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Development of complexity in motor nerve endings at the rat neuromuscular junction
Neuroscience Vol. 6. No. 8, pp. 1657 to 1662. 1981 Printed in Great Britain
03oa4522/81/081657-0su)z.oo/0 Pergamon Press Ltd 8 1981 IBRO
DEVELOPMENT OF COMPLEXITY IN MOTOR NERVE ENDINGS AT THE RAT NEUROMUSCULAR JUNCTION C. D. TWEEDLE*and K. E. STEPHENS Department of Anatomy, Michigan State University, East Lansing, MI 48824, U.S.A. Abstract-Using a combined silver-cholinesterase stain, the morphology of motor endings (axon terminals plus chohnesterase activity) was examined in both slow-twitch (soleus and adductor longus) and fast-twitch (rectus femoris and plantaris) muscles of 2,3,4,5,6 and 1l-week-old male rats. Categorization of motor endings from each muscle at each age into defined morphological classes was carried out. The differences between their distribution among these classes between muscles and between ages was then analyzed statistically. Polyneuronal innervation of endplates dropped dramatically between 2 and 3 weeks. The number of ‘branched’ endings (two muscle fibers innervated by a single terminal axon) significantly decreased between 3 and 4 weeks in all the muscle groups and did not change thereafter. At 4 and 5 weeks, the predominant appearance of the motor endings of all the muscles was similar-being basically a single ‘bare’ axon going into the endplate area (with one axon being associated with one endplate per muscle fiber). Complexity of the motor endings then developed with maturation. At 6 weeks of age, in the slow-twitch muscles only, there was first seen a significantly greater percentage of double endplates (one terminal axon giving rise to two endplates on a single muscle fiber). This difference increased significantly between 6-11 weeks. The number of more complex motor endings with accessory axonal branches to the endplate from the parent axon increased steadily throughout maturation in all the muscle groups. At 3 weeks these made up less than 5% of the endings. However, by 11 weeks they were seen in 2540% of the endings. This study indicates that (1) there is still synaptic rearrangement (loss of branched endings) between 3 and 4 weeks in all muscle groups; (2) there develops a significantly greater number of double endplates in slow-twitch muscles around 6 weeks; and (3) complexity of motor ending structure increases with growth of the animal by adaptations of the terminal axon. Thus, even under normal conditions, young adult rats spontaneously display significant evidence of synaptic remodeling and plasticity.
IN NEW-BORN mammals skeletal muscle is polyneuronally innervated, i.e. more than one axon terminates at the single endplate region of the muscle (JAN~EN, THOMPSON & KUFFLER,1978, for review). In the rat, during the first few post-natal weeks all but one axon is eliminated, leaving the adult pattern of unineuronal innervation. In the adult rat, as well as the other mammals, there are indications that the structure of this single motor ending may differ in size and arrangement on muscle fibers of different histochemical types (Co&s, 1955; NYSTR&, 1968; PADYKULA& GAUTHIER,1970; KORNFUUSSEN& WAERMUG, 1973) and may also become considerably more complex in older animals my, 1971X apparently by nerve sprouting from the parent axon. BARKER& IP (1966) also reported morphological evidence of sprouting and axonal elaboration in normal ‘young adult’ mammalian muscle and suggested that it was indicative of a continual turnover of the terminal portion of the axon. Recently, it has been reported that nerve regression and sprouting normally occur in frog neuro-
ively and compare the temporal changes in the structure of motor endings (axon terminals plus cholinesterase activity as revealed by combined nerve and cholinesterase staining) in both predominantly slowtwitch and fast-twitch muscles. Such data provide information on the evolution of complexity at the neuromuscular junction and on the differences in the terminal innervation pattern of different muscle fiber types.
EXPERIMEIVAL
PROCEDURES
Normal male Sprague-Dawley rats, ranging in age from 2 to 11 weeks, were used. The animals were housed in sedentary conditions in group cages (2-3 rats per 38 x 33 x 16.8 cm cage) and were killed at 2 (n = 6), 3 (n = 4). 5 (n = 6), 6 (n = 6) and 11 (n = 6) weeks of age. At these times the plantaris, rectus femoris, soleus, and adductor longus muscles were removed. The plantaris and rectus femoris muscles were selected as being representative of ‘fast-twitch’ muscles, while the soleus and adductor longus muscles were chosen to represent ‘slow-twitch’ muscles muscular junctions and that this may be modulated (ARIANO, ARMSTRONG & EDGERTON,1973). by seasonal factors and muscle activity (WERNIG, Rats were killed by decapitation, the left hind-limb was F%COT-DECHAVASSINE & STiivw, 1980). skinned, the muscles were removed immediately and The present study was designed to follow quantitatplaced in a 10% formal-saline solution containing 0.5% dimethyl sulfoxide for 6 h. Following several distilled water * Author to whom reprint requests should be sent. rinses over a period of 16 h, the fixed muscle blocks were 1657
C. D. TWEEDLEand K. E.
1658
quick-frozen in 2-methylbutane (isopentane), pre-cooled by liquid nitrogen. Longitudinal serial sections from the midportion of the muscles, cut 60 pm thick, were stained using a combined Bielchowsky-cholinesterase nerve stain (GWYN & HEARDMAN,1965). Slides containing 4-6 sections from a single muscle were coded and the morphology of the motor endings was evaluated independently by each author. A minimum of 100 endplates per slide was counted. Only clearly defined endplates and terminal axons which could be traced back 2 mm or into a nerve bundle were evaluated and the numbers of simple, double, accessory, branched, multiple and polyneuronally-innervated endplates (see Figs 1 and 2) were tabulated for each sIide. Use of 60pm sections enabled following of axons for considerable distances deep into the muscle. As in squash techniques abruptly-ending, ‘broken’ axons were eliminated from any data collection or analysis. The data obtained by the two examiners were averaged to give values for the final analysis. Records of nerve sprouting (nodal, preterminal and ultraterminal) were also kept. Because of the difficulty in absolutely dassifying endings in the 2-week-old animals due to the extremely small muscle fiber diameter, only the percentage of endplates showing polyneuronal innervation was determined at this age. Comparative analysis was done using the Mann-Whitney U test (SIEGEL,1956).A statistical probability (P) of less than 0.05 was considered to indicate significant difference between means. RESULTS Polyneuronal
innervation
As can be seen in Fig. 3, there remained evidence of polyneuronal innervation at 2 weeks, but this decreased significantly by 3 weeks in all muscle groups (P < 0.01). In the muscles from the 2-week-old animals a number of possible ‘retraction bulbs’ were seen (Fig. 2f) as described by RILEY (1977). At 3 and 4 weeks, the rectus femoris muscle continued to have a low, but sigaificantly greater, number of polyneuronally innervated muscle fibers than the other muscles (P -=z0.05). M&t of the polyneuronally innervated endplates observed at 2 weeks had only two axons (Fig. 2e) leading into them, although axonal numbers up to five were occasionally seen. Diflerent types of nerve endings Complex endings. This category of nerve endings (which we defined to include everything except ‘simple’ endings) was greater in slow-twitch than in fasttwitch muscles at all ages examined (Fig 4) (P < 0.01). The percentage of complex q in all the different muscles remained co-t uhti‘betw~~ 5-6 weeks when it increased (P < 001); Qompbx endings continued to increase furt& behvoen 6 and 11 weeks (P < 0.01). The temporal iWea6eincornpla
endings is due largely to the dcvcbpmcnt of additional accessory and double endings (Figs 5 and 6). Accessory nerve endings. The pazuktrp of w ory nerve endings (Fig. 2b) 4&h t@e in &ll muscles examined (Fig. 5). In the dand 11-w&&-old animals, there were more accessory endings found in
STEPHENS
FIG. 1. A diagram showing the categories into which motor nerve terininals were classified. (a) Simple ending (endplatej-one axon terminating at one endplate and innervating a tingle *uscle fiber. (see also Fig. 2a). (b) ACcesdiory ending-an axon with one or .more thin bran&es originatihg either from nodes of Ranvier or at fh+ end of the my&n sheat% which inserts into one e&plate on a single muscle fiber (see also Fig. 2b). (c) ljoutlk-ertding-a branched terminal axon which ends in 2 d&M endp&es on one mu_&! fiber (see also Fig. 2c)_ (d) Lanchcd ending-a bra&ed &z-m&11axon which culminat~a in 2 endplates on 2 separate muscle fibers (see also Fig. 26). [e) Multiple end@-3 or more banches from a single terminal axon which form more than 2 en&&a& structures on a single mtsscIe f&r. (f) Polyneuronal endpWe-a single end+& structure on one muscle fiber a*entIy innervated by 2 or more distinct terminal axons (see also Fig. 2~). (g) Sprout-a fine unmyelinated fiber orig&ating either from nodes of Ranvier, the end of the myelin sheath, or from the endplate itself.
the slow-twitch muscles than in the fast-t&& muscles (P < 0.03). In 11-week-old rats, they made up a su~ris~y large percentage of the nerve end&s. Double endings. The formation of a second qndplate on a single muscle fiber by a single term&~al axon (Fig. 2c) was found to increase only in Fhe st&vtwitch muscles (Fig. 6).. This increase was seen to occur mainly between 6 and 1I weeks although there were @nitkantly more double endings seen in slowtwitch than fast-twitch muscles in animals at 5 and 6 weeks of age (P < 0.001). Preliminary evidence (authors’ unpublished) indicates that an increase in percentage of double endings continues in slow musck fibers throughout adult life. At 11 weeks, the adductor muscle had more double endings than the soleus muscle (P < 0.03). Branched endings. Branched endings are indicative of the innarvption of more than one mu&e fiber by a siDgkaxon(Fii2d).ThepeKerJtllgeofthistypeof en&g ru&ncd i&ta constant level %&wing an initial s&c&ant (P < 0.01) drop b&men 3 and 4 weeks in arftthe mu&es (see Fig. 7). Multiple endws. Multiple endings (Fig. I) were seen only at 11 weeks and in just the soleus and adductor muscks (1.5 and 2.7”/, rezqectively). Nerve sprouts Nodal, ultraterminal
and preterminal nerve sprouts
FIG. 2. (a) Photomicrograph of simple nerve endings, 4 weeks; plantaris. x 200. (b) Photomicrograph showing 4 accessory and one double (d) nerve terminal, 6 weeks; adductor longus. x 200. (c) Phatomicrograph of a double nerve ending, 6 weeks; soleus. x 200. (d) Photomicrograph of a branched nerve ending, 6 weeks; rectus femoris. x 290. (e) Photomicrograph of 2 endplates from the soleus muscle of a 2-week-old rat. The innervation of one endplate is by 2 axons (arrow). x 1200. (f) Photomicrograph of a possible retracting axon (arrow), 2 weeks; soleus. + 1800.
16.59
1661
Development of complexity of neuromuscular junctions P--Q e---v
lO.Or
ADWCTOR SOLEUS RECTUS FEMORIS PLANTARIS
M
WEEKS
FIG. 3. Percentage of endplates showing polyneuronal innervation. Note the drop between 2 and 3 weeks seen in all the muscles. *---a x-x e---s
50-
ACDUCTOR
*---a soLElJs
o.oL
60-
w
3
4
5
RECTUS
6
FEMORIS
II
FIG. 7. Percentage of branched endings. Note the drop between 3 and 4 weeks in all the muscles.
(Fig. 1) were seen at between 1 and 3% of nerve endings in all the muscles analyzed and this percentage
ACOUCTOR SOLEUS RECTUS FEMCftlS PLANTARIS
did not change with time.
E 40 -
DISCUSSION
E
Polyneuronal innervation
30.
3
5
4
II
6 WEEKS
FIG. 4. Percentage of complex nerve endings. The slowtwitch muscles had more complex nerve endings than fasttwitch muscles. In both of these groups a greater number of complex endings appeared after 5 weeks of age. u c---d x--x *--I
i
ADDUCTOR SOLEUS RECTUS FEMORIS PLANTARIS
i
5
,,,’ / / I’
I’
II
6
WEEKS
FIG. 5. Percentage of accessory nerve endings. Accessory nerve endings increased with time. At 6 and 11 weeks, the slow-twitch muscles had significantly more of them. At 11 weeks, the values for the plantaris and rectus femoris were identical. c~ P--Q M *--a
3
4
Branched endings
ADDUCTOR SGiEUS RECTUS FEMORIS PLANTARIS
5
6
Our findings indicate that the structure of the motor ending in rat skeletal muscle is continually changing throughout the early life of the animal and some of these changes appear to be specific to muscles of a certain fiber composition. Other workers (JANSEN et al., 1978; JACOBSON, 1978, for review) have shown that ‘redundant’ nerves to skeletal muscle fibers are eliminated in the first few post-natal weeks, leaving the adult pattern of one axon to one motor endplate per muscle fiber. We have also observed this in the present study, wherein most polyneuronal innervation disappears between 2 and 3 weeks. The general pattern of this loss of extra axons was similar in all the muscles examined. The presence of many possible axonal ‘retraction bulbs’ during the time when the supernumerary axons are being lost supports the idea that at least some of them disappear by resorption (KORNELIUSSEN & JANSEN, 1976; RILEY, 1977). The rectus femoris was seen’to maintain a small number of polyneuronally-innervated muscle fibers even in 4-week-old animals. The reason for this is not known, although one might speculate that in this muscle there lingers a small number of undifferentiated muscle fibers.
II
WEEKS
F:G. 6. Percentage of double endings. At 5 weeks and after, the number of double endings was greater in the slowtwitch muscles.
A somewhat unexpected finding was the presence of a relatively large number of branched nerve endings in all muscles examined in 3-week-old rats. These branched nerve endings disappear by 4 weeks. As branched endings reflect the innervation of more than one muscle fiber by a single terminal axon, their presence in 3-week-old rats and subsequent reduction suggests that synaptic reorganization may be taking place at this time period. Perhaps synaptic competition is still occurring, whereby the axons associated with branched endings lose out to terminal axons (previously displaced from polyneuronal endplates?)
1662
C. D. TWEEDLEand K. E. STEPHENS
which would only have one muscle fiber to support instead of two. Development of complexity in neuromuscular junctions
‘Complex’ nerve endings were significantly greater in number in the slow-twitch muscles. The percentage of complex endings remained stable during the period from 3 to 5 weeks of age and thereafter increased. Accessory and double nerve endings showed this same general pattern. As most muscle fiber differentiation in the rat has taken place by 5 weeks, we originally though that the stimulus for the formation of an increased number of complex endings was probably related to increases in muscle fiber size or in response to increased workload. This latter parameter might be especially important in postural muscles affected by increases in animal weight. However, we have performed preliminary experiments in which the adductor longus muscle has been tenotomized and the effect of this on the development of neuromuscular complexity analyzed. Results to date indicate that while the accessory endings increase normally, double endings do not (C. D. TWEEDLE &
K. E. STEPHENS, unpublished). Thus, some aspects of the elaboration of motor endings appear to be independent of use of the muscle while others may be more dependent on neuromuscular activity. TUFFERY(1971) suggested that increased complexity in the neuromuscular junctions of aged cats was due
to an increasing workload as well as to a decline in the trophic function of the neuron and loss of muscle fibers in senility. That signs of complexity at axonal terminals may be seen developing in the muscles of rats which are only a few months old indicates that the loss of muscle fibers or other old-age-related disorders are not required for endplate elaboration. In Tuffery’s study, it was also found that accessory endings increase with age of the animal, but these changes were observed over a period of years, not weeks. Acknowledgements--Supported by a grant from NSF (BNS 76-81406)and NIH BRSG funds. We are indebted to Mr S. PAZDZIORKOfor skilled technical assistance and MS C. MEISTERfor typing the manuscript. Drs G. HATTON,M. RHEUBEN,WM. FALLSand C. WILSONkindly read an earlier draft of this manuscript. We are indebted to Mr D. ANDERSONfor providing Figs 37.
REFERENCES ARIANOM. A., ARMSTRONG R. B. & EDGER~~NV. R. (1973) Hind-limb muscle fiber populations of five mammals. J. Histochem. Cytochem.21, 51-55. BARKERD. 8s IP M. C. (1966) Sprouting and degeneration of mammalian motor axons in normal and de-a&rented skeletal muscle. Proc. R. Sot. B 163, 538-554. Cotis C. (1955) Les variations structurelles normales et pathologiques de la jonction neuromusculaire. Acta neural. be&. 55, 741-866. CO&U C. N., TELERMAN-TOPPET N. & GERARDJ. M. (1973) Terminal innervation ratio in neuromuscular disease. Archs. Neurol., Paris 29, 210-222. GWYND. G. & HEARDMAN V. A. (1965) A cholinesterase-Bielchowsky staining method for mammalian motor endplates. Stain Technol. 40, 15-18. JACOBSON M. (1978) DevelopmentalNeurobiology.Plenum, New York. JANSENJ. K. S., THOMPSONW. & KUFFLERD. P. (1978) The formation and maintenance of synaptic connnections as illustrated by studies of the neuromuscular junction. In Progress in Brain Research (eck CORER M. A., BAKERR. E., VANDE POLL E., SWAABB. F. & EWLINGSH. A. M.) Vol. 48, pp. 3-18. Elsevier, Amsterdam. KORNELN~SENH. L JANSENJ. K. S. (1976) Morphological aspects of the elimination of polyneuronal innervation of skeletal muscle fibres in new-born rats. J. Neurocytol. 5, 591-604. KORNELUS~NH. & WAERHAUG0. (1973) Three morphological types of motor nerve terminals in the rat diaphragm and their possible innervation of different muscle fiber types. Z. Anat. EntwGesch.14@,73-84. NYSTR~~M B. (1968) Post-natal development of motor nerve terminals in ‘slow-red’ and ‘fast-white’ cat muscles. Acta neural, stand. 44, 363-383. PADYKULAH. & GAUTHIERG. (1970) The ultrastructure of the neuromuscular junction of mammalian red, white and intermediate muscle fibers. J. Cell Biol. 46, 2741. RILEYD. A. (1977) Spontaneous elimination of nerve terminals from the endplates of developing skeletal myofibers. Brain Res. 134,279285. SIEGELS. (1956) Non-parametricStatistics.McGraw-Hill, New York. TUPFERYA. R. (1971) Growth and degeneration of motor endplates in normal cat hind-limb mu&es. .r. Anut. 119 221-247. WERNIGA., ~ccrr-DECHAVASS~NE M. & STavw H. (1980) Sprouting and regression of the nerve at the frog neuromuscular junction in normal conditions and after prolonged paralysis with curare. J. Nenrocylof. 9, 277-303. (Accepted 18 February 1981)
03oa4522/81/081657-0su)z.oo/0 Pergamon Press Ltd 8 1981 IBRO
DEVELOPMENT OF COMPLEXITY IN MOTOR NERVE ENDINGS AT THE RAT NEUROMUSCULAR JUNCTION C. D. TWEEDLE*and K. E. STEPHENS Department of Anatomy, Michigan State University, East Lansing, MI 48824, U.S.A. Abstract-Using a combined silver-cholinesterase stain, the morphology of motor endings (axon terminals plus chohnesterase activity) was examined in both slow-twitch (soleus and adductor longus) and fast-twitch (rectus femoris and plantaris) muscles of 2,3,4,5,6 and 1l-week-old male rats. Categorization of motor endings from each muscle at each age into defined morphological classes was carried out. The differences between their distribution among these classes between muscles and between ages was then analyzed statistically. Polyneuronal innervation of endplates dropped dramatically between 2 and 3 weeks. The number of ‘branched’ endings (two muscle fibers innervated by a single terminal axon) significantly decreased between 3 and 4 weeks in all the muscle groups and did not change thereafter. At 4 and 5 weeks, the predominant appearance of the motor endings of all the muscles was similar-being basically a single ‘bare’ axon going into the endplate area (with one axon being associated with one endplate per muscle fiber). Complexity of the motor endings then developed with maturation. At 6 weeks of age, in the slow-twitch muscles only, there was first seen a significantly greater percentage of double endplates (one terminal axon giving rise to two endplates on a single muscle fiber). This difference increased significantly between 6-11 weeks. The number of more complex motor endings with accessory axonal branches to the endplate from the parent axon increased steadily throughout maturation in all the muscle groups. At 3 weeks these made up less than 5% of the endings. However, by 11 weeks they were seen in 2540% of the endings. This study indicates that (1) there is still synaptic rearrangement (loss of branched endings) between 3 and 4 weeks in all muscle groups; (2) there develops a significantly greater number of double endplates in slow-twitch muscles around 6 weeks; and (3) complexity of motor ending structure increases with growth of the animal by adaptations of the terminal axon. Thus, even under normal conditions, young adult rats spontaneously display significant evidence of synaptic remodeling and plasticity.
IN NEW-BORN mammals skeletal muscle is polyneuronally innervated, i.e. more than one axon terminates at the single endplate region of the muscle (JAN~EN, THOMPSON & KUFFLER,1978, for review). In the rat, during the first few post-natal weeks all but one axon is eliminated, leaving the adult pattern of unineuronal innervation. In the adult rat, as well as the other mammals, there are indications that the structure of this single motor ending may differ in size and arrangement on muscle fibers of different histochemical types (Co&s, 1955; NYSTR&, 1968; PADYKULA& GAUTHIER,1970; KORNFUUSSEN& WAERMUG, 1973) and may also become considerably more complex in older animals my, 1971X apparently by nerve sprouting from the parent axon. BARKER& IP (1966) also reported morphological evidence of sprouting and axonal elaboration in normal ‘young adult’ mammalian muscle and suggested that it was indicative of a continual turnover of the terminal portion of the axon. Recently, it has been reported that nerve regression and sprouting normally occur in frog neuro-
ively and compare the temporal changes in the structure of motor endings (axon terminals plus cholinesterase activity as revealed by combined nerve and cholinesterase staining) in both predominantly slowtwitch and fast-twitch muscles. Such data provide information on the evolution of complexity at the neuromuscular junction and on the differences in the terminal innervation pattern of different muscle fiber types.
EXPERIMEIVAL
PROCEDURES
Normal male Sprague-Dawley rats, ranging in age from 2 to 11 weeks, were used. The animals were housed in sedentary conditions in group cages (2-3 rats per 38 x 33 x 16.8 cm cage) and were killed at 2 (n = 6), 3 (n = 4). 5 (n = 6), 6 (n = 6) and 11 (n = 6) weeks of age. At these times the plantaris, rectus femoris, soleus, and adductor longus muscles were removed. The plantaris and rectus femoris muscles were selected as being representative of ‘fast-twitch’ muscles, while the soleus and adductor longus muscles were chosen to represent ‘slow-twitch’ muscles muscular junctions and that this may be modulated (ARIANO, ARMSTRONG & EDGERTON,1973). by seasonal factors and muscle activity (WERNIG, Rats were killed by decapitation, the left hind-limb was F%COT-DECHAVASSINE & STiivw, 1980). skinned, the muscles were removed immediately and The present study was designed to follow quantitatplaced in a 10% formal-saline solution containing 0.5% dimethyl sulfoxide for 6 h. Following several distilled water * Author to whom reprint requests should be sent. rinses over a period of 16 h, the fixed muscle blocks were 1657
C. D. TWEEDLEand K. E.
1658
quick-frozen in 2-methylbutane (isopentane), pre-cooled by liquid nitrogen. Longitudinal serial sections from the midportion of the muscles, cut 60 pm thick, were stained using a combined Bielchowsky-cholinesterase nerve stain (GWYN & HEARDMAN,1965). Slides containing 4-6 sections from a single muscle were coded and the morphology of the motor endings was evaluated independently by each author. A minimum of 100 endplates per slide was counted. Only clearly defined endplates and terminal axons which could be traced back 2 mm or into a nerve bundle were evaluated and the numbers of simple, double, accessory, branched, multiple and polyneuronally-innervated endplates (see Figs 1 and 2) were tabulated for each sIide. Use of 60pm sections enabled following of axons for considerable distances deep into the muscle. As in squash techniques abruptly-ending, ‘broken’ axons were eliminated from any data collection or analysis. The data obtained by the two examiners were averaged to give values for the final analysis. Records of nerve sprouting (nodal, preterminal and ultraterminal) were also kept. Because of the difficulty in absolutely dassifying endings in the 2-week-old animals due to the extremely small muscle fiber diameter, only the percentage of endplates showing polyneuronal innervation was determined at this age. Comparative analysis was done using the Mann-Whitney U test (SIEGEL,1956).A statistical probability (P) of less than 0.05 was considered to indicate significant difference between means. RESULTS Polyneuronal
innervation
As can be seen in Fig. 3, there remained evidence of polyneuronal innervation at 2 weeks, but this decreased significantly by 3 weeks in all muscle groups (P < 0.01). In the muscles from the 2-week-old animals a number of possible ‘retraction bulbs’ were seen (Fig. 2f) as described by RILEY (1977). At 3 and 4 weeks, the rectus femoris muscle continued to have a low, but sigaificantly greater, number of polyneuronally innervated muscle fibers than the other muscles (P -=z0.05). M&t of the polyneuronally innervated endplates observed at 2 weeks had only two axons (Fig. 2e) leading into them, although axonal numbers up to five were occasionally seen. Diflerent types of nerve endings Complex endings. This category of nerve endings (which we defined to include everything except ‘simple’ endings) was greater in slow-twitch than in fasttwitch muscles at all ages examined (Fig 4) (P < 0.01). The percentage of complex q in all the different muscles remained co-t uhti‘betw~~ 5-6 weeks when it increased (P < 001); Qompbx endings continued to increase furt& behvoen 6 and 11 weeks (P < 0.01). The temporal iWea6eincornpla
endings is due largely to the dcvcbpmcnt of additional accessory and double endings (Figs 5 and 6). Accessory nerve endings. The pazuktrp of w ory nerve endings (Fig. 2b) 4&h t@e in &ll muscles examined (Fig. 5). In the dand 11-w&&-old animals, there were more accessory endings found in
STEPHENS
FIG. 1. A diagram showing the categories into which motor nerve terininals were classified. (a) Simple ending (endplatej-one axon terminating at one endplate and innervating a tingle *uscle fiber. (see also Fig. 2a). (b) ACcesdiory ending-an axon with one or .more thin bran&es originatihg either from nodes of Ranvier or at fh+ end of the my&n sheat% which inserts into one e&plate on a single muscle fiber (see also Fig. 2b). (c) ljoutlk-ertding-a branched terminal axon which ends in 2 d&M endp&es on one mu_&! fiber (see also Fig. 2c)_ (d) Lanchcd ending-a bra&ed &z-m&11axon which culminat~a in 2 endplates on 2 separate muscle fibers (see also Fig. 26). [e) Multiple end@-3 or more banches from a single terminal axon which form more than 2 en&&a& structures on a single mtsscIe f&r. (f) Polyneuronal endpWe-a single end+& structure on one muscle fiber a*entIy innervated by 2 or more distinct terminal axons (see also Fig. 2~). (g) Sprout-a fine unmyelinated fiber orig&ating either from nodes of Ranvier, the end of the myelin sheath, or from the endplate itself.
the slow-twitch muscles than in the fast-t&& muscles (P < 0.03). In 11-week-old rats, they made up a su~ris~y large percentage of the nerve end&s. Double endings. The formation of a second qndplate on a single muscle fiber by a single term&~al axon (Fig. 2c) was found to increase only in Fhe st&vtwitch muscles (Fig. 6).. This increase was seen to occur mainly between 6 and 1I weeks although there were @nitkantly more double endings seen in slowtwitch than fast-twitch muscles in animals at 5 and 6 weeks of age (P < 0.001). Preliminary evidence (authors’ unpublished) indicates that an increase in percentage of double endings continues in slow musck fibers throughout adult life. At 11 weeks, the adductor muscle had more double endings than the soleus muscle (P < 0.03). Branched endings. Branched endings are indicative of the innarvption of more than one mu&e fiber by a siDgkaxon(Fii2d).ThepeKerJtllgeofthistypeof en&g ru&ncd i&ta constant level %&wing an initial s&c&ant (P < 0.01) drop b&men 3 and 4 weeks in arftthe mu&es (see Fig. 7). Multiple endws. Multiple endings (Fig. I) were seen only at 11 weeks and in just the soleus and adductor muscks (1.5 and 2.7”/, rezqectively). Nerve sprouts Nodal, ultraterminal
and preterminal nerve sprouts
FIG. 2. (a) Photomicrograph of simple nerve endings, 4 weeks; plantaris. x 200. (b) Photomicrograph showing 4 accessory and one double (d) nerve terminal, 6 weeks; adductor longus. x 200. (c) Phatomicrograph of a double nerve ending, 6 weeks; soleus. x 200. (d) Photomicrograph of a branched nerve ending, 6 weeks; rectus femoris. x 290. (e) Photomicrograph of 2 endplates from the soleus muscle of a 2-week-old rat. The innervation of one endplate is by 2 axons (arrow). x 1200. (f) Photomicrograph of a possible retracting axon (arrow), 2 weeks; soleus. + 1800.
16.59
1661
Development of complexity of neuromuscular junctions P--Q e---v
lO.Or
ADWCTOR SOLEUS RECTUS FEMORIS PLANTARIS
M
WEEKS
FIG. 3. Percentage of endplates showing polyneuronal innervation. Note the drop between 2 and 3 weeks seen in all the muscles. *---a x-x e---s
50-
ACDUCTOR
*---a soLElJs
o.oL
60-
w
3
4
5
RECTUS
6
FEMORIS
II
FIG. 7. Percentage of branched endings. Note the drop between 3 and 4 weeks in all the muscles.
(Fig. 1) were seen at between 1 and 3% of nerve endings in all the muscles analyzed and this percentage
ACOUCTOR SOLEUS RECTUS FEMCftlS PLANTARIS
did not change with time.
E 40 -
DISCUSSION
E
Polyneuronal innervation
30.
3
5
4
II
6 WEEKS
FIG. 4. Percentage of complex nerve endings. The slowtwitch muscles had more complex nerve endings than fasttwitch muscles. In both of these groups a greater number of complex endings appeared after 5 weeks of age. u c---d x--x *--I
i
ADDUCTOR SOLEUS RECTUS FEMORIS PLANTARIS
i
5
,,,’ / / I’
I’
II
6
WEEKS
FIG. 5. Percentage of accessory nerve endings. Accessory nerve endings increased with time. At 6 and 11 weeks, the slow-twitch muscles had significantly more of them. At 11 weeks, the values for the plantaris and rectus femoris were identical. c~ P--Q M *--a
3
4
Branched endings
ADDUCTOR SGiEUS RECTUS FEMORIS PLANTARIS
5
6
Our findings indicate that the structure of the motor ending in rat skeletal muscle is continually changing throughout the early life of the animal and some of these changes appear to be specific to muscles of a certain fiber composition. Other workers (JANSEN et al., 1978; JACOBSON, 1978, for review) have shown that ‘redundant’ nerves to skeletal muscle fibers are eliminated in the first few post-natal weeks, leaving the adult pattern of one axon to one motor endplate per muscle fiber. We have also observed this in the present study, wherein most polyneuronal innervation disappears between 2 and 3 weeks. The general pattern of this loss of extra axons was similar in all the muscles examined. The presence of many possible axonal ‘retraction bulbs’ during the time when the supernumerary axons are being lost supports the idea that at least some of them disappear by resorption (KORNELIUSSEN & JANSEN, 1976; RILEY, 1977). The rectus femoris was seen’to maintain a small number of polyneuronally-innervated muscle fibers even in 4-week-old animals. The reason for this is not known, although one might speculate that in this muscle there lingers a small number of undifferentiated muscle fibers.
II
WEEKS
F:G. 6. Percentage of double endings. At 5 weeks and after, the number of double endings was greater in the slowtwitch muscles.
A somewhat unexpected finding was the presence of a relatively large number of branched nerve endings in all muscles examined in 3-week-old rats. These branched nerve endings disappear by 4 weeks. As branched endings reflect the innervation of more than one muscle fiber by a single terminal axon, their presence in 3-week-old rats and subsequent reduction suggests that synaptic reorganization may be taking place at this time period. Perhaps synaptic competition is still occurring, whereby the axons associated with branched endings lose out to terminal axons (previously displaced from polyneuronal endplates?)
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C. D. TWEEDLEand K. E. STEPHENS
which would only have one muscle fiber to support instead of two. Development of complexity in neuromuscular junctions
‘Complex’ nerve endings were significantly greater in number in the slow-twitch muscles. The percentage of complex endings remained stable during the period from 3 to 5 weeks of age and thereafter increased. Accessory and double nerve endings showed this same general pattern. As most muscle fiber differentiation in the rat has taken place by 5 weeks, we originally though that the stimulus for the formation of an increased number of complex endings was probably related to increases in muscle fiber size or in response to increased workload. This latter parameter might be especially important in postural muscles affected by increases in animal weight. However, we have performed preliminary experiments in which the adductor longus muscle has been tenotomized and the effect of this on the development of neuromuscular complexity analyzed. Results to date indicate that while the accessory endings increase normally, double endings do not (C. D. TWEEDLE &
K. E. STEPHENS, unpublished). Thus, some aspects of the elaboration of motor endings appear to be independent of use of the muscle while others may be more dependent on neuromuscular activity. TUFFERY(1971) suggested that increased complexity in the neuromuscular junctions of aged cats was due
to an increasing workload as well as to a decline in the trophic function of the neuron and loss of muscle fibers in senility. That signs of complexity at axonal terminals may be seen developing in the muscles of rats which are only a few months old indicates that the loss of muscle fibers or other old-age-related disorders are not required for endplate elaboration. In Tuffery’s study, it was also found that accessory endings increase with age of the animal, but these changes were observed over a period of years, not weeks. Acknowledgements--Supported by a grant from NSF (BNS 76-81406)and NIH BRSG funds. We are indebted to Mr S. PAZDZIORKOfor skilled technical assistance and MS C. MEISTERfor typing the manuscript. Drs G. HATTON,M. RHEUBEN,WM. FALLSand C. WILSONkindly read an earlier draft of this manuscript. We are indebted to Mr D. ANDERSONfor providing Figs 37.
REFERENCES ARIANOM. A., ARMSTRONG R. B. & EDGER~~NV. R. (1973) Hind-limb muscle fiber populations of five mammals. J. Histochem. Cytochem.21, 51-55. BARKERD. 8s IP M. C. (1966) Sprouting and degeneration of mammalian motor axons in normal and de-a&rented skeletal muscle. Proc. R. Sot. B 163, 538-554. Cotis C. (1955) Les variations structurelles normales et pathologiques de la jonction neuromusculaire. Acta neural. be&. 55, 741-866. CO&U C. N., TELERMAN-TOPPET N. & GERARDJ. M. (1973) Terminal innervation ratio in neuromuscular disease. Archs. Neurol., Paris 29, 210-222. GWYND. G. & HEARDMAN V. A. (1965) A cholinesterase-Bielchowsky staining method for mammalian motor endplates. Stain Technol. 40, 15-18. JACOBSON M. (1978) DevelopmentalNeurobiology.Plenum, New York. JANSENJ. K. S., THOMPSONW. & KUFFLERD. P. (1978) The formation and maintenance of synaptic connnections as illustrated by studies of the neuromuscular junction. In Progress in Brain Research (eck CORER M. A., BAKERR. E., VANDE POLL E., SWAABB. F. & EWLINGSH. A. M.) Vol. 48, pp. 3-18. Elsevier, Amsterdam. KORNELN~SENH. L JANSENJ. K. S. (1976) Morphological aspects of the elimination of polyneuronal innervation of skeletal muscle fibres in new-born rats. J. Neurocytol. 5, 591-604. KORNELUS~NH. & WAERHAUG0. (1973) Three morphological types of motor nerve terminals in the rat diaphragm and their possible innervation of different muscle fiber types. Z. Anat. EntwGesch.14@,73-84. NYSTR~~M B. (1968) Post-natal development of motor nerve terminals in ‘slow-red’ and ‘fast-white’ cat muscles. Acta neural, stand. 44, 363-383. PADYKULAH. & GAUTHIERG. (1970) The ultrastructure of the neuromuscular junction of mammalian red, white and intermediate muscle fibers. J. Cell Biol. 46, 2741. RILEYD. A. (1977) Spontaneous elimination of nerve terminals from the endplates of developing skeletal myofibers. Brain Res. 134,279285. SIEGELS. (1956) Non-parametricStatistics.McGraw-Hill, New York. TUPFERYA. R. (1971) Growth and degeneration of motor endplates in normal cat hind-limb mu&es. .r. Anut. 119 221-247. WERNIGA., ~ccrr-DECHAVASS~NE M. & STavw H. (1980) Sprouting and regression of the nerve at the frog neuromuscular junction in normal conditions and after prolonged paralysis with curare. J. Nenrocylof. 9, 277-303. (Accepted 18 February 1981)