Mechanisms of Elimination, Remodeling, and Competition at Frog Neuromuscular Junctions Albert A. Herrera* and Michael J. Werlet Neurobiology Section, Department of Biological Sciences, and Program in Neural, Informational, and Behavioral Sciences, University of Southern California, Los Angeles, California 90089
SUMMARY Mechanisms governing synapse elimination, synaptic remodeling, and polyneuronal innervation were examined in anatomical and electrophysiological studies of frog neuromuscular junctions. There was a substantial level of polyneuronal innervation in adult junctions and this varied seasonally. Nerve terminal retraction and synapse elimination occurred during normal growth and following reinnervation. Synapse elimination was not inevitable, however. Repeated in vivo observations of some identified junctions showed that polyneuronal innervation could persist for over a year, while a t other junctions it arose de nuvu by terminal sprouting. W e concluded that polyneuronal innervation in adult muscles was governed by an equilibrium between processes of retraction and elimination on one hand, and sprouting and synaptogenesis on the other. Other observations revealed that structural remodeling was a common feature of adult
junctions. Most often, remodeling involved the simultaneous growth and retraction of different parts of the same junction. The net result was usually junctional growth that, in small frogs, appeared to provide a good match between synaptic size and the electrical demands of transmission. In larger animals, pre- and postsynaptic sizes were not as well matched, providing morphological evidence for a growth-associated decline in synaptic efficacy. Finally, electrophysiology was used to describe some of the functional correlates and consequences of competitive interactions between the terminals of different axons. These results are explained by a hypothetical mechanism that involves trophic support provided by the muscle to the motoneuron, the overall level of nervemuscle activity, and the synchrony of pre- and postsynaptic activity.
INTRODUCTION
underlying mechanisms. At neuromuscular junctions of small laboratory mammals, synapse elimination results in the establishment of mononeuronal innervation at most junctions shortly after the time of birth (reviewed by Van Essen. 1982; Thompson, 1985; Betz, 1987). At the frog neuromuscular junction a similar period of synapse elimination occurs at about the time of metamorphosis (Letinsky and Morrison-Graham, 1980; Morrison-Graham, 1983). Unlike mammals, the process slows before reaching completion, and a substantial fraction of the junctions in adult frogs are polyneuronally innervated (reviewed in Wernig and Herrerd, 1986; see also Werle and Herrera, 1987: Diaz and Pecot-Dechavassine, 1988). Our laboratory has been studying mechanisms go\ierning synapse elimination, synaptic remodeling, and polyneuronal innervation at frog neuro-
Synapse elimination is a collective term for those processes by which the excess synapses characteristic of embryos and neonates are reduced and the adult numbcr is established. Although synapse elimination occurs in many areas of the developing nervous system, it has been particularly well studied at neuromuscular junctions, where experimental accessibility and a wealth of background information has made possible detailed studies of Received June 12, 1989; accepted July 17. 1989 Journal of Neurobiology, Vol. 21, No. I , pp. 73-98 (1990) 0 1990 John Wiley & Sons, lnc. CCC 0022-3034/90/010073-26$04.00 * TOwhom correspondence should be addressed. t Present address: Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305.
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muscular junctions. We find that, as in development, adult motoneurons and muscles retain the capacity to express dramatic structural plasticity. During normal adult life, this plasticity results in continual synapse formation and elimination. Sprouting and synapse formation can cause the de novu establishment of polyneuronal innervation. We have concluded that the physical state of the junction reflects an equilibrium between processes favoring growth and those causing retraction. We speculate that these opposing processes are driven by at least three factors: the metabolic support that motoneurons supply to their terminals, competition between axons for synaptic connections, and activity-dependent nerve-muscle interactions. In this article we will review these findings and conclusions, with our goals being to better understand the frog nerve-muscle system, appreciate differences between frog and mammalian junctions, and discern mechanisms governing these forms of plasticity at neuromuscular junctions and other synapses. We will not attempt an overall review of the field since this has been done by others in this issue (Betz, Ridge, and Ribchester. in press; Lichtman and Balice-Gordon, in press; Thompson, in press; Van Essen, Gordon, and Fraser, in press).
METHODS Unless otherwise noted, experiments were performed on sartorius and cutaneous pectoris nerve-muscle preparations of Rana pipiens (northern variety). Tissues were immersed in or irrigated with frog Ringer solution (in mM: NaCl, 116; KCl, 2; CaCI,, 1.8; N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate,5 ; pH 7.2). Some experiments were done with the pectoral muscle of Xenopus luevis. In some cases sartorius muscles were denervated weeks or months before acute experiments by anesthetizing frogs (immersion in 0.1% aqueous tricaine methanesulfonate, Sigma) and crushing the sartorius nerve where it enters the muscle. For histological observations, neuromuscular junctions were stained either with a nitroblue tetrazolium nerve terminal stain plus cholinesterase stain (Letinsky and DeCino, 1980) or with a silver nerve terminal stain plus cholinesterase stain (Pecot-Dechavassine, Wernig, and Stover, 1979). Methods for dissecting and mounting single identified fibers for high-resolution light microscopy have been described (Werle and Herrera, 1987). For visualization during electrophysiological experiments or for repeated, in vivo observation of living nerve terminals, the fluorescent mitochondria1 dyes DlOC (3,3’diethyloxadicarbocyanine iodide) or 4-Di-2-Asp [4-(4-diethylaminostyry1)-N-methylpyridinium iodide] were used. Methods for dye application, low-light fluorescence imaging, and
image proccssing as well as physiological and morphological controls for obscrvation-induced damage are thoroughly described in Herrera and Banner (in press). For electrophysiology, excised nerve-muscle preparations were superfused with Ringer containing the minimal concentration of d-tubocurarine necessary to prevent twitching upon nerve stimulation. Previously described methods were used for focal extracellular recording of presynaptic spikes and postsynaptic endplate currents (EPCs) and for intracellular recording of endplate potentials (EPPs) from junctions that were later analyzed histologically (Banner and Herrera, 1986; W a l e and Herrera, 1987). Junctions were identified by attaching a video camera to the microscope and recording verbal notes and images of landmarks onto videotape.
RESULTS 1. Methods of Estimating Polyneuronal innervation
The histological appearance of multiple axons converging on a single junctional site has long been used as an indicator of polyneuronal innervation both in tadpoles and normal adult frogs (Letinsky and Morrison-Graham, 1980; Mallart, Angaut-Petit, Zilber-Gachelin, Tomas i Ferre, and Haimann, 1980; Werle and Herrera, 1987; Diaz and Pecot-Dechavassine, 1988). Figure l(A1) shows a typical example from an adult cutaneous pectoris muscle stained with the silver/cholinesterase method. In this case. the upper fiber is innervated by two inputs. The first is a myelinated axon (Ax 1) that exits the intramuscular nerve bundle just above the center of the photograph. The second input is a small terminal sprout [arrow; enlarged in Fig. 1(A2)] that originates from the junction on the adjacent fiber. This adjacent junction is supplied by another myelinated axon (Ax 2). Whether or not the upper fiber is indeed polyneuronally innervated, or simply innervated in a circuitous manner by the same axon, depends on whether Ax 1 and Ax 2 are branches of the same motoneuron. This is difficult to determine morphologically, since each motor axon branches profusely to contact many different junctions (Trussell and Grinnell, 1985). This convergence of two or more axons from different motoneurons onto the same synaptic site is defined as focal polyneuronal innervation. A second morphological feature, the doubly innervated synaptic gutter, is highly correlated with focal polyneuronal innervation in reinnervated and normal muscles (Herrera and Werle, in preparation). Doubly inner-
Figure 1 Characteristic histological signs of polyneuronal innervation in normal adult junctions stained with silver terminal stain and cholinesterase stain. ( A l ) The junction on the upper fiber is innervated by two inputs. One input (Ax 1) is a myelinated axon that supplies most of the terminal arborization. The second input arises as a terminal sprout (arrow) from the junction on the lower fiber. This lower junction is supplied by another myelinated axon (Ax 2). (A2) View of the part of the lower junction that gives rise to the terminal sprout (arrow). Arrowheads indicate a doubly innervated gutter. (B) Doubly innervated gutters (arrowheads). Scale bar = 60 pm for A l , 30 pm for A2 and B.
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vated gutters are single cholinesterase-outlined postsynaptic specializations occupied by two closely apposed nerve terminals. Examplcs are indicated by arrowheads in Figure l(A2,B). Certain frog muscles exhibit a second form of polyneuronal innervation. When fibers are examined along their entire length. multiple junctional sites spaced several millimeters apart can be seen. Multiple junctions are more common in muscles with longer fibers. This multisite innervation makes it possible that different axons can contact the same fiber at distant sites. We will refer to this nonfocal form of polyneuronal innervation as distributed. The presence of multiple axonal inputs and doubly innervated gutters suggests that each of the several distinct junctions on a single fiber may also be focally polyneuronally innervated. Electrophysiology provides additional methods for detecting polyneuronal innervation. With intracellular or extracellular recording, focal polyneuronal innervation can be detected as a stepwise variation in the amplitude of EPPs or EPCs as thc multiple axons are separately recruited by grading the strength of a stimulus applied to the nerve. As would be expected for the focal convergence of two axons, the rise times of the separate EPP components are usually identical. In most cases, distributed polyneuronal innervation cannot be detected by recording at a single site on the fiber because the spacing between junctions usually far exceeds the range of passively conducting potentials. In rare cases an intracellular microelectrode positioncd at one junctional site will record an EPP arising at a distant site, but electrotonic conduction greatly attenuates the amplitude and prolongs the time course of the distant EPP. Distributed polyneuronal innervation is best studied by the simultaneous use of several microclectrodes, with care taken that they are applied to the same fiber. Another physiological method, commonly used to estimate polyneuronal innervation in mammalian muscles, is the extent of overlap between the tension generated by separate motor units (Brown and Matthews, 1960). If two motor axons innervate some of the same fibers, the tension generated by these motor units will not sum linearly. The interpretation of tension overlap is more complex in frog muscles because of the multisite innervation of single fibers (Luff and Proske, 1976). Each of the methods for estimating polyneuronal innervation has certain limitations, some of which have been discussed (Betz, 1987). Counting the number of junctions showing multiple axonal inputs certainly leads to overestimates because
many singly innervated junctions, especially when they are large, are supplied by two myelinated branches of the same axon. Whether these are scored as one or two inputs depends on whether the branch point is visible, and in many cases it is not. The incidence of junctions with doubly innervated gutters is a certain underestimate because not all polyneuronally innervated junctions show this form of close spatial overlap. When identified polyneuronally innervated junctions in normal cutaneous pectoris muscles were examined histologically, the cxtent of overlap between nerve terminals was found to vary between extensive [Fig. 2(A)], moderate [Fig. 2(B)], and slight [Fig. 2(C)]. Physiological methods that measure the incidence of multiple EPP steps yield underestimates for several reasons. First, multiple axonal inputs might not be separable by stimulus threshold. This problem can be ameliorated by changing the polarity as well as the amplitude of the stimulus and by stimulating at multiple sites along the nerve. Second, if the weaker input at a polyneuronally innervated junction has the higher threshold, its contribution to the EPP may be shunted or obscured by the amplitude variations of the stronger input. Finally, most physiological methods require that contraction be blocked by adding curare (Redfern, 1970), treating with formamide or glycerol (Herrera. 1984), or cutting fibers (Barstad, 1962). When these trcatments are used to the minimal extent necessary to block the strongest junctions, weak polyneuronal inputs may be attenuated to undetectable levels. VaIues obtained with each of these three methods are probably directly proportional to the truc incidence of polyneuronal innervation, but this true level probably lies somewhere between the high and low estimates.
2. Polyneuronal Innervation at Normal Adult Neuromuscular Junctions When the methods described above are applied to muscles of normal adult frogs, one finds a considerable leveI of focal polyneuronal innervation. In histologically stained cutaneous pectoris muscles, the incidence of junctions with multiple axonal inputs ranged from 22 to 53% and that of doubly innervated gutters from 8 to 24% (see Results Section 4). In nearly all cases, the terminal arbors of the two axonal inputs were colocalized within a compact area no larger than 5,000-20,000 pm2. Intracellular recording in curare-blocked muscles detected multiple EPP steps in an average of 9% of the junctions in the sartorius and 36% in the cuta-
Plasticity and Growih of'Frog Endplutcs
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Figure 2 Cumera lucida drawings of polyneuronally innervated sartorius junctions showing differing degrees of spatial overlap between ncrve terminals. One terminal is drawn as a solid process and the other is dashed. Cholinesterdse reactivity is shown as a dotted outline. (A) Extreme overlap, with inputs mostly sharing the same synaptic gutters. (B) Moderate overlap, with a few doubly innervated synaptic gutters. (C) Extreme separation within a single junctional site. Scale bar = 30 pm.
neous pectoris (Fig. 3). More sensitive methods, such as blocking contraction with the excitationcontraction uncoupler formamide, revealed cven higher levels (Herrera, 1984). In adult cutaneous pectons muscles, 96% (269/277, 10 muscles) ofthe polyneuronally innervated junctions had only two inputs, with the remainder having three. It may be noted that the three methods for estimating polyneuronal innervation in normal muscles yielded values that differed considerably. These discrepancies were further explored by examining a different group of normal and reinnervated sartorius junctions. Reinnervated muscles are known to have higher than normal levels of polyneuronal innervation (see Results Section 3). Figure 4 shows the incidence of multiple EPP steps, doubly innervated gutters, and multiple axonal inputs for normal sartorius junctions and for reinnervated junctions 1.7-2.4 years after nerve
crush. All three parameters for reinnervated muscles were elevated to similar extents relative to levels measured in normal muscles. There was good agreement between the absolute values of the physiological estimate and doubly innervated gutters for both reinnervated (27% versus 28%) and normal muscles (10% versus 8%). Measuring the percentage of junctions with more than one axonal input, however, yielded values 2.5-3.5-fold higher than the other two parameters. The polyneuronal innervation present at adult neuromuscular junctions could arise from three
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Figure 3 Incidence of physiological pol yneuronal innervation in 7 sartorius (SART) and 13 cutaneous pectons (CP) muscles tested in summer, expressed as the percent ofjunctions showing multiple EPP steps as stimulus strength was varied. Transmission was partially blocked with d-tubocurarine. Each point is the mean for 40-60 junctions in a single muscle. Lengths of bars represent means.
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MULTIPLE EPP STEPS
DOUBLY INNERVATED GUTERS
MULTIPLE AXONAL INPUTS
Figure 4 Physiological and morphological estimates of the incidence of polyneuronal innervation in reinnervated sartorius muscles 1.7-2.4 years after nerve crush (solid bars) and normal muscles (open bars). Vertical lines are standard errors of proportions.
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sources. First, it may be left over from earlier developmental stages due to incomplete synapse elimination. Second, polyneuronal innervation may be formed de MOVO as a result of synaptic remodeling. Third, synapses that are added as muscle fibers grow in length may recapitulate the developmental stages of synaptogenesis and thus express a transient period of polyneuronal innervation. These possibilities are discussed in Results Sections 3 , 6, and 7, respectively. 3. Synapse Elimination following Reinnervation of Adult Muscles A simple explanation for thc presence of polyneu-
ronal innervation in adult frog muscles is that it is residual, i.e., left over from earlier developmental stages. Studies of developing frogs have shown that the rate of elimination slows at about the time of metamorphosis while there is still a substantial amount of polyneuronal innervation (MomsonGraham, 1983). It was therefore of interest to determine whether the adult nerve-muscle system retains the capacity to express synapse elimination under any circumstances. In studies in which muscles of adult mammals were denervated, it was found that polyneuronal innervation was established during reinnervation and, with time, was reduced by a form of synapse elimination similar to that which occurs during development (McArdle, 1975; Benoit and Changeux, 1978; Brown and Ironton, 1978;Thompson, 1978; Gorio, Carmignoto, Finesso, Polato, and Nunzi, 1983; Taxt, 1983; Hopkins, Liang, and Barrett, 1986; Rich and Lichtman, 1989). We found that the same sequence of events occurs in reinnervated frog muscles (Werle and Herrera, 1988; Herrera and Werle, In preparation). Figure 5 shows the incidence of physiological polyneuronal innervation in 53 reinnervated sartorlus muscles tested 14-877 days after crushing the nerve. Functional reinnervation first occurs at 7-1 0 days (DeCino, 1981; Ding, 1982). As early as physiologicai methods could be used reliably (14-21 days), polyneuronal innervation was found to be about 5 times the normal adult level. Synapse elimination progressed over the next several weeks. Both the starting level of polyneuronal innervation and the rate of elimination seemed to vary in different preparations. Over several months, pol yneuronal innervation was reduced to a steady-state level that approximated, but still significantly exceeded, that found in normal adult muscles (Herrera and Werle, in preparation). Despite this hilure to re-
store the normal pattern, these findings demonstrated that the adult neuromuscular system retains the capacity for synapse elimination. 4. Synapse Elimination at Normal Adult Neuromuscular Junctions
The demonstration that synapse elimination occurs following reinnervation does not directly address the question of whether synapse elimination can also occur under normal conditions. AXotoniy may cause pre- and postsynaptic components to regress to a less mature state. In muscle, denervation causes the reappearance of several embryonic and neonatal proteins (Fambrough, 1979; Pappone, 1980; Booth, Brown, Keynes, and Barclay, 1984; Covault and Sanes, 1985, 1986; Schiaffino, Gorza, Pitton. Saggin, Ausoni, Sartore, and Lomo, 1988). The first evidence that synapse elimination does indeed occur in adult frog muscles was the finding of seasonal variation in the incidence of junctions with multiple EPP steps (Herrera and Grinnell, 1981; Herrera, 1984; Diaz and Pecot-Dechavassine, 1988) and histological signs of remodeling (Wernig, Pecot-Dechavassine, and Stover. 1980a.b; Jans, Salzmann, and Wernig, 1986: Diaz and Pecot-Dechavassine, 1988). For examplc, the incidence of multiple EPP steps was about twice as high in sartorius muscles of winter frogs ( 1 9%) compared with summer frogs (9%; Herrera. 1984). These data suggest that there is an annual cycle of synapse formation and elimination in adult frog muscles. Synapse elimination has been shown to involve the retraction of nerve terminals (Bixby, 1981). Therefore, the occurrence of retraction in normal muscles may be considered a necessary condition for the demonstration of synapse elimination in normal adult frogs. We (Herrera and Scott. 1985; Herrera et al., in press) and others (Wernig et al., 1980a,b; Anzil, Bieser, and Wernig, 1984; see Wernig and Herrera, 1986, for review) found that nerve terminal retraction is a common feature of neuromuscular junctions in adult frogs. The typical histological appearance of a retracted nerve terminal is shown in Fig. 6(A).* Exposed, cholinesterase-stained postsynaptic folds can be seen distal [to the right of the arrowhead in Fig. 6(A)] and lateral to the terminal. Since these persistent postsynaptic specializations are normally found only directly opposite presynaptic active zones (Peper, * See color plate section at end of issue
Pla.Pticity and Growth of Frog Endplates
torius. Polyneuronally innervated junctions were identified as such if in vivo observations revealed at least one of the two histologically defined characteristics, doubly innervated gutters, or multiple axonal inputs. With the cutaneous pectoris, position within the muscle could also be used to judge the presence of polyneuronal innervation. We earlier showed that junctions near the medial edge of the cutaneous pectoris have a high probability of being polyneuronally innervated (Herrera, Werle, and Banner, 1986). A typical polyneuronally innervated junction near the medial edge of the cutaneous pectoris is shown in Fig. 7. The initial in vivo observation revealed both doubly innervated gutters [Fig. 7(A), arrowheads] and dual axonal inputs [Fig. 7(A), arrow]. When reexamined 1.2 years (438 days) later, this junction had grown considerably but still appeared to be polyneuronally innervated, as judged by the same morphological criteria [Fig. 7(B-D)]. The symbols in Fig. 7(B) point to features similar to those shown in Fig. 7(A). Most junctions showed persistence of apparent polyneuronal innervation over the observation interval. In a few cases of persistence, however, one of the two inputs suffered disproportionate retraction. An example from a sartorius junction is shown in Fig. 8. At the initial observation, the junction was supplied by two axonal inputs which are out of the focal plane and so cannot be seen in the fluorescence photographs. As shown by the filled and open outlines, the terminal arborization of each of the inputs could be traced [Fig. 8(A,B)].When the
Dreyer, Sandri, Akert, and Moor, 1974), they precisely mark former sites of synaptic contact. Further studies of junctional remodeling by the use of repeated, in vivo observation directly confirmed the occurrence of nerve terminal retraction at junctions in adult frogs (Herrera and Banner, 1987; Herrera, et al., in press; Chen and KO, 1988). Shortening of at least 1 terminal branch was found in 60%of 243 sartoriusjunctions observed twice at intervals of 4-6 months. In 12%of these 243 junctions, at least 1 branch completely disappeared. At one junction, terminal retraction led to the complete abandonment of the junction by the original input, and the junction was partial reinnervated by a sprout from a nearby junction (Herrera et al., in press). These results clearly demonstrate that nerve terminal retraction, a necessary step in synapse elimination, occurs at neuromuscular junctions of normal adult frogs.
5. Rate of Synapse Elimination at Adult Neuromuscular Junctions Although the demonstration of seasonal differences in polyneuronal innervation and of terminal retraction suggested that synapse elimination was possible, it remained to directly test whether the phenomenon occurred in adult muscles. We therefore made repeated, in vivo observations of polyneuronally innervated junctions over intervals of approximately 50 or 420 days in the cutaneous pectoris or approximately 180 days in the sar-
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Figure 5 Incidence of physiological polyneuronal innervation in 53 reinnervated sartorius muscles tested 14-877 days after crushing the nerve. Polyneuronal innervation is established upon reinnervation and then eliminated over the next few months. Data for 10 normal sartorius muscles are shown at the far right (short horizontal lines indicate mean). Synapse elimination does not reestablish the normal level of polyneuronal innervation.
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Figure 6 Neuromuscular junctions in normal frog sartorius muscle. The NBT-stained nerve terminal (purple) is surrounded by periodic synaptic folds filled with cholinesterase reaction product (brown). (A) Nerve terminal retraction is indicated by exposed synaptic folds beyond the distal tip of the terminal. Distal tip of terminal is indicated by the arrowhead. Exposed folds are to the right. (B) Lower magnification view of a junction showing several short terminal sprouts (arrowheads). (C) High magnification view of one of the branches in B, showing filopodial sprouts extending from the distal tip of the terminal. Scale bars = 10 Fm in A and C, 20 pm in B. (See color plate section at end of issue.)
junction was reexamined 183 days later, it was still apparently polyneuronally innervated. One of the inputs, however, had retracted almost completely [Fig. S(C,D)]. After histological staining, abandoned synaptic gutter lined with junctional folds seemed to mark the former extent of the retracted
terminal [Fig. 8(D)]. It is interesting that the terminal supplied by the other input grew considerably [compare open outlines in Fig. 8(B) and (D)].Despite this growth there was 27 p m of abandoned gutter beyond the distal tips of this terminal, indicating that the terminal was even longer at some
Plasticity and Growth Ctf'FrogEndplates
time during the observation interval. The finding of growth and retraction within single junctions is discussed further in Results Section 6, below.
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Additional evidence for the rate of synapse elimination was obtained by using morphology and physiology to estimate the incidence of polyneuronai innervation in frogs of various ages. We examined the cutaneous pectoris, a muscle that has a high level of polyneuronal innervation (Fig. 3). Since frogs grow throughout life (Gibbons and McCarthy, 1983), body size was used as a rough index of age. Figure 9 shows the incidence of junctions with multiple axonal inputs (solid circles, solid line), doubly innervated gutters (open circles, large dashed line), and multiple EPP steps (open squares, small dashed line) for groups of muscles from small (young) and large (old) frogs. As explained in Results Section 1, the different estimation methods yielded different results. With each method, however, there was a highly significant decrease in polyneuronal innervation with increasing age. We consider it likely that this progressive reduction of polyneuronal innervation represents continuation of the slow rate of synapse elimination described by Morrison-Graham (1983). An alternative explanation is discussed in Results Section 7. 6. The Equilibrium between Synaptic Growth and Retraction: Synaptic Remodeling
D
Figure 7 Repeated, in vivo observation of the persistence of poiyneurond innervation in a normal cutaneous pectoris junction. (A) Initial in vivo observation of terminal stained with the tluorescent mitochondria1 dye 4-Di-2-Asp. Video image was obtained with very dim illumination and digitally enhanced. Arrow indicates two axonal inputs, and arrowheads point to doubly innervated synaptic gutters (DIGs). (B) Final histological observation 438 days later. Junction stained with the NBT method, which stains only unmyelinated processes, and with cholinesterase stain. Arrow indicates two axonal inputs. The unmyelinated preterminal axon of one input stains with NBT and so can be clearly seen. The other input remains myelinated until it contacts the fiber. Thus it does not stain with NBT and can only be faintly seen in this photograph. Arrowheads point to DIGS.(C, D) Higher magnification views of intertwined terminals and DIGs in the left portion of the junction in B. Scale bar = 30 Frn for A and B, 15 fim for C and D.
Estimates of the rate of synapse elimination made from in vivo observations or by measurement of polyneuronal innervation in young and old frogs would be in error if polyneuronal innervation could be generated de novo. Histology indicated that this might occur. As others have described (reviewed in Wernig and Herrera, 1986), many junctions showed nerve terminal sprouts. These were usually evident as elongations of terminal processes distal to the area of postsynaptic specialization. Fine filopodial processes, which we suppose are an early stage of terminal sprouting (see also Robbins and Polak, 1988), can be seen extending from several points along one terminal branch in Fig. 6(B, arrowheads). The distal tip of this sprouting branch is enlarged in Fig. 6(C).It is interesting that the other branches, which are clearly part of the same terminal arborization, were not sprouting at the time of fixation. In an earlier study, we found that about 9% of normal sartorius junctions had such sprouts (Herrera and Scott, 1985) and others have found even higher levels (Wernig et al., 1980a). These sprouts result in junctional growth when they terminate on the fiber of origin. Both neuromuscular junctions and
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Figure 8 Rcpeated, in vivo observation of disproportionate retraction of one input at a douhly innervated ,junction in a normal sartorius muscle. (A, B) Photograph and drawing of initial in viva observation using 4-Di-2-Asp. Open and solid outlines in B indicate terminal branches that. in other focal planes, could be seen to arise from different axons. Crossing of terminals is a characteristic feature of polyneuronally innervated junctions. (C, D) Final in vivo observation 183 days later. Drawing made from histologically stained junction (NBT/ ChE method). Terminal shown by open outline had grown substantially. Terminal shown by filled outline had retracted, yet sprouted a new branch at right. Abandoned synaptic folds (vertical hatching) mark the former extent of the retracted terminal. Paradoxically. the input that grew also had abandoned synaptic folds beyond its distal tips, suggesting that growth was followed by partial retraction. Scale bar = 30 pm for A-D.
muscle fibers grow substantially throughout the life of a frog (Jans et al., 1986: see Results Section 7). The actual rates and patterns of growth in adult junctions could be better appreciated by repeated, in vivo observation (Herrera et al.. in press). Among 243 sartorius junctions imaged twice over periods of 4-6 months, 89% showed a net increase in total nerve terminal length. The average increase in terminal length of the junctions that grew was 35% (54 pm). Most (56%) of the 2 I6 junctions
that showed net growth also had a t least one branch that retracted. Eleven percent of the 243 junctions showed net retraction, with an average decrease in total terminal length of 15% (36 pm). Thesc 27 retracting junctions did not shrink uniformly, most ( I 9) had at least 1 terminal branch that grew. We concluded from these studies that most junctions grow in adult muscles and that growth is the net result of differing degrees of elongation and retraction of individual nerve terminal branches (Herrera et al., in press). This finding of
Plasticity and Growth of Frog Endplates
simultaneous growth and retraction of terminal branches of the same motoneuron at the same synapse also suggested that growth and retraction may not be completely controlled at the level of' the entire synapse but also locally, by very shortrange nerve-muscle interactions. An in vivo observation that added greatly to our understanding of polyneuronal innervation in adult muscles was the finding that sprouting terminals did not always synapse onto the fiber of origin. Sprouts occasionally jumped to the adjacent fiber where, as in Fig. l(A), they could be seen to form synaptic contacts outlined by cholinesterase reactivity. An example is shown in Fig. 10. Over an interval of 18 1 days, the leftmost branch of this terminal elongated 2.8-fold (from 39 to 1 10 pm) and bifurcated. The lower ramus was a 45 pm sprout that terminated in a bright bouton on the fiber below. As was usual in such cases, the new contact was established within the perijunctional area of the neighboring fiber. The junction on the neighboring fiber was on its deep surface and so is not shown. This acquisition of a second input by the lower fiber would represent the de n o w formation of focal polyneuronal innervation if the axon that originally supplied the neighboring fiber was from a different motoneuron. Other parts of the 60 1 I\
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Figure 9 Growth-associatedreduction in polyneuronal innervation in cutaneous pectoris muscles of normal adult frogs. For each of the 3 measures, values in large frogs are significantly lower than values in small frogs ( p < 0.0002). Vertical lines through points are standard errors of proportions, horizontal lines are standard errors of mean for body weight. Data on histologically visible multiple inputs (solid circles) are from 202 junctions in 3 large frogs and 972 junctions in 16 small frogs. Data on multiple EPP steps (open squares) are from 572 junctions in 5 large frogs and 1039 junctions in 8 small
frogs. Data on doubly innervated gutters (open circles) arc from 199junctions in 3 large frogs and 980 junctions in 16 small frogs.
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junction in Fig. 10 from which the sprout originated also grew substantially. Overall, the terminal increased 74% in total length (from 300 to 521 pm). It is interesting to note that differences in the sites of origin and termination of terminal sprouts can lead to dramatically different junctional restructuring. Sprouts originating from the distal tips of terminals and terminating within a few micrometers on the same fiber would result in simple elongation of preexisting branches. If postsynaptic differentiation could keep pace with presynaptic elongation, the incidence of sprouting terminals would be substantially underestimated by single time point observations such as those made with histology or electron microscopy. Sprouts originating from the side of a terminal branch and terminating on the same fiber would also result in junctional remodeling and, as described above, may not be recognized as new branches with single time point observations. If either type of sprout terminated in the perijunctional region of a different fiber, polyneuronal innervation may be formed. Finally, termination of a sprout far from a preexisting junction on a different fiber would result in the addition of a new synaptic site on that fiber (see Results Section 7). The view that has emerged from these findings is that there is constant turnover of polyneuronal innervation in adult frog muscles. At any time the level of polyneuronal innervation is the net result of an equilibrium between the processes of retraction and elimination on one hand and growth and synaptogenesis on the other. Terminal retraction results in junctional remodeling and can lead to synapse elimination. Terminal growth, the generation of new synaptic contacts, can result in junctional enlargement, remodeling, new formation of polyneuronal innervation, or synapse addition.
7. Size Matching and Synapse Addition during Growth Frogs grow throughout life (Gibbons and McCarthy, 1983), and body growth is accompanied by muscle fiber hypertrophy (Sperry, 1981; Jans et al., 1986). Within muscles, fiber size and junctional side are correlated (Kuno, Turkanis, and Weakly, 197 1; reviewed in Wernig and Herrera, 1986). These findings, and direct in vivo observations (Herrera et al., in press), suggest that synaptic growth maintains physiological efficacy despite the increase in postsynaptic input capacitance and decrease in input resistance caused by
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Figure 10 Repeated, in vivo observation of the apparent de nova establishment of polyneuronal innervation in adult sartorius muscle. (A) Initial in vivo observation of 4-Di-2-Aspstained terminal, supplied by a single axonal input (ax). Arrow indicates distal tip of leftmost terminal branch. Asterisk marks the distal tips of the two rightmost branches. (B) Final in vivo observation 181 days later. The junction was still supplied by a single input (ax). Arrow again indicates leftmost terminal tip. which has grown to the left. The three arrowheads point to a faintly stained terminal sprout that jumps to the lower fiber and terminates there i n a bright bouton. Original junction of the lower fiber was on the deep surface and so cannot be seen in these images. Note that the rightmost tips of the terminal (asterisk) have also grown considerably. Scale bar = 30 Fm for both panels.
fiber hypertrophy. The amplitude of the EPP is probably determined more by the capacitive properties of the fiber membrane than the steady-state resistive properties, since the endplate current is brief (approximately 1 msj compared with the fiber time constant (approximately 20 ms). To explore these questions further, we examined the relationships between presynaptic and postsynaptic size in sartorius muscles from 16 frogs representing a wide range of body size (4-404 g body weight). Figure 11 shows that growth in muscle length is accompanied by hypertrophy in fiber diameter [Fig. 1 l(A)] and by junctional enlargement [Fig. 11(Bj]. These data may also be
used to predict how synaptic efficacy changes during growth. The points in Fig. I 1(C) show thc relationship between total terminal length and muscle fiber diameter. If one assumes that endplate current flows through a region that is small relative to the length constant of the fiber, then the postsynaptic voltage response may be approximated by that of an infinite cable when charge is instantaneously applied at a point (equation 3.48 in Jack, Noble, and Tsien, 1975). The prediction is that the EPP would be inversely proportional to the 3/2 power of fiber diameter. If terminal length is proportional to transmitter release and therefore endplate current (Kuno et al., I97 1; Herrera, Grinnell,
Plasticity and Growth of Frog Endplates
and Wolowske, 1985; Propst, Herrera, and KO, 1986; Propst and KO, 1987), then terminal length must increase as the 3 / 2 power of fiber diameter to maintain EPP size as the fiber grows. The curve in Fig. 1 1(C) is a 3/2 power function drawn by eye to fit data from the 9 smallest frogs (4-74 g body weight). Synaptic efficacy appears to be well maintained in this range of body size. The 7 points on the right, representing the largest frogs (77-404 g), clearly fall below the curve, suggesting that synaptic efficacy falls as frogs grow very large. Such a growth-associated decline in efficacy at frog junctions has been reported (see Trussell and Grinnell, 1985). The assumptions and predictions of this theoretical approach should be further tested by direct measurement of transmitter release, postsynaptic transmitter sensitivity, and the active and passive electrical properties of the muscle fiber in frogs of various sizes. We tentatively conclude, however, that there are mechanisms that tend to match presynaptic and postsynaptic size during growth in frog muscles. The matching is not perfect, since there appears to be a progressive decrease in synaptic efficacy in large animals. In addition to hypertrophy, the growth of some frog muscles involves the addition of new synaptic
sites as muscle fibers elongate (Bennett and Pettigrew, 1975; Nudell and Grinnell, 1983). In fibers without propagating action potentials, such as frog tonic fibers (Kuffler and Vaughan Williams, 1953a,b; Gilly and Hui, 1980a,b)and many twitch fibers in fish (Mos, Maslam, and Armee-Horvath, 1988), multiple junctional sites serve to distribute depolarization for efficient tension production. Frog twitch fibers are like mammalian fibers in that they support propagating action potentials. In such fibers, the functional significance of multiple junctional sites is unclear. The synapses on each fiber are almost always many length constants apart so they do not interact electrically, nor does simultaneous generation of action potentials at several locations seem to confer an advantage in tension production (Nudell and Grinnell, 1983). It is possible that the formation of multiple junctional sites is involved in trophic interactions between nerve and muscle, but these are poorly understood. Whatever their function, an interesting area for future research will be the extent to which the newly added synapses recapitulate the normal developmental sequence of presynaptic and postsynaptic differentiation, polyneuronal innervation, synapse elimination, and synaptic matura-
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Figure 11 Postmetamorphic growth of sartorius muscles and neuromuscular junctions in 16 frogs ranging from 4 to 404 g body weight. Each point is the mean ( 5 SEM) of 15-49 junctions in a muscle. Error bars smaller than diameter of points are not shown. Straight lines fit by least squares regression. Growth in muscle length is accompanied by muscle fiber hypertrophy (A; r = 0.94, p < 0.0001) and nerve terminal elongation (B; r = 0.89, p < 0.0001). ( C )The relation between nerve terminal length and muscle fiber diameter. The curve is a 3/2 power function drawn by eye to fit the 9 points on the left, which are from the smallest frogs (4-74 g). This curve represents the terminal length that would be necessary to maintain EPP size as input capacitance increases with fiber growth (see text). The 7 points on the right, which are from the largest frogs (77-404 g) fall well below the curve, suggesting that synaptic efficacy decreases in large animals.
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tion. If newly added synapses progress through typical developmental stages, much of the polyneuronal innervation observed in normal adult muscles could be explained. If newly added synapses tend not to be polyneuronally innervated, the life-long reduction in polyneuronal innervation we found in normal muscles (Fig. 9) may not represent synapse elimination but rather the selective addition of singly innervated junctions. Despite these uncertainties, it seems a reasonable working hypothesis that lifelong retention of the capacities for sprouting, synapse formation, and synapse elimination in frog muscles is an adaptation to ensure the maintenance of synaptic efficacy and the orderly addition of new synaptic sites during growth. 8. Measurement of Competitive Interactions
It has been demonstrated that synapse elimination involves competitive interactions between nerve terminals at the same synaptic site (Brown, Jansen, and Van Essen, 1976; Thompson and Jansen, 1977; Betz, Caldwell, and Ribchester, 1979, 1980). We have speculated that similar competitive interactions are involved in synaptic remodeling in adult junctions (Werle and Herrera, 1987). Close examination of the anatomical and physiological correlates of polyneuronal innervation in normal muscles and synapse elimination in reinnervated muscles has provided clues to the nature these competitive mechanisms.
a. Relative Synaptic Eficacy as an Index of Compefition. In a previous study (Werle and Herrera, 1988), we compared the properties of two groups of doubly reinnervated sartorius junctions. The first group was examined 40-80 days after crushing the nerve, a time when most junctions were still polyneuronally innervated (Fig. 5 ) . The second group was taken more than 250 days after nerve crush, which was after the main period of synapse elimination (Fig. 5). The junctions in the latter group were considered to be those that survived the elimination process. By using a combination of vital fluorescent staining, simultaneous intracellular and focal extracellular recording, and histological examination of the junctions from which recordings were made, we determined the size, position, and physiological efficacy of both inputs at doubly innervated synapses. By comparing the two competing inputs at junctions before and after synapse elimination, we were able to make strong inferences regarding the mechanisms
of elimination. A number of properties were not different. For example, relative endplate potential size, nerve terminal length, and number of terminal branch points did not differ between the competing inputs before and after synapse elimination. This finding suggested that inputs were not eliniinated on the basis of these properties, and thus argued against models that assume simply that EPP amplitude, terminal length, or terminal complexity are important determinants of elimination or persistence. Although differences in synaptic efficacy alone did not correlate with elimination, there was a striking correlation when efficacy was normalized to nerve terminal size. Relative differences in this normalized measure of efficacy were expressed as a parameter called ratio of synaptic efficacy (RSE). This dimensionless parameter wdS calculated as EPP size per unit nerve terminal length of the weaker input divided by EPP size per unit length of the stronger input. The values of RSE thus ranged from near zero, when inputs were very different in EPP/terminal length, to near one as they approached equality. When polyneuronally innervated junctions were examined before synapse elimination had occurred, RSE was broadly distributed, with a median value of 0.42 (Fig. 12, top). After synapse elimination was complete, the synaptic efficacies of competing inputs at surviving polyneuronally innervated junctions were significantly more similar, with median RSE = 0.64 (Fig. 12. middle). Our interpretation of these results is that inputs with inherently different release efficacies come to be randomly associated at polyneuronally innervated junctions during reinnervation. Synapse elimination then occurs selectively at those junctions wherc the two inputs differ the most, and polyneuronal innervation will persist at those junctions where the efficacy of the two inputs is similar. We also determined RSE for doubly innervated junctions in normal sartorius muscles. As discussed above, the majority of these doubly innervated junctions were considered to be the survivors of developmental and ongoing synapse elimination. As shown at the bottom of Fig. 12, we found that these normal polyneuronally innervated junctions had the same tendency toward equality in EPP size per unit terminal length (median RSE = 0.67) as did reinnervated junctions after synapse elimination (Fig. 12, middle). These findings support the view that during normal development and growth, as well as following reinnervation, synapse elimination occurs primarily at
Plasticity and Growh ojFrog Endplates
junctions where the competing inputs have vastly different normalized synaptic efficacies. In other studies we have explored the physiological consequences of synaptic competition. If synapse elimination involves mutually deleterious competitive interactions, we reasoned that polyneuronally innervated junctions may be smaller, weaker, or both compared with singly innervated junctions in the same muscle. The literature provides conflicting evidence on this point. In the pectoral muscle of Xenopus, the sum of the separate EPPs at doubly innervated junctions averaged 30% less than EPP amplitude at singly innervated junctions (Angaut-Petit and Mallart, 1979; Haimann, Mallart, Tomas i Ferre, and Zilber-Gachelin, 1981a). In cutaneous pectoris and sartorius muscles of Rana pipiens, no significant difference was found, although EPPs at doubly innervated cutaneous pectoris junctions were 10% smaller than at singly innervated junctions (Weakly and Yao, 1983). We reexamined this question using
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Figure I 2 Distributions of ratio of synaptic efficacy (RSE) at dually innervated sartoriusjunctions. RSE defined as EPP size per unit terminal length of weaker input divided by EPP size per unit length of stronger input. Arrowheads indicate medians. Top: Data for 87 reinnervated junctions 40-80 days after nerve crush. which was before the major period of synapse elimination. Middle: A different group of 76 reinnervated junctions at more than 250 days after nerve crush, after synapse elimination. Note significant ( p < 0.001, MannWhitney U test) shift of distribution to the right. Bottom: Data for 46 junctions in a normal unopcrdted
muscle.
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data from the cutaneous pectoris. In each of 19 curare-blocked muscles, EPPs were recorded at 31-164 junctions, and each EPP was normalized by dividing it by the average EPP amplitude for that muscle. This normalization allowed data from different muscles to be combined despite differences in curare concentration or sensitivity to receptor block. Nerve stimulus strength was varied to determine whether junctions were singly or doubly innervatcd. All dual innervation was focal. A preliminary analysis showed that EPP size at 1042 singly innervated junctions averaged 5% above the mean of alljunctions, whereas total EPP size at 464 doubly innervated junctions was 11% below the mean of all junctions, a highly significant difference (p < 0.002). It thus appeared that competitive interactions reduced the efficacy of one or both inputs at doubly innervated cutaneous pcctoris junctions. As yet we do not know whether this diminished efficacy is due to presynaptic or postsynaptic factors, or whether it has a physiological or structural basis. 6 . Competition within and between Synaptic Sites. It has been shown that the elimination of multiple synaptic sites from rat muscle fibers is enhanced if the sites are closely spaced (200 days), there was significantly less focal polyneuronal innervation in muscles with reduced motor units (Werle and Herrera, 1988). Data for the period after the initial wave of synapse elimination (>60 days) are presented in Fig. 14. This figure shows three different estimates of focal polyneuronal innervation in reinnervated muscles with large and small motor units and in normal muscles. By the methods of counting EPP steps and doubly innervated gutters, there was significantly less polyneuronal innervation in small motor unit muscles compared with large. The method of counting multiple axonal inputs did not yield a significant difference, confirming that the tendency of this method to overestimate polyneuronal innervation (see Results Section 1) makes it less reliable in this situation. By all three methods, normal muscles showed significantly less polyneuronal innervation than both types of reinnervated muscles, confirming that synapse elimination after reinnervation does not restore the normal low level of polyneuronal innervation (Fig. 5 ; Herrera and Werle, in preparation). We concluded that decreasing motor unit size increases the ability of regenerated terminals to compete for and dominate synaptic sites.
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We speculate that this increased level of competition is fueled by the enhanced metabolic support received by terminals in reduced motor units. DISCUSSION 1. Introduction
The results presented here, and those of similar studies by others (reviewed in Wernig and Herrera, 1986; Robbins, 1988), demonstrate that neuromuscular junctions of adult frogs are highly dynamic synapses. Within a single junction there is often growth of some nerve terminal branches while others are retracting. With time, this remodeling usually results in net synaptic enlargement. In a similar sense, synapse formation and elimination can occur simultaneously in different parts of the same muscle. These opposing processes wax and wane in an annual cycle, giving rise to seasonal differences. The equilibrium progressively shifts towards elimination, resulting in a net reduction in polyneuronal innervation over a lifelong time course. Within the muscle as a whole, there is a progressive increase in the number of synapses, with a conscquent expansion of each motoneuron’s peripheral field.
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Figure 14 Estimates of the incidence of focal polyneuronal innervation using a physiological method (multiple EPP steps) and two morphological methods (doubly innervated gutters. multiple axonal inputs). Data are for reinnervated sartorius muscles with large motor units (solid bars; sample size for each method was 1919, 245, and 163 junctions, respectively), reinnervated sartorius muscles with small motor units (hatched bars; sample size 656, 190, and 139 junctions), and normal muscles (open bars; sample size 585,25 1, and 25 1 junctions). Vertical lines are standard errors of the mean. Reinnervated muscles were denervated by nerve crush at least 60 days earlier. Within each cluster of bars, values for the 3 types of preparations were significantly different ( p < 0.01) except for the solid and hatched bars in rightmost cluster. which did not differ significantly from each other ( p > 0.05).
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The view that emerges from these findings is that, at any one time. the structural and functional state of the neuromuscular junction represents an equilibrium between processes favoring growth and those favoring retraction. This discussion is organized to consider three major factors that may influence this equilibrium: metabolic support nerve terminals receive from their somata, competition between terminals of different motoneurons, and activity-dependent interaction between nerve and muscle. We will then discuss the differences in synaptic plasticity and synapse elimination in neuromuscular junctions of frogs and mammals. With the goal of posing testable hypotheses, our approach will be purposely speculative. 2. Metabolic Support It is a well-established principle that the materials needed for maintenance and growth of motor nerve terminals are synthesized in and exported from the soma. The metabolic load and transport distances involved can be considerable. Using a published value of 12 mm for dendrite length (Bregman and Cruce, 1980) and our own measurements of 35 mm for typical axon length and 57 mm for total nerve terminal length (Herrera, unpublished), it can be calculated that a typical spinal motoneuron in a 20 g frog must support approximately 10 cm of processes, with much of this located at a considerable distance. Consistent with the view that the ability to provide metabolic suppod is matched to the demand, positive correlations have been found between soma size and motor unit size (Yao and Weakly, 1986) and between motor unit size and synaptic efficacy (Grinnell and Trussell, 1983; Trussell and Grinnell, 1985). Since metabolic support must be shared among all terminals within a motoneuron’s arborization, the level of support each terminal receives might be expected to change when motor unit size is changed. The maintenance of synapses and the ability of terminals to compete for synaptic connections is likely to be altered by changes in the level of metabolic support that terminals receive. Experimental studies using adult nerve-muscle preparations confirmed these predictions. Artificial enlargement of motor unit size resulted in decreased transmitter release and synaptic efficacy (Bennctt and Raftos, 1977; Bennett et al., 1979; Wigston, 1980; Hopkins et al.. 1980; Haimann et al., 198la; Slack and Hopkins. 1982; Luff,
Hatcher, and Torkko, 1988; Rochel and Robbins, 1988; Regnier and Herrera, 1988). Conversely, a decrease in motor unit size caused increased release (Herrera and Grinnell, 1980, 1985; Pockett and Slack, 1982a). Over time, the enhanced release was reduced to normal levels probably because metabolic support was down-regulated in response to reduced demand or reduced supply of trophic factor (Pockett and Slack, 1983; Herrera and Grinnell, 1985). Manipulations of the size of the peripheral arborizations of motoneurons also affect the ability of terminals to compete for synaptic connections. Regenerating motor axons, which initially have few synapses to support, have been shown to displace the terminals of motoneurons that have expanded their fieIds by sprouting (Brown et al., 1976; Genat and Mark, 1977; Brown and lronton, 1978; Dennis and Yip, 1978; Thompson, 1978; Wigston, 1980; Haimann et al., 1981b; Lowrie, O’Brien, and Vrbova, 1985; Ribchester, 1988). The finding that terminals in overextended motor units are at a competitive disadvantage suggests the following principle. During synapse elimination and perhaps throughout adult life, local changes in one part of a motoneuron’s arborization can affect synaptic maintenance and competition elsewhere in the motor unit. However, it is difficult to envision how presynaptic and postsynaptic size could bc matched at individual junctions unless local influences predominated. Although similar studies are considerably more difficult with developing nerve-muscle preparations, it is interesting to speculate that the level of metabolic support may also determine the outcome of synapse elimination. For example, a reduction in metabolic support may be involved in the retraction of axonal arborizations. When the rat soleus was partially denervated at birth so that only a few motor axons remained, there was still some reduction in motor unit size by the end of the synapse elimination period (Brown et al., 1976;Thompson and Jansen, 1977;but see Betz et al., 1980). Similar results were found in experiments on the mouse soleus (Fladby and Jansen, 1987). These findings suggest that motoneurons have an intrinsic tendency to retract their terminals during the peak period of synapse elimination, and this is likely to involve diminished metabolic support. It seems unlikely that the ability of motoneurons to provide metabolic support would remain fixed. In developing rat muscle, secondary myogenesis occurs during the penod of synapse elimi-
Plasticity and Growth of Frog Eidplatt>y
nation, so that the number of fibers and therefore the number of synapses is actually increasing (Betz et al., 1979). This finding suggests that the ability of motoneurons to support terminals should increase as motoneurons mature. This maturation may in turn be guided by trophic influences that motoneurons receive from the fibers they innervate (see Discussion Section 4). We speculate that at the embryonic stage at which synapse elimination begins. there are inherent differences in the ability of motoneurons to support terminal arborizations. At each polyneuronally innervated junction, nerve terminals that are supported to a greater extent have a competitive advantage and are more likely to survivc the elimination process. Thus the more robust motoneurons will develop into larger motor units. The level of support each terminal receives is not fixed, however. As the arborization of weaker motoneurons is pruned to a smaller size, the support received by each terminal might increase to the point where they are equally able to compete with terminals of larger and stronger motoneurons. In addition, there may be simultaneous maturation of motoneurons driven by trophic feedback. In this way each motoneuron would establish a motor unit size appropriate to that neuron’s ability to provide support, and, as synapses increase in size and number in adult life, there would be a commensurate increase in the ability of motoneurons to support their enlarged arborizations.
3. Competition Since the pioneering studies of Brown et al. (1976) and Betz et al. (1980), it has been widely accepted that synapse elimination can be at least partly explained by competition between axons that converge on the same junctional site. Thesc interactions result in the stabilization or withdrawal of (synapticcontacts. It is likely that the object of the competition is the acquisition of trophic support (Thompson, 1985: see Discussion Section 4). Building on this foundation, we will discuss how recent work has advanced our understanding of Gynaptic competition at frog neuromuscular junctions. The fact that synapse elimination does not proceed to completion in either normal or reinncrvated frog junctions offers an important advantage for the study of synaptic competition. Competitive mechanisms can be inferred by comparing the properties of synapses where polyneuronal innervation is and is not eliminated. Using this ap-
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proach, we discovered two important principles governing the elimination of focal polyneuronal innervation (Werle and Herrera, 1987, 1988). First, spatial proximity does not affect the outcome of synapse elimination. Competing terminals were equally intertwined or segregated before and after the main period of synapse elimination. Second, junctions at which polyneuronal innervation was eliminated were those in which competing inputs differed in EPP size per unit terminal length. If EPP size per unit terminal length was similar. polyneuronal innervation persisted. Our interpretation of these findings rested on the assumption that this measure of physiological efficacy also reflected the ability of terminals to compete for synaptic connections. Physiological and competitive efficacy have been shown to covary in a variety of experimental situations, probably because both depend on metabolic support from the motoneuron soma (see Discussion Section 2; also, Wcrle and Herrera, 1987). We concluded that supernumerary inputs are displaced from a junction only when those inputs compete with others of higher competitive strength. Com petiti on between inputs of equivalcnt strength leads to a stalemate in which polyneuronal innervation persists. Comparisons of synaptic efficacy at singly and focally doubly innervated junctions in normal frog muscles indicate that competitive interactions impair synaptic function. In the cutaneous pectoris muscle, we found that total EPP amplitude was 16% lower in doubly innervated junctions compared with singly inncrvated junctions. Others havc also found that polyneuronal innervation at frogjunctions is associated with deficits in synaptic cfficacy (Angaut-Petit and Mallart, 1979;Grinncll, Letinsky, and Rheuben, 1979; Haimann et al., 198 1 a, Weakly and Yao, 1983; Trussell and Grinnell, 1985). Upon considering these and other results on the nature of synaptic competition at frog neuromuscular junctions, we have formulated a two-part hypothesis that we are currently testing. First, wc propose that under normal conditions. competition is a heteroncuronal interaction. Different parts of the same motor axon, including different terminal branches at a singly innervated junction, do not compete with each other. Second, we propose that it makes little difference whether competing heteroneuronal inputs terminate at the same junctional site or at different sites on the same fiber. Focal and distributed polyneuronal innervation would provoke equivalent competition. The first statement, that competition occurs
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only between different neurons, conveys an appealing sense of efficiency in that it would prevent a terminal from competing with itself. More importantly, the hypothesis is supported by a preliminary analysis of growth-associated changes in the pattern of muscle fiber innervation (see Results Section 8b and Fig. 13). We found that postmetamorphic growth in the pectoral muscle ofXenopus is associated with a progressive conversion of the a/b innervation pattern to the a/a pattern. We speculate that competition in a/b fibers would cause functional or structural deficits at one or both junctions. The resulting decrease in activity or the partial uncovering of postsynaptic sites would render the junctions susceptible to additional innervation by nearby axons and terminals. Histological [Fig. 1(A)] and repeated in vivo observations (Fig. 10) have shown that interjunctional sprouting occurs. If this sprouting generated additional polyneuronal innervation, e.g., addition of a c input, the new input would be similarly destabilized. When, by chance, the same axon came to innervate both sites, decreased competition would lead to synaptic stability. The second hypothesis, that competition occurs to an equivalent extent regardless of the distance between heteroneuronal inputs, also has some support. By mapping the exact size and position of competing inputs at doubly innervated junctions in normal muscles (Werle and Herrera, 1987) and reinnervated muscles (Werle and Herrera, 1988), we found evidence that spatial proximity did not intensify competition. Further tests of this hypothesis could be made by relating synaptic function and structure with the amount and type of polyneuronal innervation at identified junctions in several different muscles. One would predict that physiological efficacy and morphological signs of structural stability (large junctional size, lack of abandoned synaptic gutter, etc.) should relate inversely to the incidence of total polyneuronal innervation, both focal and distributed. Muscles with more distributed polyneuronal innervation should have less focal polyneuronal innervation because increased competition would have driven synapse elimination to a more complete state. Such appears to be the case when the sartorius and cutaneous pectoris are compared. The sartorius averages about three times as many junctional sites per fiber and has about one-third the focal polyneuronal innervation of the cutaneous pectoris. Although the reciprocal nature of these numerical relationships may be coincidental, these findings are consistent with the hypothesis. 6'
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Following the lead of an early theoretical approach (Stent, 1973), we propose that competition between motor axons is governed by a Hebbian rule (Hebb, 1949)in which an important determinant of synaptic stability is the timing of presynaptic and postsynaptic activity (see also Nudell and Grinnell, 1983). In mononeuronally innervated fibers with two junctions (a/a), the destructive collision of muscle fiber action potentials in the region between junctions would insure that presynaptic and postsynaptic spikes always occurred synchronously at each junction. In the same way, the different terminal branches of the same motoneuron at a singly innervated junction could avoid mutual competition by virtue of their synchronous activity. In fibers innervated with the a/b pattern, however, it is likely that the two axons would not be synchronously active. On average, a postsynaptic spike could pass beneath an inactive presynaptic terminal about half the time. This presynaptic and postsynaptic asynchrony would destabilize both inputs, with the advantage going to the input better able to withstand the detrimental influence. The advantage may be determined by the relative level of metabolic support each terminal receives. Competition is clearly involved in the developmental process of synapse elimination, and similar interactions occur in reinnervated adult muscle. The simplest view is that the tendency of motor axons to compete for synaptic connections does not cease at the end of embryonic life. The same processes probably continue to remodel synaptic structure and function throughout adult life. 4. Activity-Dependent Nerve-Muscle
Interactions Alterations in neuromuscular activity have profound effects on synapse elimination. Generally, synapse elimination is delayed by blocking activity and accelerated by increasing activity via direct stimulation (reviewed by Thompson, 1985; Betz 1987). Thompson (1985) has proposed an attractive hypothesis for the mechanism of synapse elimination that conceptually unites these effects of activity with growing evidence for muscle-derived motoneuronotrophic factors (Henderson, Huchet, and Changeux, 1983; Slack and Pockett, 1982; Pockett and Slack, 1982b: Smith and Appel. 1983; Hill and Bennett, 1986; Dohrman, Edgar, and Thoenen, 1987; Heaton, 1988). The hypothesis is that during developmental synapse elimination motoneurons depend upon trophic factors that are
Pluslicily und Growth of Frog Endplates
produced by muscle in amounts inversely related to activity. A similar mechanism was the subject of an earlier theoretical analysis (Gouze, Lasry, and Changeux, 1983). Our purpose here will be to examine whether the same mechanism can also explain the postmetdmorphic growth and plasticity of frog junctions that we have described. It is important to note that in frogs, as in mammals, various methods of inducing inactivity trigger the production of a nerve terminal growth-promoting stimulus (Wernig et al., 1980a; Wines and Letinsky, 1988; Diaz and Pecot-Dechavassine, 1989). Both histological and repeated in vivo observdtions have shown that frog neuromuscular junctions grow throughout life. Presynaptic and postsynaptic growth is matched in such a way that physiological efficacy tends to be maintained. The maintenance of efficacy may be better in younger animals. Thcse findings can be explained by the hypothesis that mature motoneurons are as dependent on muscle-derived trophic factors as dcveloping motoneurons, and that the release of those factors in both development and adult life is an inverse function of activity. This would provide a negative feedback mechanism that explains the matching of presynaptic and postsynaptic size during growth in adults. Synaptic efficacy would be regulated through its effect on activity. The activity signal that regulates trophic support may not only involve propagating action potentials, but also more local influences. In frogs, and in other lower vertebrates (Morgan and Proske, 1984; Mos et al.. 1988), muscle growth involves the addition of new synaptic sites. In mammals, certain of the influences of nerve on muscle, such as concentration of acetylcholine receptors (Fambrough, 1979)and voltage-dependent Na channels (Caldwell, Campbell, and Beam, 1986), diminish sharply with increasing distance from the junction. If the suppression of trophic factor production by activity in frog muscle also diminishes with increasing distance from the junction, growth in muscle fiber length may trigger sufficient production of trophic substance to attract and support new synapses at the growing ends of the fiber. In response to this additional source of trophic support, the adult motoneuron may hypertrophy to restore the balance between metabolic supply and demand. A mechanism similar to that proposed by Thompson (1985) for developmental synapse elimination may also explain the synapse elimination that follows reinnervation of adult muscle. Axotomy would interrupt the retrograde flow of
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trophic substances. This loss of support, together with regenerative axonal elongation, may severely compromise the metabolic capacity of motoneurons. This crisis would continue until two conditions were fulfilled. First, motoneurons must reestablish trophic support by regenerating a sufficient amount of synaptic contact. Second, those trophic molecules must be transported and have time to exert their effects on the soma. Until these conditions were met, nerve terminal retraction would predominate over growth, and synapse elimination would result. One would predict from this scheme that once enough trophic support was secured, the rate of elimination would slow dramatically. In fact, both our results with frogs (Herrera and Werle, in preparation) and studies by others in mammals (McArdle. 1975; Benoit and Changeux, 1978; Brown and Ironton, 1978; Gorio et al., 1983; Taxt, 1983: Rich and Lichtman, 1989) have shown that synapse elimination after reinnervation never quite restores the low level of polyneuronal innervation found in normal adult muscle. As with competition, the role for motoneuronotrophic factors probably does not cease when the developmental periods of neuronal cell death and synapse elimination are over. Instead, such factors probably play a vital role in the normal growth, maintenance, plasticity, and senescence of adult neuromuscular junctions. They may also determine the ability of adult motoneurons to recover from injury or disease.
5. Differences between Frog and Mammalian Neuromuscular Junctions Differences in synapse elimination at neuromuscular junctions in frogs and small laboratory mammals can be easily summarized. In frogs, embryonic polyneuronal innervation is not climinated completely, and new polyneuronal innervation can be generated in adults. In mammals, developmental synapse elimination is essentially complete, and there is little evidence for residual polyneuronal innervation in adults. Morphological studies have also revealed differences in the growth and remodelling of adult junctions in frogs and mammals. Since the results of studies using fixed tissue have been reviewed recently (Wernig and Herrera, 1986; Robbins, 1988), we will focus on studies using new methods of repeated in vivo observation. Junctional growth in frogs is accompanied by considerable remodeling of presynaptic and postsynaptic structures (Herrera and Banner, 1987;
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Herrera et al. 1988, in press; Chen and KO, 1988). Both retraction of branches within junctions that are growing and elongation of branches within junctions showing net retraction are common. Complete retraction and the de now appearance of new branches also occurs. In fast-twitch muscles of the mouse. in vivo observations show that an equivalent amount of growth occurs over similar time periods, but growth mostly involves the proportionate enlargement of preexisting branches (Lichtman, Magrassi, and Purves, 1987; Wigston, 1988; Balice-Gordon and Lichtman, in press). Disproportionate growth or shrinkage is somewhat more common at junctions in the mouse soleus, a muscle with approximately equal numbers of fast and slow fibers (Wigston, 1987, 1989). Terminals in the slow-twitch mouse pectineus extend small but highly dynamic pseudopodial processes (Hill and Robbins, 1987, 1988; Robbins and Polak, 1987, 1988). Since there are so many similarities in the overall structure and function of frog and mammalian junctions, a thorough understanding of the basis of these differences in synaptic plasticity may provide insights to mechanisms common to both systems. Much may be explained by interspecific differences in activity. Frogs are much less active than mice, and inactivity has been shown to stimulate motor axon sprouting in frogs (see Discussion Section 4) as it does in mammals (reviewed in Brown, Holland, and Hopkins, 1981; Robbins, 1988). Unlike laboratory mammals, frogs are poikilothermic and, from winter to summer, experience a wide range of body temperature and activity. Seasonal differences in the structure and function of frog junctions have been noted (Wernig et al., 1980a,b; Herrera, 1984;Jans et al., 1986; Diaz and Pecot-Dechavassine, 1988; older literature reviewed in Grinnell and Herrera, 198 1). In particular, higher levels of sprouting and polyneuronal innervation are found in winter frogs, which are less active. In light of Thompson’s (1985) trophic factor hypothesis and the discussion above, we speculate that these changes are due to inactivityinduced increase in the production of motoneuronotrophic factors during winter (see also Wernig and Herrera, 1986). Of equal or greater importance in explaining differences in junctional plasticity between frogs and mice may be the pattern of body growth. Unlike mice, frogs grow throughout life. Growth in body size is accompanied by muscle fiber hypertrophy in both diameter and length and by muscle fiber addition (Sperry, 1981). Because of this in-
definite growth, a presumably fixed number of spinal motoneurons are called upon to support a peripheral arborization that is constantly expanding in two ways. First, the size of each terminal must increase to compensate for the hypertrophy-induced postsynaptic electrical changes. Second, entirely new junctional sites are innervated as fibers elongate and new fibers appear. The greater capacity for synaptic plasticity in the frog nerve-muscle system would clearly seem to be an adaptation to meet demands not encountered by mice. This hypothesis could be tested by examining junctional plasticity in other animals with fixed and indeterminate growth.
CONCLUSIONS
We conclude that the neuromuscular junction of the normal adult frog is a highly dynamic synapse and that much of this plasticity can be explained by the same mechanisms that govern synaptogenesis during development. The fact that the nervous system of the frog never reaches a static state of maturity is probably an adaptation to accommodate continual growth. Although there are many differences between neuromuscular junctions and synapses in the brain, and there are ccntral synaptic mechanisms that have no counterpart in the periphery, we are optimistic that this simple peripheral synapse will be useful as a model for studying plasticity in the brain. This work was supported by grants from NIH (NS24805) and NSF (BNS85 18232) and by an NIH Research Career Development Award (WS0095 1). We are grateful to Dr. Richard Dunia, Michael Regnier, and Naomi Nagaya for providing unpublished data and for commenting on the manuscript and to Christa Mulkey and Julie Houser for technical assistance. This article is dedicated to Jonathan and Meredith Herrera.
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Plasticity and Growth qfFrog Endplates expansion of existing motor nerve terminals and acetylcholine receptor sites without continual remodelling. J. Neurosci. BANNER,L. R., and HERRERA, A. A. (1986). Differences in synaptic efficacy at neurornuscular junctions in frog twitch muscles. J. Physiol. 379:205-2 15. BARSTAD, J. A. B. (1962). Presynaptic effect of the neuromuscular transmitter. Experientia 18:579-58 1. BENNETT, M. R., MCGRATH,P. A,, and DAVEY,D. F. ( 1979).The regression of synapses formed by a foreign nerve in mature axolotl striated muscle. Brain Res. 173:45 1-469. BENNETT,M. R., and PETTIGREW,A. G. (1975). The formation of synapses in amphibian striated muscle during development. J. Physiol. 252:203-239. BENNETT, M. R., and RAFTOS,J. (1977). The formation and regression of synapses during the re-innervation of axolotl striated muscles. J. Physiol. 265261-295. BENOIT,P., and CHANGEUX, J. P. (1 978). Consequences of blocking the nerve with a local anaesthetic on the evolution of multiinnervation at the regenerating neuromuscular junction of the rat. Bruin Res. 149:89-96. BETZ, W. J. (1987). Motoneuron death and synapse elimination in vertebrates. In The Vertebrute Neuromuscular Junction, M. M. Salpeter (Ed.). Alan Liss, New York, Neurology and Neurobiology Series, Vol. 2.3, pp. 117- 162. BETZ,W. J., CALDWELL, J. H., and RIBCHESTER, R. R. (1979). The size of motor units during post-natal development of rat lumbrical muscle. J . Physiol. 29746 3-47 8. BETZ,W. J., CALDWELL, J. H., and RIBCHESTER, R. R. (1980). Sprouting of active nerve terminals in partially inactive muscles of the rat. J. Physiol. 303:28 1297. BETZ,W. J., RIDGE,R. M. A. P., and RIBCHESTER, R. R. (in press). Competition and the molding of synaptic connections in rat lumbricdl muscle. J. Neuro-
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in new-born rats and its elimination during maturation. J. Physiol. 261:387-422. BROWN,M. C., and MATTHEWS, P. B. C. (1960). An investigation into the possible existence of polyneuronal innervation of individual skeletal muscle fibres in certain hind-limb muscles of the cat. J . Physiol. 151:436-457. CALDWELL, J. H., CAMPBELL, D. T., and BEAM,K. G. (1986). Sodium channel distribution in vertebrate skeletal muscle. J. Gen. Physiol. 87:907-932. CHEN,L., and KO, C.-P. (1988). Dynamic relationship between synaptic extracellular matrix and motor nerve terminals in living frogs. Soc. Neurosci. Abstr. 14: 1209. COVAULT, J., and SANES,J. R. (1985). Neural cell adhesion molecule (N-CAM) accumulates in denervated and paralyzed skeletal muscles. Proc. Nutl. Acud. Sci. (USA) 82:4544-4548, COVAULT, J., and SANES,J. R. (1986). Distribution of N-CAM in synaptic and extrasynaptic regions of developing and adult skeletal muscle. J . Cell Biol. 102:7 16-730. DECINO,P. (198 I). Transmitter release properties along regenerated processes at the frog neuromuscular junction. J. Neiirosci. 1:308-3 17. DENNIS,M. J., and YIP, J. W. (1978). Formation and elimination of foreign synapses on adult salamander muscle. J. Physiol. 274:299-3 10. DIAZ,J., and PECOT-DECHAVASSINE, M. ( 1988). Electrophysiological and morphological study of polyneuronal innervation in the cutaneous pectoris muscle of adult frog. J. Neurocytol. 17:279-292. DIAZ,J., and PECOT-DECHAVASSINE, M. ( 1989). Terminal nerve sprouting at the frog neuromuscular junction induced by prolonged tetrodotoxin blockade of ncrve conduction. J . Neurocytol. 18:39-46. DING,R. (1 982). Lack of correlation between physiological and morphological features of regenerating frog neuromuscular junctions. Brain Res. 253:47-55. DOHRMANN, U., EDGAR,D., and THOENEN, H. (1 987). Distinct neurotrophic factors from skeletal muscle and the central nervous system interact synergistically to support the survival of cultured embryonic spinal motor neurons. Dev. Biol. 124: 145- 1 52. DUNIA,R., and HERRERA, A. A. (1989). Morphological and physiological correlates of synaptic competition at frog neuromuscular junctions. Soc. Neurosci.Ahstr. 15299. FAMBROUGH, D. M. (1979). Control of acetylcholine receptors in skeletal muscle. Physiol. Rev. 59:165227. FLADBY,T., and JANSEN, J. K. S. (1987). Postnatal loss of synaptic terminals in the partially denervated mouse soleus muscle. Acta Physiol. Scand. 129:239246. FOREHAND, C. J. (1987). Ultrastructural analysis of the distribution of synaptic boutons from labeled preganglionic axons on rabbit ciliary neurons. J . Neurosci. 7:3274-328 1.
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GENAT,B. R., and MARK,R. F. (1977). Electrophysiological experiments on the mechanism and accuracy of neuromuscular specificity in the axolotl. Phil. Trans. Roy. Soc. Lond. B 278:335-347. GIBBONS,M. M., and MCCARTHY,T. K. (1983). Age determination of frogs and toads (Amphibia, Anura) from northwestern Europe. 2001.Scr. 12: 145- 151. GILLY,W. F., and HUI, C. S. (1 980a). Mechanical activation in slow and twitch skeletal muscle fibres of the frog. J. Physiol. 301:137-156. GILLY,W. F., and HUI, C. S. (1980b). Membrane electrical properties of frog slow muscle fibres. J. Physiol. 301:157-173. GORIO,A., CARMIGNOTO, G., FINESSO,M., POLATO, P., and NUNZI,M. G. (1983). Muscle reinnervation -11. Sprouting, synapse formation and repression, Neuroscience 8:403-4 16. GOUZE,J.-L., LASRY,J.-M., and CHANGEUX, J.-P. (1983). Selective stabilization of muscle innervation during development: A mathematical model. Bid. Cybern. 46:207-2 15. GRINNELL, A. D., and HERRERA, A. A. ( I 98 1). Specificity and plasticity of neuromuscular connections, long-term regulation of motoneuron function. Prog Neurobiol. 17~203-282. GRINNELL, A. D., LETINSKY,M. S., and RHEUBEN, M. B. ( 1 979). Competitive interaction between foreign nerves innervating frog skeletal muscle. J. Physiol. 289:241-262. GRINNELL, A. D., and TRUSSELL, L. 0.(1983). Synaptic strength as a function of motor unit size in the normal frog sartorius. J. Physiol. 338:22 1-24 1. HATMANN, C., MALLART,A., TOMASI FERRE,J., and ZILBER-GACHELIN, N. F. (1981a). Patterns of motor innervation in the pectoral muscle of adult Xenopus laevis, evidence for possible synaptic remodelling. J. Physiol. 31024 1-256. HAIMANN, C., MALLART,A., TOMASI FERRE, J., and ZILBER-GACHELIN, N. F. (1981b). Interaction between motor axons from two different nerves reinnervating the pectoral muscle of Xenopus laevis. J. Physiol. 310~257-272. HEATON,M. B. (1988). Some characteristics of muscleconditioned media with excitatory and inhibitory influences on neurite outgrowth from early neural tube explants. J. Neurosci. Res. 1957-61. HEBB,D. 0. ( 1 949). The Organization of Behavior. John Wiley & Sons, New York. HENDERSON, C. E., HUCHET,M., and CHANGEUX, J. P. ( 1983). Denervation increases a neurite-promoting activity in extracts of skeletal muscle. Nature 302509-6 1 1. HERRERA, A. A. ( 1 984). Polyneuronal innervation and quanta1 transmitter release in formamide-treated frog sartorius muscles. J. Physinl. 355267-280. HERRERA.A. A., and BANNER,L. R. (1987). Direct observation of motor nerve terminal remodelling in living frogs. Soc. Neurosci. Abstr. 13:1665. HERRERA, A. A., and BANNER,L. R. (in press). The use
and effects of vital fluorescent dyes: Observation of motor nerve terminals and satellite cells in living frog muscles. J. Neurocytol. 19. HERRERA,A. A,, BANNER,L. R., and NAGAYA,N . ( 1 988). Discrepancies between histological and in vivo observations of motor nerve terminal remodelling. Soc. Neurosci. Ahstr. 14:1209. HERRERA,A. A., BANNER,L. R., and NAGAYA, N. (in press). Repeated, in viva observation of frog neuromuscular junctions: Remodelling involves concurrent growth and retraction. J. Neurocytol. 19. HERRERA, A. A., and GRINNELL, A. D. (1980). Transmitter release from frog motor nerve terminals depends on motor unit size. Nature 287~649-65I . HERRERA, A. A., and GRINNELL, A. D. (1981). Contralateral denervation causes enhanced transmitter release from frog motor nerve terminals. Nature 291 :495-497. HERRERA, A. A., and GRINNELL, A. D. (1985). Effects of changes in motor unit size on transmitter release at the frog neuromuscular junction. J . Neurosci. 5:1896- 1900. HERRERA, A. A., GRINNELL, A. D., and WOLOWSKE, B. ( 1985). Ultrastructural correlates of experimentally altered transmitter release efficacy in frog motor nerve terminals. Neuroscience 16:49 1-500. HERRERA, A. A., and SCOTT,D. R. (1985). Motor axon sprouting in frog sartorius muscles is not altered by contralateral axotomy. J. Neurocytol. 14: 145- 156. HERRERA,A. A., and WERLE,M. J. (in preparation). Elevated levels of polyneuronal innervation persist for as long as two years in reinnervated frog neuromuscular junctions. HERRERA, A. A., WERLE,M. J., and BANNER,L. R. (1986). Gradients in polyneuronal innervation of neuromuscular junctions within frog cutaneous pectoris muscles. SOC.Neurosci. Abstr. 12547. HILL,M. A., and BENNETT, M. R. (1986). Motoneurone survival activity in extracts of denervated muscle reduced by prior stimulation of the muscle. Bruin Rex 389:305-308. HILL,R., and ROBBINS, N. (1 987). Transient filopodiallike structures observed at thc living mammalian neuromuscular junction. SOC.Neurosci. Abstr. 13:1007. HILL, R., and ROBBINS,N. (1988). Dynamic structural rearrangements at the growing and adult mouse neuromuscular junction. Soc. Neurosci. Abstr. 14: 1208. HOPJCINS, W. G., LIANG,J., and BARRETT,E. J. (1986). Effectsof age and muscle type on regeneration of neuromuscular synapses in mice. Brain Rex 372: 163166. HOPKINS,W. G., SINGH,A. D., and SLACK,J. R. (1980). Elimination of polyaxonal innervation in adult mammalian muscle. J. Physiol. 303, 67P. JACK,J. J. B., NOBLE,D., and TSIEN,R. W. (1975). Electric Current Flow in Excitable Cells. Clarendon Press, Oxford, p. 46. JANS,H., SALZMANN, R., and WERNIG,A. (1986). Sprouting and nerve retraction in frog neuromuscular
junction during ontogenesis and environmental changes. Neuroscience 18:773-78 1. KUFFLER,D., THOMPSON,W., and JANSEN,J. K. S. ( 1 980). The fate of foreign endplates in cross-innervated rat soleus muscle. Proc. R. Soc. Lond. B 208: 189-222. KUFFLER,S. W., and VAUGHANWILLIAMS,E. M . ( 1953a). Small-nerve junctional potentials. The distribution of small motor nerves to frog skeletal muscle, and the membrane characteristics of the fibres they innervate. . I Physiul. 121:289-3 17. KUFFLER,S. W., and VAUCHANWILLIAMS,E. M . (1953b). Properties of the slow skeIetal muscle fibres of the frog. J. Physiol. 121:3 18-340. KUNO, M., TURKANIS, S. A., and WEAKLY,J. N. (1 97 1). Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog. J. Phvsiol. 213545556, LETINSKY,M., and DECINO,P. ( I 980). Histological staining of pre- and postsynaptic components of amphibian neuromuscular junctions. J. Neirrocytnl. 9~305-320. LETINSKY, M. S., and MORRISON-GRAHAM, K. ( 1 980). Structure of developing frog neuromuscular junctions. J. .&-2*roc9tol.9 3 2 1-342. LICHTMAN, J . W., and BALICE-GORDON, R. (in press). Synaptic growth and elimination at the neuromuscular junction. J . Neurobiol. LICIiTMAN, J. W., MAGRASSI,L., and PLJRVES, D. (1987). Visualization of neuromuscular junctions over periods of several months i n living mice. J . Nrurosci. 7:1215-1222. LOWRIE,M. B., O’BRIEN,R. A. D., and VRBOVA,G. (1985). The effect of altered pcripheral field on motoneuronc function in developing rat soleus muscles. J. Physiol. 3685 13-524. LUFF, A. R.,HATCHER,D. D., and TORKKO. K. ( I 988). Enlarged motor units resulting from dcnervation of cat hindlimb muscles. J. Nmrophysiol. 59: 13771394. LUFF, A. R., and PROSKE,U. (1976). Properties of motor units of the frog sartorius muscle. J . Physiol. 258:673-685. MALLART,A., APU’GAUT-PETIT, D., ZILBER-GACHELIN, N. F., TOMASI FERRE, J., and HAIMANN, C. (1980). Synaptic efficacy and turnover of endings in pauci-innervated muscle fibres of Xenopus luevis. In Ontogrnesis and Functional Mechanisms gf Peripheral Synapses, J. Taxi (Ed.). Elsevier/North-Holland. Amsterdam, pp. 2 13-223. MCARDLE,J. J . (1975). Complex end-plate potentials at the regenerating neuromuscular junction of the rat. Exp. Neurol. 49:29-638. MORGAN,D. L., and PKOSKE,LJ. (1984). Vertebrate slow muscle: Its structure, pattern of innervation, and mechanical properties. Physiul. Rev. 64: 103-1 69. MORRISON-GRAHAM, K. ( 1983). Sprouting and regeneration of frog motoneurons during synapse climination. Dev. Bid. 99:3 12-3 17.
s.. and A R M ~ E - H O R V A TE.I I , (1988). Changes in the distribution of synapses during growth: A quantitative morphological study of the neuromuscular system of fishes. Nei.~roscienre 24:106l-1069. NUDELL.B. M., and GRINNELL, A. D. ( 1 983). Regulation of synaptic position, size, and strength in anuran skeletal muscle. J. Neurosci. 3: 16 1-176. NUDELL, B. M., and GRINNELL, A. D. ( 1 985). Specificity of innervation among X-enopus twitch muscle fibers. Bsain Rex 330:353-357. PAPPONE,P. A. (1980). Voltage clamp experiments in normal and dcnervated mammalian skeletal muscle fibres. J. l’!iysiol. 306:337-410. PECOT-DECHAV.4SSINe, M., WERNIG, A., and STOVER, H. (1979). A combined silvcr and cholinesterase method for studying cxact relations between pre- and postsynaptic elements at the frog neuromuscular junction. Stain Technol. S4:25-28. PEER, K., DREYER,F. SANDRI,C.. A u R I , K.: and MOOR. H. (1974). Structure and ultrastructure of thc frog motor endplate. Cell Tiss. R m . 149:437-455. POCKETT,S.? and SLACK,J. R. ( I 982a). Pruning of axonal trees results in increased efficacy of surviving nerve terminals. Bruin Res. 243:350-353. POCKETT,S., and SLACK,5. R. (1982b). Source of the stirnuhis for nerve terminal sprouting in partially denervated muscle. Neuroscience 7:3 173-3 176. POCKETT,S., and SLACK,J. R. (1983). Ability of motoneurons to regulate quanta1 release and terminal growth after reduction in motor unit size. Brain Res. 258:296-298. PROPST,J. W., HEKRERA. A. A.. and KO, C.-P. (1 986). A comparison of active zone structure in frog neuromuscular junctions from two fast muscles with different synaptic efficacy.
SUMMARY Mechanisms governing synapse elimination, synaptic remodeling, and polyneuronal innervation were examined in anatomical and electrophysiological studies of frog neuromuscular junctions. There was a substantial level of polyneuronal innervation in adult junctions and this varied seasonally. Nerve terminal retraction and synapse elimination occurred during normal growth and following reinnervation. Synapse elimination was not inevitable, however. Repeated in vivo observations of some identified junctions showed that polyneuronal innervation could persist for over a year, while a t other junctions it arose de nuvu by terminal sprouting. W e concluded that polyneuronal innervation in adult muscles was governed by an equilibrium between processes of retraction and elimination on one hand, and sprouting and synaptogenesis on the other. Other observations revealed that structural remodeling was a common feature of adult
junctions. Most often, remodeling involved the simultaneous growth and retraction of different parts of the same junction. The net result was usually junctional growth that, in small frogs, appeared to provide a good match between synaptic size and the electrical demands of transmission. In larger animals, pre- and postsynaptic sizes were not as well matched, providing morphological evidence for a growth-associated decline in synaptic efficacy. Finally, electrophysiology was used to describe some of the functional correlates and consequences of competitive interactions between the terminals of different axons. These results are explained by a hypothetical mechanism that involves trophic support provided by the muscle to the motoneuron, the overall level of nervemuscle activity, and the synchrony of pre- and postsynaptic activity.
INTRODUCTION
underlying mechanisms. At neuromuscular junctions of small laboratory mammals, synapse elimination results in the establishment of mononeuronal innervation at most junctions shortly after the time of birth (reviewed by Van Essen. 1982; Thompson, 1985; Betz, 1987). At the frog neuromuscular junction a similar period of synapse elimination occurs at about the time of metamorphosis (Letinsky and Morrison-Graham, 1980; Morrison-Graham, 1983). Unlike mammals, the process slows before reaching completion, and a substantial fraction of the junctions in adult frogs are polyneuronally innervated (reviewed in Wernig and Herrerd, 1986; see also Werle and Herrera, 1987: Diaz and Pecot-Dechavassine, 1988). Our laboratory has been studying mechanisms go\ierning synapse elimination, synaptic remodeling, and polyneuronal innervation at frog neuro-
Synapse elimination is a collective term for those processes by which the excess synapses characteristic of embryos and neonates are reduced and the adult numbcr is established. Although synapse elimination occurs in many areas of the developing nervous system, it has been particularly well studied at neuromuscular junctions, where experimental accessibility and a wealth of background information has made possible detailed studies of Received June 12, 1989; accepted July 17. 1989 Journal of Neurobiology, Vol. 21, No. I , pp. 73-98 (1990) 0 1990 John Wiley & Sons, lnc. CCC 0022-3034/90/010073-26$04.00 * TOwhom correspondence should be addressed. t Present address: Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305.
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muscular junctions. We find that, as in development, adult motoneurons and muscles retain the capacity to express dramatic structural plasticity. During normal adult life, this plasticity results in continual synapse formation and elimination. Sprouting and synapse formation can cause the de novu establishment of polyneuronal innervation. We have concluded that the physical state of the junction reflects an equilibrium between processes favoring growth and those causing retraction. We speculate that these opposing processes are driven by at least three factors: the metabolic support that motoneurons supply to their terminals, competition between axons for synaptic connections, and activity-dependent nerve-muscle interactions. In this article we will review these findings and conclusions, with our goals being to better understand the frog nerve-muscle system, appreciate differences between frog and mammalian junctions, and discern mechanisms governing these forms of plasticity at neuromuscular junctions and other synapses. We will not attempt an overall review of the field since this has been done by others in this issue (Betz, Ridge, and Ribchester. in press; Lichtman and Balice-Gordon, in press; Thompson, in press; Van Essen, Gordon, and Fraser, in press).
METHODS Unless otherwise noted, experiments were performed on sartorius and cutaneous pectoris nerve-muscle preparations of Rana pipiens (northern variety). Tissues were immersed in or irrigated with frog Ringer solution (in mM: NaCl, 116; KCl, 2; CaCI,, 1.8; N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate,5 ; pH 7.2). Some experiments were done with the pectoral muscle of Xenopus luevis. In some cases sartorius muscles were denervated weeks or months before acute experiments by anesthetizing frogs (immersion in 0.1% aqueous tricaine methanesulfonate, Sigma) and crushing the sartorius nerve where it enters the muscle. For histological observations, neuromuscular junctions were stained either with a nitroblue tetrazolium nerve terminal stain plus cholinesterase stain (Letinsky and DeCino, 1980) or with a silver nerve terminal stain plus cholinesterase stain (Pecot-Dechavassine, Wernig, and Stover, 1979). Methods for dissecting and mounting single identified fibers for high-resolution light microscopy have been described (Werle and Herrera, 1987). For visualization during electrophysiological experiments or for repeated, in vivo observation of living nerve terminals, the fluorescent mitochondria1 dyes DlOC (3,3’diethyloxadicarbocyanine iodide) or 4-Di-2-Asp [4-(4-diethylaminostyry1)-N-methylpyridinium iodide] were used. Methods for dye application, low-light fluorescence imaging, and
image proccssing as well as physiological and morphological controls for obscrvation-induced damage are thoroughly described in Herrera and Banner (in press). For electrophysiology, excised nerve-muscle preparations were superfused with Ringer containing the minimal concentration of d-tubocurarine necessary to prevent twitching upon nerve stimulation. Previously described methods were used for focal extracellular recording of presynaptic spikes and postsynaptic endplate currents (EPCs) and for intracellular recording of endplate potentials (EPPs) from junctions that were later analyzed histologically (Banner and Herrera, 1986; W a l e and Herrera, 1987). Junctions were identified by attaching a video camera to the microscope and recording verbal notes and images of landmarks onto videotape.
RESULTS 1. Methods of Estimating Polyneuronal innervation
The histological appearance of multiple axons converging on a single junctional site has long been used as an indicator of polyneuronal innervation both in tadpoles and normal adult frogs (Letinsky and Morrison-Graham, 1980; Mallart, Angaut-Petit, Zilber-Gachelin, Tomas i Ferre, and Haimann, 1980; Werle and Herrera, 1987; Diaz and Pecot-Dechavassine, 1988). Figure l(A1) shows a typical example from an adult cutaneous pectoris muscle stained with the silver/cholinesterase method. In this case. the upper fiber is innervated by two inputs. The first is a myelinated axon (Ax 1) that exits the intramuscular nerve bundle just above the center of the photograph. The second input is a small terminal sprout [arrow; enlarged in Fig. 1(A2)] that originates from the junction on the adjacent fiber. This adjacent junction is supplied by another myelinated axon (Ax 2). Whether or not the upper fiber is indeed polyneuronally innervated, or simply innervated in a circuitous manner by the same axon, depends on whether Ax 1 and Ax 2 are branches of the same motoneuron. This is difficult to determine morphologically, since each motor axon branches profusely to contact many different junctions (Trussell and Grinnell, 1985). This convergence of two or more axons from different motoneurons onto the same synaptic site is defined as focal polyneuronal innervation. A second morphological feature, the doubly innervated synaptic gutter, is highly correlated with focal polyneuronal innervation in reinnervated and normal muscles (Herrera and Werle, in preparation). Doubly inner-
Figure 1 Characteristic histological signs of polyneuronal innervation in normal adult junctions stained with silver terminal stain and cholinesterase stain. ( A l ) The junction on the upper fiber is innervated by two inputs. One input (Ax 1) is a myelinated axon that supplies most of the terminal arborization. The second input arises as a terminal sprout (arrow) from the junction on the lower fiber. This lower junction is supplied by another myelinated axon (Ax 2). (A2) View of the part of the lower junction that gives rise to the terminal sprout (arrow). Arrowheads indicate a doubly innervated gutter. (B) Doubly innervated gutters (arrowheads). Scale bar = 60 pm for A l , 30 pm for A2 and B.
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vated gutters are single cholinesterase-outlined postsynaptic specializations occupied by two closely apposed nerve terminals. Examplcs are indicated by arrowheads in Figure l(A2,B). Certain frog muscles exhibit a second form of polyneuronal innervation. When fibers are examined along their entire length. multiple junctional sites spaced several millimeters apart can be seen. Multiple junctions are more common in muscles with longer fibers. This multisite innervation makes it possible that different axons can contact the same fiber at distant sites. We will refer to this nonfocal form of polyneuronal innervation as distributed. The presence of multiple axonal inputs and doubly innervated gutters suggests that each of the several distinct junctions on a single fiber may also be focally polyneuronally innervated. Electrophysiology provides additional methods for detecting polyneuronal innervation. With intracellular or extracellular recording, focal polyneuronal innervation can be detected as a stepwise variation in the amplitude of EPPs or EPCs as thc multiple axons are separately recruited by grading the strength of a stimulus applied to the nerve. As would be expected for the focal convergence of two axons, the rise times of the separate EPP components are usually identical. In most cases, distributed polyneuronal innervation cannot be detected by recording at a single site on the fiber because the spacing between junctions usually far exceeds the range of passively conducting potentials. In rare cases an intracellular microelectrode positioncd at one junctional site will record an EPP arising at a distant site, but electrotonic conduction greatly attenuates the amplitude and prolongs the time course of the distant EPP. Distributed polyneuronal innervation is best studied by the simultaneous use of several microclectrodes, with care taken that they are applied to the same fiber. Another physiological method, commonly used to estimate polyneuronal innervation in mammalian muscles, is the extent of overlap between the tension generated by separate motor units (Brown and Matthews, 1960). If two motor axons innervate some of the same fibers, the tension generated by these motor units will not sum linearly. The interpretation of tension overlap is more complex in frog muscles because of the multisite innervation of single fibers (Luff and Proske, 1976). Each of the methods for estimating polyneuronal innervation has certain limitations, some of which have been discussed (Betz, 1987). Counting the number of junctions showing multiple axonal inputs certainly leads to overestimates because
many singly innervated junctions, especially when they are large, are supplied by two myelinated branches of the same axon. Whether these are scored as one or two inputs depends on whether the branch point is visible, and in many cases it is not. The incidence of junctions with doubly innervated gutters is a certain underestimate because not all polyneuronally innervated junctions show this form of close spatial overlap. When identified polyneuronally innervated junctions in normal cutaneous pectoris muscles were examined histologically, the cxtent of overlap between nerve terminals was found to vary between extensive [Fig. 2(A)], moderate [Fig. 2(B)], and slight [Fig. 2(C)]. Physiological methods that measure the incidence of multiple EPP steps yield underestimates for several reasons. First, multiple axonal inputs might not be separable by stimulus threshold. This problem can be ameliorated by changing the polarity as well as the amplitude of the stimulus and by stimulating at multiple sites along the nerve. Second, if the weaker input at a polyneuronally innervated junction has the higher threshold, its contribution to the EPP may be shunted or obscured by the amplitude variations of the stronger input. Finally, most physiological methods require that contraction be blocked by adding curare (Redfern, 1970), treating with formamide or glycerol (Herrera. 1984), or cutting fibers (Barstad, 1962). When these trcatments are used to the minimal extent necessary to block the strongest junctions, weak polyneuronal inputs may be attenuated to undetectable levels. VaIues obtained with each of these three methods are probably directly proportional to the truc incidence of polyneuronal innervation, but this true level probably lies somewhere between the high and low estimates.
2. Polyneuronal Innervation at Normal Adult Neuromuscular Junctions When the methods described above are applied to muscles of normal adult frogs, one finds a considerable leveI of focal polyneuronal innervation. In histologically stained cutaneous pectoris muscles, the incidence of junctions with multiple axonal inputs ranged from 22 to 53% and that of doubly innervated gutters from 8 to 24% (see Results Section 4). In nearly all cases, the terminal arbors of the two axonal inputs were colocalized within a compact area no larger than 5,000-20,000 pm2. Intracellular recording in curare-blocked muscles detected multiple EPP steps in an average of 9% of the junctions in the sartorius and 36% in the cuta-
Plasticity and Growih of'Frog Endplutcs
77
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Figure 2 Cumera lucida drawings of polyneuronally innervated sartorius junctions showing differing degrees of spatial overlap between ncrve terminals. One terminal is drawn as a solid process and the other is dashed. Cholinesterdse reactivity is shown as a dotted outline. (A) Extreme overlap, with inputs mostly sharing the same synaptic gutters. (B) Moderate overlap, with a few doubly innervated synaptic gutters. (C) Extreme separation within a single junctional site. Scale bar = 30 pm.
neous pectoris (Fig. 3). More sensitive methods, such as blocking contraction with the excitationcontraction uncoupler formamide, revealed cven higher levels (Herrera, 1984). In adult cutaneous pectons muscles, 96% (269/277, 10 muscles) ofthe polyneuronally innervated junctions had only two inputs, with the remainder having three. It may be noted that the three methods for estimating polyneuronal innervation in normal muscles yielded values that differed considerably. These discrepancies were further explored by examining a different group of normal and reinnervated sartorius junctions. Reinnervated muscles are known to have higher than normal levels of polyneuronal innervation (see Results Section 3). Figure 4 shows the incidence of multiple EPP steps, doubly innervated gutters, and multiple axonal inputs for normal sartorius junctions and for reinnervated junctions 1.7-2.4 years after nerve
crush. All three parameters for reinnervated muscles were elevated to similar extents relative to levels measured in normal muscles. There was good agreement between the absolute values of the physiological estimate and doubly innervated gutters for both reinnervated (27% versus 28%) and normal muscles (10% versus 8%). Measuring the percentage of junctions with more than one axonal input, however, yielded values 2.5-3.5-fold higher than the other two parameters. The polyneuronal innervation present at adult neuromuscular junctions could arise from three
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Figure 3 Incidence of physiological pol yneuronal innervation in 7 sartorius (SART) and 13 cutaneous pectons (CP) muscles tested in summer, expressed as the percent ofjunctions showing multiple EPP steps as stimulus strength was varied. Transmission was partially blocked with d-tubocurarine. Each point is the mean for 40-60 junctions in a single muscle. Lengths of bars represent means.
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MULTIPLE EPP STEPS
DOUBLY INNERVATED GUTERS
MULTIPLE AXONAL INPUTS
Figure 4 Physiological and morphological estimates of the incidence of polyneuronal innervation in reinnervated sartorius muscles 1.7-2.4 years after nerve crush (solid bars) and normal muscles (open bars). Vertical lines are standard errors of proportions.
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Herreru and Werle
sources. First, it may be left over from earlier developmental stages due to incomplete synapse elimination. Second, polyneuronal innervation may be formed de MOVO as a result of synaptic remodeling. Third, synapses that are added as muscle fibers grow in length may recapitulate the developmental stages of synaptogenesis and thus express a transient period of polyneuronal innervation. These possibilities are discussed in Results Sections 3 , 6, and 7, respectively. 3. Synapse Elimination following Reinnervation of Adult Muscles A simple explanation for thc presence of polyneu-
ronal innervation in adult frog muscles is that it is residual, i.e., left over from earlier developmental stages. Studies of developing frogs have shown that the rate of elimination slows at about the time of metamorphosis while there is still a substantial amount of polyneuronal innervation (MomsonGraham, 1983). It was therefore of interest to determine whether the adult nerve-muscle system retains the capacity to express synapse elimination under any circumstances. In studies in which muscles of adult mammals were denervated, it was found that polyneuronal innervation was established during reinnervation and, with time, was reduced by a form of synapse elimination similar to that which occurs during development (McArdle, 1975; Benoit and Changeux, 1978; Brown and Ironton, 1978;Thompson, 1978; Gorio, Carmignoto, Finesso, Polato, and Nunzi, 1983; Taxt, 1983; Hopkins, Liang, and Barrett, 1986; Rich and Lichtman, 1989). We found that the same sequence of events occurs in reinnervated frog muscles (Werle and Herrera, 1988; Herrera and Werle, In preparation). Figure 5 shows the incidence of physiological polyneuronal innervation in 53 reinnervated sartorlus muscles tested 14-877 days after crushing the nerve. Functional reinnervation first occurs at 7-1 0 days (DeCino, 1981; Ding, 1982). As early as physiologicai methods could be used reliably (14-21 days), polyneuronal innervation was found to be about 5 times the normal adult level. Synapse elimination progressed over the next several weeks. Both the starting level of polyneuronal innervation and the rate of elimination seemed to vary in different preparations. Over several months, pol yneuronal innervation was reduced to a steady-state level that approximated, but still significantly exceeded, that found in normal adult muscles (Herrera and Werle, in preparation). Despite this hilure to re-
store the normal pattern, these findings demonstrated that the adult neuromuscular system retains the capacity for synapse elimination. 4. Synapse Elimination at Normal Adult Neuromuscular Junctions
The demonstration that synapse elimination occurs following reinnervation does not directly address the question of whether synapse elimination can also occur under normal conditions. AXotoniy may cause pre- and postsynaptic components to regress to a less mature state. In muscle, denervation causes the reappearance of several embryonic and neonatal proteins (Fambrough, 1979; Pappone, 1980; Booth, Brown, Keynes, and Barclay, 1984; Covault and Sanes, 1985, 1986; Schiaffino, Gorza, Pitton. Saggin, Ausoni, Sartore, and Lomo, 1988). The first evidence that synapse elimination does indeed occur in adult frog muscles was the finding of seasonal variation in the incidence of junctions with multiple EPP steps (Herrera and Grinnell, 1981; Herrera, 1984; Diaz and Pecot-Dechavassine, 1988) and histological signs of remodeling (Wernig, Pecot-Dechavassine, and Stover. 1980a.b; Jans, Salzmann, and Wernig, 1986: Diaz and Pecot-Dechavassine, 1988). For examplc, the incidence of multiple EPP steps was about twice as high in sartorius muscles of winter frogs ( 1 9%) compared with summer frogs (9%; Herrera. 1984). These data suggest that there is an annual cycle of synapse formation and elimination in adult frog muscles. Synapse elimination has been shown to involve the retraction of nerve terminals (Bixby, 1981). Therefore, the occurrence of retraction in normal muscles may be considered a necessary condition for the demonstration of synapse elimination in normal adult frogs. We (Herrera and Scott. 1985; Herrera et al., in press) and others (Wernig et al., 1980a,b; Anzil, Bieser, and Wernig, 1984; see Wernig and Herrera, 1986, for review) found that nerve terminal retraction is a common feature of neuromuscular junctions in adult frogs. The typical histological appearance of a retracted nerve terminal is shown in Fig. 6(A).* Exposed, cholinesterase-stained postsynaptic folds can be seen distal [to the right of the arrowhead in Fig. 6(A)] and lateral to the terminal. Since these persistent postsynaptic specializations are normally found only directly opposite presynaptic active zones (Peper, * See color plate section at end of issue
Pla.Pticity and Growth of Frog Endplates
torius. Polyneuronally innervated junctions were identified as such if in vivo observations revealed at least one of the two histologically defined characteristics, doubly innervated gutters, or multiple axonal inputs. With the cutaneous pectoris, position within the muscle could also be used to judge the presence of polyneuronal innervation. We earlier showed that junctions near the medial edge of the cutaneous pectoris have a high probability of being polyneuronally innervated (Herrera, Werle, and Banner, 1986). A typical polyneuronally innervated junction near the medial edge of the cutaneous pectoris is shown in Fig. 7. The initial in vivo observation revealed both doubly innervated gutters [Fig. 7(A), arrowheads] and dual axonal inputs [Fig. 7(A), arrow]. When reexamined 1.2 years (438 days) later, this junction had grown considerably but still appeared to be polyneuronally innervated, as judged by the same morphological criteria [Fig. 7(B-D)]. The symbols in Fig. 7(B) point to features similar to those shown in Fig. 7(A). Most junctions showed persistence of apparent polyneuronal innervation over the observation interval. In a few cases of persistence, however, one of the two inputs suffered disproportionate retraction. An example from a sartorius junction is shown in Fig. 8. At the initial observation, the junction was supplied by two axonal inputs which are out of the focal plane and so cannot be seen in the fluorescence photographs. As shown by the filled and open outlines, the terminal arborization of each of the inputs could be traced [Fig. 8(A,B)].When the
Dreyer, Sandri, Akert, and Moor, 1974), they precisely mark former sites of synaptic contact. Further studies of junctional remodeling by the use of repeated, in vivo observation directly confirmed the occurrence of nerve terminal retraction at junctions in adult frogs (Herrera and Banner, 1987; Herrera, et al., in press; Chen and KO, 1988). Shortening of at least 1 terminal branch was found in 60%of 243 sartoriusjunctions observed twice at intervals of 4-6 months. In 12%of these 243 junctions, at least 1 branch completely disappeared. At one junction, terminal retraction led to the complete abandonment of the junction by the original input, and the junction was partial reinnervated by a sprout from a nearby junction (Herrera et al., in press). These results clearly demonstrate that nerve terminal retraction, a necessary step in synapse elimination, occurs at neuromuscular junctions of normal adult frogs.
5. Rate of Synapse Elimination at Adult Neuromuscular Junctions Although the demonstration of seasonal differences in polyneuronal innervation and of terminal retraction suggested that synapse elimination was possible, it remained to directly test whether the phenomenon occurred in adult muscles. We therefore made repeated, in vivo observations of polyneuronally innervated junctions over intervals of approximately 50 or 420 days in the cutaneous pectoris or approximately 180 days in the sar-
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Figure 5 Incidence of physiological polyneuronal innervation in 53 reinnervated sartorius muscles tested 14-877 days after crushing the nerve. Polyneuronal innervation is established upon reinnervation and then eliminated over the next few months. Data for 10 normal sartorius muscles are shown at the far right (short horizontal lines indicate mean). Synapse elimination does not reestablish the normal level of polyneuronal innervation.
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Figure 6 Neuromuscular junctions in normal frog sartorius muscle. The NBT-stained nerve terminal (purple) is surrounded by periodic synaptic folds filled with cholinesterase reaction product (brown). (A) Nerve terminal retraction is indicated by exposed synaptic folds beyond the distal tip of the terminal. Distal tip of terminal is indicated by the arrowhead. Exposed folds are to the right. (B) Lower magnification view of a junction showing several short terminal sprouts (arrowheads). (C) High magnification view of one of the branches in B, showing filopodial sprouts extending from the distal tip of the terminal. Scale bars = 10 Fm in A and C, 20 pm in B. (See color plate section at end of issue.)
junction was reexamined 183 days later, it was still apparently polyneuronally innervated. One of the inputs, however, had retracted almost completely [Fig. S(C,D)]. After histological staining, abandoned synaptic gutter lined with junctional folds seemed to mark the former extent of the retracted
terminal [Fig. 8(D)]. It is interesting that the terminal supplied by the other input grew considerably [compare open outlines in Fig. 8(B) and (D)].Despite this growth there was 27 p m of abandoned gutter beyond the distal tips of this terminal, indicating that the terminal was even longer at some
Plasticity and Growth Ctf'FrogEndplates
time during the observation interval. The finding of growth and retraction within single junctions is discussed further in Results Section 6, below.
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Additional evidence for the rate of synapse elimination was obtained by using morphology and physiology to estimate the incidence of polyneuronai innervation in frogs of various ages. We examined the cutaneous pectoris, a muscle that has a high level of polyneuronal innervation (Fig. 3). Since frogs grow throughout life (Gibbons and McCarthy, 1983), body size was used as a rough index of age. Figure 9 shows the incidence of junctions with multiple axonal inputs (solid circles, solid line), doubly innervated gutters (open circles, large dashed line), and multiple EPP steps (open squares, small dashed line) for groups of muscles from small (young) and large (old) frogs. As explained in Results Section 1, the different estimation methods yielded different results. With each method, however, there was a highly significant decrease in polyneuronal innervation with increasing age. We consider it likely that this progressive reduction of polyneuronal innervation represents continuation of the slow rate of synapse elimination described by Morrison-Graham (1983). An alternative explanation is discussed in Results Section 7. 6. The Equilibrium between Synaptic Growth and Retraction: Synaptic Remodeling
D
Figure 7 Repeated, in vivo observation of the persistence of poiyneurond innervation in a normal cutaneous pectoris junction. (A) Initial in vivo observation of terminal stained with the tluorescent mitochondria1 dye 4-Di-2-Asp. Video image was obtained with very dim illumination and digitally enhanced. Arrow indicates two axonal inputs, and arrowheads point to doubly innervated synaptic gutters (DIGs). (B) Final histological observation 438 days later. Junction stained with the NBT method, which stains only unmyelinated processes, and with cholinesterase stain. Arrow indicates two axonal inputs. The unmyelinated preterminal axon of one input stains with NBT and so can be clearly seen. The other input remains myelinated until it contacts the fiber. Thus it does not stain with NBT and can only be faintly seen in this photograph. Arrowheads point to DIGS.(C, D) Higher magnification views of intertwined terminals and DIGs in the left portion of the junction in B. Scale bar = 30 Frn for A and B, 15 fim for C and D.
Estimates of the rate of synapse elimination made from in vivo observations or by measurement of polyneuronal innervation in young and old frogs would be in error if polyneuronal innervation could be generated de novo. Histology indicated that this might occur. As others have described (reviewed in Wernig and Herrera, 1986), many junctions showed nerve terminal sprouts. These were usually evident as elongations of terminal processes distal to the area of postsynaptic specialization. Fine filopodial processes, which we suppose are an early stage of terminal sprouting (see also Robbins and Polak, 1988), can be seen extending from several points along one terminal branch in Fig. 6(B, arrowheads). The distal tip of this sprouting branch is enlarged in Fig. 6(C).It is interesting that the other branches, which are clearly part of the same terminal arborization, were not sprouting at the time of fixation. In an earlier study, we found that about 9% of normal sartorius junctions had such sprouts (Herrera and Scott, 1985) and others have found even higher levels (Wernig et al., 1980a). These sprouts result in junctional growth when they terminate on the fiber of origin. Both neuromuscular junctions and
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Figure 8 Rcpeated, in vivo observation of disproportionate retraction of one input at a douhly innervated ,junction in a normal sartorius muscle. (A, B) Photograph and drawing of initial in viva observation using 4-Di-2-Asp. Open and solid outlines in B indicate terminal branches that. in other focal planes, could be seen to arise from different axons. Crossing of terminals is a characteristic feature of polyneuronally innervated junctions. (C, D) Final in vivo observation 183 days later. Drawing made from histologically stained junction (NBT/ ChE method). Terminal shown by open outline had grown substantially. Terminal shown by filled outline had retracted, yet sprouted a new branch at right. Abandoned synaptic folds (vertical hatching) mark the former extent of the retracted terminal. Paradoxically. the input that grew also had abandoned synaptic folds beyond its distal tips, suggesting that growth was followed by partial retraction. Scale bar = 30 pm for A-D.
muscle fibers grow substantially throughout the life of a frog (Jans et al., 1986: see Results Section 7). The actual rates and patterns of growth in adult junctions could be better appreciated by repeated, in vivo observation (Herrera et al.. in press). Among 243 sartorius junctions imaged twice over periods of 4-6 months, 89% showed a net increase in total nerve terminal length. The average increase in terminal length of the junctions that grew was 35% (54 pm). Most (56%) of the 2 I6 junctions
that showed net growth also had a t least one branch that retracted. Eleven percent of the 243 junctions showed net retraction, with an average decrease in total terminal length of 15% (36 pm). Thesc 27 retracting junctions did not shrink uniformly, most ( I 9) had at least 1 terminal branch that grew. We concluded from these studies that most junctions grow in adult muscles and that growth is the net result of differing degrees of elongation and retraction of individual nerve terminal branches (Herrera et al., in press). This finding of
Plasticity and Growth of Frog Endplates
simultaneous growth and retraction of terminal branches of the same motoneuron at the same synapse also suggested that growth and retraction may not be completely controlled at the level of' the entire synapse but also locally, by very shortrange nerve-muscle interactions. An in vivo observation that added greatly to our understanding of polyneuronal innervation in adult muscles was the finding that sprouting terminals did not always synapse onto the fiber of origin. Sprouts occasionally jumped to the adjacent fiber where, as in Fig. l(A), they could be seen to form synaptic contacts outlined by cholinesterase reactivity. An example is shown in Fig. 10. Over an interval of 18 1 days, the leftmost branch of this terminal elongated 2.8-fold (from 39 to 1 10 pm) and bifurcated. The lower ramus was a 45 pm sprout that terminated in a bright bouton on the fiber below. As was usual in such cases, the new contact was established within the perijunctional area of the neighboring fiber. The junction on the neighboring fiber was on its deep surface and so is not shown. This acquisition of a second input by the lower fiber would represent the de n o w formation of focal polyneuronal innervation if the axon that originally supplied the neighboring fiber was from a different motoneuron. Other parts of the 60 1 I\
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Figure 9 Growth-associatedreduction in polyneuronal innervation in cutaneous pectoris muscles of normal adult frogs. For each of the 3 measures, values in large frogs are significantly lower than values in small frogs ( p < 0.0002). Vertical lines through points are standard errors of proportions, horizontal lines are standard errors of mean for body weight. Data on histologically visible multiple inputs (solid circles) are from 202 junctions in 3 large frogs and 972 junctions in 16 small frogs. Data on multiple EPP steps (open squares) are from 572 junctions in 5 large frogs and 1039 junctions in 8 small
frogs. Data on doubly innervated gutters (open circles) arc from 199junctions in 3 large frogs and 980 junctions in 16 small frogs.
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junction in Fig. 10 from which the sprout originated also grew substantially. Overall, the terminal increased 74% in total length (from 300 to 521 pm). It is interesting to note that differences in the sites of origin and termination of terminal sprouts can lead to dramatically different junctional restructuring. Sprouts originating from the distal tips of terminals and terminating within a few micrometers on the same fiber would result in simple elongation of preexisting branches. If postsynaptic differentiation could keep pace with presynaptic elongation, the incidence of sprouting terminals would be substantially underestimated by single time point observations such as those made with histology or electron microscopy. Sprouts originating from the side of a terminal branch and terminating on the same fiber would also result in junctional remodeling and, as described above, may not be recognized as new branches with single time point observations. If either type of sprout terminated in the perijunctional region of a different fiber, polyneuronal innervation may be formed. Finally, termination of a sprout far from a preexisting junction on a different fiber would result in the addition of a new synaptic site on that fiber (see Results Section 7). The view that has emerged from these findings is that there is constant turnover of polyneuronal innervation in adult frog muscles. At any time the level of polyneuronal innervation is the net result of an equilibrium between the processes of retraction and elimination on one hand and growth and synaptogenesis on the other. Terminal retraction results in junctional remodeling and can lead to synapse elimination. Terminal growth, the generation of new synaptic contacts, can result in junctional enlargement, remodeling, new formation of polyneuronal innervation, or synapse addition.
7. Size Matching and Synapse Addition during Growth Frogs grow throughout life (Gibbons and McCarthy, 1983), and body growth is accompanied by muscle fiber hypertrophy (Sperry, 1981; Jans et al., 1986). Within muscles, fiber size and junctional side are correlated (Kuno, Turkanis, and Weakly, 197 1; reviewed in Wernig and Herrera, 1986). These findings, and direct in vivo observations (Herrera et al., in press), suggest that synaptic growth maintains physiological efficacy despite the increase in postsynaptic input capacitance and decrease in input resistance caused by
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Figure 10 Repeated, in vivo observation of the apparent de nova establishment of polyneuronal innervation in adult sartorius muscle. (A) Initial in vivo observation of 4-Di-2-Aspstained terminal, supplied by a single axonal input (ax). Arrow indicates distal tip of leftmost terminal branch. Asterisk marks the distal tips of the two rightmost branches. (B) Final in vivo observation 181 days later. The junction was still supplied by a single input (ax). Arrow again indicates leftmost terminal tip. which has grown to the left. The three arrowheads point to a faintly stained terminal sprout that jumps to the lower fiber and terminates there i n a bright bouton. Original junction of the lower fiber was on the deep surface and so cannot be seen in these images. Note that the rightmost tips of the terminal (asterisk) have also grown considerably. Scale bar = 30 Fm for both panels.
fiber hypertrophy. The amplitude of the EPP is probably determined more by the capacitive properties of the fiber membrane than the steady-state resistive properties, since the endplate current is brief (approximately 1 msj compared with the fiber time constant (approximately 20 ms). To explore these questions further, we examined the relationships between presynaptic and postsynaptic size in sartorius muscles from 16 frogs representing a wide range of body size (4-404 g body weight). Figure 11 shows that growth in muscle length is accompanied by hypertrophy in fiber diameter [Fig. 1 l(A)] and by junctional enlargement [Fig. 11(Bj]. These data may also be
used to predict how synaptic efficacy changes during growth. The points in Fig. I 1(C) show thc relationship between total terminal length and muscle fiber diameter. If one assumes that endplate current flows through a region that is small relative to the length constant of the fiber, then the postsynaptic voltage response may be approximated by that of an infinite cable when charge is instantaneously applied at a point (equation 3.48 in Jack, Noble, and Tsien, 1975). The prediction is that the EPP would be inversely proportional to the 3/2 power of fiber diameter. If terminal length is proportional to transmitter release and therefore endplate current (Kuno et al., I97 1; Herrera, Grinnell,
Plasticity and Growth of Frog Endplates
and Wolowske, 1985; Propst, Herrera, and KO, 1986; Propst and KO, 1987), then terminal length must increase as the 3 / 2 power of fiber diameter to maintain EPP size as the fiber grows. The curve in Fig. 1 1(C) is a 3/2 power function drawn by eye to fit data from the 9 smallest frogs (4-74 g body weight). Synaptic efficacy appears to be well maintained in this range of body size. The 7 points on the right, representing the largest frogs (77-404 g), clearly fall below the curve, suggesting that synaptic efficacy falls as frogs grow very large. Such a growth-associated decline in efficacy at frog junctions has been reported (see Trussell and Grinnell, 1985). The assumptions and predictions of this theoretical approach should be further tested by direct measurement of transmitter release, postsynaptic transmitter sensitivity, and the active and passive electrical properties of the muscle fiber in frogs of various sizes. We tentatively conclude, however, that there are mechanisms that tend to match presynaptic and postsynaptic size during growth in frog muscles. The matching is not perfect, since there appears to be a progressive decrease in synaptic efficacy in large animals. In addition to hypertrophy, the growth of some frog muscles involves the addition of new synaptic
sites as muscle fibers elongate (Bennett and Pettigrew, 1975; Nudell and Grinnell, 1983). In fibers without propagating action potentials, such as frog tonic fibers (Kuffler and Vaughan Williams, 1953a,b; Gilly and Hui, 1980a,b)and many twitch fibers in fish (Mos, Maslam, and Armee-Horvath, 1988), multiple junctional sites serve to distribute depolarization for efficient tension production. Frog twitch fibers are like mammalian fibers in that they support propagating action potentials. In such fibers, the functional significance of multiple junctional sites is unclear. The synapses on each fiber are almost always many length constants apart so they do not interact electrically, nor does simultaneous generation of action potentials at several locations seem to confer an advantage in tension production (Nudell and Grinnell, 1983). It is possible that the formation of multiple junctional sites is involved in trophic interactions between nerve and muscle, but these are poorly understood. Whatever their function, an interesting area for future research will be the extent to which the newly added synapses recapitulate the normal developmental sequence of presynaptic and postsynaptic differentiation, polyneuronal innervation, synapse elimination, and synaptic matura-
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Figure 11 Postmetamorphic growth of sartorius muscles and neuromuscular junctions in 16 frogs ranging from 4 to 404 g body weight. Each point is the mean ( 5 SEM) of 15-49 junctions in a muscle. Error bars smaller than diameter of points are not shown. Straight lines fit by least squares regression. Growth in muscle length is accompanied by muscle fiber hypertrophy (A; r = 0.94, p < 0.0001) and nerve terminal elongation (B; r = 0.89, p < 0.0001). ( C )The relation between nerve terminal length and muscle fiber diameter. The curve is a 3/2 power function drawn by eye to fit the 9 points on the left, which are from the smallest frogs (4-74 g). This curve represents the terminal length that would be necessary to maintain EPP size as input capacitance increases with fiber growth (see text). The 7 points on the right, which are from the largest frogs (77-404 g) fall well below the curve, suggesting that synaptic efficacy decreases in large animals.
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tion. If newly added synapses progress through typical developmental stages, much of the polyneuronal innervation observed in normal adult muscles could be explained. If newly added synapses tend not to be polyneuronally innervated, the life-long reduction in polyneuronal innervation we found in normal muscles (Fig. 9) may not represent synapse elimination but rather the selective addition of singly innervated junctions. Despite these uncertainties, it seems a reasonable working hypothesis that lifelong retention of the capacities for sprouting, synapse formation, and synapse elimination in frog muscles is an adaptation to ensure the maintenance of synaptic efficacy and the orderly addition of new synaptic sites during growth. 8. Measurement of Competitive Interactions
It has been demonstrated that synapse elimination involves competitive interactions between nerve terminals at the same synaptic site (Brown, Jansen, and Van Essen, 1976; Thompson and Jansen, 1977; Betz, Caldwell, and Ribchester, 1979, 1980). We have speculated that similar competitive interactions are involved in synaptic remodeling in adult junctions (Werle and Herrera, 1987). Close examination of the anatomical and physiological correlates of polyneuronal innervation in normal muscles and synapse elimination in reinnervated muscles has provided clues to the nature these competitive mechanisms.
a. Relative Synaptic Eficacy as an Index of Compefition. In a previous study (Werle and Herrera, 1988), we compared the properties of two groups of doubly reinnervated sartorius junctions. The first group was examined 40-80 days after crushing the nerve, a time when most junctions were still polyneuronally innervated (Fig. 5 ) . The second group was taken more than 250 days after nerve crush, which was after the main period of synapse elimination (Fig. 5). The junctions in the latter group were considered to be those that survived the elimination process. By using a combination of vital fluorescent staining, simultaneous intracellular and focal extracellular recording, and histological examination of the junctions from which recordings were made, we determined the size, position, and physiological efficacy of both inputs at doubly innervated synapses. By comparing the two competing inputs at junctions before and after synapse elimination, we were able to make strong inferences regarding the mechanisms
of elimination. A number of properties were not different. For example, relative endplate potential size, nerve terminal length, and number of terminal branch points did not differ between the competing inputs before and after synapse elimination. This finding suggested that inputs were not eliniinated on the basis of these properties, and thus argued against models that assume simply that EPP amplitude, terminal length, or terminal complexity are important determinants of elimination or persistence. Although differences in synaptic efficacy alone did not correlate with elimination, there was a striking correlation when efficacy was normalized to nerve terminal size. Relative differences in this normalized measure of efficacy were expressed as a parameter called ratio of synaptic efficacy (RSE). This dimensionless parameter wdS calculated as EPP size per unit nerve terminal length of the weaker input divided by EPP size per unit length of the stronger input. The values of RSE thus ranged from near zero, when inputs were very different in EPP/terminal length, to near one as they approached equality. When polyneuronally innervated junctions were examined before synapse elimination had occurred, RSE was broadly distributed, with a median value of 0.42 (Fig. 12, top). After synapse elimination was complete, the synaptic efficacies of competing inputs at surviving polyneuronally innervated junctions were significantly more similar, with median RSE = 0.64 (Fig. 12. middle). Our interpretation of these results is that inputs with inherently different release efficacies come to be randomly associated at polyneuronally innervated junctions during reinnervation. Synapse elimination then occurs selectively at those junctions wherc the two inputs differ the most, and polyneuronal innervation will persist at those junctions where the efficacy of the two inputs is similar. We also determined RSE for doubly innervated junctions in normal sartorius muscles. As discussed above, the majority of these doubly innervated junctions were considered to be the survivors of developmental and ongoing synapse elimination. As shown at the bottom of Fig. 12, we found that these normal polyneuronally innervated junctions had the same tendency toward equality in EPP size per unit terminal length (median RSE = 0.67) as did reinnervated junctions after synapse elimination (Fig. 12, middle). These findings support the view that during normal development and growth, as well as following reinnervation, synapse elimination occurs primarily at
Plasticity and Growh ojFrog Endplates
junctions where the competing inputs have vastly different normalized synaptic efficacies. In other studies we have explored the physiological consequences of synaptic competition. If synapse elimination involves mutually deleterious competitive interactions, we reasoned that polyneuronally innervated junctions may be smaller, weaker, or both compared with singly innervated junctions in the same muscle. The literature provides conflicting evidence on this point. In the pectoral muscle of Xenopus, the sum of the separate EPPs at doubly innervated junctions averaged 30% less than EPP amplitude at singly innervated junctions (Angaut-Petit and Mallart, 1979; Haimann, Mallart, Tomas i Ferre, and Zilber-Gachelin, 1981a). In cutaneous pectoris and sartorius muscles of Rana pipiens, no significant difference was found, although EPPs at doubly innervated cutaneous pectoris junctions were 10% smaller than at singly innervated junctions (Weakly and Yao, 1983). We reexamined this question using
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Figure I 2 Distributions of ratio of synaptic efficacy (RSE) at dually innervated sartoriusjunctions. RSE defined as EPP size per unit terminal length of weaker input divided by EPP size per unit length of stronger input. Arrowheads indicate medians. Top: Data for 87 reinnervated junctions 40-80 days after nerve crush. which was before the major period of synapse elimination. Middle: A different group of 76 reinnervated junctions at more than 250 days after nerve crush, after synapse elimination. Note significant ( p < 0.001, MannWhitney U test) shift of distribution to the right. Bottom: Data for 46 junctions in a normal unopcrdted
muscle.
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data from the cutaneous pectoris. In each of 19 curare-blocked muscles, EPPs were recorded at 31-164 junctions, and each EPP was normalized by dividing it by the average EPP amplitude for that muscle. This normalization allowed data from different muscles to be combined despite differences in curare concentration or sensitivity to receptor block. Nerve stimulus strength was varied to determine whether junctions were singly or doubly innervatcd. All dual innervation was focal. A preliminary analysis showed that EPP size at 1042 singly innervated junctions averaged 5% above the mean of alljunctions, whereas total EPP size at 464 doubly innervated junctions was 11% below the mean of all junctions, a highly significant difference (p < 0.002). It thus appeared that competitive interactions reduced the efficacy of one or both inputs at doubly innervated cutaneous pcctoris junctions. As yet we do not know whether this diminished efficacy is due to presynaptic or postsynaptic factors, or whether it has a physiological or structural basis. 6 . Competition within and between Synaptic Sites. It has been shown that the elimination of multiple synaptic sites from rat muscle fibers is enhanced if the sites are closely spaced (200 days), there was significantly less focal polyneuronal innervation in muscles with reduced motor units (Werle and Herrera, 1988). Data for the period after the initial wave of synapse elimination (>60 days) are presented in Fig. 14. This figure shows three different estimates of focal polyneuronal innervation in reinnervated muscles with large and small motor units and in normal muscles. By the methods of counting EPP steps and doubly innervated gutters, there was significantly less polyneuronal innervation in small motor unit muscles compared with large. The method of counting multiple axonal inputs did not yield a significant difference, confirming that the tendency of this method to overestimate polyneuronal innervation (see Results Section 1) makes it less reliable in this situation. By all three methods, normal muscles showed significantly less polyneuronal innervation than both types of reinnervated muscles, confirming that synapse elimination after reinnervation does not restore the normal low level of polyneuronal innervation (Fig. 5 ; Herrera and Werle, in preparation). We concluded that decreasing motor unit size increases the ability of regenerated terminals to compete for and dominate synaptic sites.
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We speculate that this increased level of competition is fueled by the enhanced metabolic support received by terminals in reduced motor units. DISCUSSION 1. Introduction
The results presented here, and those of similar studies by others (reviewed in Wernig and Herrera, 1986; Robbins, 1988), demonstrate that neuromuscular junctions of adult frogs are highly dynamic synapses. Within a single junction there is often growth of some nerve terminal branches while others are retracting. With time, this remodeling usually results in net synaptic enlargement. In a similar sense, synapse formation and elimination can occur simultaneously in different parts of the same muscle. These opposing processes wax and wane in an annual cycle, giving rise to seasonal differences. The equilibrium progressively shifts towards elimination, resulting in a net reduction in polyneuronal innervation over a lifelong time course. Within the muscle as a whole, there is a progressive increase in the number of synapses, with a conscquent expansion of each motoneuron’s peripheral field.
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Figure 14 Estimates of the incidence of focal polyneuronal innervation using a physiological method (multiple EPP steps) and two morphological methods (doubly innervated gutters. multiple axonal inputs). Data are for reinnervated sartorius muscles with large motor units (solid bars; sample size for each method was 1919, 245, and 163 junctions, respectively), reinnervated sartorius muscles with small motor units (hatched bars; sample size 656, 190, and 139 junctions), and normal muscles (open bars; sample size 585,25 1, and 25 1 junctions). Vertical lines are standard errors of the mean. Reinnervated muscles were denervated by nerve crush at least 60 days earlier. Within each cluster of bars, values for the 3 types of preparations were significantly different ( p < 0.01) except for the solid and hatched bars in rightmost cluster. which did not differ significantly from each other ( p > 0.05).
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The view that emerges from these findings is that, at any one time. the structural and functional state of the neuromuscular junction represents an equilibrium between processes favoring growth and those favoring retraction. This discussion is organized to consider three major factors that may influence this equilibrium: metabolic support nerve terminals receive from their somata, competition between terminals of different motoneurons, and activity-dependent interaction between nerve and muscle. We will then discuss the differences in synaptic plasticity and synapse elimination in neuromuscular junctions of frogs and mammals. With the goal of posing testable hypotheses, our approach will be purposely speculative. 2. Metabolic Support It is a well-established principle that the materials needed for maintenance and growth of motor nerve terminals are synthesized in and exported from the soma. The metabolic load and transport distances involved can be considerable. Using a published value of 12 mm for dendrite length (Bregman and Cruce, 1980) and our own measurements of 35 mm for typical axon length and 57 mm for total nerve terminal length (Herrera, unpublished), it can be calculated that a typical spinal motoneuron in a 20 g frog must support approximately 10 cm of processes, with much of this located at a considerable distance. Consistent with the view that the ability to provide metabolic suppod is matched to the demand, positive correlations have been found between soma size and motor unit size (Yao and Weakly, 1986) and between motor unit size and synaptic efficacy (Grinnell and Trussell, 1983; Trussell and Grinnell, 1985). Since metabolic support must be shared among all terminals within a motoneuron’s arborization, the level of support each terminal receives might be expected to change when motor unit size is changed. The maintenance of synapses and the ability of terminals to compete for synaptic connections is likely to be altered by changes in the level of metabolic support that terminals receive. Experimental studies using adult nerve-muscle preparations confirmed these predictions. Artificial enlargement of motor unit size resulted in decreased transmitter release and synaptic efficacy (Bennctt and Raftos, 1977; Bennett et al., 1979; Wigston, 1980; Hopkins et al.. 1980; Haimann et al., 198la; Slack and Hopkins. 1982; Luff,
Hatcher, and Torkko, 1988; Rochel and Robbins, 1988; Regnier and Herrera, 1988). Conversely, a decrease in motor unit size caused increased release (Herrera and Grinnell, 1980, 1985; Pockett and Slack, 1982a). Over time, the enhanced release was reduced to normal levels probably because metabolic support was down-regulated in response to reduced demand or reduced supply of trophic factor (Pockett and Slack, 1983; Herrera and Grinnell, 1985). Manipulations of the size of the peripheral arborizations of motoneurons also affect the ability of terminals to compete for synaptic connections. Regenerating motor axons, which initially have few synapses to support, have been shown to displace the terminals of motoneurons that have expanded their fieIds by sprouting (Brown et al., 1976; Genat and Mark, 1977; Brown and lronton, 1978; Dennis and Yip, 1978; Thompson, 1978; Wigston, 1980; Haimann et al., 1981b; Lowrie, O’Brien, and Vrbova, 1985; Ribchester, 1988). The finding that terminals in overextended motor units are at a competitive disadvantage suggests the following principle. During synapse elimination and perhaps throughout adult life, local changes in one part of a motoneuron’s arborization can affect synaptic maintenance and competition elsewhere in the motor unit. However, it is difficult to envision how presynaptic and postsynaptic size could bc matched at individual junctions unless local influences predominated. Although similar studies are considerably more difficult with developing nerve-muscle preparations, it is interesting to speculate that the level of metabolic support may also determine the outcome of synapse elimination. For example, a reduction in metabolic support may be involved in the retraction of axonal arborizations. When the rat soleus was partially denervated at birth so that only a few motor axons remained, there was still some reduction in motor unit size by the end of the synapse elimination period (Brown et al., 1976;Thompson and Jansen, 1977;but see Betz et al., 1980). Similar results were found in experiments on the mouse soleus (Fladby and Jansen, 1987). These findings suggest that motoneurons have an intrinsic tendency to retract their terminals during the peak period of synapse elimination, and this is likely to involve diminished metabolic support. It seems unlikely that the ability of motoneurons to provide metabolic support would remain fixed. In developing rat muscle, secondary myogenesis occurs during the penod of synapse elimi-
Plasticity and Growth of Frog Eidplatt>y
nation, so that the number of fibers and therefore the number of synapses is actually increasing (Betz et al., 1979). This finding suggests that the ability of motoneurons to support terminals should increase as motoneurons mature. This maturation may in turn be guided by trophic influences that motoneurons receive from the fibers they innervate (see Discussion Section 4). We speculate that at the embryonic stage at which synapse elimination begins. there are inherent differences in the ability of motoneurons to support terminal arborizations. At each polyneuronally innervated junction, nerve terminals that are supported to a greater extent have a competitive advantage and are more likely to survivc the elimination process. Thus the more robust motoneurons will develop into larger motor units. The level of support each terminal receives is not fixed, however. As the arborization of weaker motoneurons is pruned to a smaller size, the support received by each terminal might increase to the point where they are equally able to compete with terminals of larger and stronger motoneurons. In addition, there may be simultaneous maturation of motoneurons driven by trophic feedback. In this way each motoneuron would establish a motor unit size appropriate to that neuron’s ability to provide support, and, as synapses increase in size and number in adult life, there would be a commensurate increase in the ability of motoneurons to support their enlarged arborizations.
3. Competition Since the pioneering studies of Brown et al. (1976) and Betz et al. (1980), it has been widely accepted that synapse elimination can be at least partly explained by competition between axons that converge on the same junctional site. Thesc interactions result in the stabilization or withdrawal of (synapticcontacts. It is likely that the object of the competition is the acquisition of trophic support (Thompson, 1985: see Discussion Section 4). Building on this foundation, we will discuss how recent work has advanced our understanding of Gynaptic competition at frog neuromuscular junctions. The fact that synapse elimination does not proceed to completion in either normal or reinncrvated frog junctions offers an important advantage for the study of synaptic competition. Competitive mechanisms can be inferred by comparing the properties of synapses where polyneuronal innervation is and is not eliminated. Using this ap-
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proach, we discovered two important principles governing the elimination of focal polyneuronal innervation (Werle and Herrera, 1987, 1988). First, spatial proximity does not affect the outcome of synapse elimination. Competing terminals were equally intertwined or segregated before and after the main period of synapse elimination. Second, junctions at which polyneuronal innervation was eliminated were those in which competing inputs differed in EPP size per unit terminal length. If EPP size per unit terminal length was similar. polyneuronal innervation persisted. Our interpretation of these findings rested on the assumption that this measure of physiological efficacy also reflected the ability of terminals to compete for synaptic connections. Physiological and competitive efficacy have been shown to covary in a variety of experimental situations, probably because both depend on metabolic support from the motoneuron soma (see Discussion Section 2; also, Wcrle and Herrera, 1987). We concluded that supernumerary inputs are displaced from a junction only when those inputs compete with others of higher competitive strength. Com petiti on between inputs of equivalcnt strength leads to a stalemate in which polyneuronal innervation persists. Comparisons of synaptic efficacy at singly and focally doubly innervated junctions in normal frog muscles indicate that competitive interactions impair synaptic function. In the cutaneous pectoris muscle, we found that total EPP amplitude was 16% lower in doubly innervated junctions compared with singly inncrvated junctions. Others havc also found that polyneuronal innervation at frogjunctions is associated with deficits in synaptic cfficacy (Angaut-Petit and Mallart, 1979;Grinncll, Letinsky, and Rheuben, 1979; Haimann et al., 198 1 a, Weakly and Yao, 1983; Trussell and Grinnell, 1985). Upon considering these and other results on the nature of synaptic competition at frog neuromuscular junctions, we have formulated a two-part hypothesis that we are currently testing. First, wc propose that under normal conditions. competition is a heteroncuronal interaction. Different parts of the same motor axon, including different terminal branches at a singly innervated junction, do not compete with each other. Second, we propose that it makes little difference whether competing heteroneuronal inputs terminate at the same junctional site or at different sites on the same fiber. Focal and distributed polyneuronal innervation would provoke equivalent competition. The first statement, that competition occurs
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only between different neurons, conveys an appealing sense of efficiency in that it would prevent a terminal from competing with itself. More importantly, the hypothesis is supported by a preliminary analysis of growth-associated changes in the pattern of muscle fiber innervation (see Results Section 8b and Fig. 13). We found that postmetamorphic growth in the pectoral muscle ofXenopus is associated with a progressive conversion of the a/b innervation pattern to the a/a pattern. We speculate that competition in a/b fibers would cause functional or structural deficits at one or both junctions. The resulting decrease in activity or the partial uncovering of postsynaptic sites would render the junctions susceptible to additional innervation by nearby axons and terminals. Histological [Fig. 1(A)] and repeated in vivo observations (Fig. 10) have shown that interjunctional sprouting occurs. If this sprouting generated additional polyneuronal innervation, e.g., addition of a c input, the new input would be similarly destabilized. When, by chance, the same axon came to innervate both sites, decreased competition would lead to synaptic stability. The second hypothesis, that competition occurs to an equivalent extent regardless of the distance between heteroneuronal inputs, also has some support. By mapping the exact size and position of competing inputs at doubly innervated junctions in normal muscles (Werle and Herrera, 1987) and reinnervated muscles (Werle and Herrera, 1988), we found evidence that spatial proximity did not intensify competition. Further tests of this hypothesis could be made by relating synaptic function and structure with the amount and type of polyneuronal innervation at identified junctions in several different muscles. One would predict that physiological efficacy and morphological signs of structural stability (large junctional size, lack of abandoned synaptic gutter, etc.) should relate inversely to the incidence of total polyneuronal innervation, both focal and distributed. Muscles with more distributed polyneuronal innervation should have less focal polyneuronal innervation because increased competition would have driven synapse elimination to a more complete state. Such appears to be the case when the sartorius and cutaneous pectoris are compared. The sartorius averages about three times as many junctional sites per fiber and has about one-third the focal polyneuronal innervation of the cutaneous pectoris. Although the reciprocal nature of these numerical relationships may be coincidental, these findings are consistent with the hypothesis. 6'
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Following the lead of an early theoretical approach (Stent, 1973), we propose that competition between motor axons is governed by a Hebbian rule (Hebb, 1949)in which an important determinant of synaptic stability is the timing of presynaptic and postsynaptic activity (see also Nudell and Grinnell, 1983). In mononeuronally innervated fibers with two junctions (a/a), the destructive collision of muscle fiber action potentials in the region between junctions would insure that presynaptic and postsynaptic spikes always occurred synchronously at each junction. In the same way, the different terminal branches of the same motoneuron at a singly innervated junction could avoid mutual competition by virtue of their synchronous activity. In fibers innervated with the a/b pattern, however, it is likely that the two axons would not be synchronously active. On average, a postsynaptic spike could pass beneath an inactive presynaptic terminal about half the time. This presynaptic and postsynaptic asynchrony would destabilize both inputs, with the advantage going to the input better able to withstand the detrimental influence. The advantage may be determined by the relative level of metabolic support each terminal receives. Competition is clearly involved in the developmental process of synapse elimination, and similar interactions occur in reinnervated adult muscle. The simplest view is that the tendency of motor axons to compete for synaptic connections does not cease at the end of embryonic life. The same processes probably continue to remodel synaptic structure and function throughout adult life. 4. Activity-Dependent Nerve-Muscle
Interactions Alterations in neuromuscular activity have profound effects on synapse elimination. Generally, synapse elimination is delayed by blocking activity and accelerated by increasing activity via direct stimulation (reviewed by Thompson, 1985; Betz 1987). Thompson (1985) has proposed an attractive hypothesis for the mechanism of synapse elimination that conceptually unites these effects of activity with growing evidence for muscle-derived motoneuronotrophic factors (Henderson, Huchet, and Changeux, 1983; Slack and Pockett, 1982; Pockett and Slack, 1982b: Smith and Appel. 1983; Hill and Bennett, 1986; Dohrman, Edgar, and Thoenen, 1987; Heaton, 1988). The hypothesis is that during developmental synapse elimination motoneurons depend upon trophic factors that are
Pluslicily und Growth of Frog Endplates
produced by muscle in amounts inversely related to activity. A similar mechanism was the subject of an earlier theoretical analysis (Gouze, Lasry, and Changeux, 1983). Our purpose here will be to examine whether the same mechanism can also explain the postmetdmorphic growth and plasticity of frog junctions that we have described. It is important to note that in frogs, as in mammals, various methods of inducing inactivity trigger the production of a nerve terminal growth-promoting stimulus (Wernig et al., 1980a; Wines and Letinsky, 1988; Diaz and Pecot-Dechavassine, 1989). Both histological and repeated in vivo observdtions have shown that frog neuromuscular junctions grow throughout life. Presynaptic and postsynaptic growth is matched in such a way that physiological efficacy tends to be maintained. The maintenance of efficacy may be better in younger animals. Thcse findings can be explained by the hypothesis that mature motoneurons are as dependent on muscle-derived trophic factors as dcveloping motoneurons, and that the release of those factors in both development and adult life is an inverse function of activity. This would provide a negative feedback mechanism that explains the matching of presynaptic and postsynaptic size during growth in adults. Synaptic efficacy would be regulated through its effect on activity. The activity signal that regulates trophic support may not only involve propagating action potentials, but also more local influences. In frogs, and in other lower vertebrates (Morgan and Proske, 1984; Mos et al.. 1988), muscle growth involves the addition of new synaptic sites. In mammals, certain of the influences of nerve on muscle, such as concentration of acetylcholine receptors (Fambrough, 1979)and voltage-dependent Na channels (Caldwell, Campbell, and Beam, 1986), diminish sharply with increasing distance from the junction. If the suppression of trophic factor production by activity in frog muscle also diminishes with increasing distance from the junction, growth in muscle fiber length may trigger sufficient production of trophic substance to attract and support new synapses at the growing ends of the fiber. In response to this additional source of trophic support, the adult motoneuron may hypertrophy to restore the balance between metabolic supply and demand. A mechanism similar to that proposed by Thompson (1985) for developmental synapse elimination may also explain the synapse elimination that follows reinnervation of adult muscle. Axotomy would interrupt the retrograde flow of
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trophic substances. This loss of support, together with regenerative axonal elongation, may severely compromise the metabolic capacity of motoneurons. This crisis would continue until two conditions were fulfilled. First, motoneurons must reestablish trophic support by regenerating a sufficient amount of synaptic contact. Second, those trophic molecules must be transported and have time to exert their effects on the soma. Until these conditions were met, nerve terminal retraction would predominate over growth, and synapse elimination would result. One would predict from this scheme that once enough trophic support was secured, the rate of elimination would slow dramatically. In fact, both our results with frogs (Herrera and Werle, in preparation) and studies by others in mammals (McArdle. 1975; Benoit and Changeux, 1978; Brown and Ironton, 1978; Gorio et al., 1983; Taxt, 1983: Rich and Lichtman, 1989) have shown that synapse elimination after reinnervation never quite restores the low level of polyneuronal innervation found in normal adult muscle. As with competition, the role for motoneuronotrophic factors probably does not cease when the developmental periods of neuronal cell death and synapse elimination are over. Instead, such factors probably play a vital role in the normal growth, maintenance, plasticity, and senescence of adult neuromuscular junctions. They may also determine the ability of adult motoneurons to recover from injury or disease.
5. Differences between Frog and Mammalian Neuromuscular Junctions Differences in synapse elimination at neuromuscular junctions in frogs and small laboratory mammals can be easily summarized. In frogs, embryonic polyneuronal innervation is not climinated completely, and new polyneuronal innervation can be generated in adults. In mammals, developmental synapse elimination is essentially complete, and there is little evidence for residual polyneuronal innervation in adults. Morphological studies have also revealed differences in the growth and remodelling of adult junctions in frogs and mammals. Since the results of studies using fixed tissue have been reviewed recently (Wernig and Herrera, 1986; Robbins, 1988), we will focus on studies using new methods of repeated in vivo observation. Junctional growth in frogs is accompanied by considerable remodeling of presynaptic and postsynaptic structures (Herrera and Banner, 1987;
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Herrera et al. 1988, in press; Chen and KO, 1988). Both retraction of branches within junctions that are growing and elongation of branches within junctions showing net retraction are common. Complete retraction and the de now appearance of new branches also occurs. In fast-twitch muscles of the mouse. in vivo observations show that an equivalent amount of growth occurs over similar time periods, but growth mostly involves the proportionate enlargement of preexisting branches (Lichtman, Magrassi, and Purves, 1987; Wigston, 1988; Balice-Gordon and Lichtman, in press). Disproportionate growth or shrinkage is somewhat more common at junctions in the mouse soleus, a muscle with approximately equal numbers of fast and slow fibers (Wigston, 1987, 1989). Terminals in the slow-twitch mouse pectineus extend small but highly dynamic pseudopodial processes (Hill and Robbins, 1987, 1988; Robbins and Polak, 1987, 1988). Since there are so many similarities in the overall structure and function of frog and mammalian junctions, a thorough understanding of the basis of these differences in synaptic plasticity may provide insights to mechanisms common to both systems. Much may be explained by interspecific differences in activity. Frogs are much less active than mice, and inactivity has been shown to stimulate motor axon sprouting in frogs (see Discussion Section 4) as it does in mammals (reviewed in Brown, Holland, and Hopkins, 1981; Robbins, 1988). Unlike laboratory mammals, frogs are poikilothermic and, from winter to summer, experience a wide range of body temperature and activity. Seasonal differences in the structure and function of frog junctions have been noted (Wernig et al., 1980a,b; Herrera, 1984;Jans et al., 1986; Diaz and Pecot-Dechavassine, 1988; older literature reviewed in Grinnell and Herrera, 198 1). In particular, higher levels of sprouting and polyneuronal innervation are found in winter frogs, which are less active. In light of Thompson’s (1985) trophic factor hypothesis and the discussion above, we speculate that these changes are due to inactivityinduced increase in the production of motoneuronotrophic factors during winter (see also Wernig and Herrera, 1986). Of equal or greater importance in explaining differences in junctional plasticity between frogs and mice may be the pattern of body growth. Unlike mice, frogs grow throughout life. Growth in body size is accompanied by muscle fiber hypertrophy in both diameter and length and by muscle fiber addition (Sperry, 1981). Because of this in-
definite growth, a presumably fixed number of spinal motoneurons are called upon to support a peripheral arborization that is constantly expanding in two ways. First, the size of each terminal must increase to compensate for the hypertrophy-induced postsynaptic electrical changes. Second, entirely new junctional sites are innervated as fibers elongate and new fibers appear. The greater capacity for synaptic plasticity in the frog nerve-muscle system would clearly seem to be an adaptation to meet demands not encountered by mice. This hypothesis could be tested by examining junctional plasticity in other animals with fixed and indeterminate growth.
CONCLUSIONS
We conclude that the neuromuscular junction of the normal adult frog is a highly dynamic synapse and that much of this plasticity can be explained by the same mechanisms that govern synaptogenesis during development. The fact that the nervous system of the frog never reaches a static state of maturity is probably an adaptation to accommodate continual growth. Although there are many differences between neuromuscular junctions and synapses in the brain, and there are ccntral synaptic mechanisms that have no counterpart in the periphery, we are optimistic that this simple peripheral synapse will be useful as a model for studying plasticity in the brain. This work was supported by grants from NIH (NS24805) and NSF (BNS85 18232) and by an NIH Research Career Development Award (WS0095 1). We are grateful to Dr. Richard Dunia, Michael Regnier, and Naomi Nagaya for providing unpublished data and for commenting on the manuscript and to Christa Mulkey and Julie Houser for technical assistance. This article is dedicated to Jonathan and Meredith Herrera.
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