and a dendrite (Den) (triangles). The synaptic clefts are filled with granular material. Mit = mitochondria; sV = synaptic vesicles.

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Fig. 74. Axo-axonic and axo-dendritic contacts Rat, spinal cord, substantia gelatinosa. A dendrite (Den) is covered by presynaptic E faces (pr EF) with distinct active zones of different shape. An axon (Ax) shows an active zone (triangles) in contact with a branch of the same dendrite (Den). This axon is further in contact with an axon terminal (Axt'). Axt' shows the P face of an active zone. Axt3 is broken through and filled with synaptic vesicles.

156 2. Neuromuscular junction

The frog motor endplate represents perhaps the best paradigm of chemical synapses because (1) its physiological and pharmacological properties are relatively well known and (2) the structural organization is characterized by a distinct geometry of the major components (Figs. 75-77). It consists, in its simplest form, of an elongated nerve ending embedded in a gutter-like depression of the sarcolemma (Peper et al., 1974). This nerve terminal contains the usual presynaptic organelles: synaptic vesicles, mitochondria and smooth endoplasmic reticulum; glycogen granules are frequently present but difficult to see in freeze-etch preparations (Fig. 78). The nerve terminal is covered by a thin sheath of Schwann cell which embraces the terminal with finger-like processes from both sides (Fig. 79), thereby subdividing it by tiny constrictions into regularly spaced compartments along the length of the fiber. These compartments contain the active zones (Couteaux and Pecot-Dechavassine, 1973) and are thus comparable to the varicosities of the terminal sympathetic network or the “en passant” boutons seen in the central nervous system (Fig. 54, see p. 119). Each compartment contains one or more bar-like densities (called dense projections) of the presynaptic membrane, which separate two rows of attachment sites between synaptic vesicles and plasmalemma. This configuration - designated “active zones” - has been fully recognized by Couteaux and PCcot-Dechavassine in 1970; its appearance in freeze-etch replicas was established by Dreyer et al. (1973), Peper et al. (1974) and by Heuser et al. (1974). Active zones ( = dense bars with a row of vesicle attachment sites on either side) have a regular transverse arrangement (Fig. 80) and occur in register with and parallel to the sarcolemmal junctional folds (Fig. 83); they are often fragmented and the frequency of vesicle attachment sites is highly variable (Figs. 81, 82).

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Fig. 75. Neuromusculur junction, diagram Frog, motor endplate. A nerve terminal in the synaptic gutter of a muscle fiber has been lifted out and turned back to expose the underside which lies in contact with the muscle fiber. This exposes (in the enlargement of the P face, PF) the prolongations of the Schwann cell (Sch) which form the glial fingers (f). These partially encircle the nerve terminal and separate the active zones (az) at fairly regular intervals. The lower portion of the drawing is enlarged to show the active zones in cross-section with the dense bar (dp) surrounded by synaptic vesicles (sV). My = myelin; Sch Nc = Schwann cell nucleus.

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Fig. 76. Motor endplate, diagram

The membrane faces as seen in freeze-etch replicas are viewed with an orientation from the nerve terminal downwards to the muscle. The cytoplasmic surface (PS) of the nerve terminal with the dense projection (dp) and synaptic vesicles (sV) (althouph not exposed by freeze-etching) is also shown. The intercellular space (ics) at the junctional region is not drawn to scale. Particles at the P face of the sarcolemma occupy the region of the entrance to the primary folds (so-called juxtaneural lips) and the adjacent interfold areas. Note that the active zone (az) of the presynaptic membrane lies opposite the junctional fold and the specific sites where particle aggregations are localized (see also Fig. 77).

Fig. 77. Motor endplate, diagram

This diagram presents the membrane faces which are complementary to those depicted in Fig. 76. They are viewed with an opposite orientation from the muscle down towards the nerve ending. The main features are the specific sites at the sarcoplasmic E face characterized by circumscribed patches of particle aggregations lying near the entrance to the primary folds (JF with arrow), and the double-double row of particles lying alongside the dense projection (dp, slightly elevated segment of the P face of the nerve terminal membrane); active zone (az).

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a. Presynaptic membrane

The freeze-etch aspects of the presynaptic active zone are illustrated in a number of photographs of the P face and the E face. A general orientation on fracture faces of junctional membranes is provided in Figs. 76 and 77. The P face aspect of the active zone is characterized by a slightly elevated bar which is approximately 50 nm wide and 100-2000 nm long. These bars correspond to the dense projections seen in thin-section electron microscopy (Akert et al., 1969). Regularly spaced pairs of 10 nm particles are localized in intervals of about 10 nm on either side of the bar (Fig. 83). Immediately outside to the double row of particles one may detect the vesicle attachment sites, i.e. stomata or circular invaginations of the membrane which have a diameter of 20-50 nm. In many preparations the stomata are scarce or absent (Figs. 81, 82a), occasionally, they occur in large numbers (Fig. 82b). Heuser et al. (1974) were able to correlate the number of vesicle attachment sites to functional changes at the junction. This interpretation is in keeping with observations in thin sections made under various states of stimulation by Ceccarelli et al. (1973) and by Heuser and Reese (1973, 1974). The E face aspect of the presynaptic active zone (Figs. 80, 84) reveals a shallow groove corresponding in width and length to the bars described in the P face. The grooves are often lined by tiny pits (Fig. 84) which appear to be the complementary sites of the double row of particles seen on the P face (Heuser et al., 1974). Furthermore, the grooves are lined by bumps and protuberances which characterize the vesicle attachment sites. Crater-like openings are occasionally seen (Ceccarelli et al., 1979 a,b). The motor endplates of mice and rats have a much more complicated configuration due to the irregularity of the postsynaptic junctional folds. Due to these irregularities, the active zones are very small but the presynaptic P face clearly shows the double row of 10-nm particles on each side of an elevated bar (Fig. 85). Ellisman et al., (1976) published the first freeze-etch illustrations of mammalian motor endplates. However, it appeared that the orientation of the active zones to the junctional folds was different, i.e. rotated 90°, thereby spanning the interval between two adjacent postsynaptic folds. Subsequent studies of our own material, however, demonstrated a similar relationship in frogs and rats or mice, i.e.

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Fig. 78. Neuromuscular junction, longitudinal profile Frog, motor endplate. Axon terminal (Axt) shows synaptic vesicles (sV), glycogen granules (GLy) and mitochondria (Mit); it is covered with a thin Schwann cell coating (Sch) and overlies several junctional folds (JF) of the postsynaptic sarcolemma. Between the synaptic cleft (Syc) and the axon terminal are two Schwann cell fingers (asterisk). Col = collagen fibres; MF = myofilaments; NcM = nucleus of muscle cell. (from Peper et al., Cell Tiss. Res., 149: 441, Springer-Verlag, 1974).

162 the longitudinal axis of active zones and junctional folds were parallel. In recent years several investigators (Heuser et al., 1979; Ceccarelli and Hurlbut, 1980) have made a major effort to correlate the anatomical findings with the actual process of transmitter release. It is now well established that synaptic vesicles undergo attachment and exocytosis close to the double row of particles in the active zone and that the synaptic vesicles are involved in the quantal release of acetylcholine (Ceccarelli and Hurlbut, 1980). Exocytosis has been stimulated by Black Widow spider venom (Ceccarelli et al., 1979a) and by 4-aminopyridine and electrical stimulation as demonstrated by quick freezing (Heuser et al., 1979). With indirect electrical stimulation it was found that 30 seconds after stimulation a large number of dimples appeared along the double rows of particles on the P face of motor endplates. Thirty minutes later, most of the dimples had gone and the membrane had the usual resting appearance. In another interesting experiment, the region of exocytotic dimpling was found to spread away from the usual site near the active zones under the influence of a high concentration of potassium in the muscle (Ceccarelli et al., 1979b). Pesce et al. (1980) have confirmed that the distribution of large and small particles in the active zone and surrounding membrane is roughly similar to that reported by Venzin et al. (1977) for chemical synapses in the rat spinal cord. They postulate that the loss of small particles is compatible with the incorporation of the relatively smooth synaptic vesicular membrane during exocytosis. In summarizing the main features of the membrane-vesicle complex, designated as the “active zone”, one remains with two specific relationships: (1) between vesicle and dense projection and (2) between vesicle and membrane-associated particles on the P face. Both relationships seem related to the geometrical locus of vesicle attachment to plasmalemma, and it may well be that one or both of these contingencies are essential for the mechanism of endo-exocytosis.

Fig. 79. Presynaptic membrane with active zones Frog, motor endplate. A presynaptic region (pr PF) is viewed from the synaptic cleft. Active zones (az) appear in regular linear arrangement perpendicular to the longitudinal axis of the axon terminal (Axt). Note the juxtaposition of active zones and postsynaptic specific sites at the juxtaneurai lip (arrow) of the junctional fold which is fractured tangentially. The postsynaptic membrane (POPF) shows the coarse particles of the specific site. Schwann cell (Sch) fingers (asterisks) embrace the presynaptic region. P = pinocytotic sites. (from Peper et al., Cell Tiss. Res., 149: 450, Springer-Verlag, 1974).

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Fig. 80. Presynaptic membrane with active zones

Frog, motor endplate. Active zones (az) of an axonal ending viewed from within the terminal (Axt). The outer leaflet (EF) shows a series of parallel grooves, perpendicular to the longitudinal axis of the terminal. The membrane is bulging between grooves due to Schwann cell fingers (asterisks) which embrace the terminal from both sides and thereby compartmentalize the active zones. Note small fragments of cytoplasm of the nerve terminal containing synaptic vesicles (sV). (from Peper et al., Cell Tiss. Res., 149: 443, Springer-Verlag, 1974).

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Fig. 81. Presynaptic active zones Frog, motor endplate. Presynaptic membrane (pr PF) and its relationship to the postsynaptic junctional folds (JF). The active zones (az) are precisely juxtaposed to the openings of the primary folds. Note also the separation of active zones by intervening Schwann cell fingers in the lower half of the picture (arrows). Vesicle attachment sites are barely visible in this preparation. The postsynaptic region of the muscle cell (M) appears in a cross-fracture (to the right), while the presynaptic nerve terminal exposes the membrane face (to the left). Note the cisterns of the sarcoplasmic reticulum adjacent to the secondary folds (triangles).

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Fig. 82. Neuromuscular junctions, active compared with inactive junctions. Frog, motor endplates. a: Two quiescent active zones (az) display regular parallel double rows of large particles on the presynaptic P face (pr PF). The Schwann cell (Sch) on the right shows a fingerlike process which is broken off at the asterisk. b: The presynaptic P face (pr PF) shows an activated endplate. The paired double rows of large particles of the active zones (az) are thrown into disarray by vesicular attachment sites (vas). Schwann cell (Sch) borders the endplate on either side. On the left, a finger-like process of the Schwann cell is broken off (asterisk).

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Fig. 83. Presynaptic active zones juxtaposed to junctional folds Frog, motor endplate. “Precision fit” between the presynaptic P face (pr PF) active zones (az) and the junctional folds (JF) at higher magnification; same orientation as in Fig. 81. (from Akert et al., In: P. G. Waser (Ed.), Cholinergic Mechanisms, p. 46, Raven Press, 1975).

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172 b. Postsynaptic membrane En face views of the postsynaptic membrane are complicated by the deep invaginations designated as postjunctional folds. Complete views of the folded membrane can obviously not be obtained from a single replica. However, from a large number of replicas one can piece together the structural features of the postsynaptic membrane. The reconstruction reveals that the specific sites are localized at the proximal entrance to the primary folds in the membranes of the juxtaneural lips and adjacent interfold areas (Peper et al., 1974; Akert et al., 1975). These sites are distinguished by the presence of dense aggregates of membrane-associated particles of various contours and sizes. Particle aggregations on the E face stand out sharply against the particle-poor background of the surrounding sarcolemma (Fig. 86). No clearcut lattice arrangement of particles can be found within these patches, the density being about 7500 pm2. Comparable particle aggregations were also identified at the P faces (Figs. 79, 84b, 85). In the rat Ellisman and Rash (1977) illustrate that the juxtajunctional particles are often arranged on the lips of junctional folds in irregular rows perpendicular to the longitudinal axis of the fold. After denervating the muscle they found that the particles did not disperse. They reported corresponding pits on the E face of the sarcolemma which would be in agreement with the observations in Torpedo postsynaptic membrane (Rosenbluth, 1975; Cartaud et al., 1978; Heuser and Salpeter, 1979). Conceivably, these membrane-associated particles may correspond to postjunctional membrane specializations described in thin section material by Rosenbluth (1974). In amphibian motor endplates this author was able to demonstrate convincingly that the outer dense lamina of the juxtaneural lip is thickened by the presence of granular elements

Fig. 84. Postsynaptic specific site and active zone Frog, motor endplate. a: A higher magnification of the E face of the outer leaflet of the presynaptic membrane (pr EF) in the active zone (az). The shallow grooves are lined by a double row of pits sometimes alternating with particles. This is the complementary aspect of the active zone seen on the P face. Cross-fractured synaptic cleft (Syc) and postsynaptic sarcolemma (PO PF) are seen at the right. b: Active zone and specific site in juxtaposition. The groove (az, active zone) in the presynaptic membrane (pr EF) and the junctional fold (JF) are precisely in register. Note the coarse particles on the P face of the sarcolemma at the juxtaneural lip (arrows). Pinocytosis (P) characterizes both faces (EF, PF) of sarcolemma. Syc = synaptic cleft. (from Akert et al., In: P. G. Waser (Ed.), Cholinergic Mechanisms, p. 51, Raven Press, 1975).

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174 approximately 6-12 nm in diameter which are spaced semiregularly at 10-15 nm intervals and which border the junctional cleft directly. In these regions the concentration of granules was found to be of the order of 104 ym2, which is in the same range as the estimated concentration of cholinergic receptor sites (Cartaud et al., 1973; Nickel and Potter, 1973; Fertuck and Salpeter, 1974; Barnard et al., 1975). This is not much greater than the density of particles as seen in freeze-etch replicas. It is tempting to speculate that the granular component projecting from the outer surface of the membrane into the synaptic cleft and the membrane-associated particles seen at the interior membrane faces by the freeze-etch technique may be identical. Rash and Ellisman (1974) have identified a similar structural differentiation of the postjunctional sarcolemma in the rat implicating the relationship between the granules in thin section and the freeze-etch particles as well as their functional significance as cholinergic receptor sites. Finally, it should be pointed out that the postsynaptic membrane of central synapses is similary studded with particles (Sandri et al., 1972; Akert et al., 1975) on the E face. Although the functional significance of these particles has not been fully established either in the motor endplate or in the central synapse, it seems noteworthy that the particle aggregations at the postsynaptic site are in register with the clustering of presynaptic vesicle attachment sites. This orderly arrangement between what we believe are the sites of release and chemical interaction of neurotransmitters seems to indicate that both specializations are intimately related to synaptic transfer mechanisms. The matter has been further clarified by Cohen and Pumplin (1979) who studied neuromuscular contacts established in cultured chick myotubules. They found an exact correspondence between the location of patches of large angular particles and acetylcholine as indicated by binding to

Fig. 85. Neuromuscular junction of mammals Mouse, omohyoid muscle (preparation by K. Peper, Homburg/Saar, F.R.G.). a: A break through an axon terminal (Axt) resembles a conventional thin section. Synaptic vesicles (sV) are somewhat more numerous in the region of entrance of the postsynaptic junctional folds (JF). The P face of the juxtaneural lip of one fold is studded with postsynaptic particles (PO PF). A synaptic cleft (Syc) is seen above. The axon terminal is surrounded by Schwann by cell (Sch). Mit = mitochondrion. b: The P face of an axon terminal (Axt PF) shows multiple active zones (az) which are seen as paired double rows of large particles. The active zones are very short and change direction to correspond to the irregular arrangement of junctional folds (JF). Postsynaptic particles are concentrated on the juxtaneural lips of the folds (PO PF).

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fluorescent a-bungarotoxin. On complementary replicas, they also found patches of particles on postsynaptic E faces but they were less numerous. They suggest that the clusters of particles may include proteins other than acetylcholine receptors. Some might represent the sites of insertion of a cytoskeletal system.

Fig. 86. Postsynaptic specifrc sites Frog, motor endplate. a: Enface view of the postsynaptic membrane (POEF) from within the endplate with orientation towards the overlying nerve terminal (cf. Fig. 77, p. 159). The junctional folds (JF) are cross-fractured in several places. Arrow points to a T-profile of primary fold branching into secondary folds. The intact membrane faces represent the juxtaneural lips and the interfold area. Asterisk marks an interfold fragment. Particle aggregations form irregular patches (triangles) sparing the interfold area and covering mainly the juxtaneural lips and adjacent interfold areas (from Akert et al., In: P. G . Waser (Ed.), Cholinergic Mechanisms, p. 52, Raven Press, 1975). b: Similar view upon postsynaptic membrane (PO EF) exposing mainly an interfold area which contains several sharply circumscribed patches with densely packed particles (triangles).

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178 3. Electrical synapses

The history of the discovery of synaptic transmission is of dramatic interest because the two concepts of electrical and chemical mediation of nerve impulses were the subject of violent controversies ever since chemical transmitters had been identified. Recent interdisciplinary and comparative studies have made it clear that both concepts are fully justified since both chemical and electrical synapses exist. Cogent descriptions of the two forms are found in a review by Sotelo (1971) and in the report of a symposium on the structure and function of synapses (Bennett, 1972; Pappas and Waxman, 1972). The crucial ultrastructural features of electrical synapses as seen in thin-section electron microscopy were described by Robertson (1963) in the club endings of the Mauthner cell lateral dendrite in the goldfish. The presence of electrical transmission at this synapse was established by Furshpan (1964) (see also review by Sotelo, 1975). Combined electrophysiological and electron-microscopic studies in large relay neurons and spinal motoneurons of the teleost electromotor system made it increasingly clear (Bennett et al., 1967a-d) that the “gap junction” represents the ultrastructural correlate of electrotonic coupling between neurons. Direct evidence that gap junctions are sites of electrotonic coupling was provided by Pappas et al. (1971) in the lateral giant fiber of the crayfish by demonstrating that experimentally induced changes in coupling resistance were associated with separation of the junctional membranes by interposed Schwann cell processes. Much of the early work on electrotonic transmission was carried out in invertebrates and low vertebrates. The general thin-section and freeze-etch aspects of gap junctions have been amply described and discussed (McNutt and Weinstein, 1973; Staehelin, 1974, mainly for non-neural tissues). Only a few regions exist in the mammalian central nervous system where physiologically and morphologically identified electrical synapses could be demonstrated. These are the rat lateral vestibular nucleus (Sotelo and Palay, 1970), the mouse mesencephalic trigeminal nucleus (Hinrichsen and Larramendi, 1970) and the cat inferior olive (Sotelo et al., 1974). In other regions, gap junctions have been found, but electrophysiological evidence of electrotonic coupling is lacking (e.g. the primate cerebral cortex as shown by Sloper, 1972).

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Fig. 87. Electrotonic coupling between Ranvier node and soma of neuron, morphological correlate, diagram Contact between descending axon (Ax) (node of Ranvier, nR) and soma ( S ) of spinal electromotoneuron in Gymnotids, based on thin section and freeze-etching electron microscopy. The nodal zone is bulging towards the apposed cell body. The area of contact consists of numerous macular gap junctions (GJ) alternating with intermediate junctions (iJ). My = myelin. Note that the gap junctions are asymmetrical.

Freeze-etch studies of electrophysiologically identified sites of electrotonic transmission have been performed by Perracchia (1973) in the giant motor synapse of the crayfish, by Zampighi and Robertson (1973) in the club ending synapse of the goldfish Mauthner cell, by Pfenninger and Rovainen (1974) in the lamprey spinal cord, by Cantino and Mugnaini (1975) in the avian ciliary ganglion and by Raviola and Gilula (1973, 1975) in the retina of monkey and rabbit. It has turned out that the same particle arrangement (hexagonal array with a 90-nm center-to-center spacing) is found in all examples of gap junctions. The particles observed in freeze-etched preparations adhere almost exclusively to the P face. A question arises with respect to the problem of symmetry since rectifying electrical synapses have been found (see Bennett, 1972); as yet specific morphological differences between the rectifying and the nonrectifying variety have not been demonstrated with certainty. Freeze-etch replicas of electrical junctions (Figs. 88-91) were obtained from the electromotor system in several gymnotid species, especially sternarchids, whose neurogenic electric organ is known to discharge between 700 and at least 1500 impulses/sec (Bennett, 1971a). The

Fig. 88. Electrotonic coupling by prefiber, morphological correlate

Knifefish (Eigenmannia virescens), medullary pacemaker nucleus. The node of Ranvier (nR) is obliquely fractured by exposing the glial loops (GL) on one side. It bulges towards the soma of a closely apposed neuron (S) and forms at least three macular gap junctions (triangles). The P face of the soma is exposed. Note that the uppermost gap junction is surrounded by a membrane district which is indented and contains very few particles (arrows). This area may represent intermediate junctions. Mit = mitochondria; nf = neurofilaments; sER = smooth endoplasmic reticulum.

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182 electromotor control pathway of gymnotids consists of three stages: a pacemaker nucleus in the medulla oblongata which sets the frequency and transmits to a medullary relay nucleus which in turn transmits to the spinal motoneurons. In the species studied, transmission seem to be electrotonic at all three stages (Bennett et al., 1967d). Coupling between neurons may be effected in two ways: (1) directly by electrical synapses between dendrites and/or cell bodies, (2) by way of presynaptic axons forming electric synapses with two or more of the coupled cells (Meszler et al., 1972). Of particular interest is the prefiber coupling between axons of the descending spinal tract and the perikarya of several motoneurons found in the electric eel (Bennett et al., 1964; Meszler et al., 1974). Morphological evidence for prefiber coupling of electromotoneurons was also in Sternarchus (Pappas et al., 1975). It turned out that gap junctions exist between the nodal membrane of the descending axons terminating in the spinal electromotor nucleus and the soma membrane of the target neurons. This situation is schematically represented in Fig. 87. Multiple punctate gap junctions alternate with intermediate junctions over the extended contact area. Freeze-etched replicas of electric synapses in axonodal-somatic contacts are depicted in Figs. 88-91.

Fig. 89. Electrical synapses

Knifefish (Sternarchus albifrons), spinal cord. Contacts between nodes of Ranvier and electromotoneurons. a: The electrical synapses appear as large gap junctions with regular arrays of particles on P faces (triangles) or pits on E faces. Around the junctions a low convexity on the postsynaptic E face of the electromotoneuron (N PO EF) may represent intermediate junctions. b: Around the junction on the P face of a presynaptic node of Ranvier (nR pr PF) there is a shallow trench (? intermediate junction). c: Node of Ranvier (nR PF) with two gap junctions (triangles) shows the glial loops (GL) at its border.

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From experience gained by careful study of many thin sections and replicas from Sternarchus albifrons (Bennett et al., 1978; Tokunaga et al., 1980) we have become convinced that the electrical synapse is a special electrotonic gap junction. The surrounding intermediate junctions and the “postsynaptic” density on the neuronal soma side of gap junctions are particularly well seen after negative staining with bismuth iodide (Fig. 91a). In freeze-etch replicas the membrane surrounding the gap junctions (thought to contain the intermediate junctions) is characterized by slight indentation on the P face which is nearly free of particles and with slight bulging on the E face of the corresponding region. It is noteworthy that the hexagonal mode of packing of 10-nm particles is particularly clear in these junctions. One may speculate that the surrounding intermediate junctions impede the movement of particles during fixation and cryoprotection.

Fig. 90. Electrical synapses Knifefish (Sternarchus albifrons). Electrical synapses on a giant neuron in the medullary nucleus. a: Neuronal soma, postsynaptic face (N PO PF). The slightly concave rings (triangles) around the gap junctions (GJ) represent intermediate junctions and are distinguished by a relative paucity of particles. b: The E face of a presynaptic node of Ranvier (nR pr EF) is applied to a neuronal soma (N). A probable intermediate junction is outlined by triangles but the designation is uncertain. c: A thin section shows the intermediate junctions (iJ) surrounding the gap junctions (GJ). Note the postsynaptic density in the neuronal soma (N) which underlies both the gap and intermediate junction. nR = node of Ranvier.

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4. Mixed chemical and electrical synapses

Mixed synapses occur in the peripheral as well as in the central nervous system of vertebrates. Their functional significance is not clear. Morphological evidence for the coexistence of chemical synapses and gap junctions has been reported by Sotelo and Palay (1970) in the lateral vestibular nucleus of the rat. Other examples are the synapses between the club endings and the lateral dendrite of the goldfish Mauthner cell (Nakajima, 1974) and the synapses between mossy fibers and granule cell dendrites in the gymnotid and amphibian cerebellar cortex (Sotelo and Llinas, 1972) and on frog spinal motoneurons and their large dendrites (Taugner et al., 1978). Physiological evidence for a dual mechanism of synaptic transmission has only been obtained by Martin and Pilar (1963) in the avian ciliary ganglion. Occulomotor nerve stimulation elicited in the so-called ciliary neurons an electrotonic coupling potential which was followed by a curare-sensitive postsynaptic potential. The morphological correlates of this mixed synaptic complex were provided by Cantino and Mugnaini (1975) in thin sections and freeze-fracture replicas. The area involved in chemical transmission was found to be more than 40 times larger than that reserved for gap junctions. Mixed synapses also occur in the suprasegmental electromotor control nuclei of gymnotid fish (Bennett et al., 1967d). Corresponding junctional complexes have been observed in freeze-etched specimens derived from medullary electromotor neurons; a few examples are illustrated in Fig. 92. Sites of chemical transmission are identified by the presence of circumscribed aggregations of large particles at the postsynaptic E face. These contacts lie side-by-side with gap junctions characterized by regular hexagonal arrays of pits. One should distinguish between mixed synapses formed by separate chemical and electrotonic nerve terminals converging Fig. 91. Electrical synapse between axon and soma, morphological correlate Electric eel (Electrophorus electricus), spinal electromotoneuron. Thin section and freeze-etch aspect of a large gap junction (GJ). a: Contrast accentuated by bismuth iodide incubation. The membranes are spared (as in negative staining) and the membrane coats are enhanced in this picture. The gap junction is asymmetrical since the thickening of the soma membrane (SM) is more pronounced than that of the axon (Ax). Two intermediate junctions (iJ) are seen next to the gap junction; they are characterized by a wide cleft and symmetrical membrane thickenings. R = ribosomes; Mit = mitochondrion (swollen). b: Gap junction at axonal E face (Ax EF). The junctional membrane area is protruding and occupied by tiny pits in typical arrangement. The perijunctional zone is particle-poor and bulging in the opposite direction, possibly representing the site of an intermediate junction. A second gap junction is depicted at the right lower corner with the complementary membrane face exposed.

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upon a single postsynaptic cell body or dendrite and truly mixed synapses occurring in one and the same presynaptic element. Only presynaptic E and P faces are suitable for the identification of truly mixed synapses. The presynaptic membrane of one nerve terminal containing both vesicle attachment sites and a nearby gap junction is shown in Fig. 92c. Intermediate junctions clearly shown t o be a frequent if not obligatory component of mixed synapses (Peters et al., 1976) are not reliably identified in the freeze-etched junctional membranes (see discussion on intermediate junctions in Chapter 11, Sect. D, p. 32).

Fig. 92. Mixed synapses in Gymnotid electromotor neurons a: Hypopomus artedi, medullary relay nucleus. At least two macular gap junctions (GJ) are exposed at the postsynaptic E face (POEF). In their immediate vicinity two particle aggregations are found (triangles). The particles are of relatively large size and are thought to belong to the specific site of a chemical synapse. Note that the gap junctions are characterized by regularly arranged pits in contrast to the clustered particles typically seen in the chemical junctions. b: Sternarchus albifrons, medullary nucleus. An axon terminal (Axt) in cross-fracture displays synaptic vesicles (sV). The postsynaptic E face of a neuron (N PO EF) with which it makes contact shows both a gap junction (GJ) and a chemical synapse (triangles). Some of the presynaptic particles of the gap junction can be seen on the P face of the terminal (Axt pr PF) where the fracture passed through the junction. In the chemical synapse the postsynaptic particles appear to cross the synaptic cleft (arrow) (cf. Fig. 69). c: Eigenmannia virescens, medullary pacemaker nucleus. Four macular gap junctions (GJ) with nearly hexagonal contours are exposed at the postsynaptic P face (PO PF). They are surrounded, by a region relatively poor in particles (intermediate junctions?). Several presynaptic membrane fragments (pr EF) contain vesicular attachment sites representing chemical synapses. The membrane fragment at the left belongs to an axon; both chemical (above) and electrical synapses (below) are seen on the same membrane face (pr EF). The gap junction is marked by triangle. The hole in the postsynaptic membrane (above, indicated by arrow) may represent the site of a chemical synapse since particle aggregations are seen at the intact margins. vas = vesicle attachment sites. d: Eigenmannia virescens, medullary pacemaker nucleus. A chemical synapse (triangles) marked by an aggregation of large particles lies side-by-side with an electrical synapse, gap junction (GJ) at the postsynaptic E face (POEF).

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190 5. Neurohaemal junction

Neurosecretory systems are known to send their axons to the pericapillary spaces where the hormones and other secretory materials are released. A prototype of a neurosecretory system in vertebrates is represented by the supraoptic and paraventricular nuclei whose axons form the hypothalamo-hypophyseal tract and end in the immediate vicinity of the neurohypophyseal capillaries (Bargmann and Scharrer, 195 1). The axon terminals found in the posterior lobe have been described electronmicroscopically by Palay (1957). Their similarity to presynaptic terminals is striking. Not only do they contain secretory granules (120-190 nm) and microvesicles (50 nm), but presynaptic dense projections are also seen (Rufener, personal communication), although the latter fail to form a triagonal grid. Since these terminals end at the perivascular space and not against another neuron or effector cell, the term, neurohaemal junction is used to characterize the situation. The secretory granules apparently release their content by exocytosis (Dreifuss et al., 1974) into the perivascular space. Considerable efforts have been made to demonstrate exocytotic membrane events during hormonal release. A variety of tissues have been studied (rat parotid acinar cells, De Camilli et al., 1976; rat pancreatic 0-cells, Orci et al., 1977; rat peritonea1 mast cells, Burwen and Satir, 1977; Lawson et al., 1977; hibernating rodent hypophysis, Theodosis et al., 1978a; rat hypophyseal pars intermedia, Saland, 1978; bovine adrenal chromaffin

Fig. 93. Neurohaemal junction Rat, neurohypophysis. The abluminal P face (PF) of a fenestrated capillary (Cap) is seen above with fenestrae. Several pituicytes (Pit'-3) surround the capillary. The finger-like processes Pit' and Pit3 are characteristic of these cells. A gap junction (GJ) joins a pituicyte and the underlying cell. Axon terminal (Axt2) contains neurosecretory granules (NSG). Fib = a possible fibroblast; BL = basal lamina. Triangles mark exo-endocytotic sites.

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cells, Aunis et al., 1979; rabbit and mouse anterior pituitary, Ishimura et al., 1980) in a very careful study Tanaka et al. (1980) postulated that the particle-free zones thought to play a role in the final events of exocytosis (fusion process) might simply-be fixation artifacts. Abraham et al. (1979) found in the pituitary of teleosts that, despite the widespread belief that hormonal release occurs by exocytosis, there is little morphological evidence of such a mechanism. It is thus possible that the exocytosis is a very rapid event similar to the transmitter release in chemical synapses. Further efforts to improve the rapidity of fixation may prove fruitful. From the perivascular space the secretory products are transported into the capillary lumen and carried in the bloodstream to the target organs. Exocytosis may be partly spontaneous, partly triggered by electrical activity (“neurosecretory impulse”, Cross, 1974) of the supraoptic and paraventricular neurons (Kandel, 1964; Dreifuss, 1975). The features of freeze-etch preparations of the neurohaemal junction of the neurohypophysis are shown in Figs. 93-96. Cross-fractured axon terminals contain microvesicles which bear striking resemblance to synaptic vesicles. Their functional significance is not yet fully understood, although there is evidence (Nagasawa et al., 1971) that microvesicles arise at sites of exocytosis by inward budding of the membrane (Figs. 94-96), and it has been argued that this endocytotic process may serve the recycling of granular membrane and indirectly the homeostasis of plasmalemmal surface area. The sites of exo-endocytosis are particularly well seen in the freezeetched membrane faces of neurosecretory axon terminals. They appear as small stomata on P faces (Figs. 94-96) and protuberances with or without crater-like openings in E faces (Fig. 95b). Orderly arranged aggregates of

Fig. 94. Neurohaemal junction Rat, neurohypophysis. a: Five axon terminals (Axt) are seen at the level of the pericapillary extracellular space (ecs). They contain secretory granules (SG) as well as synaptic vesicles (sV). Note the exo-endocytotic sites (arrows) in Axt’. b: Two axon terminals (Axt) at the level of the pericapillary space (ecs). The capillary endothelium (End) is identified at the left. Note the vast number of fenestrae (F). Ax = axon, Col = collagen fibers, Pit = pituicyte. Arrows mark exo-endocytotic sites.

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194 membrane-associated particles occur in regions where fragments are being added or taken away from the plasmalemma during secretion (Dreifuss et al., 1976); these aggregates form rosette-like and necklace-like patterns (Figs. 95, 96) similar t o those described at sites of exo-endocytosis in other systems (Orci and Perrelet, 1973; Satir et al., 1973). The pericapillary space (Fig. 94) contains collagen fibrils. The neurohypophyseal capillaries are composed of endothelial cells which display a high degree of pinocytotic activity and fenestration (Dreifuss et al., 1973). The description of the neurohaemal junction is incomplete without mentioning the third element: the pituicyte and its processes which form bell-like sheets for each individual nerve terminal (see Chapter VI, Sect. C , p. 296). 6. Some remarks on the freeze-etch appearance of exo-endocytosis

Recent studies of the dynamics of vesicular release have emphasized the plasticity of distribution of membrane particles in synaptic terminals. In the frog neuromuscular junction, Heuser et al. (1979) have demonstrated that the lines of particles bordering the active zones are thrown into disarray by the exocytotic stomata which appear within or close to these lines. As the vesicles disgorge their contents and merge with the surrounding membrane the site is marked by a small cluster of several large particles in a shallow depression on the axonal P face. Ceccarelli et al. (1979a,b) hoped to distinguish between exo- and endocytotic events in the same preparation by contrasting modes of stimulation (20 mM K + and Black Widow spider venom). Although exoand endocytosis were considered to occur in the same region about the active zone, they were unable to find distinguishing morphological features (cf. studies of the rat median eminence by Rohlich and Halasz, 1978). In the rat neurohypophysis, Theodosis et al. (1978b) concluded the two Fig. 95. Neurohaemal junction, arrangement of membrane particles

Rat, neurohypophysis. a: The viewer is faced with the palisade formed by multiple neurosecretory nerve endings (Axt). Endocytotic sites are indicated by triangles and exocytotic depressions are outlined by dashed lines. Note the short double rows of large particles which are related to the exocytotic sites (arrow). b: A neurosecretory ending shows the E face (Axt EF) and some cross-fractured cytoplasm. Note the neurosecretory granule with satellite microvesicles (arrow). An exocytotic site is surrounded by a dashed line and endocytotic stomata are indicated by triangles. The arrangement of particles about the endocytotic sites is well seen in the inset.

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196 processes could be distinguished. On the plasmalemma P face, endocytosis was characterized by small invaginations lacking a granular core with clusters of larger (k 12 nm) particles on the membrane and in depressions. Exocytosis, on the other hand, showed larger depressions with a granular core with a surrounding reduction in the population of k 8 nm particles which were seen on the rest of the P face. They also illustrated particle-free bulges in the axonal membrane P face which might represent prefusion sites for synaptic vesicles. Care in interpretation of particle-free areas of the P face is warranted since Tanaka et al. (1980) demonstrated that such areas might be related simply to the difficulties attending aldehyde fixation. In the vesicular attachment sites on axon terminals in rat spinal motoneurons, Tokunaga et al. (1979a) were able to distinguish 3 types of sites on the presynaptic P face. Type 1 lacked intramembranous particles, type 2 had particles attached around the edge of the micropit but with a central particle-free area, and type 3 contained 1-4 large (over 10.5 nm) or small (less than 10.5 nm) particles. Activation of the synapses by 4-aminopyridine markedly increased the total number of vesicular attachment sites but not the frequency of any of the 3 types. On the basis of other evidence, exocytosis was linked with types 1 and 2 and endocytosis with type 3 but the matter remains conjectural.

Fig. 96. Neurohaemal junction, exo-endocytotic sites

Rat, neurohypophysis. a and b: The perivascular aspect of the P face of secreting endings showing the typical characteristics of exo-endocytosis. Triangles indicate particle aggregations partly in small pits and thought to characterize endocytosis. Other pits, often with a core and always a smooth aspect are enclosed by dashed lines. They probably correspond to exocytotic remnants. Arrows point to small double rows of large particles.

198 7. Special sensory synapses

In certain sensory cells, the presynaptic dense bodies become clearly evident and may undergo considerable specialization. A round or oval presynaptic dense body is found in constant relationship to an asymmetrical synapse in the organ of Corti (Smith and Sjostrand, 1961; Lowenstein et al., 1964, 1968; Spoendlin, 1968, 1969, 1971; Takasaka and Smith, 1971), the lateral line organ of fish and amphibia (Flock, 1965; Hama, 1965; Jande, 1966; koberts and Ryan, 1971) and mechanoreceptors of Torpedo (Nickel and Fuchs, 1974). A similar presynaptic dense body is seen in electroreceptors of certain fish (Lissmann and Mullinger, 1968; Bennett, 1971b; Szamier and Bennett, 1974). Perhaps the most striking example of this is the invaginated synapse of the photoreceptor cells of the mammalian retina. In this junction, a ridge (the synaptic ridge) of receptor cell membrane separates the processes of the horizontal cells and is the site of specialized synaptic contacts. Within the (pre)synaptic ridge, a plate (“ribbon” in cross-section) of dense material maintains a constant relationship within the apex of the ridge and is separated from it by a second trough-shaped density, the “arciform density’’ (Raviola and Gilula, 1975). Synaptic vesicles around the ribbon are oriented in an open hexagonal pattern. Tenuous fibrils appear to connect the vesicles to the ribbon (Gray and Pease, 1971). Microtubules lie close to the ribbons and occasionally are inserted into the ribbon (Gray, 1976a; Glees and Spoerri, 1977). Similar synaptic specializations with presynaptic ribbons have been found during the early development of pinealocytes in the neonatal rat (Zimmerman and Tso, 1975). A recent study by Gulley and Reese (1977) has shown a similar synapse in the organ of Corti. In the electroreceptors of the skin of the weakly electric fish (Sternarchus albifrons) we found structures resembling the retinal synapses. The sensory cells of the tuberous organ make invaginating synapses into the margins of the terminal bulbs of a myelinated nerve. An example is shown in Fig. 97. Arrays of large particles on the E face of the postsynaptic membrane are seen which resemble those shown in a process of the retinal outer plexiform layer (Raviola and Gilula, 1975).

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Fig. 97. Sensory synapses

Sternarchus albifrons, skin electroreceptor (tuberous organ). a and b: In thin sections the presynaptic element is seen to form a deep pocket in the postsynaptic membrane. The tip of an electron-dense bar (arrow), which is surrounded by small vesicles (sV) lies in the orifice of this pocket. Floccular densities and larger vesicles (V) lie just below the synapse at the apex of the pocket on the postsynaptic side. c: In replicas the presynaptic dense bar is faintly indicated by fine particles (triangles) in cross-fracture. Large vesicles (V) appear in the postsynaptic element. d: Regularly arranged particles are seen on the postsynaptic E face (PO EF).