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What controls the development of neuromuscular junctions?
TINS - May 1980
126 release of bound calcium ions (see cartoon). It is intriguing that neurons are, apparently by a variety of influences, able to control the synthesis of specific m e m b r a n e proteins in their target tissues. This points to a subtle and sophisticated regulation by nerves of the chemical sensitivity of innervated cells, one purpose being the maintenance of the tissue in a condition in which it responds maximally to existing synaptic inputs but is unresponsive to accessory innervation. The supersensitivity resulting from denervation illustrates only the effects of total removal of nervous influences. The physiological 'value' of denervation supersensitivity is presumably to
make the tissue responsive to reinnervation by increasing its chemical sensitivity and its general excitability to a level where even immature nerve sprouts, irrespective of their site of attachment, have a chance to form functioning synaptic contacts and to re-establish neurotrophic control.
Reading list 1 Cannon, W. B. (1939) Am. J. Med. Sci. 198,
5 FLeming, W. W., McPhillips, J.J. and Westfall, D. P. (1973) Rev. Physiol. 68, 55-119
6 Greengard, P. (1978) Science 199, 146-152 7 Gutmann, E. (1976) Annu. Rev. PhysioL 38, 177-216 8 LCmo, T. (1976) in Motor innervation of muscle (Thesleff,S. ed.), Academic Press 9 Thesleff,S. (1960) Physiol. Rev. 40, 734-752 10 Thesleff, S. (1974) Ann. N.Y. Acad. Sei. 228, 89-104 I 1 Thesleff,S., Libelius, R. and Lundquist,1. (1979) in Muscle, nerve and brain degeneration. Internation Congress Series, 473 (Kidman, A. D. and Tomkins, J. K. eds), Excerpta Medica, Amsterdam 12 Trendelenburg, U. (1966) Pharmacol. Rev. 18, 629-640
737-750 2 Cannon, W. B. and Rosenblueth, A. (1949) Supersensitivity of denervated structures, Macmillan, New York 3 Dolly, J. O. (1979) Int. Rev. Biochem. 26, 257-309 4 Fambrough, D. M. (1979) Physiol. Rev. 59, The authors" are at the Department of Pharmacology, University of Lund, Lund, Sweden. 165-227
has not yet appeared and the time course of the acetylcholine (ACh) activated ionic channels is presumably still as prolonged as it is at neonatal junctions. T h e early e.p.p.s are therefore relatively large and longlasting. Furthermore, each fibre is often innervated by up to four or more axons (Fig. 2). These give multiple e.p.p.s which sum to evoke action potentials and contractions at an early time despite junctional immaturity. Several observations help to explain the Terje Lemo triggering effect of denervation on synapse formation. First of all, denervation elicits The formation o f new synapses on a denervated muscle recapitulates many of the processes the synthesis of acetylcholine receptors observed during normal development. Here Terje LCmo outlines the physiologieal (AChRs) which begin to appear in the mechanisms underlying synapse formation, and suggests how the early multiple junctional entire muscle fibre m e m b r a n e 1-2 days contacts may be eliminated leading to the formation o f a single mature synapse. after denervation (Fig. 2). This change precedes synapse formation and fibres which To understand the wiring pattern and func- neuromuscular junctions (n.m.j.s) with the first become highly sensitive to A C h are tion of the nervous system it is important to nearby foreign axons (Fig. 1). This system also first innervated, suggesting that the know how synapses are formed and how is advantageous because n.m.j.s can be appearance of A C h R s is important for the their n u m b e r and distribution on single triggered by denervation to form nearly development of receptivity to innervation. cells are controlled. Ideally one would synchronously on large muscle fibres. Secondly, denervation elicits sprouting in study this during normal development but Furthermore, it is relatively easy to man- adjacent foreign axons. This is likely to be this is often difficult because the small size ipulate experimentally both the nerve and caused by substance(s) released from the of the system and the complexity of the the muscle. Below is a brief account of denervated fibres because foreign axons many simultaneously occurring develop- some insights and speculations arising from are not observed closer than several mental processes impose severe experi- work 3,7,~,9on this preparation. hundred nanometres to the muscle fibre m e m b r a n e before denervation 6. Finally, it mental limitations. Other systems for studying synapse formation have therefore Denervation triggers formation of ectopic has been shown that if activity of the been developed. O n e of these is obtained n . m . j . s denervated muscle is maintained by direct in adult animals by cutting a nerve (the In 1967 Fex and TheslefP first reported stimulation of the muscle through chronisuperficial fibular nerve) in the leg of rats that ectopic synapses form amazingly fast if cally implanted electrodes, neither and transplanting it to another muscle (the the transplanted nerve is allowed to grow supersensitivity to ACh, sprouting 1 nor soleus). Within 2-3 weeks numerous close to the muscle fibres before the muscle ectopic synapse formation will occur. O n e branches from the transplanted nerve grow is denervated. Already 2½-3 days after the may suppose, therefore, that denervation close to the soleus muscle fibres usually at a denervation endplate potentials (e.p.p.s) or any of the other procedures which block distance of some millimetres from the orig- are evoked by stimulating the foreign muscle activity, initiate synapse formation inal endplates. No synapses form, however, nerve. Initially the junctions are very by eliciting changes which make the fibre unless the soleus is paralysed, for example, immature. The e.p.p.s have low quantal receptive to innervation and at the same by cutting its original nerve. The denerva- content and the frequency of the spontan- time cause the release of substances which tion elicits n u m e r o u s changes in the muscle eous miniature e.p.p.s is low. However, have a stimulating and attractive influence and rapid formation of new ectopic junctional acetylcholinesterase (ACHE) on adjacent axons.
What controls the development of
neuromuscular junctions?
@ Elsevier/North-HollandBiomedicalPress 1980
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Acetylcholine receptors are quickly concentrated and stabilized underneath the nerve terminal O n e of the earliest events in the formation of n.m.j.s is accumulation of A C h R s in the muscle m e m b r a n e immediately underneath the nerve terminal. W h e n transmission is first detected A C h R or A C h sensitive 'hot spots', rising above background denervation levels, are already present at sites of transmission. This has been demonstrated both by testing the sensitivity to locally applied A C h and by autoradiography after incubation with radioactive c~-bungarotnxin which binds specifically and irreversibly to the A C h R (Fig. 2). Equally early the hot spots become resistant to the effccts of muscle activity. This has been demonstrated in experiments where the foreign nerve is cut early so that it degenerates a few hours after the A C h R hot spots appear. The muscle is then stimulated directly. Within 4-5 days the stimulation, as usual, removes all extrajunctional A C h R s but not the newly induced A C h R hot spots. These persist as discrete spots with a sensitivity comparable to that of normal endplates. The mechanisms behind A C h R aggregation and stabilization are unknown. Postjunctional specializations consisting of filamentous material appear very early and may be involved in fixing the receptors in the membrane. In nervemuscle cultures A C h R s have been shown to move rapidly in the m e m b r a n e towards sites of contact with the nerve 4 and a similar m e c h a n i s m may operate in the present preparation. Denervation may therefore be seen as eliciting useful adaptive responses which favour later reinnervation by causing the appearance of loosely dispersed A C h R s in the entire m e m b r a n e ready to be mobilized, concentrated and stabilized upon contact with an appropriate nerve. Both the nerve and evoked muscle activity influence junctional acetylcholinesterase A n important property of the mature n.m.j, is the presence of highly concentrated A C h E in the synaptic cleft. This junctional A C h E shortens e.p.p, duration and is essential for faithful impulse transmission and reuse of choline. In the present preparation junctional A C h E appears 3-4 days after the onset of transmission. Extrajunctional A C h sensitivity disappears at the same time suggesting that junctional A C h E and extrajunctional A C h sensitivity are reciprocally affected by the evoked activity. The importance of muscle activity for the control of junctional A C h E is
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Fig. 1. Part o f adult rat soleus muscle 35 days after transplantation of foreign superficial fibular nerve and 14 days after cutting original soleus nerve fibres. To the left are denervated original n.m.j.s as revealed by treating A C h E activity. To the right and below are new ectopic n.m.j.s underneath extensive branches o f foreign nerve axons stained by methylene blue.
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IOms
°'
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Fig. 2. Transmission sites and A C h R hot spots at immature ectopic n.m.j.s. The illustrated muscle fibre is from a muscle examined electrophysialogically 3½ days after cutting the original soleus nerve. A weak stimulus to the foreign nerve evoked an endplate potential (e.p.p. marked 1) which was largest approximately 150 ~ to the right of recording position a. A slightly stronger stimulus evoked a second superimposed e.p.p. (2) which had a variable maximum between 200 and 600 pen to the right of recording position b. A small third e.p.p. (not shown) was maximal approximately 1300 tam to the right of recording position c. The positions o f the three axonal inputs (double arrow heads') are based on simultaneous recordings with three separate electrodes from positions indicated by the dots. [Mg 2] and [Ca z"] were 5 and 2 mM respectively. The muscle fibre was marked by dye injected from the recording electrode at a. After incubation in radioactive e~-bungarotoxin and fixation, the marked fibre was teased from the muscle and treated for autoradiography. The dye mark as well as accumulations o f grains (A ChR hot spots) are seen in the lower photographs which were taken from the regions indicated by cross hatching. Note multiple inputs, long lasting e.p.p.s, presence o f uniformly distributed extra]unctional A ChRs, and presence o f multiple A ChR hot spots coinciding with the axonal inputs.
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c
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a
o
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.
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Fig. 3. Transmission site and A ChR hot spots at mature ectopic n.m.j. The muscle fibre is from a muscle examined electrophysiologically 240 days after cutting the original soleus nerve. Dye was injected at recording position a and the mark is shown in the lower lefi photograph. [Mg 2"] and [Ca~"] were 8 and 2 mM respectively. See legend to Fig. 2 for further details. Note single input, short lasting e.p.p., lack ofextrajunetional A ChRs and two discrete A ChR hot spots, 40 pen apart, coinciding with the single input determined electrophysiologically.
shown by findings obtained in preparations where the foreign nerve is cut at the time when the new junctions are just beginning to form. If muscle activity is also imposed
Days a f t e r cut ting original
by stimulating the muscle directly, intense A C h E activity develops precisely at the A C h sensitive hot spots induced by the nerve before it degenerated. Stimulation
Or iginal ne rye
starting after the foreign nerve has degenerated also causes A C h E to appear. In the absence of stimulation no A C h E appears. These results indicate first that the nerve determines where A C h E will appear on the muscle surface and secondly that muscle activity is required for A C h E to actually appear at these sites. In agreement with this, very little junctional A C h E appears at ectopic junctions made by a foreign nerve in which all impulse conduction is chronically blocked. A reasonable hypothesis is that the nerve terminals not only induce persistent A C h R hot spots in the underlying plasma m e m b r a n e but also permanent binding (or aggregation) sites for A C h E in the basal lamina where the junctional A C h E is normally located. Later when muscle activity is evoked, A C h E synthesis is turned on and the A C h E released into the synaptic cleft where it accumulates at predetermined sites in the basal lamina. This hypothesis is consistent with much recent work '°,n but does exclude the possibility that junctional A C h E may also come from the nerve and be subject to influences which are independent of muscle activity.
Important processes develop independently of impulse activity Although impulse activity is important for many aspects of n.m.j, formation it
Inactivity
of
muscle
norve
Appearance of AChRs and r e c e p t i v i t y to innervation in non-junctional membrane.
o
1
Release of nerve sprouting f a c t o r .
{I
Contact between nerve terminals and receptive muscle membrane.
Accumulations of AChRs (hot spots) and induction of AChE binding s i t e s at Differentiation of nerve terminals. 12 mission. Evnked muscle activity.
} ~
•:=:"
Acetylcholine receptors (AChRs)
III
Acetylcholinesterase
(ACHE)
Fig. 4. Sequence ofeven~ underlyingformationofectopicneuromuscu~rjunctions.
.
l
Disappearance of nonjunctional AChRs and r e c e p t i v i t y tn inner-
L Elimination,
[
ration. Appearance of AChE at nerve induced binding sites.
of n . m . j . s .
I
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does not appear to be involved in the binding process itself or the induction of preand post-synaptic specializations necessary for impulse transmission. For example, impulse conduction can be completely blocked in the foreign nerve and yet the 'silent' nerve terminals go on to make n u m e r o u s ectopic junctions with the paralysed muscle. Ectopic junctions also form in the presence of vigorous muscle activity imposed by direct stimulation of the muscle. The requirement is that stimulation starts about 2 days after denervation (if denervation and stimulation start at the same time synapse formation is completely blocked). At 2 days new junctions have not yet formed and A C h supersensitivity has just started to develop. This extrajunctional A C h sensitivity continues to rise for another day after the onset of stimulation, reaches 'full values on the third day and only then begins to decline to disappear completely after a further few days of stimulation. Evidently the muscle responds both to denervation and to subsequent resumption of activity with considerable delays. Therefore, transient changes develop which induce synapse formation at a time when vigorous muscle activity is imposed. These results suggest that n.m.j .s form by processes which do not require impulse activity and which are intrinsically resistant to the effects of muscle activity• This is as it should be since immature junctions must survive the effects of the activity they themselves generate.
Factors controlling number and distribution of n.m.j.s T h e synapse formation studied here recapitulates most of the processes observed in normal development. In both cases post junctional aggregation of A C h R s and transmission appear first, and are followed by disappearance of extrajunctional A C h sensitivity, appearance of junctional ACHE, postjunctional folds and a gradual increase in synaptic size and efficacy. Initially several axons form functional contact with individual muscle fibres in both situations and over the next 1-2 weeks many of these contacts are eliminated (Fig. 3). However, in the adult preparation these contacts are not all.crammed at one site as they are in normal m a m m a l i a n development but are found at many separate sites underneath the foreign nerve. This feature, which m a y simply be due to a more extensive nerve-muscle overlap in the adult preparation, presents an opportunity to study the rules which govern n u m b e r and distribution of synapses on single fibres.
In recent experiments Sue Pockett, Henrich Sommerschild and 1 have determined the position of different inputs on single muscle fibres electrophysiologically and found that 2-3 days or more after the onset of transmission the inputs are either close together (within 300/xm) or 6 0 0 / * m or more apart. The empty 'gap' is reflected in a corresponding gap in the distribution of the A C h R hot spots as determined by autoradiography after incubation with l=q-c~-bungarotoxin. The gap in the hot spot distribution, however, appears relatively late and is not present at 2-3 days when large numbers of hot spots are present on innervated but not on nearby noninnervated fibres. One interpretation of this is that initially the hot spots are induced at random by numerous sprouts from extensively branching foreign axons. Very soon afterwards, however, competitive interactions occur and terminals near more successful sites withdraw and leave 'empty' hot spots which disappear later. Over the next 1-2 weeks further elimination occurs possibly because motoneurones have an inherent tendency to withdraw some of their terminals during synaptic growth = or because nerve growth stimulating substances released by inactive muscle disappear when the muscle becomes active. Closely adjacent inputs may be preferentially eliminated perhaps because they compete for available 'synaptic space' during growth of the individual endplate. These findings may be relevant to how the normal innervation pattern of muscle is established. In normal development the first successfully innervated site may generate similar refractory zones which will cover the rest of the fibre because of its short length. This will force other available inputs to converge to form a single multiply innervated site. If propagated activity develops, further innervation is prevented as the fibre grows and innervation is restricted to the one site. If propagated activity does not develop multiple junctions may form at intervals determined by the length of the refractory zones.
(2) The nerve terminals induce rapid accumulation and stabilization of A C h R s in the underlying plasma membrane and binding sites for A C h E in the underlying basal lamina• These processes are intrinsically resistant to the effects of muscle activity. (3) The n.m.j.s are initially immature with e.p.p.s of low quantal content. Nevertheless, muscle activity is evoked early because synaptic transmission is facilitated by multiple inputs, absence of A C h E and presumably long ionic channel open times. (4) T h e evoked muscle activity turns off the synthesis of A C h R s in non-synaptic regions and makes the muscle refractory to further innervation. It also turns on the synthesis and the release of A C h E which accumulates at the nerve induced binding sites in the basal lamina within the synaptic cleft. (5) Initially up to four or more axons make junctional contacts with the same fibre. Of these inputs one to two (rarely three) develop into stable, mature n.m.j.s • while others are eliminated within 2-3 weeks. Part of the elimination occurs early and is related to the creation of refractory zones extending up to 700/xm to either side of successful sites. Subsequent elimination occurs everywhere and may be related to an intrinsic tendency in motoneurones to withdraw some of their terminals, to the disappearance of a sprouting stimulus or to competitive interactions between closely adjacent axons for 'synaptic space'.
Reading list l Brown, M, C. and Holland, R. L. (1979) Nature (London) 282, 724 726 2 Brown, M. C., Jansen, J. K. S. and van Essen, D. (1976) J. PhysioL (London) 261,387~422 3 Cangiano, A., Lcmo, T., Lutzemberger, L. and Sveen, O. (1980) Acta Physiol. Scan& (in press) 4 Cohen, M. W., Anderson, M. J., Zorychta, E. and Weidon, P.R. (1979) Progr. Brain Res. 49, 335-349 5 Fex, S. and Thesleff, S. (1967) Life Sci. 6, 635-639 6 Korneliussen, H. and Sommerschild,H. (1976) Cell. Tissue Res. 167, 439452 7 L0mo, T. and Slater, C. R. (1978) J. Physiol. (London) 275,391M02 8 LC,mo, T. and Slater, C. R. (1980a) J. Physiol. (London) (in press) 9 Lomo, T. and Slater, C. R. (1980b) J. Physiol. (London) (in press) 10 Rubin, L. L., Schuetze, S. M., Weill, C. k. and Fischbach, G. D. (1980) Nature (London) 283, 264-267 l 1 Weinberg,C. B. and Hail,Z. W. (1979) Dev. Biol. 68, 631 635
A scheme for the formation of n.m.j.s The results obtained in the present preparation suggest the following sequence of events in the formation of n.m.j.s (Fig. 4). (1) Muscle inactivity elicits the synthesis of A C h R s and other molecules in the muscle fibre.' Some of these molecules appear in the entire m e m b r a n e and make it receptive to innervation. Others are released to stimulate adjacent axons to sprout and make contact with the receptive 72 LOmo is at the Nevrofysiologisk lnstitutt, Karl membrane. Johans Gt. 47, Oslo 1, Norway.
126 release of bound calcium ions (see cartoon). It is intriguing that neurons are, apparently by a variety of influences, able to control the synthesis of specific m e m b r a n e proteins in their target tissues. This points to a subtle and sophisticated regulation by nerves of the chemical sensitivity of innervated cells, one purpose being the maintenance of the tissue in a condition in which it responds maximally to existing synaptic inputs but is unresponsive to accessory innervation. The supersensitivity resulting from denervation illustrates only the effects of total removal of nervous influences. The physiological 'value' of denervation supersensitivity is presumably to
make the tissue responsive to reinnervation by increasing its chemical sensitivity and its general excitability to a level where even immature nerve sprouts, irrespective of their site of attachment, have a chance to form functioning synaptic contacts and to re-establish neurotrophic control.
Reading list 1 Cannon, W. B. (1939) Am. J. Med. Sci. 198,
5 FLeming, W. W., McPhillips, J.J. and Westfall, D. P. (1973) Rev. Physiol. 68, 55-119
6 Greengard, P. (1978) Science 199, 146-152 7 Gutmann, E. (1976) Annu. Rev. PhysioL 38, 177-216 8 LCmo, T. (1976) in Motor innervation of muscle (Thesleff,S. ed.), Academic Press 9 Thesleff,S. (1960) Physiol. Rev. 40, 734-752 10 Thesleff, S. (1974) Ann. N.Y. Acad. Sei. 228, 89-104 I 1 Thesleff,S., Libelius, R. and Lundquist,1. (1979) in Muscle, nerve and brain degeneration. Internation Congress Series, 473 (Kidman, A. D. and Tomkins, J. K. eds), Excerpta Medica, Amsterdam 12 Trendelenburg, U. (1966) Pharmacol. Rev. 18, 629-640
737-750 2 Cannon, W. B. and Rosenblueth, A. (1949) Supersensitivity of denervated structures, Macmillan, New York 3 Dolly, J. O. (1979) Int. Rev. Biochem. 26, 257-309 4 Fambrough, D. M. (1979) Physiol. Rev. 59, The authors" are at the Department of Pharmacology, University of Lund, Lund, Sweden. 165-227
has not yet appeared and the time course of the acetylcholine (ACh) activated ionic channels is presumably still as prolonged as it is at neonatal junctions. T h e early e.p.p.s are therefore relatively large and longlasting. Furthermore, each fibre is often innervated by up to four or more axons (Fig. 2). These give multiple e.p.p.s which sum to evoke action potentials and contractions at an early time despite junctional immaturity. Several observations help to explain the Terje Lemo triggering effect of denervation on synapse formation. First of all, denervation elicits The formation o f new synapses on a denervated muscle recapitulates many of the processes the synthesis of acetylcholine receptors observed during normal development. Here Terje LCmo outlines the physiologieal (AChRs) which begin to appear in the mechanisms underlying synapse formation, and suggests how the early multiple junctional entire muscle fibre m e m b r a n e 1-2 days contacts may be eliminated leading to the formation o f a single mature synapse. after denervation (Fig. 2). This change precedes synapse formation and fibres which To understand the wiring pattern and func- neuromuscular junctions (n.m.j.s) with the first become highly sensitive to A C h are tion of the nervous system it is important to nearby foreign axons (Fig. 1). This system also first innervated, suggesting that the know how synapses are formed and how is advantageous because n.m.j.s can be appearance of A C h R s is important for the their n u m b e r and distribution on single triggered by denervation to form nearly development of receptivity to innervation. cells are controlled. Ideally one would synchronously on large muscle fibres. Secondly, denervation elicits sprouting in study this during normal development but Furthermore, it is relatively easy to man- adjacent foreign axons. This is likely to be this is often difficult because the small size ipulate experimentally both the nerve and caused by substance(s) released from the of the system and the complexity of the the muscle. Below is a brief account of denervated fibres because foreign axons many simultaneously occurring develop- some insights and speculations arising from are not observed closer than several mental processes impose severe experi- work 3,7,~,9on this preparation. hundred nanometres to the muscle fibre m e m b r a n e before denervation 6. Finally, it mental limitations. Other systems for studying synapse formation have therefore Denervation triggers formation of ectopic has been shown that if activity of the been developed. O n e of these is obtained n . m . j . s denervated muscle is maintained by direct in adult animals by cutting a nerve (the In 1967 Fex and TheslefP first reported stimulation of the muscle through chronisuperficial fibular nerve) in the leg of rats that ectopic synapses form amazingly fast if cally implanted electrodes, neither and transplanting it to another muscle (the the transplanted nerve is allowed to grow supersensitivity to ACh, sprouting 1 nor soleus). Within 2-3 weeks numerous close to the muscle fibres before the muscle ectopic synapse formation will occur. O n e branches from the transplanted nerve grow is denervated. Already 2½-3 days after the may suppose, therefore, that denervation close to the soleus muscle fibres usually at a denervation endplate potentials (e.p.p.s) or any of the other procedures which block distance of some millimetres from the orig- are evoked by stimulating the foreign muscle activity, initiate synapse formation inal endplates. No synapses form, however, nerve. Initially the junctions are very by eliciting changes which make the fibre unless the soleus is paralysed, for example, immature. The e.p.p.s have low quantal receptive to innervation and at the same by cutting its original nerve. The denerva- content and the frequency of the spontan- time cause the release of substances which tion elicits n u m e r o u s changes in the muscle eous miniature e.p.p.s is low. However, have a stimulating and attractive influence and rapid formation of new ectopic junctional acetylcholinesterase (ACHE) on adjacent axons.
What controls the development of
neuromuscular junctions?
@ Elsevier/North-HollandBiomedicalPress 1980
127
TINS - May 1980
Acetylcholine receptors are quickly concentrated and stabilized underneath the nerve terminal O n e of the earliest events in the formation of n.m.j.s is accumulation of A C h R s in the muscle m e m b r a n e immediately underneath the nerve terminal. W h e n transmission is first detected A C h R or A C h sensitive 'hot spots', rising above background denervation levels, are already present at sites of transmission. This has been demonstrated both by testing the sensitivity to locally applied A C h and by autoradiography after incubation with radioactive c~-bungarotnxin which binds specifically and irreversibly to the A C h R (Fig. 2). Equally early the hot spots become resistant to the effccts of muscle activity. This has been demonstrated in experiments where the foreign nerve is cut early so that it degenerates a few hours after the A C h R hot spots appear. The muscle is then stimulated directly. Within 4-5 days the stimulation, as usual, removes all extrajunctional A C h R s but not the newly induced A C h R hot spots. These persist as discrete spots with a sensitivity comparable to that of normal endplates. The mechanisms behind A C h R aggregation and stabilization are unknown. Postjunctional specializations consisting of filamentous material appear very early and may be involved in fixing the receptors in the membrane. In nervemuscle cultures A C h R s have been shown to move rapidly in the m e m b r a n e towards sites of contact with the nerve 4 and a similar m e c h a n i s m may operate in the present preparation. Denervation may therefore be seen as eliciting useful adaptive responses which favour later reinnervation by causing the appearance of loosely dispersed A C h R s in the entire m e m b r a n e ready to be mobilized, concentrated and stabilized upon contact with an appropriate nerve. Both the nerve and evoked muscle activity influence junctional acetylcholinesterase A n important property of the mature n.m.j, is the presence of highly concentrated A C h E in the synaptic cleft. This junctional A C h E shortens e.p.p, duration and is essential for faithful impulse transmission and reuse of choline. In the present preparation junctional A C h E appears 3-4 days after the onset of transmission. Extrajunctional A C h sensitivity disappears at the same time suggesting that junctional A C h E and extrajunctional A C h sensitivity are reciprocally affected by the evoked activity. The importance of muscle activity for the control of junctional A C h E is
.................
:
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Fig. 1. Part o f adult rat soleus muscle 35 days after transplantation of foreign superficial fibular nerve and 14 days after cutting original soleus nerve fibres. To the left are denervated original n.m.j.s as revealed by treating A C h E activity. To the right and below are new ectopic n.m.j.s underneath extensive branches o f foreign nerve axons stained by methylene blue.
I
IOms
°'
2
3
500um
Fig. 2. Transmission sites and A C h R hot spots at immature ectopic n.m.j.s. The illustrated muscle fibre is from a muscle examined electrophysialogically 3½ days after cutting the original soleus nerve. A weak stimulus to the foreign nerve evoked an endplate potential (e.p.p. marked 1) which was largest approximately 150 ~ to the right of recording position a. A slightly stronger stimulus evoked a second superimposed e.p.p. (2) which had a variable maximum between 200 and 600 pen to the right of recording position b. A small third e.p.p. (not shown) was maximal approximately 1300 tam to the right of recording position c. The positions o f the three axonal inputs (double arrow heads') are based on simultaneous recordings with three separate electrodes from positions indicated by the dots. [Mg 2] and [Ca z"] were 5 and 2 mM respectively. The muscle fibre was marked by dye injected from the recording electrode at a. After incubation in radioactive e~-bungarotoxin and fixation, the marked fibre was teased from the muscle and treated for autoradiography. The dye mark as well as accumulations o f grains (A ChR hot spots) are seen in the lower photographs which were taken from the regions indicated by cross hatching. Note multiple inputs, long lasting e.p.p.s, presence o f uniformly distributed extra]unctional A ChRs, and presence o f multiple A ChR hot spots coinciding with the axonal inputs.
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c
!
a
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b
.
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5ms
c
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• •
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Fig. 3. Transmission site and A ChR hot spots at mature ectopic n.m.j. The muscle fibre is from a muscle examined electrophysiologically 240 days after cutting the original soleus nerve. Dye was injected at recording position a and the mark is shown in the lower lefi photograph. [Mg 2"] and [Ca~"] were 8 and 2 mM respectively. See legend to Fig. 2 for further details. Note single input, short lasting e.p.p., lack ofextrajunetional A ChRs and two discrete A ChR hot spots, 40 pen apart, coinciding with the single input determined electrophysiologically.
shown by findings obtained in preparations where the foreign nerve is cut at the time when the new junctions are just beginning to form. If muscle activity is also imposed
Days a f t e r cut ting original
by stimulating the muscle directly, intense A C h E activity develops precisely at the A C h sensitive hot spots induced by the nerve before it degenerated. Stimulation
Or iginal ne rye
starting after the foreign nerve has degenerated also causes A C h E to appear. In the absence of stimulation no A C h E appears. These results indicate first that the nerve determines where A C h E will appear on the muscle surface and secondly that muscle activity is required for A C h E to actually appear at these sites. In agreement with this, very little junctional A C h E appears at ectopic junctions made by a foreign nerve in which all impulse conduction is chronically blocked. A reasonable hypothesis is that the nerve terminals not only induce persistent A C h R hot spots in the underlying plasma m e m b r a n e but also permanent binding (or aggregation) sites for A C h E in the basal lamina where the junctional A C h E is normally located. Later when muscle activity is evoked, A C h E synthesis is turned on and the A C h E released into the synaptic cleft where it accumulates at predetermined sites in the basal lamina. This hypothesis is consistent with much recent work '°,n but does exclude the possibility that junctional A C h E may also come from the nerve and be subject to influences which are independent of muscle activity.
Important processes develop independently of impulse activity Although impulse activity is important for many aspects of n.m.j, formation it
Inactivity
of
muscle
norve
Appearance of AChRs and r e c e p t i v i t y to innervation in non-junctional membrane.
o
1
Release of nerve sprouting f a c t o r .
{I
Contact between nerve terminals and receptive muscle membrane.
Accumulations of AChRs (hot spots) and induction of AChE binding s i t e s at Differentiation of nerve terminals. 12 mission. Evnked muscle activity.
} ~
•:=:"
Acetylcholine receptors (AChRs)
III
Acetylcholinesterase
(ACHE)
Fig. 4. Sequence ofeven~ underlyingformationofectopicneuromuscu~rjunctions.
.
l
Disappearance of nonjunctional AChRs and r e c e p t i v i t y tn inner-
L Elimination,
[
ration. Appearance of AChE at nerve induced binding sites.
of n . m . j . s .
I
129
TINS - May 1980
does not appear to be involved in the binding process itself or the induction of preand post-synaptic specializations necessary for impulse transmission. For example, impulse conduction can be completely blocked in the foreign nerve and yet the 'silent' nerve terminals go on to make n u m e r o u s ectopic junctions with the paralysed muscle. Ectopic junctions also form in the presence of vigorous muscle activity imposed by direct stimulation of the muscle. The requirement is that stimulation starts about 2 days after denervation (if denervation and stimulation start at the same time synapse formation is completely blocked). At 2 days new junctions have not yet formed and A C h supersensitivity has just started to develop. This extrajunctional A C h sensitivity continues to rise for another day after the onset of stimulation, reaches 'full values on the third day and only then begins to decline to disappear completely after a further few days of stimulation. Evidently the muscle responds both to denervation and to subsequent resumption of activity with considerable delays. Therefore, transient changes develop which induce synapse formation at a time when vigorous muscle activity is imposed. These results suggest that n.m.j .s form by processes which do not require impulse activity and which are intrinsically resistant to the effects of muscle activity• This is as it should be since immature junctions must survive the effects of the activity they themselves generate.
Factors controlling number and distribution of n.m.j.s T h e synapse formation studied here recapitulates most of the processes observed in normal development. In both cases post junctional aggregation of A C h R s and transmission appear first, and are followed by disappearance of extrajunctional A C h sensitivity, appearance of junctional ACHE, postjunctional folds and a gradual increase in synaptic size and efficacy. Initially several axons form functional contact with individual muscle fibres in both situations and over the next 1-2 weeks many of these contacts are eliminated (Fig. 3). However, in the adult preparation these contacts are not all.crammed at one site as they are in normal m a m m a l i a n development but are found at many separate sites underneath the foreign nerve. This feature, which m a y simply be due to a more extensive nerve-muscle overlap in the adult preparation, presents an opportunity to study the rules which govern n u m b e r and distribution of synapses on single fibres.
In recent experiments Sue Pockett, Henrich Sommerschild and 1 have determined the position of different inputs on single muscle fibres electrophysiologically and found that 2-3 days or more after the onset of transmission the inputs are either close together (within 300/xm) or 6 0 0 / * m or more apart. The empty 'gap' is reflected in a corresponding gap in the distribution of the A C h R hot spots as determined by autoradiography after incubation with l=q-c~-bungarotoxin. The gap in the hot spot distribution, however, appears relatively late and is not present at 2-3 days when large numbers of hot spots are present on innervated but not on nearby noninnervated fibres. One interpretation of this is that initially the hot spots are induced at random by numerous sprouts from extensively branching foreign axons. Very soon afterwards, however, competitive interactions occur and terminals near more successful sites withdraw and leave 'empty' hot spots which disappear later. Over the next 1-2 weeks further elimination occurs possibly because motoneurones have an inherent tendency to withdraw some of their terminals during synaptic growth = or because nerve growth stimulating substances released by inactive muscle disappear when the muscle becomes active. Closely adjacent inputs may be preferentially eliminated perhaps because they compete for available 'synaptic space' during growth of the individual endplate. These findings may be relevant to how the normal innervation pattern of muscle is established. In normal development the first successfully innervated site may generate similar refractory zones which will cover the rest of the fibre because of its short length. This will force other available inputs to converge to form a single multiply innervated site. If propagated activity develops, further innervation is prevented as the fibre grows and innervation is restricted to the one site. If propagated activity does not develop multiple junctions may form at intervals determined by the length of the refractory zones.
(2) The nerve terminals induce rapid accumulation and stabilization of A C h R s in the underlying plasma membrane and binding sites for A C h E in the underlying basal lamina• These processes are intrinsically resistant to the effects of muscle activity. (3) The n.m.j.s are initially immature with e.p.p.s of low quantal content. Nevertheless, muscle activity is evoked early because synaptic transmission is facilitated by multiple inputs, absence of A C h E and presumably long ionic channel open times. (4) T h e evoked muscle activity turns off the synthesis of A C h R s in non-synaptic regions and makes the muscle refractory to further innervation. It also turns on the synthesis and the release of A C h E which accumulates at the nerve induced binding sites in the basal lamina within the synaptic cleft. (5) Initially up to four or more axons make junctional contacts with the same fibre. Of these inputs one to two (rarely three) develop into stable, mature n.m.j.s • while others are eliminated within 2-3 weeks. Part of the elimination occurs early and is related to the creation of refractory zones extending up to 700/xm to either side of successful sites. Subsequent elimination occurs everywhere and may be related to an intrinsic tendency in motoneurones to withdraw some of their terminals, to the disappearance of a sprouting stimulus or to competitive interactions between closely adjacent axons for 'synaptic space'.
Reading list l Brown, M, C. and Holland, R. L. (1979) Nature (London) 282, 724 726 2 Brown, M. C., Jansen, J. K. S. and van Essen, D. (1976) J. PhysioL (London) 261,387~422 3 Cangiano, A., Lcmo, T., Lutzemberger, L. and Sveen, O. (1980) Acta Physiol. Scan& (in press) 4 Cohen, M. W., Anderson, M. J., Zorychta, E. and Weidon, P.R. (1979) Progr. Brain Res. 49, 335-349 5 Fex, S. and Thesleff, S. (1967) Life Sci. 6, 635-639 6 Korneliussen, H. and Sommerschild,H. (1976) Cell. Tissue Res. 167, 439452 7 L0mo, T. and Slater, C. R. (1978) J. Physiol. (London) 275,391M02 8 LC,mo, T. and Slater, C. R. (1980a) J. Physiol. (London) (in press) 9 Lomo, T. and Slater, C. R. (1980b) J. Physiol. (London) (in press) 10 Rubin, L. L., Schuetze, S. M., Weill, C. k. and Fischbach, G. D. (1980) Nature (London) 283, 264-267 l 1 Weinberg,C. B. and Hail,Z. W. (1979) Dev. Biol. 68, 631 635
A scheme for the formation of n.m.j.s The results obtained in the present preparation suggest the following sequence of events in the formation of n.m.j.s (Fig. 4). (1) Muscle inactivity elicits the synthesis of A C h R s and other molecules in the muscle fibre.' Some of these molecules appear in the entire m e m b r a n e and make it receptive to innervation. Others are released to stimulate adjacent axons to sprout and make contact with the receptive 72 LOmo is at the Nevrofysiologisk lnstitutt, Karl membrane. Johans Gt. 47, Oslo 1, Norway.