Brain Research, 560 (1991) 311-314 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939124849N
311
BRES 24849
Nerve sprouting and endplate growth induced in normal muscle by contralateral partial denervation of rat plantaris Bruce R. Pachter and Arthur Eberstein Department of Rehabilitation Medicine, New York University Medical Center, New York, NY 10016 (U.S.A.)
(Accepted 18 June 1991) Key words: Rat plantaris muscle; Partial denervation; Motor endplate; Nerve sprouting; Induced contralateral sprouting; Terminal sprouting
The incidence of motor nerve and terminal sprouting was quantitatively analyzed in normal unoperated muscles, in homologous muscles contralateral to muscles which have been partially denervated, in partially denervated muscles, and in sham-operated muscles. Muscles were studied by light microscopy after staining motor endplates by a combined silver-cholinesterase stain. In addition, the incidence of endplates containing terminal sprouts, the number of terminal branch points per endplate, and endplate size were also assessed in the various groups examined. We observed that following section of the L 4 spinal nerve, the incidence of sprouting (preterminal and intranodal) in the contralateral muscle exhibited a 2-fold increase over sham-operated controls. We also found a correlation between nerve terminal sprouting, terminal branch point number and endplate size. All of these parameters were significantly increased in the contralateral m u s c l e s as compared to the sham-operated control muscles. These findings suggest that normal muscles undergo sprouting which can be enhanced by contralateral partial denervation. The possible underlying mechanism may be the transneuronal induction of sprouting. In recent years, a n u m b e r of studies have r e p o r t e d that a x o t o m y on one side will induce a variety of changes in intact m o t o n e u r o n s on the opposite side 6'17-21. Observations that sprouting and synapse formation at the neuromuscular junction can be enhanced in n o r m a l innervated muscle by contralateral a x o t o m y has generated the most controversy. R o t s h e n k e r 1s'19 and Elizalde et al. 6 found in frog, and R o t s h e n k e r and Ta121 in the mouse, that sprouting is e n h a n c e d after contralateral denervation. H o w e v e r , H e r r e r a and Grinnel113 and H e r r e r a and Scott 14 r e p o r t e d no effect on sprouting after contralateral a x o t o m y in the frog, and Brown and I r o n t o n 3 arrived at the same conclusion in mice. In the above studies, a c o m p l e t e a x o t o m y was p e r f o r m e d and the muscles were thus completely denervated. In a study of partial denervation in rat soleus muscle, Fagg et al. 7 r e p o r t e d that after 2 weeks of such partial denervation, no enh a n c e m e n t of axonal sprouting was seen in the contralateral soleus muscle. While studying partially d e n e r v a t e d plantaris muscles in the rat, we had the o p p o r t u n i t y to investigate sprouting and e n d p l a t e growth in homologous muscles on the opposite side. Partial denervation was accomplished by section of the t 4 spinal nerve, a p r o c e d u r e which does not prevent the rats from using the affected limb. G a r diner and Faltus 8 found that by day 4 postsurgery, activity of the partially d e n e r v a t e d rats could not be dif-
ferentiated from that of the normal ones. Using a c o m b i n e d silver-cholinesterase stain 16, we obtained evidence that partial denervation enhances nerve sprouting and e n d p l a t e growth in the contralateral plantaris muscle following 1 m o n t h post-surgery. Young adult female Wistar rats were divided into 3 groups; partially denervated, s h a m - o p e r a t e d , and normal n o n - o p e r a t e d . A l l rats were 11 weeks old at the time of surgery. Rats were anesthetized with sodium pentobarbital (36 mg/kg, i.p.) and chloral hydrate (160 mg/kg, i.p.). The p r o c e d u r e e m p l o y e d to expose the spinal nerves was described by G u t h et al. 4 Using aseptic techniques, the right spinal n e r v e s L 4 and L 5 were exposed from the intervertebral foramina to whdre they unite and give rise to the sciatic nerve. A 1 m m segment of the L 4 nerve was excised close to the vertebral column and both the proximal and distal stumps were ligated with a silk thread. The distal stump was then turned on itself and tied again to prevent reinnervation. F o r s h a m - o p e r a t e d rats, the same p r o c e d u r e was followed except that the r i g h t L 4 was not cut. O n e month post-surgery, 8 animals p e r experimental group were anesthetized and the partially d e n e r v a t e d plantaris, along with its left contralateral muscle, was r e m o v e d as well as the plantaris muscles from the control and s h a m - o p e r a t e d animals. The animals were then sacrificed with an intracardial injection of saturated so-
Correspondence: B. Pachter, Department of Rehabilitation Medicine, IRM Room RR-720, New York University Medical Center, 400 East 34th Street, New York, NY 10016, U.S.A.
312 lution of KC1 while the animals were still anesthetized. The plantaris muscles were removed with a 'U'-shaped biopsy clamp. Longitudinal sections, 40/~m thick, were cut in a cryostat at -20 °C, collected on glass slides coated with 3% disodium E D T A , used to prevent contracture of the muscle fibers, and air dried. The histologic-histochemical method of Pestronk and Drachman 16 was used to display both nerve terminals and cholinesterase in the same section. By the use of this method we were able to demonstrate the preterminal axon, its terminal arborization, and any terminal, nodal, or ultraterminal outgrowth. Neuromuscular junctions were sampled only if the terminal portion of motor axons, nerve endings, and endplate regions were all visualized. All analyses were carried out 'blind', the identity of tissues from the different experimental groups was unknown and encoded, and the identifying muscles were revealed only after the analyses were completed. In each muscle, at least 300-350 endplates were examined under a total magnification of 400 or 1000x, and classified into one of the following categories: (1) no sprouting (normal endplates); (2) terminal sprouts consisting of very thin, filamentous terminal branches ending in bulbous, conical tips. These sprouts represent new terminal growth3j7; (3) ultraterminal sprouts, which are outgrowths from the terminal arborization which extend beyond the perimeter of the endplate demarcated by the cholinesterase stain1°; (4) intranodal sprouts, where their point of origin was from a node of Ranvier and where their tip was located within the endplate region z4, and; (5) preterminal sprouts, which originate from the myelin-free preterminal segment of the preterminal portion of the axon before the onset of terminal branching 25. In this study, motor axon sprouts were defined as fine supernumery, non-myelinated nerve processes. We determined the percentage of all endplates that showed evidence of sprouting from the total number of endplates sampled. In addition to the above, at least 100 endplates in each muscle were quantitatively measured by counting the number of terminal branch points within each endplate area 17. Endplate length was also determined and was calculated by measuring the length of each cholinesterase-stained endplate area with the aid of an ocular micrometer ~6-~7. This measurement is made parallel to the length of the muscle fiber. Data were analyzed with a one-way A N O V A , and if necessary, a posthoc test (Tukey HSD) on pairs of means. A difference of P < 0.05 was considered significant. For each type of sprouting analyzed, no significant difference was found between the contralateral control muscle from the sham-operated animals and normal, non-operated muscles. Thus, only the results from the former animals are shown in Tables I and II. These re-
TABLE 1 The percentage (mean -+ S.D.) of preterminal (PT), intranodal (IN), ultraterminal (UT), and terminal sprouts (TS) in shamoperated control (Control), partially denervaled (1.4) and contralateral muscles following 1 month post-surgery
Per muscle 300-350 endplates were examined. Each experimental group contained a total of 8 animals. PT
Control L4
Contralateral
IN
9.1_+2.1 1.4-+0.7 11.5_+3.2 3.0-+1.8 20.6_+2.4* 2.3_+0.6**
UT
7S
0 9.6±2.2 2.4-+1.4 24.6_+8.5 0 31.4-+ 12.0"
* Significantly different (P < 1).001) from sham-operated control and L4 muscles. ** Significantly different (P < 0.001) from sham-operated controls.
suits are similar to those found by Herrera and Scott ~4 and Rotshenker and Ta121 in frog and mouse muscles, respectively. The incidence of sprouting observed in the sham-operated muscles can be seen in Table I. The majority of sprouts arose from either preterminal branches (9.1 2.1%) or nodes of Ranvier (1.4 +-- 0.7%). We did not observe any evidence of ultraterminal sprouts in these muscles. Terminal sprouts within the endplate proper accounted for 9.6 -+ 2.2% of the endplates examined. These endplates were found to contain an average of 5.2 --- 2.1 terminal branch points per endplate. The incidence of sprouting observed in the partially denervated muscles was significantly increased for each category of sprouting (Table I). In addition 2.4 -+ 1.4% of endplates contained ultraterminal sprouts. The incidence of terminal sprouts in the partially denervated muscles was found to be 2.5 times more (24.6 -+ 8.5%) than that observed in the muscles from the sham-operated control animals (9.6 - 2.2%). The mean number of
TABLE II Number (mean +- S.D.) of terminal branch points per endplate and endplate length (in microns) in sham-operated control (Control), partially denervated (L4), and contralateral muscles following one month post-surgery
Per muscle 100 endplates were examined. Each experimental group contains 8 animals.
Control L4 Contralateral
Number of branch points per endplate
Endplate length
5.2 -+ 2.1 4.3 -+ 2.1 6.0 +- 1.3"
27.5 -+ 7.2 30.3 _+ 6.8 32.1 -+ 1.3"*
* Significantly different (P < 0.01) from sham-operated control endplates and significantly different (P < 0.001) from L 4 endplates. ** Significantly different (P < 0.001) from sham-operated endplates.
313 terminal branch points in the endplates from the partially denervated muscles was 4.3 +-- 2.1 (see Table II), significantly less (P < 0.01) than that found in control muscles. On average, partial denervation in one hind limb was followed in the muscles of the opposite limb by an almost two-fold increase in preterminal plus intranodal sprouting and a 3-fold increase in the number of terminal sprouts over control (Table I). Both preterminal and terminal sprouting were mostly affected; ultraterminal sprouting was not observed. It is of interest to note that the incidence of preterminal sprouting was significantly higher (P < 0.001) in the contralateral (20.6 - 2.4%) than in the partially denervated muscles (11.5 _+ 3.2%). Additionally, the percentage of endplates exhibiting terminal sprouts was also significantly higher (P < 0.001) in the contralateral muscles than in the partially denervated muscles. The number of terminal branch points per endplate was also found to be significantly increased in the contralateral muscles when compared to endplates in control and partially denervated muscles (see Table II). This increase in sprouting in the contralateral muscles was reflected in their having significantly larger (P < 0.001) endplates as compared to sham-operated control muscles (Table II). The results from the present study demonstrate that partial denervation of rat plantaris muscle induced an increase in nerve sprouting in the intact endplates of the contralateral plantaris muscle. The enhancement of nerve sprouting in the contralateral muscle was significantly greater than that found in the sham-operated controls and the partially denervated muscles. This increase in endplate sprouting resulted in the enlargement of the endplate length. It is important to note that the increase of sprouting in the normal plantaris muscle refers only to sprouting occurring within the endplate area of the muscle fiber (preterminal, intranodal, and terminal sprouts) and not to collateral sprouting. The latter type of sprouting is the growth of an intact axon beyond its original endplate to innervate a neighboring muscle fiber. In the partially denervated muscle there is considerable collateral sprouting 3. In this study collateral sprouting was not observed in normal plantaris muscle. The present results are in accord with those of Rotshenker and Za121 who found an increase in nodal and preterminal sprouting in the contralateral peroneal and extensor digitorum longus muscles after complete section of the sciatic nerve in mice. In our study following L4 nerve section, the incidence of sprouting (preterminal plus intranodal) on the contralateral side increased to 23.5 - 1.9%, a 2-fold increase over sham-operated control muscles. In mouse, Rotshenker and Ta121 reported that the sprouting rate was 20.7 -+ 2.1% in the contralat-
eral muscles. In contrast, Brown et al. 4 did not find any significant enhancement of sprouting in the contralateral peroneus tertius muscles in mice after section of the sciatic nerve. The muscles were examined 4-25 days after surgery. Rotshenker and Ta121 explained the discrepancy in results as possibly being due to a lower resolution of sprouts and to some of the muscles not having sufficient time to develop sprouts. Additionally, Fagg et al. 7 examined partially denervated soleus muscle from rat induced by transection of the L 4 spinal nerve. They found no increase in axonal sprouting occurring in the contralateral soleus muscle. The possible discrepancy in our results and those of Fagg et al. 7 may be due to the fact that their time interval following partial denervation of only 2 weeks might be insufficient for sprouting to be initiated. In our study, however, the muscles were examined 1 month post-surgery. It is also possible that the degree of denervation was too low, since transection of the L 4 spinal nerve in rat has been shown to denervate only approximately 20% of the soleus muscle, whereas, in our study, L 4 spinal nerve transection denervates approximately 50-80% of the plantaris muscle 9. It is quite possible that a sufficient number of motoneurons must be injured before the induction of contralateral sprouting. Preliminary data at 3 months of partial denervation indicates that enhancement of axonal sprouting in the homologous contralateral muscle is still apparent. Herrera and Grinnel113 showed that contralateral denervation in frog sartorius muscle caused enhanced transmitter release from the intact homologous muscle endplates. They measured endplate size in contralateral and normal muscles and concluded that there were no size differences. We, however, found a correlation between nerve terminal sprouting, terminal branch point number and endplate length. All of these parameters were significantly increased in the contralateral muscles as compared to the sham-operated control muscles. In support of our findings, Steinbach 22 observed an increase in the mean junctional length following contralateral axotomy in cats. Thus, the enhanced transmitter release observed in contralateral endplates following axotomy may be due to the enhanced availability of more endplate presynaptic active zone sites brought about by increased nerve sprouting and subsequent enlargement of the nerve terminal. A positive correlation between terminal size and transmitter release has also been noted by a number of authors 1"12a5. Additionally, Rosenheimer and Smith 17 noted a correlation between an increase in quantal release and an increased number of terminal branches at the diaphragm endplates of old rats. We observed a 3-fold increase in the percentage of endplates exhibiting terminal sprouts in both the partially denervated and contralateral muscles as compared
314 to sham-operated control muscles. Terminal sprouts or buds are forms of nerve terminal sprouting 1°'26. The
cles (10.6 -----2.4%) of the present study is similar to that reported by Barker and lp 2 and Tuffery 24 in cat and
nerve terminal sprouts observed in the present study
rabbit muscles, respectively.
were similar in appearance to the sprouts, representing new terminal growth, seen in partially denervated muscles 3, botulinum toxin-treated muscles 5, and normal muscles during aging 17"25. Torigoe 23 has visualized these ter-
sprouting and synapse formation occurs in muscles of normal animals 26. In addition, we observed that sprout-
minal sprouts by scanning electron microscope following partial denervation in mouse muscle and has referred to the bulbous tip as a growth cone. In this regard, the increase in terminal sprouts was reflected by an increase in the n u m b e r of branch points per endplate in the contralateral muscles as compared to sham-operated control and partially denervated muscles. The branch point count indicates an increasing complexity of the terminal arborization in contralateral muscles. The presence of axonal sprouting in normal control muscles supports the observation that the structure of neuromuscular junctions is continually remodelled during normal growth and development. The present data confirm previous studies 2'24. In this regard, it is of interest to note that the incidence of sprouts (preterminal and intranodal) observed in the sham-operated control mus-
1 Angaut-Petit and Mallart, A., Dual innervation of endplate sites
and its consequencesfor neuromuscular transmissionin musclesof adult Xenopus laevis, J. Physiol., 289 (1979) 203-218. 2 Barker, D. and Ip, M.C., Sprouting and degeneration of mammalian motor axons in normal and deafferented skeletal muscle, Proc. R. Soc. Lond. B, 163 (1966) 538-553. 3 Brown, M.C. and Ironton, R., Sprouting and regression of neuromuscular synapse in partially denervated mammalian muscles, J. Physiol., 278 (1978) 325-348. 4 Brown, M.C., Holland, R.L. and Ironton, R., Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse, J. Physiol., 306 (1980) 493-510. 5 Duchen, L.W., An EM study of the changes induced by botulinum toxin in the motor end-plates of slow and fast skeletal muscle fibers of the mouse, J. Neurol. Sci., 14 (1971) 47-60. 6 Elizalde, A.M., Huerta, M. and Stefani, E., Selective reinnervation of twitch and tonic muscle fibers of the frog, J. Physiol., 340 (1983) 513-524. 7 Fagg, G.E., Scheff, S.W. and Cotman, C.W., Axonal sprouting at the neuromuscular junction of adult and aged rats, Exp. Neurol., 74 (1981) 847-854. 8 Gardiner, P.F. and Faitus, R.E., Contractile responses of rat plantaris muscles following partial denervation, and the influence of daily exercise, Pfl~gers Arch., 406 (1986) 51-56. 9 Gardiner, P.E, Michel, R.N., Olha, A.E. and Pettigrew, F., Force and fatiguability of sprouting motor units in partially denervated rat plantaris, Exp. Brain Res., 66 (1987) 597-606. 10 Gorio, A., Marinin, P. and Zanoi, R., Muscle reinnervation. II. Motoneuron capacity, enhancement by exogenous gangliosides, Neuroscience, 8 (1983) 417-429. 11 Guth, L., Smith, S., Donati, J. and Albuquerque, E.X., Induction of intramuscular collateral nerve sprouting by neurally applied colchicine, Exp. Neurol, 67 (1980) 513-523. 12 Harris, J.B. and Ribchester, R.R., The relationship between end-plate size and transmitter release in normal and dystrophic muscles of the mouse, J. Physiol., 296 (1979) 245-265. 13 Herrera, A.A. and Grinnell, A.D., Contralateral denervation causes enhanced transmitter release from frog motor nerve ter-
It is apparent from the above that some degree of
ing was enhanced in muscles by partial denervation of the contralateral homologous muscle. The mechanism involved in inducing sprouting activity on the normally innervated side after such contralateral nerve injury is u n k n o w n at present. However, it has been suggested by R o t s h e n k e r and Ta121 that axotomy initiates in the cell bodies of the injured m o t o n e u r o n s a signal for growth which is then transferred transneuronally across the spinal cord to the intact m o t o n e u r o n s which, in turn, respond by inducing sprouting in the homologous muscle. In this regard, it should be emphasized that contralateral muscles following either complete or partial denervation should not be used as normal controls.
The authors wish to acknowledge the skillful technical assistancc of Ruth Johnston and Barbara Zimmer. This work was supported by NIH Grant NS25624.
minals, Nature, 291 (1981) 495-497. 14 Herrera, A.A. and Scott, D.R., Motor axon sprouting in frog satorius muscles is not altered by contralateral axotomy, J. Neurocytol., 14 (1985) 145-156. 15 Kuno, M., Turkanis, S.A. and Weakly, J.N., Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog, J. Physiol., 213 (1971) 545-556. 16 Pestronk, A. and Drachman, D.B., A new stain for quantitative measurement of sprouting at neuromuscular junctions, Muscle Nerve, 1 (1978) 70-74. 17 Rosenheimer, J.L. and Smith, D.O., Differential changes in the end-plate architecture of functionally diverse muscles during aging, J. Neurosci., 53 (1985) 1567-1581. 18 Rotshenker, S., Synapse formation in intact innervated cutaneous-pectoris muscles of the frog following denervation of opposite muscles, J. Physiol., 292 (1979) 535-547. 19 Rotshenker, S., Transneuronal and peripheral mechanisms for the induction of motor neuron sprouting, J. Neurosci., 2 (1982) 1359-1368. 20 Rotshenker, S., Multiple modes and sites for the induction of axonal growth, Trends Neurosci., 11 (1988) 363-366. 21 Rotshenker, S. and Tal, M., The transneuronal induction of sprouting and synapse formation in intact mouse muscles, J. Physiol., 360 (1985) 387-396. 22 Steinbach, H.J., Neuromuscular junctions and ct-bungarotoxin binding sites in denervated and contralateral cat skeletal muscles, J. Physiol., 313 (1981) 513-528. 23 Torigoe, K., Terminal sprouting in partially denervated muscle of the mouse: a scanning electron microscopic study, J. Neurocytol., 17 (1988) 563-571. 24 Tuffery, A.R., Growth and degeneration of motor end-plates in normal cat hind limb muscles, J. Anat., 110 (1971) 221-247. 25 Wernig, A., Carmody, J.J., Anzil, A.P., Hansert, E., Marciniok, M. and Zucker, H., Persistence of nerve sprouting with features of synapse remodelling in soleus muscles of adult mice, Neuroscience, 11 (1984) 241-253. 26 Wernig, A. and Herrera, A.A., Sprouting and remodelling at the nerve-muscle junction, Prog. Neurobiol., 27 (1986) 251-291.
311
BRES 24849
Nerve sprouting and endplate growth induced in normal muscle by contralateral partial denervation of rat plantaris Bruce R. Pachter and Arthur Eberstein Department of Rehabilitation Medicine, New York University Medical Center, New York, NY 10016 (U.S.A.)
(Accepted 18 June 1991) Key words: Rat plantaris muscle; Partial denervation; Motor endplate; Nerve sprouting; Induced contralateral sprouting; Terminal sprouting
The incidence of motor nerve and terminal sprouting was quantitatively analyzed in normal unoperated muscles, in homologous muscles contralateral to muscles which have been partially denervated, in partially denervated muscles, and in sham-operated muscles. Muscles were studied by light microscopy after staining motor endplates by a combined silver-cholinesterase stain. In addition, the incidence of endplates containing terminal sprouts, the number of terminal branch points per endplate, and endplate size were also assessed in the various groups examined. We observed that following section of the L 4 spinal nerve, the incidence of sprouting (preterminal and intranodal) in the contralateral muscle exhibited a 2-fold increase over sham-operated controls. We also found a correlation between nerve terminal sprouting, terminal branch point number and endplate size. All of these parameters were significantly increased in the contralateral m u s c l e s as compared to the sham-operated control muscles. These findings suggest that normal muscles undergo sprouting which can be enhanced by contralateral partial denervation. The possible underlying mechanism may be the transneuronal induction of sprouting. In recent years, a n u m b e r of studies have r e p o r t e d that a x o t o m y on one side will induce a variety of changes in intact m o t o n e u r o n s on the opposite side 6'17-21. Observations that sprouting and synapse formation at the neuromuscular junction can be enhanced in n o r m a l innervated muscle by contralateral a x o t o m y has generated the most controversy. R o t s h e n k e r 1s'19 and Elizalde et al. 6 found in frog, and R o t s h e n k e r and Ta121 in the mouse, that sprouting is e n h a n c e d after contralateral denervation. H o w e v e r , H e r r e r a and Grinnel113 and H e r r e r a and Scott 14 r e p o r t e d no effect on sprouting after contralateral a x o t o m y in the frog, and Brown and I r o n t o n 3 arrived at the same conclusion in mice. In the above studies, a c o m p l e t e a x o t o m y was p e r f o r m e d and the muscles were thus completely denervated. In a study of partial denervation in rat soleus muscle, Fagg et al. 7 r e p o r t e d that after 2 weeks of such partial denervation, no enh a n c e m e n t of axonal sprouting was seen in the contralateral soleus muscle. While studying partially d e n e r v a t e d plantaris muscles in the rat, we had the o p p o r t u n i t y to investigate sprouting and e n d p l a t e growth in homologous muscles on the opposite side. Partial denervation was accomplished by section of the t 4 spinal nerve, a p r o c e d u r e which does not prevent the rats from using the affected limb. G a r diner and Faltus 8 found that by day 4 postsurgery, activity of the partially d e n e r v a t e d rats could not be dif-
ferentiated from that of the normal ones. Using a c o m b i n e d silver-cholinesterase stain 16, we obtained evidence that partial denervation enhances nerve sprouting and e n d p l a t e growth in the contralateral plantaris muscle following 1 m o n t h post-surgery. Young adult female Wistar rats were divided into 3 groups; partially denervated, s h a m - o p e r a t e d , and normal n o n - o p e r a t e d . A l l rats were 11 weeks old at the time of surgery. Rats were anesthetized with sodium pentobarbital (36 mg/kg, i.p.) and chloral hydrate (160 mg/kg, i.p.). The p r o c e d u r e e m p l o y e d to expose the spinal nerves was described by G u t h et al. 4 Using aseptic techniques, the right spinal n e r v e s L 4 and L 5 were exposed from the intervertebral foramina to whdre they unite and give rise to the sciatic nerve. A 1 m m segment of the L 4 nerve was excised close to the vertebral column and both the proximal and distal stumps were ligated with a silk thread. The distal stump was then turned on itself and tied again to prevent reinnervation. F o r s h a m - o p e r a t e d rats, the same p r o c e d u r e was followed except that the r i g h t L 4 was not cut. O n e month post-surgery, 8 animals p e r experimental group were anesthetized and the partially d e n e r v a t e d plantaris, along with its left contralateral muscle, was r e m o v e d as well as the plantaris muscles from the control and s h a m - o p e r a t e d animals. The animals were then sacrificed with an intracardial injection of saturated so-
Correspondence: B. Pachter, Department of Rehabilitation Medicine, IRM Room RR-720, New York University Medical Center, 400 East 34th Street, New York, NY 10016, U.S.A.
312 lution of KC1 while the animals were still anesthetized. The plantaris muscles were removed with a 'U'-shaped biopsy clamp. Longitudinal sections, 40/~m thick, were cut in a cryostat at -20 °C, collected on glass slides coated with 3% disodium E D T A , used to prevent contracture of the muscle fibers, and air dried. The histologic-histochemical method of Pestronk and Drachman 16 was used to display both nerve terminals and cholinesterase in the same section. By the use of this method we were able to demonstrate the preterminal axon, its terminal arborization, and any terminal, nodal, or ultraterminal outgrowth. Neuromuscular junctions were sampled only if the terminal portion of motor axons, nerve endings, and endplate regions were all visualized. All analyses were carried out 'blind', the identity of tissues from the different experimental groups was unknown and encoded, and the identifying muscles were revealed only after the analyses were completed. In each muscle, at least 300-350 endplates were examined under a total magnification of 400 or 1000x, and classified into one of the following categories: (1) no sprouting (normal endplates); (2) terminal sprouts consisting of very thin, filamentous terminal branches ending in bulbous, conical tips. These sprouts represent new terminal growth3j7; (3) ultraterminal sprouts, which are outgrowths from the terminal arborization which extend beyond the perimeter of the endplate demarcated by the cholinesterase stain1°; (4) intranodal sprouts, where their point of origin was from a node of Ranvier and where their tip was located within the endplate region z4, and; (5) preterminal sprouts, which originate from the myelin-free preterminal segment of the preterminal portion of the axon before the onset of terminal branching 25. In this study, motor axon sprouts were defined as fine supernumery, non-myelinated nerve processes. We determined the percentage of all endplates that showed evidence of sprouting from the total number of endplates sampled. In addition to the above, at least 100 endplates in each muscle were quantitatively measured by counting the number of terminal branch points within each endplate area 17. Endplate length was also determined and was calculated by measuring the length of each cholinesterase-stained endplate area with the aid of an ocular micrometer ~6-~7. This measurement is made parallel to the length of the muscle fiber. Data were analyzed with a one-way A N O V A , and if necessary, a posthoc test (Tukey HSD) on pairs of means. A difference of P < 0.05 was considered significant. For each type of sprouting analyzed, no significant difference was found between the contralateral control muscle from the sham-operated animals and normal, non-operated muscles. Thus, only the results from the former animals are shown in Tables I and II. These re-
TABLE 1 The percentage (mean -+ S.D.) of preterminal (PT), intranodal (IN), ultraterminal (UT), and terminal sprouts (TS) in shamoperated control (Control), partially denervaled (1.4) and contralateral muscles following 1 month post-surgery
Per muscle 300-350 endplates were examined. Each experimental group contained a total of 8 animals. PT
Control L4
Contralateral
IN
9.1_+2.1 1.4-+0.7 11.5_+3.2 3.0-+1.8 20.6_+2.4* 2.3_+0.6**
UT
7S
0 9.6±2.2 2.4-+1.4 24.6_+8.5 0 31.4-+ 12.0"
* Significantly different (P < 1).001) from sham-operated control and L4 muscles. ** Significantly different (P < 0.001) from sham-operated controls.
suits are similar to those found by Herrera and Scott ~4 and Rotshenker and Ta121 in frog and mouse muscles, respectively. The incidence of sprouting observed in the sham-operated muscles can be seen in Table I. The majority of sprouts arose from either preterminal branches (9.1 2.1%) or nodes of Ranvier (1.4 +-- 0.7%). We did not observe any evidence of ultraterminal sprouts in these muscles. Terminal sprouts within the endplate proper accounted for 9.6 -+ 2.2% of the endplates examined. These endplates were found to contain an average of 5.2 --- 2.1 terminal branch points per endplate. The incidence of sprouting observed in the partially denervated muscles was significantly increased for each category of sprouting (Table I). In addition 2.4 -+ 1.4% of endplates contained ultraterminal sprouts. The incidence of terminal sprouts in the partially denervated muscles was found to be 2.5 times more (24.6 -+ 8.5%) than that observed in the muscles from the sham-operated control animals (9.6 - 2.2%). The mean number of
TABLE II Number (mean +- S.D.) of terminal branch points per endplate and endplate length (in microns) in sham-operated control (Control), partially denervated (L4), and contralateral muscles following one month post-surgery
Per muscle 100 endplates were examined. Each experimental group contains 8 animals.
Control L4 Contralateral
Number of branch points per endplate
Endplate length
5.2 -+ 2.1 4.3 -+ 2.1 6.0 +- 1.3"
27.5 -+ 7.2 30.3 _+ 6.8 32.1 -+ 1.3"*
* Significantly different (P < 0.01) from sham-operated control endplates and significantly different (P < 0.001) from L 4 endplates. ** Significantly different (P < 0.001) from sham-operated endplates.
313 terminal branch points in the endplates from the partially denervated muscles was 4.3 +-- 2.1 (see Table II), significantly less (P < 0.01) than that found in control muscles. On average, partial denervation in one hind limb was followed in the muscles of the opposite limb by an almost two-fold increase in preterminal plus intranodal sprouting and a 3-fold increase in the number of terminal sprouts over control (Table I). Both preterminal and terminal sprouting were mostly affected; ultraterminal sprouting was not observed. It is of interest to note that the incidence of preterminal sprouting was significantly higher (P < 0.001) in the contralateral (20.6 - 2.4%) than in the partially denervated muscles (11.5 _+ 3.2%). Additionally, the percentage of endplates exhibiting terminal sprouts was also significantly higher (P < 0.001) in the contralateral muscles than in the partially denervated muscles. The number of terminal branch points per endplate was also found to be significantly increased in the contralateral muscles when compared to endplates in control and partially denervated muscles (see Table II). This increase in sprouting in the contralateral muscles was reflected in their having significantly larger (P < 0.001) endplates as compared to sham-operated control muscles (Table II). The results from the present study demonstrate that partial denervation of rat plantaris muscle induced an increase in nerve sprouting in the intact endplates of the contralateral plantaris muscle. The enhancement of nerve sprouting in the contralateral muscle was significantly greater than that found in the sham-operated controls and the partially denervated muscles. This increase in endplate sprouting resulted in the enlargement of the endplate length. It is important to note that the increase of sprouting in the normal plantaris muscle refers only to sprouting occurring within the endplate area of the muscle fiber (preterminal, intranodal, and terminal sprouts) and not to collateral sprouting. The latter type of sprouting is the growth of an intact axon beyond its original endplate to innervate a neighboring muscle fiber. In the partially denervated muscle there is considerable collateral sprouting 3. In this study collateral sprouting was not observed in normal plantaris muscle. The present results are in accord with those of Rotshenker and Za121 who found an increase in nodal and preterminal sprouting in the contralateral peroneal and extensor digitorum longus muscles after complete section of the sciatic nerve in mice. In our study following L4 nerve section, the incidence of sprouting (preterminal plus intranodal) on the contralateral side increased to 23.5 - 1.9%, a 2-fold increase over sham-operated control muscles. In mouse, Rotshenker and Ta121 reported that the sprouting rate was 20.7 -+ 2.1% in the contralat-
eral muscles. In contrast, Brown et al. 4 did not find any significant enhancement of sprouting in the contralateral peroneus tertius muscles in mice after section of the sciatic nerve. The muscles were examined 4-25 days after surgery. Rotshenker and Ta121 explained the discrepancy in results as possibly being due to a lower resolution of sprouts and to some of the muscles not having sufficient time to develop sprouts. Additionally, Fagg et al. 7 examined partially denervated soleus muscle from rat induced by transection of the L 4 spinal nerve. They found no increase in axonal sprouting occurring in the contralateral soleus muscle. The possible discrepancy in our results and those of Fagg et al. 7 may be due to the fact that their time interval following partial denervation of only 2 weeks might be insufficient for sprouting to be initiated. In our study, however, the muscles were examined 1 month post-surgery. It is also possible that the degree of denervation was too low, since transection of the L 4 spinal nerve in rat has been shown to denervate only approximately 20% of the soleus muscle, whereas, in our study, L 4 spinal nerve transection denervates approximately 50-80% of the plantaris muscle 9. It is quite possible that a sufficient number of motoneurons must be injured before the induction of contralateral sprouting. Preliminary data at 3 months of partial denervation indicates that enhancement of axonal sprouting in the homologous contralateral muscle is still apparent. Herrera and Grinnel113 showed that contralateral denervation in frog sartorius muscle caused enhanced transmitter release from the intact homologous muscle endplates. They measured endplate size in contralateral and normal muscles and concluded that there were no size differences. We, however, found a correlation between nerve terminal sprouting, terminal branch point number and endplate length. All of these parameters were significantly increased in the contralateral muscles as compared to the sham-operated control muscles. In support of our findings, Steinbach 22 observed an increase in the mean junctional length following contralateral axotomy in cats. Thus, the enhanced transmitter release observed in contralateral endplates following axotomy may be due to the enhanced availability of more endplate presynaptic active zone sites brought about by increased nerve sprouting and subsequent enlargement of the nerve terminal. A positive correlation between terminal size and transmitter release has also been noted by a number of authors 1"12a5. Additionally, Rosenheimer and Smith 17 noted a correlation between an increase in quantal release and an increased number of terminal branches at the diaphragm endplates of old rats. We observed a 3-fold increase in the percentage of endplates exhibiting terminal sprouts in both the partially denervated and contralateral muscles as compared
314 to sham-operated control muscles. Terminal sprouts or buds are forms of nerve terminal sprouting 1°'26. The
cles (10.6 -----2.4%) of the present study is similar to that reported by Barker and lp 2 and Tuffery 24 in cat and
nerve terminal sprouts observed in the present study
rabbit muscles, respectively.
were similar in appearance to the sprouts, representing new terminal growth, seen in partially denervated muscles 3, botulinum toxin-treated muscles 5, and normal muscles during aging 17"25. Torigoe 23 has visualized these ter-
sprouting and synapse formation occurs in muscles of normal animals 26. In addition, we observed that sprout-
minal sprouts by scanning electron microscope following partial denervation in mouse muscle and has referred to the bulbous tip as a growth cone. In this regard, the increase in terminal sprouts was reflected by an increase in the n u m b e r of branch points per endplate in the contralateral muscles as compared to sham-operated control and partially denervated muscles. The branch point count indicates an increasing complexity of the terminal arborization in contralateral muscles. The presence of axonal sprouting in normal control muscles supports the observation that the structure of neuromuscular junctions is continually remodelled during normal growth and development. The present data confirm previous studies 2'24. In this regard, it is of interest to note that the incidence of sprouts (preterminal and intranodal) observed in the sham-operated control mus-
1 Angaut-Petit and Mallart, A., Dual innervation of endplate sites
and its consequencesfor neuromuscular transmissionin musclesof adult Xenopus laevis, J. Physiol., 289 (1979) 203-218. 2 Barker, D. and Ip, M.C., Sprouting and degeneration of mammalian motor axons in normal and deafferented skeletal muscle, Proc. R. Soc. Lond. B, 163 (1966) 538-553. 3 Brown, M.C. and Ironton, R., Sprouting and regression of neuromuscular synapse in partially denervated mammalian muscles, J. Physiol., 278 (1978) 325-348. 4 Brown, M.C., Holland, R.L. and Ironton, R., Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse, J. Physiol., 306 (1980) 493-510. 5 Duchen, L.W., An EM study of the changes induced by botulinum toxin in the motor end-plates of slow and fast skeletal muscle fibers of the mouse, J. Neurol. Sci., 14 (1971) 47-60. 6 Elizalde, A.M., Huerta, M. and Stefani, E., Selective reinnervation of twitch and tonic muscle fibers of the frog, J. Physiol., 340 (1983) 513-524. 7 Fagg, G.E., Scheff, S.W. and Cotman, C.W., Axonal sprouting at the neuromuscular junction of adult and aged rats, Exp. Neurol., 74 (1981) 847-854. 8 Gardiner, P.F. and Faitus, R.E., Contractile responses of rat plantaris muscles following partial denervation, and the influence of daily exercise, Pfl~gers Arch., 406 (1986) 51-56. 9 Gardiner, P.E, Michel, R.N., Olha, A.E. and Pettigrew, F., Force and fatiguability of sprouting motor units in partially denervated rat plantaris, Exp. Brain Res., 66 (1987) 597-606. 10 Gorio, A., Marinin, P. and Zanoi, R., Muscle reinnervation. II. Motoneuron capacity, enhancement by exogenous gangliosides, Neuroscience, 8 (1983) 417-429. 11 Guth, L., Smith, S., Donati, J. and Albuquerque, E.X., Induction of intramuscular collateral nerve sprouting by neurally applied colchicine, Exp. Neurol, 67 (1980) 513-523. 12 Harris, J.B. and Ribchester, R.R., The relationship between end-plate size and transmitter release in normal and dystrophic muscles of the mouse, J. Physiol., 296 (1979) 245-265. 13 Herrera, A.A. and Grinnell, A.D., Contralateral denervation causes enhanced transmitter release from frog motor nerve ter-
It is apparent from the above that some degree of
ing was enhanced in muscles by partial denervation of the contralateral homologous muscle. The mechanism involved in inducing sprouting activity on the normally innervated side after such contralateral nerve injury is u n k n o w n at present. However, it has been suggested by R o t s h e n k e r and Ta121 that axotomy initiates in the cell bodies of the injured m o t o n e u r o n s a signal for growth which is then transferred transneuronally across the spinal cord to the intact m o t o n e u r o n s which, in turn, respond by inducing sprouting in the homologous muscle. In this regard, it should be emphasized that contralateral muscles following either complete or partial denervation should not be used as normal controls.
The authors wish to acknowledge the skillful technical assistancc of Ruth Johnston and Barbara Zimmer. This work was supported by NIH Grant NS25624.
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