Sprouting and remodelling at the nerve-muscle junction

Progress in Neurobiology Vol. 27, pp. 251 to 291, 1986 Printed in Great Britain. All rights reserved

0301-0082/86/$0.00+ 0.50 Copyright © 1986 Pergamon Journals Ltd

SPROUTING AND REMODELLING AT THE NERVE-MUSCLE JUNCTION ANTON WERNIG* a n d ALBERT

A. HERRERA t

*Department of Neurophysiology, University of Bonn, WilhelmstraBe 31, D-5300 Bonn I, West Germany tNeurobiology Section, Department of Biological Sciences, and Program in Neural, Informational and Behavioral Sciences, University of Southern California, Los Angeles, California, 9008949371, U.S.A.

(Received 6 January 1986)

Contents 1. Introduction 2. Nerve sprouting and retraction under physiological conditions 2.1. Light microscopic studies 2.2. Abandoned synaptic gutters in normal muscles 2.2.1. Frog muscles 2.2.1.1. ACh receptors 2.2.2. Mammalian muscles 2.3. Nerve sprouting and new synapse formation 2.3.1. Frog muscles 2.3.2. Mammalian muscles 2.4. Mechanisms of remodelling 3. Growth, age and other factors 3.1. Matching of muscle fiber and synaptic parameters 3.2. Frog muscles 3.2.1. Growth versus age changes 3.2.2. Seasonal factors 3.3. Mammalian muscles 3.3.1. Aging 3.3.2. Pathological conditions 4. Plasticity and regulation of synaptic efficacy 4.1. Different measures of synaptic transmission 4.2. Variability in synaptic efficacy at different junctions 4.3. Non-uniformity of release efficacy within single junctions 4.4. Plasticity in transmitter release per unit nerve terminal length 4.5. Ultrastructural basis of differences in transmitter release per unit terminal length 5. Multiply innervated muscle fibers 5.1. Number of junctional sites per muscle fiber 5.2. Number of presynaptic inputs per junctional site 6. Summary and Conclusions References

251 252 252 253 253 254 257 258 258 261 262 262 265 265 267 268 268 268 273 275 275 276 277 277 278 282 282 282 283 285

1. Introduction

It has long been known that intact adult motor neurons grow numerous collateral or terminal sprouts when muscles are partially denervated (van Harreveld, 1945; Weiss and Edds, 1945; Edds, 1950; Hoffman, 1950) or in response to poisoning with toxins that block transmitter release (e.g. botulinum toxin, tetanus toxin or tetrodotoxin, Duchen and Strich, 1968; Duchen, 1970; Duchen and Tonge, 1973; Brown and Ironton, 1977; Holland and Brown, 1981). Sprouting under such conditions can be considered to be "induced" sprouting, possibly involving sprout-inducing trophic factors produced by the muscle (for reviews see Purves and Lichtman, 1978; Brown et al., 1981; Cotman et al., 1981; Grinnell and Herrera, 1981; Wernig et al., 1981d). Recent morphological data indicate that nerve Dedicated to Brigitte Wernig and Peggy Herrera. Supported in part by grants from the Deutsche Forschungsgemeinschaft and the Land Nordrhein-Westfalen to A. W. and from the National Institutes of Health to A. A. H. 251

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terminal sprouting and new synapse formation, as well as nerve terminal retraction, also occur in untreated vertebrate muscles under physiological conditions. This "physiological" remodelling is the subject of the present review. While physiological remodelling p e r se might be without direct functional consequences, it does allow the architecture of junctions to change in response to different physiological and pathological conditions. The changes that we will investigate are growth, altered use, aging, and several pathological states. We will advance the hypothesis that the structural changes seen in these conditions are due to this inherent capacity for synaptic remodelling. The role of feedback mechanisms and trophic or activity-related regulation will be discussed. The subject of neurotrophic factors has recently been reviewed (McArdle, 1983; Barde et al., 1983; Berg, 1984). We begin this review with a detailed description including light and electron micrographs of the signs of junctional remodelling (Section 2). This is followed (Section 3) by an investigation of the effects of different physiological and pathological conditions on the structure and function of the neuromuscular junction. We then broaden the discussion and ask whether and by which means transmitter release per unit nerve terminal length can change over a longer time scale (Section 4). The final section (5) considers possible interactions between junctions on multiply innervated muscle fibers. Generally, we separate discussions of frog and mammalian muscles. Finally, we want to point out that in iris muscle of chick, which is not discussed in this review, structural and biochemical aspects of aging including continual junctional remodelling have recently been studied (Giacobini, 1982; Giacobini et al., 1984; Mussini, 1984).

2. Nerve Sprouting and Retraction Under Physiological Conditions In this section we will describe in detail the morphological studies of vertebrate muscles that indicate the existence of synaptic remodelling under normal physiological conditions. We will then review the evidence for remodelling under different conditions including growth (but not early development), aging, and changing external factors (Section 3). 2.1. LIGHT MICROSCOPIC STUDIES In several light microscopic studies over the last decades investigators have described unusual looking, thin nerve terminal branches in endplates of normal muscles. A well documented report based on silver stained mammalian muscles was given by Barker and Ip (1966). These authors found pre- and ultraterminal branches in untreated muscles which were unusual in that they left the circumference of the endplate. By analogy to sprouts developing after partial muscle denervation, these terminal branches were assumed to be sprouts (Brown and Ironton, 1978; Mallert et al., 1980; Brown et al., 1981; Herrera and Scott, 1985). Whether these outgrowths actually form synaptic contacts under physiological conditions has only scarcely been investigated with ultrastructural, cytochemical, or physiological techniques and is still unanswered. Sprouting was also inferred from the presence of collateral axons forming accessory or double endings on muscle fibers (Barker and Ip, 1966; Tuffery, 1971; Courtney and Steinbach, 1981). The difficulty in interpreting such observations lies in their static view, since thin nerve branches and small endplates could simply represent normal variations in size rather than recent growth. Accordingly, TeUo (1922) and Cajal (1925) presumed that accessory endplates were errors of development or remnants from fetal conditions (quoted by Barker and Ip, 1966). Clearly, one way to resolve this difficulty is to look for changes in junctional complexity with time. Tuffery (1971) investigated the complexity of endplates in four adult cats of different ages and found that the average number of muscle fibers innervated by more than one axon collateral increased with age. The same result was recently obtained in a thorough study on rats aged 50 to 900 days (Courtney and Steinbach, 1981). Only recently, however, have researchers measured age-related changes in the complexity of

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individual synapses, i.e. the total number of intraterminal branches contributing to an endplate (Pestronk and Drachman, 1978). Other studies have revealed an increase with age in the summed length of the presynaptic nerve terminal (Prestronk and Drachman, 1978; Wernig et al., 1980c; Jans et al., 1986), and endplate length or area (Nystr6m, 1968; Pestronk et al., 1980; Smith and Rosenheimer, 1982; Wernig et al., 1984). The number and length of terminal sprouts was also found to change with age (Fagg et al., 1981). While an increase in endplate size and complexity in young animals can somehow be attributed to growth of the muscle, reasons for sprouting in the adult seem less obvious (see below). Led by the observation of swollen nerve terminal branches which they assumed to be degenerating, Barker and Ip (1966) suggested that the innervation of muscle fibers becomes replaced from time to time or is continually rejuvenated (cf Tuffery, 1971). Even though swollen silver-stained axon terminals are not necessarily a sign of nerve degeneration and cogent ultrastructural evidence for nerve degeneration in normal muscle has never been provided, the observations served to introduce the idea that nerve-muscle junctions are not static structures but are undergoing some continual remodelling. 2.2. ABANDONED SYNAPTIC GUTTERS IN NORMAL MUSCLES

Long after denervation and disappearance of nerve and Schwann cell elements, the former location of synaptic contacts on the muscle fibers can be precisely defined. This is due to the preservation of postsynaptic elements like primary gutters, secondary folds, and the transmitter hydrolyzing enzyme acetylcholinesterase (ACHE), after denervation. Such abandoned gutters have recently been observed in normally innervated muscles in the vicinity of or within otherwise normal contacts. 2.2.1. Frog muscles To recognize abandoned gutters in the light microscope, one can take the advantage of the artificial accumulation of AChE reaction product at the lateral edges of nerve and/or Schwann cell-occupied gutters and the lack of visible amounts of reaction product within the occupied gutter itself (Fig. 1). In combined axon and AChE-stained preparations, abandoned gutters are even more obvious (Letinsky et al., 1976; P&ot-Dechavassine et al., 1979; Wernig et al., 1980a,b). Figure 1 shows a light micrograph of part of a junction after staining for axon and ACHE. The bars of reaction product to the right indicate an abandoned gutter. Electron microscopical examination of serial sections through such sites reveal that secondary folds filled with AChE reaction product are present (Fig. 2) while presynaptic elements usually are missing. In the section depicted in Fig. 2 it appears that a Schwann cell profile contained in an excessively large basal lamina sheath (arrows) is present at some distance above the muscle fiber. Most likely, this abundant basal lamina and a small Schwann cell process have remained after the nerve terminal has completely retracted. In normally occupied parts of the junction the muscle fiber provides a groove for the axon and Schwann cell called the primary gutter. After denervation this primary gutter flattens and eventually disappears (Verma, 1980). The abandoned site shown in Fig. 2 has apparently lost its primary gutter. Though we did not systematically measure the depth or volume of secondary folds, it appeared that these were also markedly reduced in size at some sites. This decrease in secondary folds is also seen after denervation. It is likely, therefore, that after nerve retraction the primary gutter and the secondary folds eventually disappear, or soon become reoccupied. These changes might explain why abandoned gutters in normal muscles have not been described in previous ultrastructural studies, especially when they were performed on muscles not stained for ACHE. It is important to ask whether axons retract or parts of axons become separated and degenerate. In the case of degeneration, axon fragments engulfed by or incorporated in phagocytes should be present. Such cases have so far not been reported in electron microscopic studies of normal muscles. In the light microscope one might expect to find silver-stained axon fragments within isolated Schwann cells in otherwise abandoned gutters. Such features have been found (Wernig et al., 1980b), but too rarely to account

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for such changes as the increase in abandoned gutter length seen in summer frogs (Wernig et al., 1980a,b; see below).

One can quite regularly find occupied gutters without stained axonal elements (Fig. 4 and Wernig et al., 1981a). These could be signs of previous nerve terminal retraction with the Schwann cell processes remaining in the gutters, similar to what occurs after nerve section, where Schwann cells remain in the gutter for a prolonged period of time (Birks et al., 1960; Krause and Wernig, 1985). Several observations indicate the existence of partially occupied gutters. In randomly sampled ultrathin transverse muscle sections of junctions from untreated frogs, some 6 to 13% of the gutters were partially occupied, i.e. the presynaptic elements, axon and Schwann cell, did not completely fill the primary synaptic gutter (Wernig et al., 1980a). Figure 3 shows a representative electron micrograph from a transverse section through a synaptic gutter which illustrates this occurrence; the axon occupies only a fraction of the width of the gutter and a junctional fold opens outside the contact area (asterisk). In the light microscope (Fig. 5C and inset) one finds secondary folds, outlined by AChE reaction product, extending beyond the lateral edges of normally occupied gutters. There are at least two mechanisms which can account for the discrepancy between gutter width and diameter of the occupying nerve terminal branch. First, axon profiles might be reduced in diameters. This could happen in the process of abandoning or reoccupying gutters when nerve terminals probably shrink before they retract and are thinner during sprouting. In other cases, especially in central parts of junctions (Fig. 5C), terminal branches might not shrink but move laterally to form new contacts, thereby widening the synaptic gutter. In Fig. 5C, for example, there are unoccupied areas at several places which are too wide to have been occupied by a single terminal branch at a time. Morphometric data indicate that both mechanisms occur, since, on average, partially occupied gutters are wider and contain smaller axon profiles than normally occupied gutters (Wernig et al., 1980a). Abandoned gutters can also be present without disappearance of the nerve terminal. Unoccupied gutters can often be seen within junctions that otherwise appear to be normally innervated (Fig. 5A,B; "central" abandoned gutters in Fig. 14). Most likely, the axon in these cases has lifted up and is separated from the gutter, possibly by intervening Schwann cells. Synaptic transmission does not occur at these sites since ACh receptor clusters are invariably missing (Fig. 5A,B; Krause and Wernig, 1985). In a group of summer frogs 27% of all abandoned gutters were in such a central location (Jans et al., 1986). Possibly, the maintenance of synaptic contacts may involve local feedback mechanisms. When maintenance fails, nerve terminals withdraw. In frog muscles, abandoned gutters are not rare occurrences. In one group of summer frogs abandoned gutters were found in 40-75% of the junctions in the cutaneous pectoris muscle, with average abandoned gutter lengths ranging from 30 to 70 # m (Fig. 6). When the summed length of abandoned gutters on a muscle fiber is expressed as a fraction of the total gutter length (i.e. normally occupied plus abandoned gutters) more than half of the values are larger than 10% (cf Fig. 16). Another important finding is that the amount of abandoned gutter varies with different environmental conditions (Fig. 6; see Fig. 17). Also, abandoned gutters are more frequent in larger and presumably older frogs (see Fig. 16). It appears, then, that nerve terminal retraction can be influenced by a variety of factors. One can conclude from these observations that nerve-muscle contacts are not static structures. The mere existence of abandoned gutters and their drastic change within a few months (summer-winter) strongly suggest that there is remodelling of individual neuromuscular junctions. Remodelling involves nerve terminal retraction, sprouting, reoccupation of abandoned gutters, new synapse formation, and possibly also lifting up, lateral movement and change in nerve terminal diameter. 2.2.1.1. A C h receptors Abandoned synaptic gutters lack the ACh receptor clusters that are normally present in the postsynaptic membrane (Wernig et al., 1981 b; Anzil et al., 1984; Krause and Wernig,

FIG. 1. Light micrograph of part of a neuromuscular junction in a normal frog muscle (cutaneous pectoris of Rana temporaria) after staining with nerve terminal and AChE stains. To the left, the presence of presynaptic elements causes the AChE reaction product to accumulate in 2 parallel rows that outline the lateral edges of the synaptic gutter. A, silver stained nerve terminal; S, Schwann cell nucleus. To the right, an abandoned gutter is visible. Secondary synaptic folds can be seen as a pallisade-like arrangement of bars (asterisk) because of the absence of overlying presynaptic elements. Note the absence of terminal staining in the abandoned gutter. Bar = 20/zm. From Wernig et al. (1981d). FIG. 2. Electron micrograph of a transverse section through a normal frog muscle stained for ACHE, which forms electron dense crystals of reaction product (Karnovsky and Roots, 1964). Reaction product fills a well developed secondary fold and the shallow primary gutter. The presynaptic nerve terminal is missing. The cellular element present some distance above the muscle fiber (S) might be a retracting Schwann cell process with remaining basal lamina (arrows). Bar = 0.5 lam. A. Wernig, unpublished. FIG. 3. Electron micrograph from a transverse section through a normal frog muscle. The opening of the junctional fold (asterisk to the left) is not apposed by an axonal terminal; this indicates that the axon (A) with its accompanying Schwann cell (S) now occupies only a small part of the width of the synaptic gutter. Either the nerve terminal branch was much larger before and once occupied the whole width of the gutter or the terminal branch has moved laterally and formed a new synaptic site close to the previously abandoned one. Bar = 0.5 #m. M. Prcot-Dechavassine, unpublished micrograph. FIG. 4. Light micrograph of the distalmost part of a frog neuromuscular junction after staining the nerve terminal and ACHE. The silver stained axon (thin line) enters from the right and ends within the middle ring of AChE reaction product (arrow). The nerve terminal has probably retracted, leaving the ring to the left occupied only by the Schwann cell. A. Wernig, unpublished.

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FiG. 5. (A,B) Light micrographs of part of a frog neuromuscular junction after labelling with rhodamine-alpha-bungarotoxin (B) and staining for nerve terminal and AChE (A). The axon (out of focus, stippled) enters at the arrow, parts into two branches which run on top of an abandoned gutter (asterisk). At the abandoned gutter ACh receptor clusters are missing (B). Bar = 10/~m. (C,D) Light micrographs of part of a junction in a normal frog muscle whole mount. The preparation was labelled with rhodamine-alphabungarotoxin (D) and subsequently stained for AChE (C). Secondary folds extend laterally from the occupied gutters where toxin binding sites apparently are missing. The inset corresponds to the region indicated by the rectangular box in C. Here the fluorescent receptor bars (stippled) are superimposed on the AChE staining pattern (unbroken lines) and it appears that both stains delineate one and the same junctional folds. Bar = 10 ~m. From Krause and Wernig (1985). (E,F) Part of a frog neuromuscular junction from a normal animal after labelling with FITC-alpha-bungarotoxin (F) and staining the nerve terminal and AChE (E). The abandoned gutter to the fight is free of receptor clusters. Bar = 5 #m.

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FIG. 6. Quantitative evaluations o f signs of nerve sprouting (sprouts) and nerve retraction (abandoned gutters) in freshly caught summer (S) and winter (W) frogs of 6 cm body size. The relative number (%) of synapses in the two cutaneous pectoris muscles of a frog showing the parameter under investigation is shown (full circles), the mean number of sprouts and the mean summed length of abandoned gutters on the muscle fibers which show these parameters (open circles) are indicated. Sprouts were defined as small nerve terminal branches with small contacts ( < 15 #m) and were evaluated in I1 summer frogs (total of 400 muscle fibers) and 7 winter frogs (221 muscle fibers), abandoned gutters in 7 of the summer frogs (226 muscle fibers) and 4 of the winter frogs (120 muscle fibers). In rank tests the differences between summer and winter frogs are significant (p < 1%). Data from Wernig et al. (1980a).

1985). This is true for central abandoned gutters with the nerve terminal nearby (Fig. 5A,B), gutters which are much wider than the occupying axon (shrinkage and/or lateral shift of the axon thereby expanding the gutter; Fig. 5C,D), and abandoned gutters after nerve retraction (Fig. 5E,F). This finding suggests that ACh receptor clusters are actively maintained by nerve borne factors and are mainly present opposite the actual release sites (active zones; Couteaux and P6cot-Dechavassine, 1970, 1973; Wernig, 1976; see Fig. 22); even on single junctional folds receptor clusters are missing lateral from the axon contact (inset in Fig. 5C,D). Vysko6il et al. (1981) have isolated a peptide from the rat sciatic nerve that induced ACh sensitivity on nerve terminal-free segments of innervated muscle fibers and similarly, factors have been found to be effective on cultured muscle cells (Podleski et al., 1978; Jessel et al., 1979; Bauer et al., 1981). 2.2.2. M a m m a l i a n muscles Once one accepts the idea that nerve terminals can retract from synaptic sites one finds features in normal mammalian muscles which can be interpreted this way. Abandoned gutters are shorter and less frequent than in frog muscles, which might simply be due to the smaller size of mammalian endplates or may represent a fundamental difference between homeotherms and poikilotherms. In the mouse, Wernig et al. (1984) found empty gutters in distal prolongations of nerve terminal branches. The gutters contained empty basal lamina sheaths over much of their length, which suggests previous occupation by nerve terminals and Schwann cells (arrows in Fig. 7A-F). Unoccupied secondary fold-like invaginations could be seen in the vicinity of these former synaptic contacts, but they were rare and small. Perhaps secondary folds disappear rapidly after nerve retraction in the mouse. Such rapid disappearance has been seen as early as 5 days following complete denervation of the mouse soleus (Brown et al., 1982). In very thorough studies on rat muscles which, however, were not done with serial sectioning, Cardasis and Padykula (1981) and Cardasis (1983) found gutters with secondary folds that were not apposed to presynaptic elements. The occurrence of such sites increases significantly with age of the animal (Cardasis, 1983). Abandoned gutters have previously been found in aged rats by Fujisawa (1976) and Gutmann and Hanzlikowfi (1976) and have been considered to be signs of aging (see Section 3.3.1).

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Signs of nerve degenervation (like phagocytosis) have not been found at mammalian junctions (cf Fujisawa, 1976). Thus, if the features just described represent abandoned gutters, nerves seem to retract rather than degenerate. Also similar to the frog is the appearance of terminals much thinner than the width of the gutters, which could represent retracting or regenerating branches. Abandoned gutters with AChE activity have not yet been described for mammalian junctions with the light microscope, as they have been for frogs. In disused but innervated cat muscles, Eldridge et al. (1981) found parts of the AChE-stained junction bare of ACh receptors which, by analogy to frog muscles (cf Fig. 5A,B and Krause and Wernig, 1985) might represent abandoned gutters with or without Schwann cells present. As in frog muscles there is junctional redistribution which does not necessarily lead to changes in the number or length of intraterminal nerve branches. With age, axonal diameters along single branches shrink at some spots and/or enlarge at others, suggesting a kind of redistribution of axonal volume (Robbins and Fahim, 1985). At the thin parts synaptic contacts apparently are abandoned (cf the abandoned synaptic gutters in "central" locations in frog junctions; Fig. 14), and as a consequence, previously continuous contacts become segregated into smaller regions. The significant increase in the number of discrete regions of high ACh receptor density seen in junctions from older animals (Courtney and Steinbach, 1981) is consistent with this hypothesis. This increase demonstrates that junctional sites within the endplate are abandoned and ACh receptor clusters disappear though the nerve terminal is still nearby (lifted off, wrapped by Schwann cell?). So far it is not clear whether secondary folds and AChE remain in these abandoned parts, as they do in the central abandoned gutters in frog muscle (Figs 5A and 14). In frog muscle it has also been demonstrated that ACh receptors disappear from such sites (Fig. 5A,B). 2.3. NERVE SPROUTING AND NEW SYNAPSE FORMATION

The opposite of nerve retraction, nerve sprouting and new synapse formation, has also been identified in untreated adult vertebrate muscles. While it is possible to positively identify sprouts in frog muscle (Wernig et al., 1980a,b, 1981a; Anzil et al., 1984), this proves to be more difficult in mammals (Cardasis and Padykula, 1981; Cardasis, 1983; Wernig et al., 1984). However, the increase in the number of intraterminal branches with age in mammals constitutes powerful indirect evidence for continual sprouting (see Section 3.3). 2.3.1. Frog muscles In terminal and AChE-stained muscles, small (less than 5/~m) synaptic contacts can frequently be found within neuromuscular junctions. Secondary folds, the morphological marker for mature junctions, are either poorly developed or completely missing in serial sections through such contacts (Fig. 8A-D). Since other synaptic features are present (vesicles in the axon, direct axon-muscle fiber contact, ACHE, and ACh receptors at the postsynaptic membrane), and secondary folds develop only some time after the contact has been functioning (Ter/iv/iinen, 1968; Koenig, 1973), it is concluded that these contacts have been newly formed. At other spot-like contacts secondary folds are well developed (Anzil et al., 1984) so that junctional sites in different states of maturity can be seen within a single endplate. Other observations indicate that axons also grow into previously abandoned gutters where they re-establish synaptic contacts. For example, in winter frogs abandoned gutters are shorter and seen less frequently (cf Fig. 6). In ultrastructural investigations of these same junctions, thin axons wrapped by Schwann cells but lacking synaptic vesicles can be found in disproportionately wide gutters (cf Wernig et al., 1980a,b). Frogs grow throughout life (Mtiller, 1976), and with growth there is an increase in muscle fiber diameter, synaptic length, and the number of intraterminal branches (Fig. 9; Wernig et al., 1980b, 1981c; Jans et al., 1986). Clearly, then, there is growth- and

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FIG. 7. A b a n d o n e d gutter in normal m o u s e soleus muscle. Upper part: schematic reconstruction based on serially cut ultrathin sections o f part o f a Z n l O s stained endplate. The axon (black) is surrounded by the Schwann cell (S; hatched area = Schwann cell nucleus), which reaches into a groove (stippled area) formed by the muscle fibers. The vertical lines marked A - F indicate plane and position of the sections shown below. Bar = I0 # m . Lowerpart: Selected electron micrographs. (A) The axon (black) is in contact with the muscle fiber. (B) The wide gutter contains the accompanying Schwann cell (S) without axonal profile. (C-F) The otherwise empty gutter contains redundant basal lamina sheaths (arrows) which were present in all sections from this region. Bar = 1 # m . F r o m Wernig et al. (1984).

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FIG. 8. Nerve terminal sprout and newly formed synaptic sites in normal frog cutaneous pectoris muscle. Upper part: schematic reconstruction based on serial sections of the distal part of a thin presynaptic nerve branch on a AChE stained muscle. The axon is wrapped by the Schwann cell (stippled area) along most of its length, is enlarged at several sites and some but not all enlargements (asterisk) are in contact with the muscle fiber (M) through openings in the Schwann cell sheaths. At the contacts AChE reaction product (black), high electron density in the subsynaptic membrane and synaptic vesicles in the axon can be found, all signs of a true synaptic contact. Secondary folds are missing, however, suggesting that these are newly formed contacts. A-D: position and plane of the sections shown below. Bars = 1 pm. LOUW JUZ~~:Electron micrographs from transverse sections through the nerve branch reconstructed above. N = Schwann cell nucleus, M = Muscle fiber. The axon diameter is small in A (arrow), still completeIy enveloped by the Schwann cell in B and D but is in direct contact with the muscle fiber in C. Secondary folds are missing. Asterisks: crystals of AChE reaction product or corresponding holes from which crystals have been lost from the sections. Vertical arrows: membrane thickenings. Bar = OS pm. From Anzil et al. (1984).

260

SPROUTING AND REMODELLINGAT THE NERVE-MuSCLE JUNCTION synaptic length(~uml

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FIG. 9. Mean values and standard deviations for muscle fiber diameter, synaptic length and total number of intraterminal branches in cutaneous pectoris muscles of flogs with body sizes from about 4.2 to 7.5 cm. The age range for these flogs is between 3 and 6 years. Note the large scatter of values for the resuls from a frog. With growth in body size there is a shift to larger values for all 3 parameters.

age-related sprouting. Similarly, there is a significant increase in the mean number of sprouts per junction in winter frogs as compared to equally sized summer frogs (Figs 6 and 17), with a concomitant decrease in the incidence of abandoned gutters (Figs 6 and 17). For further details and discussion see Section 3.2. 2.3.2. M a m m a l i a n muscles Growth cone-like structures filled with large clear vesicles, similar to those seen in embryonic neurons (Tennyson, 1970; Kawana et al., 1971; Vaughn and Sims, 1978), have also been found in adults, in autonomic nerves innervating the iris (Townes-Anderson and Raviola, 1976). Such structures have so far not been detected in skeletal muscles of vertebrates. Since they are apparently also missing after sprouting is induced by toxin treatment (Duchen, 1971, 1973), typical growth cones might not occur or be rare in skeletal muscles (but see Letinsky et al., 1976). Since there is also in mammalian muscle a net increase in the number of intraterminal branches with age (see below), new synaptic contacts should be present. To identify sprouts we adopted the strategy successful in frog muscles, i.e. using light microscopy to localize thin nerve terminal branches in ZnIOsstained preparations. Thin branches are the most likely candidates for newly formed contacts. These same branches are then reconstructed from electron micrographs of serially cut ultrathin sections (Wernig et al., 1984). Fifteen thin branches in five mouse soleus junctions were investigated this way. These branches were unusual in several respects (see below) but had synaptic contacts with secondary fold-like invaginations somewhere along their length. Though we did not measure them, the width, depth, and especially the number of secondary folds per unit contact length appeared smaller than in other contacts. Most strikingly, however, these branches in addition had direct axon-muscle fiber contacts without secondary folds (Fig. 10). Occasionally, presumably abandoned parts of gutters contained small axonal profiles in contact with the muscle fiber. Such a case is shown in Fig. 11, where a small axon that for unknown reasons failed to stain properly with ZnIOs contains synaptic vesicles and is in contact with the muscle fiber. A single small infolding is present at the contact (asterisk), but several large infoldings unapposed by presynaptic elements are present nearby (arrows). A mature synaptic contact can be seen on the left hand side of Fig. 11. In our investigation we found two types of branches. First, we saw thin filaments with bulbous enlargements at their distal tips, often with additional enlargements along their length. These branches were wrapped by Schwann cells along most of their length and were in direct contact with the muscle fiber only at small circumscribed areas, where the axon was considerably enlarged (Wernig et al., 1984). This arrangement of axonal enlargements

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connected by thin filaments has a surprisingly close resemblance to sprouts identified in frog muscles. The second type of thin intraterminal nerve branch investigated was usually short (less than 5/~m) and without bulbous enlargement. Occasionally we found a small, separate endplate near a larger one (cf Tuffery, 1971; Courtney and Steinbach, 1981). In one such case that we investigated by serial sectioning (Wernig et al., 1984), synaptic contacts without secondary folds were relatively frequent. It appears that secondary folds in mouse soleus muscle develop rapidly following nerve contact. This notion is supported by experiments in which extensive sprouting and new synapse formation was induced by local injection of tetanus toxin (Duchen, 1973; Duchen and Tonge, 1973; Wernig et al., 1981d). In several endplates investigated by serial sectioning, all small axonal branches in contact with the muscle fiber were at least at some spot associated with AChE-containing secondary folds as early as four days following toxin application (Fig. 12). For short distances even larger axonal profiles unapposed to secondary folds could also be found (Fig. 13, left). In summary, there are points of nerve terminal apposition in normal mammalian muscles which, because of their lack of secondary folds, are likely to be new synaptic contacts. This notion is further emphasized by the finding of other ultrastructural peculiarities in thin nerve branches like their Schwann cell wrapping and small synaptic contacts at which only few rather simple secondary folds are present. The ultrastructural evidence for abandoned gutters (see above) strongly suggests that synapses in mammalian muscles are continually remodelled. The balance between nerve sprouting and nerve retraction might change in the course of life to give net changes in the number of intraterminal branches (Section 3.3). 2.4. MECHANISMS OF REMODELLING One simple hypothesis for the control of junctional remodelling is that sprouting is inherent to nerve terminals but is counterbalanced or even reversed (nerve retraction) by activity of the junction. While these factors might control local or short term remodelling at the junction, the role of activity for the whole neuron might be different. Neuronal cell bodies in the corpus geniculatum laterale of kittens shrink within one week when presynaptic inputs are blocked and neurons are presumably less active (Kuppermann and Kasamatsu, 1983). It has been suggested that formation of neurofilaments and neurotubules, which are regularly broken down by a Ca-activated protease, is the driving force for axonal sprouting (Lasek and Hoffmann, 1976; Schlaepfer, 1979; Schlaepfer and Micko, 1979). In our studies of frog muscles, we found that abandoned gutters were more frequent in more active frogs (summer vs winter, Wernig et al., 1980a,b; and to some degree freshly caught summer frogs vs "laboratory frogs", Jans et al., 1986; see Section 3.2.2). These seasonal differences could be due to a higher Ca influx into the more active nerve terminals, causing enhanced breakdown of the cytoskeletal elements and nerve retraction. Opposite changes would occur in less active animals. Such mechanisms could account for the different architecture of endplates in slow versus fast muscles (Robbins and Fahim, 1985; Hopkins et aL, 1985). Slow soleus motor units of freely moving adult rats are manyfold more active in a 24 hr period than fast units in e.d.1. (Hennig and L6mo, 1985), and their endplates are strikingly less branched and much more compact (Waerhaug and Korneliussen, 1974). The acceleration of developmental synapse elimination by imposed activity, and its retardation when activity is blocked, is further evidence for this hypothesis (reviewed in Purves and Lichtman, 1985). The hypothesis could also be experimentally tested in adult animals by imposing high activity on motor neurons. This should in the beginning cause some reduction in nerve terminal length, but rather opposite changes in the size of the neurons' cell bodies. 3. Growth, Age and Other Factors In this section we shall consider the questions of whether and by which mechanisms synapse remodelling might be influenced. It is clear from many previous studies (see

FIG. I0. Synaptic contact without junctional folds. Transverse section through a Z n l O s stained endplate in normal soleus muscle. To the right the axon (black) is in direct contact with the muscle fiber but secondary folds are missing. S = Schwann cell. Female N M R I mouse aged 18 months. Bar = 0.5 g m . FIG. 11. Electron micrograph from a transverse section through a Z n l O s stained endplate. Unstained axonal profile at A, stained axon to the left (black). S = Schwann cell. Unoccupied secondary fold-like invaginations at arrows, small invagination near asterisk. These features were present on several consecutive sections. Female N M R I mouse aged 24 months. Bar = 0.5 # m .

263

FIGS 12, 13. Electron micrographs showing immature synaptic sites besides well developed contacts 4 days after the injection of tetanus toxin. Mouse soleus muscles were injected with a high dose of toxin (about 50 x LD 50) followed 30 min later by i.v. antitoxin administration. Muscles were stained for A C h E (Strum and Hall-Craggs, 1982). Several small axonal profiles (A) were found in contact with the muscle fiber and more often secondary folds and some A C h E reaction product were present or nearby (Fig. 12, arrows). Elsewhere, however, even larger axonal profiles with faint A C h E activity in the synaptic cleft were present. Such a contact which lacked secondary folds is visible in Fig. 13 (left). In both pictures some synaptic sites with well developed secondary folds are also present. S = Schwann cell. Bars = 0.5 # m .

264

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Section 3.1) that growth in muscle fiber size is paralleled by junctional growth. Aging, and in particular old age, has long been known to have effects on muscle structure and function (Sections 3.2 and 3.3). For mammalian muscles in particular, the question arises as to whether senile muscle atrophy is a disease of the peripheral motor system with specific structural features or can be explained by physiological variation, such as reduced levels of motor activity (Section 3.3.1). In frog muscle, marked seasonal variation in junctional structure and function has been observed and will be discussed (Section 3.2.2). Finally, there are disorders of the neuromuscular contact in mammals including man which produce structural changes resembling enhanced or distorted remodelling (Section 3.3.2). 3.1. MATCHING OF MUSCLE FIBER AND SYNAPTIC PARAMETERS

One of the most striking structural features of muscle is the diversity in fiber size present within a single muscle. This diversity is already present in the postnatal period. Nystr6m (1968) found that muscle fiber diameters in soleus muscles of newborn cats ranged from about 6 to 21 #m developing to a range of about 35 to 77 #m in the adult. Kugelberg (1976) found that in young rat muscles, fiber areas are more similar within a motor unit than for the whole muscle and that type I motor units tend to have larger fibers than type II motor units. This suggests that the synchronous activity of fibers within a motor unit somehow creates similarity in fiber diameters. Surprisingly, Trussell and Grinnell (1985) found that the range of fiber input impedance within single motor units of the Rana pipiens cutaneous pectoris was not much smaller than for the whole muscle, while the range does appear to be smaller in Xenopus pectoralis muscle (Nudell and Grinnell, 1985). In focally innervated muscle fibers synaptic parameters such as endplate area, the number and length of intraterminal branches, and contact area are impressively matched with muscle fiber size in young and adult muscles (Nystr6m, 1968; Anzenbacher and Zenker, 1963; Raberger, 1971; Kordylewski, 1979; Grinnell and Herrera, 1980; Trussell and Grinnell, 1985; Wernig et al., 1986; Jans et al., 1986). Interestingly, Anzenbacher and Zenker (1963) found that synaptic size was better correlated with muscle fiber volume than diameter. These correlations are consistent with the hypothesis that there are feedback mechanisms regulating muscle fiber and nerve terminal size (see below). Several hypotheses can be advanced to explain this matching between junctional and muscle fiber size. Growing muscle fibers may provide more of a nerve sprouting factor, possibly similar to that postulated to be released from inactive muscle fibers (see Cotman et al., 1981, for review) or factors found in muscle cell cultures (Giller et al., 1973, 1977; Henderson et al., 1983). Another hypothesis is based on the effects of muscle fiber growth on activity. Fiber growth leads to a decrease in input impedance which may cause synaptic potentials to be subthreshold more often (see Section 4). The less active fibers would then produce nerve sprouting factors. In order for junctions to grow, there must be an increase in the area of muscle fiber membrane on which new synapse formation is possible. The existence of such a permissive area is demonstrated by several observations: implanted foreign nerves do not form ectopic junctions but apparently form junctions at the existing endplate areas (see Bixby and Van Essen, 1979), and new synapse formation in the course of remodelling is confined to the endplate region. Only after denervation or synapse blockade (e.g. by botulinum toxin, see Tonge, 1977) this area spreads, possibly to include the whole muscle fiber surface. Spread of this area is prevented by direct electrical stimulation of denervated muscles (L6mo and Slater, 1978) indicating that this and other changes occurring after denervation are controlled by muscle activity rather than by nerve trophic factors (L6mo, 1976; for further discussion see Grinnell and Herrera, 1981). 3.2. FROG MUSCLES

We recently performed morphometric studies on frog cutaneous pectoris muscles aimed at distinguishing between changes due to growth, age, and external factors. We compared

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flogs o f different b o d y size (and thus different age) taken from the s a m e environment and flogs o f similar b o d y size but kept under different environmental conditions (Wernig e t a l . , 1986; Jans e t a l . , 1986). Figure 14 s h o w s the aspects o f junctional structure that were evaluated.

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3.2.1. Growth versus age changes As has been shown in a number of previous studies (see Section 2.3.2), thin muscle fibers bear smaller and less complex synapses than thick muscle fibers. This is shown in Fig. 15 where muscle fiber diameters from six frogs (two cutaneous pectoris muscles each) are plotted against synaptic length, i.e. summed length of the terminal branches on a muscle fiber, and number of intraterminal branches. Since frogs grow throughout life (Miiller, 1976) ontogenetic changes will consist of both, age- and growth-related changes. To exclude growth-related changes we compared muscle fibers of the same diameter in frogs of different age, presumably 2-6 years (Fig. 16). We found differences in synaptic structure which should thus reflect age-related changes. This approach rests on the assumption--and there is no good evidence to the contrary--that

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there is little or no turnover of muscle fibers during life. Synaptic length (contact lengths plus connecting parts of axons, see Fig. 14) and number of intraterminal branches are larger in the older animals with little differences for small muscle fibers but enormous ones for large fibers (Fig. 16a,b). Both, the absolute and the relative lengths of abandoned gutters increase in a similar way in older frogs (Fig. 16c,d). Apparently, in the course of life-long remodelling, abandoned gutters accumulate. This indicates that they eventually lose their attraction for sprouting axons, so that new synaptic contacts are formed rather than abandoned sites reoccupied. Further evidence for an accumulation effect may be the larger synaptic lengths on fibers of equal diameter in old versus young frogs. Junctions apparently grow when muscle fibers grow but they may not be reduced in length when muscle fibers shrink, e.g. due to a reduction in activity or load. In this view synaptic length would to some degree reflect the maximal size over time. The accumulation of synaptic length could also be due to formation of less active contacts during remodelling (smaller or fewer active zones) which might stimulate additional growth in nerve terminal length. This would explain why on muscle fibers of equal size, larger terminals release less transmitter per unit length of nerve terminal than smaller ones (Nudell and Grinnell, 1982). These differences in release per unit length, which were primarily tested with low frequency stimulation, tend to be reduced at higher stimulus frequencies (Nudell and Grinnell, 1982; see Section 4). None the less, it is likely that large junctions release more transmitter than smaller ones (Kuno et al., 1971). Conceptually, the structural changes could be compensatory reactions to a decline in synaptic efficacy, but unfortunately this has not been investigated in frog muscle. There are similar structural findings in mammalian muscles where more is known about functional changes with aging (Section 3.3.1). 3.2.2. Seasonal factors When freshly caught summer frogs of similar body size (6 cm) are kept under defined laboratory conditions for a period of 16 weeks, synapses changed (Fig. 17). While no changes in muscle fiber diameter and synaptic length could be detected, the number of intraterminal branches and sprouts increased by one to two per synapse (Fig. 17a,b). Jumping fiber lengths also increased while the large abandoned gutter lengths were reduced in laboratory frogs (Fig. 17c,d; Wernig et al., 1986; Jans et al., 1986). There have been several reports indicating that synaptic transmission in frog muscles is different in different seasons (Otsuka et al., 1962; Braun et al., 1966; Maeno, 1969). This was also noted in a recent study on two groups of frogs comparable to the ones reported above (Fig. 17). First, a somewhat larger concentration of d-tubocurarine (7.5/~g/ml) had to be used in summer frogs versus winter frogs (6 #g/ml) to obtain synaptic potentials (epps) of similar amplitude (Fig. 18), which reflects the reduced output of transmitter observed in winter frogs (Otsuka et al., 1962; Maeno, 1969). As expected for lower levels of release, winter frogs showed much greater facilitation of transmitter release upon tetanic stimulation (Fig. 18). The structural changes observed in the corresponding morphological investigation (Fig. 17) do not readily explain these physiological differences. Synaptic length did not decline in winter (average of 695 ~m in the freshly caught summer frogs vs 690 # m in laboratory frogs in winter) and the number of branches actually increased (Fig. 17). The change in facilitation behavior by itself suggests rather subtle changes occurring on the molecular level. Most interestingly in this context, single-channel conductance of the ACh receptor channel was found to change with seasons: it is higher in winter as compared to summer frogs (Lewis, 1984). The differences in environment and season are, of course, manyfold (food, motility, hormonal status, temperature, etc.) and it will be challenging to consider these factors separately in future studies. 3.3. MAMMALIAN MUSCLES 3.3.1. Aging There has been a number of reports over the last 30 years that described structural features of mammalian nerve muscle junction which were thought to be more or less typical

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signs of aging (for some recent results see: Fujisawa, 1974, 1975, 1976; Gutmann and Hanzlikov~t, 1965, 1976; Pestronk etal., 1980; Fagg et al., 1981; Fahim and Robbins, 1982; Fahim et al., 1983; Cardasis, 1983). When considered in the context of synapse remodelling, some of these features can be seen as typical signs of nerve terminal sprouting and retraction. Two examples are gutters or secondary folds unapposed by axonal elements (Fujisawa, 1976; Gutmann and Hanzlikov/t, 1976; Banker et al., 1983; Cardasis, 1983; Wernig et al., 1984), and changes in the complexity of junctions (Gutmann and Hanzlikovfi, 1965; Pestronk et al., 1980; Fagg et al., 1981; Smith and Rosenheimer, 1982; Fahim et al., 1983). When comparing mice aged 3, 6 and 11 months, a general shift to more complex endplates was observed in ZnlOs-stained soleus muscles, while the proportion of simple endplates declined (Fig. 19). The largest change occurred between the age of 3 and 6 months, but the increase from 6 to 11 months was still significant (Wernig et al., 1984). As endplates became more complex, their total lengths increased (Table 1). During these JPN 27/3--E

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FIG. 18. Facilitation of transmitter release in 5 summer frogs (full lines) and 3 winter frogs (dashed lines). Frogs (Rana temporaria) with a body size of 6cm were caught in April, kept under laboratory conditions till their use in June to August (summer frogs) and January to February (winter frogs). Isolated nerve-muscle preparations of cutaneous pectoris muscle containing the distal nerve stump were bathed in normal Ringer solution containing 6 #g/ml d-tubocurarine in winter and 7.5 #g/ml in summer. Conventional intracellular recordings of nerve-evoked endplate potentials (epps) were monitored upon tetanic nerve stimulation (40 Hz for 3 see). During recordings temperature was kept constant between 15 to 17°C in winter and 18 to 20°C in summer. Maximum epp in a train in percent of the first epp is plotted against the absolute value (in mV) of the first epp. Points connected by lines are average relative epp values for given ranges of values of the first epp (< 1, 1-<3, 3 - < 5 etc. mV) obtained from individual frogs. The total number of synapses evaluated was 132 in the summer frogs and 98 in the winter frogs. s a m e t i m e s t h e r e w a s a s i g n i f i c a n t i n c r e a s e in b o d y w e i g h t , w h i c h m o s t likely also c a u s e d a c h a n g e in s o l e u s activity. I n a v e r y t h o r o u g h s t u d y u s i n g light a n d s c a n n i n g e l e c t r o n m i c r o s c o p y , F a h i m et al. (1983) f o u n d i n c r e a s e s in v a r i o u s a s p e c t s o f j u n c t i o n a l c o n t a c t size in t h e e x t e n s o r d i g i t o r u m l o n g u s o f y o u n g (7 m o n t h s ) v e r s u s o l d (29 m o n t h s ) mice.

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TABLE 1. EXPERIMENTALRESULTS OF THE SYNAPSE MORPHOMETRY ON ZnlOs STAINED MOUSE SOLEUS MUSCLES FROM 3, 6 AND 11 MONTH OLD ANIMALS

3 months 6 months 11 months Number of animals 6 6 6 Weights of animals (g) 30.5 + 2.05 37.7_+1.73 46.44- 1.38 Number of synapses evaluated 433 413 558 Number of intraterminal branches 16.2 4- 7.1 24.7 4- 11.4 28.74- 11.6 Endplate length (#m) 53.8 4- 16~8 60.84- 19.6 67.24- 18.5 Muscle fiber diameter (#m) 28.3 4- 5.8 35.9 4- 7.8 37.4 + 8.0 Mean values and standard deviations of the evaluations of number of intraterminal branches per synapse, endplate length and muscle fiber diameters. In rank tests differences between the 3 and 6 month group were significant for all parameters. 11 months old mice had significantly longer and more complex terminals than 6 months old ones (p < 5%) while muscle fiber diameters were not different. From Wernig et al. (1984), see there for details of statistical analysis. In rat diaphragm, Pestronk et aL (1980) found an increase in the number of intraterminal branches from age 2 months to 10 months (most likely including a significant growth period), a trend for a further increase from l0 to 18 months, and a drastic decrease in number of branches from 18 to 28 months. Changes in endplate complexity have recently been described in several other species and muscles (Smith and Rosenheimer, 1982; Hopkins et aL, 1985; Robbins and Fahim, 1985; Rosenheimer and Smith, 1985). While these results provide evidence for new formation of terminal branches during adult life, they also indicate that in later adulthood there might be a decline in synapse complexity and that different muscles behave differently. Along with structural changes in aging muscles, there have been reports of altered transmitter release (Gutmann, et al., 1971; Kelly, 1978; Kelly and Robbins, 1983; Banker et aL, 1983) and synthesis (Tu~ek and G u t m a n n , 1973). Different muscles behave differently, e.g. mouse soleus and e.d.l. consistently show increased resistance to curare and Mg-block, while the block resistance and thus the safety factor for transmission remain the same in mouse diaphragm (Banker et aL, 1983; Kelly and Robbins, 1983). Since the diaphragm necessarily remains active in old age, while other muscles possibly are used less, one could postulate a crucial role for the amount and/or pattern of activity in governing processes of transmitter release as well as junctional architecture. The current observations favour the idea that synapse remodelling and in particular nerve terminal sprouting are continuously active, inherent processes. When sprouting is not sufficiently counterbalanced by activity-dependent terminal retraction, endplates on a longer time scale increase in size and/or complexity. The net result of remodelling might thus depend on the actual activity of a muscle or, in the strict sense, of each m o t o r unit. In most cases, including frog muscles (Fig. 16), this results in an increase in junctional complexity with age. Clearly, it will be interesting to study different muscles at different times of adult life and to relate changes in morphology and synaptic transmission to changes in use (cf Robbins and Fahim, 1985; Rosenheimer, 1985). Generally, it appears that for further progress we might have to correlate structural and functional synaptic changes with motility-related measurements on the basis of individual animals, especially those that are most similar genetically and ontogenetically. Interestingly, as in remodelling, nerve terminal degeneration with phagocytosis of axonal elements apparently does not occur during aging (Fujisawa, 1976). The claim that there is age-related loss of m o t o r neurons remains controversial (for references cf G u t m a n n and Hanzlikov~i, 1976; Caccia et al., 1979). For the question of m o t o r neuron loss it might be worth considering that inactive m o t o r neurons might shrink and thus escape detection on spinal cord or ventral root sections (cf K a w a m u r a and Dyck, 1977). The overall impression is that there are no age-specific features in junctional architecture. Rather, the emerging picture seems to represent the accumulated signs of remodelling, possibly including nerve cell loss. Another example of an age-related muscle change that appears to be governed by activity is the differential loss in overall glycolytic and oxydative enzymes in 28 to 36 months old rats (Bass et al., 1975), The decline in enzyme activity again is least in the

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continuously active diaphragm, and is different in soleus as compared to e.d.1. Age-related differences in the use of muscles might also be the cause for the shift in muscle fiber type composition observed in aged rats (Bass et al., 1975). The weight increase seen in young rats as they grow may be the driving force for the increase in type I motor units (Kugelberg, 1976, 1980). In mammalian muscles, senile atrophy of individual fibers or groups of fibers seems to be the most prominent feature of aging. The atrophy includes not only a reduction in fiber diameter but also severe structural changes with possible muscle fiber loss (Fujisawa, 1974, 1975; Jennekens et al., 1971; Rebeiz et al., 1972; Tu~ek and Gutmann, 1973). Because of the unspecific nature of these changes it is not clear if they could be caused by denervation of muscle fibers. Similar changes can be induced by tenotomy (Walker et al., 1965; Zacks et al., 1969; Shafiq et al., 1969; Tomanek and Cooper, 1972). Tenotomy, however, produces a special form of disuse, with reduced motor neuron activity (Russel, 1980) and load-free isotonic muscle contraction (see Fujisawa, 1975, for further discussion). It will be interesting to know, therefore, whether the atrophied muscle fibers described in the aged are anatomically denervated. If not, the changes seen might be due to relative inactivity. Still another factor in senile muscle atrophy might be the decline in serum testosterone level which could contribute to muscle fiber atrophy and might in addition have direct or indirect effects on motor neurons (Hanzlikovfi and Gutmann, 1978; Tu~ek et al., 1976; for reviews see Tobin and Prcot-Dechavassine, 1982; Arnold and Gorski, 1984). Other hormones such as thyroid hormones and insulin might also affect muscles (see McArdle, 1983, for a review). Quite apart from these considerations, there is little known about the possibility that muscle fibers may be continually replaced under normal conditions. There are some indications that the number of fibers in a muscle can decrease in senescence (Gutmann and Hanzlikovfi, 1965), change with age (Tu~ek and Gutmann, 1973), and increase after elevated activity (Schiattino et al., 1979). It is possible, therefore, that atrophy and even the disappearance of muscle fibers are normal events that might occur more frequently in senescence but are generally counteracted by the continual formation of new muscle fibers. Grouping of muscle fiber types apparently occurs during normal aging (Gutmann and Hanzlikov~, 1976), and is usually quoted as evidence for motor neuron death and consequent reinnervation by remaining intact motor neurons. Fiber types are not fixed, but apparently depend on the activity pattern and/or on the total amount of muscle activity imposed by the motor neuron (Lrmo et al., 1980; Kwong and Vrbova, 1981; Green et al., 1983). While fiber type grouping has otherwise been observed only after severe partial muscle denervation (Kugelberg, 1973), our new understanding of synapse remodelling might provide an alternative explanation for its occurrence under physiological conditions. Figure 20 gives a graphic representation of this hypothesis. Several observations indicate that there is not only continual intraterminal sprouting (Wernig et al., 1980a,b) but also collateral sprouting from nodes of Ranvier (Tuffery, 1971; Courtney and Steinbach, 1981) and terminal sprouting whereby sprouts leave the vicinity of the parent endplates (Barker and Ip, 1966, see Section 2.1). If we assume that such sprouts reach neighbouring endplates and form synaptic contacts there, fiber type grouping could occur without denervation. Little is known about the degree to which single endplate sites are polyneuronally innervated in aging mammalian muscles, but a sharp increase in the number of endplates (up to 40%) innervated by more than one myelinated axon has been found in old mice (Hopkins et al., 1985; see also Taxt, 1983). When additional foreign neurons reach the endplates in normally innervated muscle, they apparently can form contacts and even eliminate the original axon (Bixby and Van Essen, 1979). Thus remodelling of motor units might even occur without increase in polyneuronal innervation. In lower vertebrates like the frog, where focal polyneuronal innervation is common, its extent is variable and seems to depend on seasonal factors (Herrera, 1984). In old age less active fibers could produce the presumed sprouting signal (see Section 3.1) thus attracting the nearest axons. In this case strong (more active) motor neurons would expand their territory on the expense of weaker ones, thereby also causing an apparent reduction in the

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FIG. 20. Remodelling of motor units. Schematic representation to explain fiber-type grouping under physiological conditions (aging). One neuron (dark) grows collateral and terminal sprouts which form synaptic contacts on neighbouring muscle fibers in the old endplate area (B) or nearby (C). Previously innervating axons retract (dashed) or remain to form polyneuronally innervated endplates. It is assumed that in the course of aging some motor neurons (light) become recruited less frequently which triggers denervation-like changes in their muscle fibers (atrophy, possibly increased production of a sprouting factor, increase in the area receptive for synapse formation, etc.). Histochemical parameters of the newly innervated muscle fibers gradually change according to the new activity pattern (dark fibers) which results in fiber-type grouping visible on muscle cross sections (B and C). n u m b e r o f m o t o r units. F i b e r t y p e g r o u p i n g c o u l d thus be the result o f a c o n t i n u o u s r e m o d e l l i n g o f the m o t o r units w h e r e b y s p r o u t s f o r m synapses ( p r o b a b l y in the e n d p l a t e area) o n n e i g h b o u r i n g muscle fibers with o r w i t h o u t loss o f the preexisting terminal. W i t h o u t e l i m i n a t i o n one w o u l d p o s t u l a t e t h a t the e n z y m e p a t t e r n s o f d o u b l y i n n e r v a t e d fibers generally w o u l d be d e t e r m i n e d by the n e u r o n with the higher activity. If, d u r i n g n o r m a l a d u l t life o r d u r i n g aging, muscle fibers d e g e n e r a t e a n d new fibers are f o r m e d f r o m satellite cells (see above), these fibers m i g h t be i n n e r v a t e d b y s p r o u t s f r o m the nearest axon, which c o u l d also c o n t r i b u t e to fiber t y p e g r o u p i n g . 3.3.2. Pathological conditions The existence o f s y n a p s e r e m o d e l l i n g as a n o r m a l process m i g h t also help in the i n t e r p r e t a t i o n o f u l t r a s t r u c t u r a l c h a n g e s seen in several n e u r o m u s c u l a r diseases (Engel a n d S a n t a , 1973; Engel a n d W a r m o l t s , 1973; B o w d e n a n d D u c h e n , 1976). These changes m i g h t

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in turn reveal something of the mechanisms of remodelling. In myasthenia gravis, for example, partially or totally abandoned gutters and small axonal profiles (presumably nerve terminal sprouts) with mature or immature synaptic contacts are commonly seen (Engel and Santa, 1973). These, of course, are typical signs of synapse remodelling. As secondary effect of the disease process synapse remodelling in myasthenia gravis seems to be drastically enhanced which might be due to some combination of nerve terminal sprouting promoted by sprouting factors from less active muscle fibers, enhanced retraction due to elevated motor nerve activity (see Section 2.4), or the failure of weak contacts to become stabilized. When transmitter release is blocked by botulinum poisoning or local high concentrations of tetanus toxin, nerve terminal sprouting with new synapse formation takes place (Duchen, 1971, 1973; Duchen and Tonge, 1973; Brown et aI., 1981; Wernig et al., 1981d). Although the irreversibly blocked parent endplate apparently is abandoned (Duchen, 1970), nerve terminal retraction is not prominent. These cases can also be interpreted as enhanced remodelling, though the structural picture is quite different from myasthenia gravis. It appears, therefore, that remodelling can be influenced quite differently in different pathological situations. Future analysis of these situations may add significantly to our understanding of the mechanisms of remodelling and synaptic maintenance. For example, in myasthenia gravis (see Drachman, 1983) even the newly formed synaptic contacts would be impaired by the autoimmune attack against ACh receptors. In muscles briefly exposed to botulinum or tetanus toxin, however, new synapses could function properly (and perhaps become stabilized thereby) as soon as the toxin is eliminated from the serum. The abandoning of parent endplates following botulinum poisoning might be due to failing maintenance of these contacts due to the presynaptic block. Time factors might also be important for the different structural changes. In the acute case of botulinum poisoning muscle fibers might not be prepared to provide areas receptive for synapse formation (see Section 3.1) and thus, together with a massive signal for sprouting, enforce extensive terminal sprouting including extraterminal sprouts reaching distant muscle fibers. Myasthenia gravis, on the other hand, is a chronic disease, in which transmission at first is only partially impaired. Under these conditions muscle fibers might gradually provide larger or additional synaptic areas, the sprouting signal might be less intense but continue for longer periods of time, there may be sufficient time for nerve retraction, and sprouts could reoccupy abandoned gutters. In this context it is interesting to note that sprouting after botulinum poisoning was reduced with chronic muscle stimulation (Brown and Holland, 1979) and that ectopic endplates did not form or were reduced after denervated muscles were chronically stimulated (L6mo and Slater, 1980). Inactivity might thus be necessary for sprouting and synapse formation at least during some period of time and it might be worth considering whether these observations are of any clinical relevance. In still other nerve-muscle diseases, structural changes could be due to shifts in synaptic activity rather than representing primary defects. In myasthenic syndrome (LambertEaton) depth and complexity of secondary folds increase (Engel and Warmolts, 1973; Fardeau, 1973; Bowden and Duchen, 1976) while in hereditary motor endplate disease of the mouse (med) secondary folds are reduced or completely missing (see Bowden and Duchen, 1976). Under normal conditions secondary folds are more frequent, deeper, and more complex in fast muscle (gastrocnemius) as compared to slow muscle (soleus) (Bowden and Duchen, 1976). Though overall activity of slow muscles is higher than fast muscles, the latter are activated with much higher frequencies (Henning and L6mo, 1985). It is likely, therefore, that in fast muscles synaptic currents at a single junction sum to give higher current densities in the synaptic cleft which in turn somehow triggers the growth of secondary folds. In the myasthenic syndrome endplate potentials are often too small to evoke action potentials (Lambert and Elmqvist, 1971). The firing rates of motor neurons may generally increase as a compensatory mechanism, which would lead to higher current densities in the synaptic cleft and enlargement of secondary folds. In hereditary motor endplate disease on the other hand, in which secondary folds are reduced in size and number, action potentials often fail to invade the nerve terminal

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(Duchen and Stefani, 1971) thus certainly causing a reduction in maximum synaptic current density. In myasthenia gravis as well, secondary folds are shallow (Engel and Warmolts, 1973) which, in addition to other reasons such as junctions being newly formed or in the process of being abandoned, could be due to the reduced synaptic currents in the course of ACh receptor failure. When ACh receptors in postnatal rats were blocked with alpha-bungarotoxin, development of secondary folds was retarded (Duxson, 1982) which further supports that notion that development and maintenance of secondary folds is correlated and perhaps depended on synaptic current. The above considerations support the idea that nerve-muscle junctions have to be maintained continuously and actively. Permanently blocked parent endplates in botulinum poisoning are abandoaed and insufficiently functioning junctions in myasthenia gravis seem to have a higher rate of remodelling. It was discussed above that active maintenance might also fail under physiological conditions leading to abandoning of circumscribed central parts of junctions (Section 2.2). The underlying mechanisms are not known and might be complex. The best understood aspect is the presumed active maintenance of junctional ACh receptor clusters (Section 2.2.1.1). Mechanisms which actively maintain synaptic connections have been postulated for synapses in sympathetic and parasympathetic ganglia, spinal motor neurons and brainstem nuclei (see Watson, 1972; Purves, 1975, 1977; Purves and Njfi, 1978) and might thus occur quite generally (for further discussion see McArdle, 1983; Patterson and Purves, 1984; Purves and Lichtman, 1985).

4. Plasticity and Regulation of Synaptic Efficacy In the preceding section we presented evidence that the structure of neuromuscular junctions is continually remodelled and can be altered in different physiological and pathological situations. It has recently become clear that adult junctions also exhibit long term plasticity in physiological function, and that these physiological changes can occur along with, or entirely independent of, changes in terminal length. We will focus our discussion on synaptic efficacy. Efficacy can be functionally defined as synaptic safety margin, or the likelihood that endplate potentials will exceed threshold for generation of muscle fiber action potentials. A different measure of efficacy that is more useful for comparing single identified junctions is the level of quantal transmitter release per unit length of nerve terminal. Methods for obtaining these estimates of efficacy are given below. For present purpose, we will focus on long term changes in efficacy, ignoring the large literature on important forms of physiological plasticity that operate in the millisecond or second range, such as facilitation, augmentation, potentiation, depression, desensitization, etc. (for reviews see e.g. Kandel, 1981; Silensky, 1985). 4.1. DIFFERENT MEASURES OF SYNAPTIC TRANSMISSION

Before discussing the maintenance and plasticity of synaptic efficacy, it is useful to discuss the various methods that have been used to assess transmitter release. Each technique is limited in important ways. Of course, the most critical measure of synaptic effectiveness is the one that is the most relevant to the function of the nerve-muscle contact, i.e. the likelihood that the epp will be sufficiently large to reach action potential threshold (synaptic safety margin). This safety margin depends not only on the amount of transmitter released, but also on muscle fiber input impedance, postsynaptic ACh sensitivity, and muscle fiber action potential threshold. Safety margins can be best estimated b y quantifying the blockade of nerve stimulus-evoked twitch tension as ACh receptors are blocked (Paton and Waud, 1967; Grinnell and Herrera, 1980) or as Ca concentration is lowered (Grinnell and Herrera, 1980; Herrera and Grinnell, 1980a,b; Banker et al., 1983; Trussell, 1983; Herrera and Banner, 1986). Transmitter release can be measured as epp quantal content (m), which is estimated by applying the quantal hypothesis of del Castillo and Katz (1954), i.e. m = mean epp/mean miniature epp. Because of the problem of muscle fiber contraction, and nonlinear

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summation of epps (McLachlan and Martin, 1981), this is usually applied in the rather unphysiological conditions where transmission is partially blocked by low Ca and/or high Mg levels. Quantal content in normal physiological solutions can be estimated with this method if excitation-contraction coupling is first blocked with glycerol (Miyamoto, 1975) or formamide (Herrera, 1984) but these treatments may only be suitable for determining relative differences between identically treated preparations, since it is difficult to control for presynaptic effects of the drugs. Quantal analysis under voltage clamp conditions would overcome the problem of nonlinear summation, but one must still contend with muscle fiber twitching. Moreover, adequate space clamp may be difficult to achieve, especially for large frog junctions. Still another method is to expose junctions to very low temperatures, at which evoked release is sufficiently slow and asynchronous that individual quanta can be counted (Katz and Miledi, 1965a; Johnson and Wernig, 1971). Finally, release can be measured from restricted portions of nerve terminals by focally depolarizing in the presence of tetrodotoxin (Katz and Miledi, 1967; Wernig, 1975). Experiments using this approach must, however, be carefully designed to account for the fact that the magnitude and spatial spread of the presynaptic depolarization is unknown and might be difficult to control. Quantal content at physiological levels of Ca was previously calculated from epp amplitude variations using Poisson statistics after blocking contraction with curare (Martin, 1955). The inappropriateness of Poisson statistics at high release levels (Johnson and Wernig, 1971; Miyamoto, 1975; Wernig, 1975) however, introduces serious underestimates of m (for a review see McLachlan, 1978). A different approach employing curare is the use of epp amplitude normalized for input resistance as a measure of transmitter release (Nudell and Grinnell, 1982, 1983; Trussell and Grinnell, 1985). In both these cases interpretation is complicated by possible presynaptic effects of curare (reviewed in Grinnell and Herrera, 1981). Since the amount of transmitter released depends on nerve terminal size (see above), the latter estimate of release can be further refined by normalizing for nerve terminal length (Grinnell and Herrera, 1980; Herrera and Grinnell, 1980a, 1981; Nudell and Grinnell, 1982, 1983), if one assumes that release is relatively uniform (see below) and the curare block is sufficient to eliminate the problem of nonlinear summation. With only a few exceptions (e.g. Gertler and Robbins, 1978; Nudell and Grinnell, 1982; Wernig and Fischer, 1986; Fig. 18), the importance of the nerve stimulus frequency has not been considered in studies of synaptic efficacy yet this may have major functional significance. Also it appears, for example, that there is an inverse relationship between the size of the first epp and the degree of facilitation (Fig. 18). Many measurements are made using the stable responses obtained at very low frequencies (typically 0.5-1 Hz). In other studies, synaptic efficacy has been estimated at higher stimulus frequencies (typically 5-40 Hz), using plateau responses obtained after the early tetanic rundown. Natural firing patterns of the motor neurons differ considerably (Henning and Lrmo, 1985) such that only the first few facilitated responses are functionately important for some motor units while for others also the later depressed plateau responses are important. 4.2. VARIABILITY IN SYNAPTIC EFFICACY AT DIFFERENT JUNCTIONS The first clues that synaptic efficacy might be maintained by dynamic and modifiable processes came from studies describing substantial differences in transmitter release in different frog muscles. For example, motor nerve terminals in the frog cutaneous pectoris muscle release on the average 2-3 times more transmitter and achieve higher safety margins than terminals in the frog sartorius muscle, even though the junctions and muscle fibers are of similar size and type (Herrera and Grinnell, 1980b; Grinnell and Herrera, 1980). Even larger differences are seen between junctions in the cutaneous pectoris and those in the cutaneous dorsi. In low Ca solutions, nerve terminals in the cutaneous pectoris show a three fold higher level of evoked quantal release and a six fold higher level of spontaneous quantal release per unit nerve terminal length (Herrera and Banner, 1986). These differences in release correlate with ultrastructural differences (see Section 4.5).

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Three additional lines of investigation have revealed more of this variability in synaptic efficacy. Superimposed on the overall positive relationship between transmitter release and terminal length (Kuno et al., 1971), there is an inverse relationship between release per unit terminal length and terminal length, at least when one considers junctions on fibers of similar input impedance (Nudell and Grinnell, 1982, 1983). This observation is consistent with the hypothesis that terminals, which differ in their capabilities for release, may simply stop growing when a certain level of safety margin is achieved. On the other hand, release per unit terminal length could be down-regulated in excessively large junctions. Most interestingly, the more strongly releasing terminals in a muscle may have higher resting and impulse-evoked Ca 2÷ influx (Pawson and Grinnell, 1984; see also Section 3.2.1). Second, there is a clear correlation between the size of each motor unit and synaptic effectiveness, with average safety margins being higher in larger motor units (Grinnell and Trussell, 1983; Trussell and Grinnell, 1985). It is not clear whether these strong motor neurons were predetermined to have large synaptic fields that would be maintained at high efficacy, or whether they become larger because of some other competitive advantage and were able to sustain a higher level of effectiveness because of increased trophic feedback. Thirdly, some of the differences in efficacy between different terminals on multiply innervated fibers may be the result of competitive interactions (see Section 5). 4.3. NON-UNIFORMITY OF RELEASE EFFICACY WITHIN SINGLE JUNCTIONS Several studies on toad and frog neuromuscular junctions have shown that there can be differences in release even in different parts of the same terminal. Bennett and Lavidis (1979) reported that as much as 50% of the release in a low Ca 2÷ solution could occur within the small recording area of a single focal extracellular microelectrode. This conflicted with earlier views (Katz and Miledi, 1965) that transmitter is released all along the terminal. Vautrin and Mambrini (1980), after observing that the latency of epps recorded in low Ca 2÷ varied in a stepwise manner, concluded that release under these conditions occurred only from a few discrete sites. Bennett and Lavidis (1982) later reported that there is a steep gradient in evoked release along the length of terminals, with release high proximally and diminishing rapidly toward the distal tips. A similar profile was described for evoked release by Zefirov (1983) and for spontaneous mepp release by Tremblay et al. (1984). These questions have also been examined by D'Alonzo and Grinnell (1982, 1985), using improved recording techniques and identified junctions so that release profiles could be correlated with morphological details. They concluded that release probability varies at most only a few fold along the length of nerve terminals, that sites with very high release probability do not exist, but that release does fall off at the distal tips. This tendency for lowered release from the distal tips of terminals may, together with evidence for disorganized active zones (Pumplin, 1983), greater numbers of mitochondria (Werle et al., 1984), and sprouting at distal tips (see Section 2), suggest that these areas are frequently in the process of growth and regression, and that this may occur at the expense of release efficacy. 4.4. PLASTICITY IN TRANSMITTER RELEASE PER UNIT NERVE TERMINAL LENGTH We have shown in preceding sections that nerve terminal length at neuromuscular junctions can change, with predictable consequences for synaptic effectiveness. There is also evidence that the amount of transmitter released per unit length of nerve terminal can be regulated independently of nerve terminal length. If one decreases motor unit size in the frog sartorius by causing the nerve to reinnervate fewer muscle fibers than normal, transmitter release at neuromuscular junctions is increased for several months (Herrera and Grinnell, 1980a). Similar results were reported for a mouse muscle when motor unit size was decreased by crushing intramuscular nerves (Pockett and Slack, 1982). Release per unit length eventually returns to normal even though motor unit size remains small (Herrera and Grinnell, 1985). Apparently the mechanisms that, early in development, JPN 27/3--F

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6 5

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M 12o M9o

3 2 1 I

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FIG. 21. Enhancement of quantal content (m) by stretch at individual identified frog neuromuscularjunctions. The degree of stretch enhancement is a linear function of nerve terminal length. Ordinate is the ratio of m when the muscle is at 120% in situ length to m when the muscle is at 90% in situ length. Abscissa is the total length of nerve terminal within AChE-outlined synaptic gutters. Data from 3 cutaneous pectoris muscles bathed in a low Ca2+ Ringer solution. Werle and Herrera, unpublished. match a m o t o r neuron's transmitter output level to its peripheral load still operate in adults. Synaptic effectiveness at the frog neuromuscular junction can also be strongly influenced by contralateral denervation (Herrera and Grinnell, 1981). Cutting the nerve to one sartorius muscle causes a 3-8 fold increase in m recorded in low Ca 2+ in the contralateral unoperated muscle without a change in synaptic size (Herrera and Scott, 1985). Transmitter release from frog but not mammalian neuromuscular junctions also shows an interesting form of plasticity that depends on the degree of muscle stretch. Increasing muscle length by a few percent significantly enhances m, m e p p frequency, and safety margin (Hutter and Trautwein, 1956; Turkanis, 1973; Luff and Proske, 1979; Grinnell and Herrera, 1980; see Grinnell and Herrera, 1981, for review). Curiously, the degree to which release is enhanced by stretch is proportional to junctional size (Werle and Herrera, 1985). When large junctions were stretched, quantal content in low Ca 2+ was enhanced several fold, while for the smallest junctions tested, stretch enhancement was minimal (Fig. 21). This relation raises the possibility that the absence of a demonstrable stretch effect in mammalian junctions is not due to a fundamental difference in nerve terminal properties, but simply to the fact that mammalian junctions are much smaller than frog junctions. It is tempting to speculate that a stretch-activated channel, such as that found by Guharay and Sachs (1985) in chick muscle membrane, may be involved in the stretch enhancement of release, but there is no evidence. The significance of the stretch effect is also unclear. Since m a n y frog muscles at rest length have at least some endplates where synaptic transmission is subthreshold for a single stimulus (Grinnell and Trussell, 1983; Herrera, 1984; earlier references in Grinnell and Herrera, 1981) one might speculate that the rate at which force is developed may depend on initial muscle length. 4.5. ULTRASTRUCTURAL BASIS OF DIFFERENCES IN TRANSMITTER RELEASE PER UNIT TERMINAL LENGTH

Since there can be large differences in synaptic efficacy without corresponding morphological differences visible in the light microscope (see Section 4.2), it is important to consider whether there may be ultrastructural differences. O f particular interest are variations in presynaptic active zones, the presumed sites of synaptic vesicle exocytosis and transmitter release (Couteaux and POcot-Dechavassine, 1970; Peper et al., 1974; Wernig, 1976; Heuser et aL, 1979) and perhaps the site of voltage dependent Ca 2+ influx (Pumplin et al., 1981). Using randomly selected thin sections, Herrera et al. (1985a) investigated frog cutaneous pectoris and sartorius muscles and found that terminals that released more transmitter had larger active zones as well as some other differences. In a related study, Herrera et al. (1985b) studied the ultrastructural basis of the large increase in release seen in frog

FIG. 22. Typical freeze fracture view of a neuromuscular junction from a frog cutaneous pectoris muscle, showing P face of nerve terminal membrane (N) and E face of muscle fiber membrane (M). AZ = active zone; F = secondary postsynaptic fold. Asterisk indicates a point where an active zone is broken into 2 separate segments (defined as > 50 nm separation). Other active zones consist of single segments. Unpublished photo provided by C.-P. Ko.

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TABLE 2. MEAN AND s.e.m. OF ACTIVE ZONE MEASUREMENTS TAKEN FROM 16 RANDOMLY SELECTED CUTANEOUS PECTORIS NEUROMUSCULAR JUNCTIONS IN 6 MUSCLES AND 16 CUTANEOUS DORSl JUNCTIONS IN 9 MUSCLES

Cutaneous pectoris Cutaneousdorsi (n = 413) (n = 485) P Number of active zone segments per junctional fold 1.30 + 0.03 1.78 _+0.04 < 0.0001 Spacing between active zones (/am) 0.83 +0.01 1.04+0.02 <0.0001 Total active zone per 100/am nerve terminal (#m) 156 + 2 130 _+2 < 0.0001 See Fig. 22 for discription of morphological parameters measured. Data from Propst et al. (1986)

sartorius junctions following contralateral denervation (Herrera and Grinnell, 1981). By reconstructing nine physiologically identified junctions from semiserial thin sections, they confirmed that release was proportional to active zone size. More importantly, their results suggested that active zones are plastic structures, and that a change in active zone size was the structural basis of the long term change in transmitter release efficacy. In the preceding studies, active zone size was measured indirectly, as the length of presynaptic membrane in close apposition to the muscle fiber membrane in cross sections passing through active zones. Similar studies have recently been completed using freezefracture electron microscopy, with which active zones can be viewed directly. The first study compared randomly selected junctions from frog cutaneous pectoris and cutaneous dorsi muscles (Propst et al., 1986), two preparations whose junctions show large differences in release per unit terminal length (Herrera and Banner, 1986; see Section 4.2). A typical freeze fracture view of a frog neuromuscular junction indicating the structures that we measured is shown in Fig. 22. The first line in Table 2 shows that active zones in the more strongly releasing cutaneous pectoris terminals were significantly less disrupted, i.e., each active zone consisted of fewer separate segments (asterisk in Fig. 22). The second line of Table 2 shows that active zones in the cutaneous pectoris were more closely spaced along the terminal length. As a result of these and other differences, the total length of active zone per unit terminal length was larger in the cutaneous pectoris. Propst and Ko (1985; see also Ko and Propst, 1986) have significantly extended these findings, correlating synaptic efficacy with freeze-fracture ultrastructure in single, physiologically identified junctions from cutaneous pectoris muscles. They found a good correlation between low Ca 2+ measurements of evoked release per unit terminal length and the amount of active zone per unit terminal length. Other studies have shown correlations between transmitter release levels and active zones in normal (Heuser et al., 1979), degenerating (Ko, 1981), and regenerating (Ko, 1984) frog junctions, in diseased human junctions (Fukunaga et al., 1982), in crustacean neuromuscular junctions (Sherman and Atwood, 1972; Govind and Chiang, 1979; Govind and Meiss, 1979; Meiss and Govind, 1979, 1980), and in Aplysia (Bailey and Chen, 1983) and squid (Pumplin et al., 1981) synapses. Contrary to the assumption in most of the preceding studies, there have been several reports that the ultrastructure of motor nerve terminals is spatially non-uniform. Pumplin (1983) reported that presynaptic active zones in frog fast twitch muscles are occasionally disorganized, especially at the distal tips of terminals. Since the distal tip is a common site for sprouts to originate (see Section 2.3), it is tempting to speculate that the ultrastructural disruption is related to an enhanced involvement in synaptic remodelling at these locations. Davey and Bennett (1982) suggested that there are continuous gradients in the amount of synaptic contact along the length of toad motor nerve terminals, with greater contact proximally and less distally. However, Werle et al. (1984) were unable to find any evidence for similar morphological gradients in nine serially sectioned and physiologically identified frog terminals, except a tendency for the distal tips of terminals to contain more mitochondria. In the light microscope, receptor bearing junctional folds are evenly spaced and remarkably similar over large parts of junctions, but differ in other parts (Fig. 5; Krause and Wernig, 1985). At endplates on frog slow fibers, active zones are less complete and more disorganized than in fast muscles (Verma and Reese, 1984; Verma, 1984), possibly reflecting the low levels of transmitter released from such terminals.

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5. Multiply Innervated Muscle Fibers

In multiply innervated muscle fibers, an important aspect of growth-associated plasticity is the addition of new synaptic sites as muscle fibers increase in length. We will consider the published evidence for differences in the number of endplate sites per fiber and the number of synaptic inputs per endplate site. The possible functional significance of these cases will be discussed. 5.1. NUMBER OF JUNCTIONAL SITES PER MUSCLE FIBER

It is generally accepted that mammalian skeletal muscles are innervated at a single endplate site, located about midway along each fiber's length. Some fast muscles in frogs, such as the cutaneous pectoris, also follow this pattern (Letinsky et al., 1976). Other fast muscles in frogs start with one endplate site per fiber early in development but add new sites as muscles elongate (Bennett and Pettigrew, 1975; Nudell and Grinnell, 1983) so that in the adult, there are two to four widely separated sites per fiber (Katz and Kuffler, 1941; Haimann et al., 1981a; Herrera, Grinnell and Giancona, unpublished). Twitch muscle fibers of fish and caudate amphibians are often innervated at many sites (Lehouller and Chatelain, 1974; Wigston, 1980; see Mallart et al., 1980, for review). The question arises as to why multiple endplate sites are needed in twitch fibers, since an action potential elicited anywhere along the length of a fiber conducts without decrement to either end. Any proposed explanation must also be able to account for why most mammalian muscles lack multiple endplate sites. Four possibilities have been considered. First, if the endplate sites on a given fiber are sufficiently close for summation of epps, and if the effectiveness of any one input is low, dense multiple innervation may serve to insure adequate depolarization to trigger muscle fiber action potentials. This is likely to be the case in fish and newts (Slack and Docherty, 1978) but not in frogs, where endplates are typically separated by several muscle fiber length constants. Second, multiple sites of action potential generation might generate tension more efficiently. This was discussed by Katz and Kuffler (1941) but dismissed as being unlikely. Recent observations (Nudell and Grinnell, 1983) showed that the tension produced by single Xenopus pectoralis muscle fibers in response to intracellular current injection did not vary significantly as the site of depolarization was moved, although marginal effects have been seen with similar techniques in very long frog sartorius fibers (Trussell and Grinnell, unpublished). Third, multiple innervation of fibers would increase the amount of overlap between motor units (Luff and Proske, 1976). In muscles with relatively small numbers of motor units, a high degree of overlap would increase the smoothness with which contraction could be graded as additional motor units are recruited. These consequences would seem not to apply in the case of the Xenopus pectoralis muscle, where a large fraction (at least 50%) of the multiply innervated fibers are innervated by branches of the same axon (Nudell and Grinnell, 1983; 1985). Finally, it might be supposed that the need for multiple sites of innervation is not activity-related at all, but plays a role in trophic interactions between nerve and muscle. 5.2. NUMBER OF PRESYNAPTIC INPUTS PER JUNCTIONAL SITE Muscles also differ in the number of synaptic inputs per endplate. In mammalian muscles, after a transient period of multiple innervation in early postnatal life (Redfern, 1970; reviewed in Grinnell and Herrera, 1981), the general rule is that adult endplates are singly innervated. There has been a report, however, that as many as 25% of the endplates in adult rat lumbrical muscles may be innervated by a second, often very weak, input (Taxt, 1983). In frogs, a substantial fraction of adult endplates are multiply innervated (Dennis and Miledi, 1974; Rotshenker and McMahan, 1976; Vysko~il and Magazanik, 1977; Haimann et al., 1981a; Herrera, 1984; Miledi and Uchitel, 1984; Trussell and Grinnell, 1985). This persistent multiple innervation may be the result of incomplete developmental synapse elimination (Morrison-Graham, 1983) or new synapse formation as part of the

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ongoing process of synaptic remodelling (see Section 3.3.1). The hypothesis that the adult situation represents an equilibrium between regression on the one hand and sprouting followed by new synapse formation on the other is further supported by findings of higher levels of sprouting (Wernig et al., 1980a,b) and multiple innervation (Herrera, 1984) in winter frogs. An intriguing hypothesis, based on recent studies of developmental synapse elimination is that the difference in adult levels of polyneuronal innervation in mammalian and frog neuromuscular junctions is due to a simple difference in junctional size and geometry. Forehand and Purves (1984) analyzed the geometrical arrangement of synaptic boutons onto rabbit ciliary ganglion cells as the boutons were undergoing elimination. They found that polyneuronal innervation was more likely to persist if competing nerve terminals were spatially segregated onto separate dendrites of the postsynaptic cell. Frog neuromuscular junctions occupy a five to ten fold greater area of muscle fiber surface and the long terminal branches are more widely separated than the compact boutons of mammalian junctions. Polyneuronal innervation may persist in adult frog muscles not because of any intrinsic difference in the mechanism of synapse elimination or the capacity for remodelling and new synapse formation, but because competing terminals can achieve greater separation within the much larger junctional area. This hypothesis is being tested (Werle and Herrera, unpublished). Whatever the cause of multiple innervation in adults, it probably serves to increase overlap between motor units, with functional consequences as described in the preceding section. In addition, if a substantial fraction of the subthreshold multiple inputs can sum to evoke muscle action potentials when stimulated at high frequency, the strength of contraction could be graded not only by recruiting motor units, but also by adjusting the tension of each motor unit as a function of the firing frequency of the motor axon. Multiple innervation also seems to influence synaptic efficacy. Transmitter release efficacy is lower at dually innervated endplate sites (Weakly and Yao, 1983; Trussell and Grinnell, 1985) and at separate endplate sites on dually innervated fibers (Haimann et al., 1981a,b; Nudell and Grinnell, 1983), possibly due to competitive interactions (reviewed in Mark, 1980; Grinnell and Herrera, 1981). A similar competitive interaction is seen in frog sartorius fibers reinnervated by two different spinal nerves (Grinnell et al., 1979). 6. Summary and Conclusions There is substantial evidence that neuromuscular junctions in adult vertebrates are highly modifiable synapses, undergoing remodelling throughout life. This conclusion is clearly supported by morphological evidence of nerve terminal sprouting with new synapse formation and evidence of nerve retraction in untreated muscles (Section 2). The mechanisms of remodelling, and the rate at which it normally proceeds, are unknown. A hypothesis, which is for the most part testable, is proposed (Section 2.4): sprouting may be an "inherent" tendency of motor nerve terminals (e.g. caused by continual assembly of parts of the cytoskeleton) and nerve retraction might be caused by motor nerve activity (e.g. via Ca-activated proteases that break down microtubules and filaments). Although functional consequences of remodelling per se are not prominent, this capacity for change probably plays an important role in ontogenesis, altered use, aging, and pathological conditions. As muscle fibers grow during development, for example, input resistance falls. Through sprouting and new synapse formation, there is a simultaneous increase in junctional size, presumably releasing larger amounts of transmitter and tending to maintain the safety margin of transmission. This sprouting may be a response to nerve terminal growth promoting substances released by growing or inactive muscle fibers. Thus there exist local feedback mechanisms in which muscle fiber activity (contractile or electrical) seems the most plausible mediator (Sections 3.1, 4). The new synaptic contacts are always made within or around the former endplate region, reinforcing the conclusion from other studies that only the junctional area of normally active muscle fibers is receptive to innervation (Section 3). Whether junctions also grow when muscle hypertrophy is

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induced by increased work or anabolic substances is unknown, but seems worth investigating. The structural changes associated with aging can also be viewed as consequences of lifelong remodelling. Abandoned synaptic gutters, which are more frequently seen in aged frog and mammalian muscles, may accumulate throughout life if abandoned gutters are incompletely reoccupied. Fiber type grouping may also be explained by local remodelling of motor units and may not necessarily be initiated by motor neuron loss. Muscle fiber atrophy and presumed muscle fiber loss might be due to reduced motor activity in addition to the effects of declining serum androgen levels. Thus senile muscle atrophy can largely be explained by normal physiological processes and whether true motor neuron loss occurs should be carefully re-examined (Section 3.3.1). Remodelling helps to explain structural changes occurring in endplate diseases, like myasthenia gravis (Section 3.3.2). A possible role for synaptic current density in modulating the appearance of secondary folds in normal and diseased muscle is proposed. Recent observations suggest that long term changes in transmitter release can occur without changes in nerve terminal size. The amount of transmitter released per unit length of nerve terminal (synaptic efficacy) varies in different muscles and even at different junctions in the same muscle (Section 4.5). Ultrastructural studies suggest that these differences in synaptic efficacy are due to differences in the size and organization of individual active zones and the number of active zones per unit terminal length (Section 4.6). Thus the presynaptic membrane itself may be a plastic structure, and changes in active zones, with consequent effects on transmitter release, may play an important role in synaptic remodelling. It would be interesting to know whether the number, density, and physiological properties of ACh receptor channels in the postsynaptic membrane are also variable; there are some observations on frog muscle that single channel conductance changes with season (Sections 3.2.2 and 3.3.2), and that fast muscle fibers have higher junctional receptor densities than slow ones (Sterz et al., 1983). Possible changes in presynaptic Ca 2+ channels are not known but could possibly be inferred from transmitter release characteristics. There are mammalian muscle fibers (e.g. in external eye muscles) that are not focally and mononeuronally innervated but instead receive multiple inputs. This pattern is much more frequently seen in muscles of lower vertebrates where it has been found to have functional consequences (Section 5). Multiple innervation causes motor units to overlap, affecting the mechanics of contraction during motor unit recruitment. Multiple innervation also affects transmitter release efficacy, presumably because nerve terminals are competing for synaptic space or muscle-derived trophic substances. A great many questions about the maintenance and regulation of neuromuscular junctions have been raised here and remain to be answered. On a more general level one would like to know whether the striking diversities in several junctional aspects present within a muscle or among muscles tend to make each synapse especially well suited for its particular function. If so, to what degree is this matching controlled by genetically predetermined mechanisms and how much of it are adoptive responses of inherently plastic structures to the regulatory environment. In particular it is possible that inherent nerve terminal sprouting, which tends to increase endplate size and thereby transmitter release, is down regulated by nerve activity. If this is the case, nerve activity together with local and other mechanisms might for example be responsible for the more compact and less branched endplates usually present in the more active slow versus fast muscles. Perhaps, the increase in endplate size in some muscles observed in old age and the increase in safety margin of transmission are functionally meaningless but are allowed to develop as a consequence of reduced nerve activity. Does nerve activity on a longer time scale also influence efficacy of transmission (transmitter release per unit nerve terminal length) and in which way? What other synaptic mechanisms are regulated by nerve activity? How is the interaction with regulatory mechanisms mediated via muscle fiber activity, e.g., in regulating terminal sprouting (presumed sprouting factors from the muscle) and new synapse formation in the course of remodelling (presumed control of muscle fiber

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r e f r a c t o r i n e s s ) ? A r e t h e r e g u l a t o r y m e c h a n i s m s c a u s i n g d e v e l o p m e n t a l c h a n g e s (e.g. p o s t n a t a l loss o f p o l y n e u r o n a l i n n e r v a t i o n ) a l s o a c t i v e i n a d u l t h o o d a n d , s i m i l a r l y , h o w do both relate to mechanisms governing reinnervation of denervated adult muscles? Finally, to what extent do the remodelling capabilities shown to occur at the neurom u s c u l a r j u n c t i o n a l s o o c c u r i n t h e c e n t r a l n e r v o u s s y s t e m , a n d w h a t is t h e i r r o l e i n b r a i n and spinal cord function?

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