Sprouting and nerve retraction in frog neuromuscular junction during ontogenesis and environmental changes

Neuroscience Vol. 18, No. 3, pp. 773-781, Printed

0306-4522/86$3.00+ 0.00 Pergamon Journals Ltd 0 1986IBRO

1986

in Great Britain

SPROUTING AND NERVE RETRACTION IN FROG NEUROMUSCULAR JUNCTION DURING ONTOGENESIS AND ENVIRONMENTAL CHANGES H. JANS, R. SALZMANN and A. WERNIG* Institute of Physiology, Department of Neurophysiology, University of Bonn, Wilhelmstr. 31, D-5300 Bonn 1, West Germany At&me-Based on recent evidence for a physiological remodeling of neuromuscular contacts (Wemig et u/.42*43), a morphometric study was performed on axon- and cholinesterase-stained cutaneous pectoris muscle of frog. The aim of this investigation was to separate changes due to aging, growth, and environmental conditions. Within a single muscle, fiber diameters, synaptic lengths, number of intraterminal branches, and lengths

of abandoned gutters differ considerably (with coefficients of variation from 40 to 56%). On the other hand, these parameters are correlated and correlations hold when muscle fibers grow during ontogenesis: large muscle fibers bear larger and more complex junctions than small fibers. Obviously there exist growth regulating interactions between muscle fiber and the presynaptic nerve. To dissociate between age- and growth-related changes muscle fibers of equal diameters in frogs of different age are compared. With increase in age there is an additional increase in abandoned gutters, synaptic length, and complexity independent of muscle fiber growth. Possibly, abandoned gutters accumulate with time and synaptic length increases with age as the net outcome of continual synapse remodeling. When freshly caught frogs (October) were compared with frogs kept under laboratory conditions for a period of 16 weeks (which in addition included a change in season) the number of sprouts in a junction increased by about 2, the average length of presynaptic nerve terminals with small circumscribed contacts increased by 30-150 pm, and abandoned gutters tended to be shorter on fibers with large junctions. The hypothesis is discussed that remodeling is “inherent” to nerve terminals whereby sprouting is counterbalanced and reversed by nerve activity. Remodeling per se might not directly influence synaptic transmission but allow junctions to react to different physiological and pathological conditions.

Signs of nerve sprouting with new synapse formation on the one hand and signs of nerve retraction on the other hand have recently been observed in untreated adult frog muscles.42*43These findings have been interpreted to indicate the existence of a remodeling under normal physiological conditions of nervemuscle contacts. More recently combined light and electron microscopic studies have been performed with the result that the sites of synaptic remodeling can now be ascribed with more confidence to light microscopic features on stained preparations.‘*36*37 Features of synaptic remodeling occur with different frequencies in summer and winter frogs indicating that remodeling is influenced by external factors.4zA3 Similarly, it was previously noticed that the mean synaptic length in a muscle is positively correlated with the animal’s body size (which continuously increases with age) suggesting growth-related nerve sprouting. In the present investigation it was of particular interest to reveal age-related changes and separate these from changes due to growth. This was accomplished by comparing muscle fibers of equal di*Author to whom correspondence

should be addressed. CV, coefficient of variation; jf, jumping fibres; s, sprouts.

Abbreviations: ChE, cholinesterase;

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ameters in frogs of different body size and thus of different age. In addition it was attempted to induce changes in synaptic morphology by keeping animals under altered environmental conditions for a defined period of time. Some results of this investigation have been published elsewhere.38,41 EXPERIMENTAL PROCEDURES Frogs (Rana remporaria) were caught locally in August (mean local temperature 20°C) and within a few days worked on. To compare small vs large frogs five animals with rump-to-nose lengths of 4.2, 5.2, 5.2, 5.5 and 6.0 cm (body weights of 6,11,15,20 and 20 g) and five animals with rump-to-nose lengths of 6.1, 6.5, 7.0, 7.0 and 7.5 cm (body weights 21, 25, 36, 40, 42g) were used. These frogs were selected from a larger collection as those with the most typical body weight to body length ratios. The ages of the individual animals are not known, according to Miiller (Ref. 25 and personal communication) the most likely range is 24 years. In a collection of 31 Runu temporuriu, raised from spawn under laboratory conditions, mean body size (rumpto-nose) at an age of 530 days was 3.9 cm k 1.2 SD (range from 2 to 6 cm), mean body weight was 3.5 g f 0.72 SD (range from 2 to 7 g); in natural environment mean values most likely are higher since the smallest frogs (about l/3) would not survive (Hk. Miiller, unpublished nersonal communications). A group of frogs of-similar body size (6 cm) was obtained locally during a high temperature period in October (19°C mean maximum and 7°C mean minimum day

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temperatures). Six frogs were immediately examined (freshly caught frogs) and S frogs were examined after keeping them for 16 weeks at a water temperature of 14-16°C (laboratory frogs). They were kept in a small water tank (60 x 30cm) with dry places and were force-fed on meal worms twice a week. The laboratory was equipped with timing devices to provide daily light and dark periods (cycle: 15 h light, 9 h dark; starting at 6.00 o’clock). Staining

and morphometric

measurements

Both cutaneus pectoris muscles of a frog were dissected and pinned out in dishes. A combined axon and cholinesterase (ChE) staining was appliedz7 and muscles were subsequently mounted in toto on glass slides. Measurements were made in the light microscope using an eyepiece micrometer in a x 12.5 ocular with x 40 or x 100 objectives. For each evaluated muscle fiber the sarcomere spacing was determined and subsequently all length measurements were normalized for a sarcomere length of 3 pm. Statistical

treatments

large ranges in values for muscle fiber and synaptic parameters obtained from a single muscle, and also the differences among animals within an experimental group demanded a novel statistical approach.39 When comparing two experimental groups the occurrences of failures, small, mean, and large values were separately compared. Failures are those junctions which did not show the parameter investiga~. The limits for the small and large values were determined somewhat arbitrarily, usually according to the 10% smallest and largest values of the pooled data (without failures). Consequently, the occurrence of small and large values in the data from individual animals was determined and expressed as a fraction of the total number of observations in this animal. These numbers were then used to compare the two experimental groups in a parameter-free rank test ~~lcoxon-Mann-jitneys. The same rank test was used for mean values and failures. Generally this test procedure, in addition to point to point differences, also reveals overall trends comprising the whole distribution (from failures to large values), therefore levels of significance beyond the usual range (5%) are indicated (Tables 2 and 3). me

RESULTS

Synaptic and muscle fiber parameters were evaluated on cutaneus pectoris muscles stained in toto for axon and ChE.27 The staining

method

is important

as

it allows one to distinguish, using the light m1ct-oscope, features of nerve sprouting and nerve retraction.‘,42,4’ Figure 1 shows characteristic features that can be seen in normal junctions and defines the different parameters used in this study. The parent

axons (a) innervating single muscle fibers are drawn as simple lines. The number of axons (a) contributing to a junction was defined as all those axons crossing the borders of the muscle fiber, regardless of whether they originated from the nerve trunk or a neighboring junction. Axon terminals are followed by the parallel double lines of ChE reaction product where they contact the muscle fiber. Synaptic complexity (branches) was measured as the number of axons (a) plus the number of intraterminal branches (b). Synaptic length is defined as the summed length of the axon terminal branches, each measured from the very proximal contact to the most distal one, including parts where the axon is not associated with ChE reaction product (and most likely is not in contact with the muscle fiber).’ New synaptic contacts have previously been identified in the electron microscope from the absence of secondary folds. In the light microscope they are recognizable as small rings of ChE reaction product.‘.” Small contacts (< 5 itmf are thus regarded as features of sprouting (s, jf) in spite of the fact that an unknown fraction of them may possess more or less mature secondary folds (cf. Ref. 1). We also assume that the rare nerve terminal branches without apparent contact (i.e, without ChE association) and all short branches (to 5pm) with continuous contacts are sprouts (s). Rows of small contacts (jumping fibers, jf) can be found at the distal ends of junctions and also within junctions. For the study on aging only distal parts were counted. The

length of a jumping fiber was defined as the axon terminal length, measured from the last continuous contact or the last branch point to the next continuous contact or to the nerve end. Abandoned gutters (l-4) are recognizable from the presence of

Fig. 1. Camera Iudica drawing of a neuromuscular junction of frog cutaneus pectoris muscle after staining for axon and ChE, cltaract&~ synaptic tbatures which in reality were located at severa different junctions are shown. The drawing sarves to illustrate the d&nitions given in the text: intraterminal branches (b), sprouts (s), jump@ fibers fjf) and different locations of abandoned gutters (l-4). Bar = 20 Frn.

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Remodeling of the neuromuscular junction number of observations 10

IS00

Id00

2000 (rJm)

synaptic Length

Fig. 2. Frequency distributions of synaptic lengths from 5 small frogs (S) and 5 large frogs (L). Left hand side: results from individual animals, right hand side: cumulated distribution; n = number of junctions evaluated.

ChE reaction product, often arranged in palisades. The numbers in Fig. 1 indicate the different positions of abandoned gutters within the junction as follows: in distal prolongation of an axonal branch (1), not in obvious connection with an axonal branch and most likely resulting from retraction of a large axonal branch (2), within the junction, whereby the nerve is not in contact with the gutter but located at some distance above (3), and abandoned small contacts (4). All abandoned gutter lengths on a muscle fiber were usually summed unless this is otherwise stated. Diversity in synaptic within single muscles

and muscle fiber

parameters

It is common knowledge that in the adult, muscle fibers within a single muscle are of quite different diameters and also synaptic lengths differ considerably.20+26A large scatter in synaptic length can be seen in the frequency distributions in Fig. 2 with coefficients of variations (CV) for individual frogs ranging from 44 to 76%. A similar diversity is present for muscle fiber diameters (CV 2441%) Fig. 3a) and for the total number of axonal branches in a junction (CV 41-65%, Fig, 3b). Other striking observations are the positive correlations between muscle fiber

Table 1. Locations

of abandoned gutters within neuromuscular junction (cf. Fig. 1) Distal (1)

n = 765

Free Central (2) (3)

46 41

Frequency (%) Mean abandoned gutter length @m)

4 88

21 38

the jf (4) 23 -

Distal (1): in the dorsal prolongation of an axonal branch, free (2): not in obvious connection with a nerve terminal branch but within the junctional region; central (3): within the junction, usually with the nerve terminal nearby; jf (jumping fibers) (4): a row of spotlike abandoned gutters in distal location. n = number of junctions evaluated.

I

I

1

I

I

1000

I

I

2000

diameter and synaptic parameters. Large muscle fibers bear large synapses (Figs 3a and 4a) and large

I

I

I

I

I

I

I

2000 ‘km) synaptic length

1000

.L

synapses have more branches than smaller ones (Fig. 3b). Dzxerent locations junctional region

of abandoned

gutters

within the

There are typical locations within the junction from which the axon either retracts or presumably lifts off the synaptic gutter (Fig. 1). Abandoned gutters are most frequently (46%, Table 1) located in

1000

2000

1000 2000 Cm) synaptic Length

Fig. 3. Fiber diameters (a) and number of branches (b) are plotted against synaptic lengths for 5 small (S) frogs (n = 152) and 5 large (L) frogs (n = 216).

1%

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JANS et ul.

distal prolongation of nerve terminal branches (Position 1 in Fig. 1); their average length in this location is 41 pm. Least frequent (4%) but relatively long (average length 88 pm) are abandoned gutters which run parallel to a nerve terminal branch or are otherwise not in obvious connection with the nerve terminal arborization (Position 2 in Fig. 1). In a surprising large fraction (about 27%) of abandoned gutters the nerve has not retracted but apparently lifted off the gutter (average length 38 pm, Position 3 in Fig. 1). Acetylcholine receptors are missing at such locations’9 as they are missing in abandoned gutters from which the nerve has retracted;’ this additional peculiarity seems to exclude the notion that abandoned gutters with the nerve on top are mere staining artifacts. It is important to notice that jumping fibers and sprouts may also retract, leaving behind a row of spot-like short (< 5 pm) abandoned gutters (Position 4 in Fig. 1); on the average these account for as many as 23% of all abandoned gutter sites. Comparison of dtgerently aged frogs (small us large frogs )

Since frogs continually grow, ontogenetic comparisons always include both, age- and growthrelated changes. In this study 5 small frogs (body size 4.2-6.Ocm) were compared with 5 large frogs (body size 6.1-7.5 cm). In general, larger frogs have definite larger synaptic lengths (Fig. 2), a higher number of branches in a synapse and larger muscle fibers (Table 2). It is clear from the histograms in Fig. 2, however, that there is a general shift to the right with larger Table 2. Comparison of synaptic and muscle fiber parameters in froas of different body size Larger frogs Muscle fiber diameter Synaptic length Number of branches Number of axons per junction Number of sprouts Jumping fiber length Abandoned gutter length

Failures

Small values _ _--

Mean values

Large values

++ +++

++ +++

+++

++

synapses added, while the range of the smallest synapses remains comparable. To exclude growth-related changes muscle fibers with similar diameters are compared in the two groups of animals. This comparison should reveal age-related changes as long as a turnover of muscle fibers with age does not occur or is moderate. In Fig. 4 the average values for synaptic length (Fig. 4a) and the number of branches (Fig. 4b) are considerable for individual animals (left hand side and middle) and for the two groups (S = small frogs, L = large frogs). It appears that for most ranges of muscle fiber diameters synaptic length and the number of branches are somewhat larger in the older animals (L), the difference becoming larger with the increase in muscle fiber size. Abandoned gutter lengths (Fig. 5a. Table 2) and even fractional lengths with respect to the total gutter length (Fig. 5b) are generally larger in the larger frogs; this is true for each given range of synaptic length (Fig. 4c,d) and muscle fiber diameter (not shown). It appears, therefore, that with age there is an additional, growth independent increase in synaptic length and at the same time an accumulation of abandoned gutters. The number of sprouts, number of axons contributing to a junction and the jumping fiber length merely tend to be larger in the larger frogs (Table 2). Frogs under d@rent

In this investigation frogs freshly caught in late summer (October) were compared with frogs subsequently kept under laboratory conditions for a period of 16 weeks (which in addition includes a change in season). The body size of all frogs was the same (6 cm). No significant differences in muscle fiber diameters and synaptic lengths were detectable between the two experimental groups (Table 3). Clear

Table 3. Comparison of synaptic and muscle fiber parameters in frogs of equal body size (6 cm) but kept at diRerent environmental conditions: freshly caught frogs and laboratory frogs

+ -

<

+

>

no

no

no

+

-

-

+++

+++

The signs indicate whether the larger frogs (rump-to-nose lengths from 6.1 to 7.5 cm) have higher (+, >) or lower (-: <) values than the smaller frogs (4.2-6.01~1). The groups of animals are compared in rank tests (Witcoxor4&ann-Whitney), the marka4 scatter of values within a single muscle requires the analyses of the relative frequency of failures, small and large values (smallest and largest 10% of the cumulated histogram, see Experimental Procedures and Ref. 39). In addition, the mean values are compared. Levels of significance (two tailed test) are indicated by the nsrmber ofsyn~bok~ P), P > 20% (no).

environmental conditions

Laboratory frogs Muscle fiber diameter Synaptic length Number of branches Number of axons per junction Number of sprouts Jumping fiber length Abandoned gutter length

Small Failures values __no no _

Mean values

Large values

no no > no

no no >

- -

- -

+ + + + +

+ + + +

>

>

n0

<

The signs indicate whether the laboratory frogs have higher (+, >) or lower (-, <) values. Rank tests of the animal’s mean values, relative frequenoies of failures, small and large values (see Experimental Prooxiures and Ref. 39) were performed, the levels of sign~&+nce- are indicated by the mtrnber of symbols: P < 1% (+ + + , ---), P<5% (++, --), P), P > 20% (no).

Remodeiing of the neuromu~~r

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30 b5 60 75 90

120

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30 CS 60 75 90 f ibre diameter

300 700 1100?500 synaptic length

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(urn) Fig. 4. Mean synaptic Iengths (a) and mean numbers of branches (b) for given ranges of muscle fiber diameters, mean lengths of abandoned gutters (c) and mean relative lengths of abandoned gutters (d) for given ranges of synaptic length. Lines connect average values obtained From individual frogs. Left: 5 small frogs (S), middle: 5 large frogs (L). Right: data from the cumulated groups (0, small frogs; 0, large frogs). For the graphic representation a minimum of two observations was allowed in a class, classes in the cumulated histograms (right hand side) with only two observations are connected by broken lines. Bars indicate the standard errors of the mean and are for tbe sake of clarity drawn only to one side. Same frogs as in Figs 2 and 3.

differences, however, are apparent for the number of branches, number of sprouts and the total jumping fiber lengths (Table 3, Fig. 6). For comparable ranges of synaptic lengths there are more branches and more sprouts in the laboratory frogs, for comparable muscle fibers there are longer jumping fibers (Fig. 6). It appears that during the period of 16 weeks under laboratory conditions about two sprouts were added to each junction whereby the number of branches also increased by about this number. Abandoned gutter lengths are somewhat shorter in the laboratory

frogs (Table 3, Fig. 6) but the difference is much smaller than previously found for summer and winter frogs taken from their natural environment (cf. Refs 42 and 43). A reduction in abandoned gutter length can be explained by reoccupation of gutters. An alternative explanation would have to assume that ChE activity is so much reduced in the laboratory and winter frogs 42*43that many abandoned gutters remain undetected. Following this line it is then also conceivable that the jumping fibers (especially the central ones, see Fig. 1) are due to lifting of nerve

778

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JANS et al

traction is caused by nerve activity. It was suggested that neurofilaments and microtubules, which are

frogs n=216

large frogs n=216

large

0

60 120 180 240 300 z-3590 abandonedgutterItim1

8

16 24 32 40 AL abandoned gutterI%

Fig. 5. Frequency distributions of abandoned gutter lengths (a) and abandoned gutter length as a fraction of total gutter length (i.e. occupied plus unoccupied) (b) for 5 small frogs (n = 152) and 5 large frogs (n = 216). Failures are junctions without abandoned gutters.

terminals, leaving only a few small contacts intact. Such an occurrence cannot be excluded but is not likely either, i.e. in jumping fibers investigated in the electron microscope no signs of a former contact (unoccupied secondary folds) were found.‘.36 The occurrence of central abandoned gutters (Fig. 1) on the other hand suggests that synaptic contacts become abandoned not only by nerve terminal retraction but also by axons remaining on top of the synaptic gutter (see Discussion). DISCUSSION

The evidence for new synapse formation in adult muscles obtained from ultrastructural investigationsl342943 could be explained su@ciently by the continual growth of the junctions. The very existence of abandoned gutters and particularly their accumulation with time, however, can best be explained by synaptic remodeling. In this concept nerve terminal retraction is followed by, or goes parallel with, sprouting and reoccupation of abandoned gutters and also new synapse formation.42”3 While remodeling might primarily be without direct consequences for the function of the nerve-muscle junction, it is clear that the extent and the direction of remodeling are influenced by several factors. In the present investigation we found such changes due to growth, environment and season. At least in case of muscle fiber growth synapse enlargement with supposed increase in transmitter release seems of functional importance and necessary for maintenance of transmission. Mechanisms of remodeling

In the simplest case we can assume that sprouting is “inherent” to nerve terminals, while terminal re-

regularly broken down by a calcium-activated protease, are a driving force for axonal sprouting.“,” Signs of nerve terminal retraction are prominent during periods of elevated motor activity (summer vs winter frogs,42.43and freshly caught vs laboratory kept frogs in the present investigation). A simple hypothesis would therefore be that under elevated synaptic activity elevated calcium-influx causes nerve retraction, while a reduced breakdown during inactivity favors nerve sprouting. Additional sprouting factors seem to be produced from muscles (see below). Our observations are also compatible with the idea that the supply of substrates via the axoplasmic transport to the most distal parts of the terminal arborization is reduced in elevated synaptic activity and consequently terminals shrink. Both hypotheses, however, do not readily explain the existence of centrally located abandoned gutters (Fig. 1) for which a novel mechanism like a local failure of synapse maintenance ought to be responsible. The missing of acetylcholine receptor clusters at these sites” could be due to the lack of nerve born factors presumably actively maintaining receptor cIusters.‘3 Mechanisms which actively maintain synaptic connections have also been postulated for other different neurons28,29,34(for further discussion see Ref. 40). Age-related changes

For muscle fibers of similar diameters synaptic and abandoned gutter lengths were strikingly larger in the older frogs. Possibly, in the course of continual remodeling junctional size and complexity gradually increase, and abandoned gutters accumulate with time. Interestingly, abandoned gutters sooner or later seem to lose their attraction for growing terminals, since new synaptic sites are formed rather than all former ones reoccupied. Also the increase in synaptic length with age could simply be an accumulation effect since junctions grow when muscle fibers grow but might not retract when muscle fibers shrink. Synaptic length might thus always reflect its maximum size at any time. Accumulation of synaptic length could also be due to the formation of less effective contacts (e.g. fewer and/or smaller active zones) during remodeling yielding additional growth. This could explain why on muscle fibers of the same diameter larger terminals release less transmitter per unit length than smaller ones.26 Findings in mammalian muscles can be interpreted in a similar way. In ultrastructural investigations an increase with age in abandoned gutter occurrence was found in mouse soleus and extensor digitorum longus muscles’ and in rat soleus muscle.6 On the light microscopic level also an increase in complexity with additional intraterminal branches was found without changes in muscle fiber diameters.8*30.39Independent of changes in synaptic size there is an increase in the

Remodeling of the neurom~cular laboratory

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frogs

779

junction

freshly caught frogs

20

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10

300 700 1100lSO0

2700 300 700 11001~0

2700 2700 300 700 llW1~ synaptic length@mI

300 700 11001MO

2700 300 7OOllWlSW

2700 2700 300 7WllOO 1500 synaptic length Qtrn)

@ 2; 4

4

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9.2 4 2

1

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30 45 60 75 90

120

120 30 45 6075 90 fibre diameter Q.irnj

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300 7WllOO 1500

2700 300 7WHOOlSW

2700 300 70011001500 synaptic length

2700

(Mm)

Fig. 6. Average number of branches (a), number of sprouts (b), total jumping fiber length (c) and abandons gutter length (df for given ranges of muscle fiber diameters or synaptic lengths. Left hand side: 5 laboratory frogs, middle: 6 freshly caught frogs, right hand side: values from cumulated groups (mean, SEM). 0, Laboratory frogs; 0, freshly caught frogs.

number of isolated small contacts within single endplates in aged mammals7*30 which strongly suggests that the focal synapse abandoning with “central” abandoned gutters observed in the frog also occurs in mammals. From electrophysiological recordings it appears that the safety margin for synaptic transmission increases with age in mouse soleus and extensor digitorum longus (but not in diaphragm),2~*8 which might be due to the observed increase in junctional complexity. At the same time these observations render the notion that age-related increase in junctional size is a compensation for a decline in synaptic efficacy (i.e. transmitter release per unit length of the nerve terminal per impulse) in mammals

as less likely. Possibly, leg muscles as compared to diaphragm are less active in aging, in which case activity might “economize” synaptic transmission while inactivity allows the functional meaningless increase in safety factor (see above) and junctional size. Seasonal and environmental changes Several investigators have noted that junctions in winter frogs release less transmitter per single impulses but have more facilitation than in summer frogs.24 This was also noted in two groups of frogs comparable to the ones used in this study (body size 6cm; caught in April, kept under standard labora-

780

H. JAM et ul.

tory conditions, worked on in June to August and January to February*). In the present study we did not directly measure contact lengths, but synaptic lengths were similar in both groups of frogs. It is quite likely, therefore, that synaptic efficacy (i.e. transmitter release per unit length) and certainly facilitation behavior changes due to seasonal factors. So far we can merely point out that marked structural and functional changes occur according to seasons and we do not know whether they are related and whether one is leading to the other. Considerable diversities in efficacy in different muscles of one animal or even within a single muscle have been observed12,‘5~26~35 and synaptic efficacy seems to be modifiable by experimental means.” There are still other features in the neuromuscular junction involved in signal transmission which show a considerable diversity. Since such diversities most likely reflect ongoing changes, they might-among others-be effective in regulating signal transmission. The width of receptor-bearing junctional folds differs considerably within a single junction” as does the width of the presynaptic active zone.” Similarly, quantum size is not the same within a junction and differs at certain spots.’ The growing

muscle fiber

When muscle fibers grow during ontogenesis, nerve terminals and synaptic contacts also enlarge suggesting the existence of growth-regulating interactions. The underlying mechanisms are not known and several possibilities may be considered. In one hypothesis activity of the muscle fiber plays a crucial role. When muscle fiber diameters increase then input impedance decreases, synaptic potential amplitudes will be reduced, and some might become subthreshold. In this case contraction rates and action potential frequencies would drop. Now one has to postulate that less active muscle fibers start to produce or produce more of a sprouting factor which leads to a synapse enlargement and to a restitution of suprathreshold synaptic transmission. The prod-

uction of a sprouting factor in inactive muscle fibers has been postulated in several other experlmental conditions (for reviews see Refs 5 and 13) and myotubes seem to produce factors which promote neurite growth in cultured nerve cells.‘“~“~‘4 With increase in synaptic length, however, also the area on the muscle fiber receptive for synapse formation ought to increase. Several observations indicate that under physiological conditions there is a circumscribed area on each muscle fiber at which synapse formation is possible. There, new formation of synaptic contacts occurs in the course of synapse remodeling, and additional implanted foreign nerves can apparently form synaptic contacts.4 Interestingly, after denervation or blockade of transmission by botulinum poisoning32 this area spreads and ectopic junctions can form. Ectopic junction formation is prevented by direct muscle stimulation23 suggesting that also the spread of the receptive area on muscle fibers is controlled by muscle activity. While such activity-regulated mechanisms might cause local and short time regulations at the junctions, the role of activity for whole neurons might still be different. It may be asked whether on a larger time scale sustained elevated activity causes growth of the neuron (including the nerve terminal arborization). To our knowledge there is no direct evidence to support this notion. However, nerve cells in the corpus geniculatum laterale of kittens shrink following deprivation of visual inputs for a period of only 1 week,” which suggests some correlation between activity and growth of the nerve cell. Acknowledgements-This

work was supported by a grant from the Land Nordrhein-Westfalen. Brigitte Wernig and Gabi Conrad performed much of the histochemical work. We thank Mathilde Hau for secretarial help and Norbert Haas for the photographic work. We thank also Dr Brian Freeman from the University of New South Wales, Australia, for critical reading and improving the manuscript. Dr E. Hansert from the Max-Planck-Institute for Psychiatry, Miinchen, contributed many valuable discussions on statistical problems.

REFERENCES 1. Anzil A. P., Bieser A. and Wernig A. (1984) Light and electron microscopic identification of nerve terminal sprouting and retraction in normal adult frog muscle. J. Physiof. 350, 393-399. 2. Banker B. Q., Kelly S. S. and Robbins N. (1983) Neuromuscular transmission and correlative morphology in young and adult mice. J. Physiol. 339, 355-375. 3. Bieser A,, Wemig A. and Zucker H. (1984) Different quanta1 response within single frog neuromuscular junctions. /. Physiol. 390, 401-412. 4. Bixby J. L. and Van Essen D. C. (1979) Competition between foreign and original nerves in adult mammalian skeletal muscle. Nature 282, 726-728. 5. Brown M. C., Holland R. L. and Hopkins W. G. (1981) Motor nerve sprouting. ANI. Rev. Neurosci. 4, 17-42.

6. Cardasis C. A. (1983) Ultrastructural evidence of continued reorganization at the aging (1 l-26 months) rat Mleus neuromuscular junction. hat. Rec. ZW, 399-415. 7. Courtney J. and S&it-h J. H. (1981) Age&an@ in neuromuscular junction morphology and acetykboline receptor distribu~on on rat &e&al muscle Wea. J. physiaj. 328.43-7. 8. Fahim M. A., Ho&y J. S. and RoliMils k. (1983) Scanning and light microscopic study of age changes at a neuromuscular jut&on in the mot&. J. Neurocytol. 12, 13-25. 9. Fahim M. A. and Rabbiins N. (1982) Ul~tructural studies of young and old mouse neuromuscular junctions. J. Neurocytol. 11, 641-656.

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10. Giller E. L., Neale J. H., Bullock P. N., Schrier B. K. and Nelson P. G. (1977) Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium. J. Cell Biol. 74,1629. 11. Giller E. L., Schrier B. K., Shainberg A., Fisk H. R. and Nelson P. G. (1973) Choline acetyltransferase activity is increased in combined cultures of spinal cord and muscles cells from mice. Science 182, 588-589. 12. Grinnell A. D. and Herrera A. A. (1980) Physiological regulation of synaptic effectiveness at frog neuromuscular junctions. J. Physiol. 307, 301-317. 13. Grinnell A. D. and Herrera A. A. (1981) Specificity and plasticity of neuromuscular connections: Long-term regulation of motoneuron function. Prog. Neurobiol. 17, 203-282. 14. Henderson C. E., Huchet M. and Changeux J. P. (1973) Denervation increases a neurite-promoting activity in extracts of skeletal muscles. Nature 302, 609-611. 15. Herrera A. A. and Grinnell A. D. (1980) Differences in synaptic effectiveness at frog neuromuscular junctions: evidence for long-term physiological regulation. Brain Res. 194, 228-231. 16. Herrera A. A. and Grinnell A. D. (1981) Contralateral denervation causes enhanced transmitter release from frog motor nerve terminals. Nature 291, 495497. 17. Herrera A. A., Grinnell A. D. and Wolowske B. (1985) Ultrastructural correlates of naturally occurring differences in transmitter release efficacy in frog motor nerve terminals. J. Neurocyrol. 14, 193-202. 18. Kelly S. S. and Robbins N. (1983) Progression of age changes in synaptic transmission at mouse neuromuscular junctions. J. Physiol. 343, 375-383. 19. Krause M. and Wernig A. (1985) The distribution of acetylcholine receptors in the normal and denervated neuromuscular junction of the frog. J. Neurocytol. 14, 765-780. 20. Kuno M., Turkanis S. A. and Weakly J. N. (1971) Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the f&g. J. physiil. 213, 545-556. 21. Kuppermann B. D. and Kasamatsu T. (1983) Changes in geniculate cell size following brief monocular blockade of retinal activity in kittens. Nature 306, 465468. 22. Lasek R. J. and Hoffmann P. N. (1976) The neuronal cytoskeleton, axonal transport and axonal growth. In Cell Motility (eds Goldman R., Pollard T. and Rosenbaum J.j, p. 1021. Cold Spring garbour. 23. LGmo T. and Slater C. R. (1978) Control of acetylcholine sensitivity and synapse formation by muscle activity. J. Physiol. 275, 391-402.

24. Maeno T. (1969) Analysis of mobilization and demobilization processes in neuromuscular transmission in the frog. J. Neurophysiol. 32, 793-800.

25. MiilIer Hk. (1976) The frog as an experimental animal. In Frog Neurobiology (eds Llinas R. and Precht W.), pp. 1023-1039. Springer, Berlin. 26. Nude11 B. M. and Grinnell A. D. (1982) Inverse relationship between transmitter release and terminal length in synapses on frog muscle fibers of uniform input resistance. J. Neurosci. Res. 2, 216-224. 27. Pecot-Dechavassine M., Wernig A. and Stiiver H. (1979) A combined silver and cholinesterase staining method for studying exact relations between the pre- and the postsynaptic elements at the frog neuromuscular junction. Slain Technol. 54, 25-28.

neurones following interruption of their 28. F’urves D. (1975) Functional and structural changes in mammalian sympathetic _ _ axons. J. ihysiol. 252, 429-463. 29. Purves D. and Nja (1978) Trophic maintenance of synaptic connections in autonomic ganglia. In Neuronal Plasiicity (ed. Cotman C. W.), ou. 27-47. Raven, New York. 30. kobbins N. and Fal&M. A. (1985) Progression of age changes in mature mouse motor nerve terminals and its relation to locomotor activity. J. Neurocytol. 14, 1019-1036. 31. Schlaepfer W. W. (1979) The nature of mammalian neurofilaments and their breakdown by calcium. Prog. Neuropath. 4, 101-123.

32. Tonge D. A. (1977) Effect of implantation of an extra nerve on the recovery of neuromuscular transmission from botulinum toxin. J. Physiol. 265, 809-820. 33. Vyskocil F., Syrovy I. and Prusik Z. (1981) Induction of extrajunctional acetylcholine sensitivity of rat e.d.1. muscle by peptidic component of peripheral nerve. Pfltigers Arch. 390, 265-269. 34. Watson W. E. (1972) Some quantitative observations upon the responses of neuroglial cells which follow axotomy of adjacent neurones. J. Physiol. 225, 415-435. 35. Weakly J. N. and Yao M. (1983) Synaptic efficacy at singly- and dually-innervated junctions in the frog. Brain Res. 273, 3 19-323.

36. Wernig A., Anzil A. P. and Bieser A. (1981) Light and electron microscopic identification of a nerve sprout in muscle of normal adult frog. Neurosci. Mt. 21, 261-266. 37. Wernig A.. Anzil A. P.. Bieser A. and Schwarz U. (1981) Abandoned synaDtic - _ sites in muscles of normal adult frog. Neuroici. kr. 23, 1lo%1 110. 38. Wernig A., Anzil A. P., Marciniak M. and Bieser A. (1981) Evidence for remodelling of synaptic contacts in muscles of adult frog. In Cellular Analoaues of Conditioning and Neural Plasticitv. Advances in Phvsioloeical Science teds Fkher , Y 0. and J&F.), Vol. 36, pp. 2%33.-Pergamon Press, New York. . 39. Wernig A., Carmody J. J., Anzil A. P., Hansert E., Marciniak M. and Zucker H. (1984) Continual nerve sprouting with features of synapse remodelling in soleus muscles of adult mice. Neuroscience 11, 241-253. 40. Wernig A. and Herrera A. A. (1986) Sprouting and remodelling at the nerve-muscle junction. Prog. Neurobiol. in press. 41. Wernig A., Jans and Zucker H. (1986) A parametric study of the neuromuscular junction during ontogenesis and under different external conditions. In Calcium, Neuronal Function and Transmitter Release (eds Katz B. and Rahamimoff R.). Martinus Nijhoff Publ., Boston, in press. 42. Wernig A., Pecot-Dechavassine M. and Stdver H. (1980) Sprouting and regression of the nerve at the frog neuromuscular junction in normal conditions and after prolonged paralysis with curare. J. Neurocyrol. 9, 277-303. 43. Wemig A., Pecot-Dechavassine M. and Stiiver H. (1980) Signs of nerve regression and sprouting in the frog neuromuscular synapse. In Onrogenesis and Functional Mechanisms of Peripheral Synapses (ed. Taxi J.), pp. 225-238. Elsevier, Amsterdam. (Accepted 8 January 1986)