354
Brain Research, 155 (1978) 354--356 ~,!~Elsevier/North-Holland Biomedical Press
Sprouting of intact motor neurons induced by neuronal lesion in the absence of denervated muscle fibers and degenerating axons
SHLOMO ROTSHENKER Dept. of Neurobiology, HarvardMedical School, Boston, Mass. 02115 (U.S.A.) and Dept. of Anatomy and Embryology*, Hebrew University-Hadassah Medical School, Jerusalem (Israel)
(Accepted June 15th, 1978)
Sprouting of intact nerve fibers and synapse formation constitute a response to injury common to both peripheral and central nervous systems (for reviews see refs. 2, 4-7, 11 and 15). For example, in a muscle, following partial denervation, the remaining intact motor axons sprout and form synaptic connections with the denervated muscle fibersS,4, s. However, sprouting and synapse formation are not restricted to the denervated structure only; denervation of one muscle can induce sprouting in another muscle; after crushing the motor nerve to one cutaneous-pectoris muscle of the frog, the motor nerve to the opposite muscle sprouts and forms additional synaptic connections with intact and already innervated muscle fibers 13,14. A question arises regarding the source of the stimulus for this sprouting and synapse formation. The stimulus could arise from the denervated muscle fibers or degenerating axons, as it has been commonly suggested to occur in partially denervated muscles 4,s,9,16. Alternatively, however, the injured nerve cells could be the source of the stimulus for sprouting. The unique situation where denervation and sprouting occur in two distinctly different pairs of nerve and muscle allows one to determine the role that injured nerve cells play in the induction of sprouting and synapse formation. In the present study I examined the dependence of sprouting and synapse formation in one cutaneouspectoris muscle on the removal of the opposite muscle. The removal of a muscle would thus result in the elimination of the postulated peripheral sources (e.g. denervated muscle fibers or degenerating axons) of the stimulus for sprouting, leaving behind a potential central source for sprouting the injured nerve cells. Frogs (Rana pipiens) were anesthetized (0.1 ~ tricaine methanesulphonate, Ayerst) and their left cutaneous-pectoris muscle removed. Surgery was performed under a dissecting microscope without any damage to the opposite muscle and minimal injury to the neighboring muscle on the same side. Cutaneous-pectoris muscles of normal and experimental frogs were removed, curarized (2-4 × l0 -6 g/ml curare) and examined electrophysiologically to determine the proportion of muscle * Present address for correspondence.
355 fibers innervated by more than one motor neuron. Conventional intracellular recording techniques were used to record end-plate potentials evoked by various intensities of stimulation applied to the motor nerve at a distance of 1 cm from the muscle. In muscle fibers innervated by several motor neurons multiple end-plate potentials were observed as the increasing stimulus intensity recruited a greater number of axons innervating them 1,12,13. From each muscle 50-60 muscle fibers (comprising about 10 of the muscle fiber population) were examined and the incidence of polyneuronal innervation determined. These values were used to calculate the average values given in the following paragraphs. It was shown that in normal frogs about 16 % of the cutaneous-pectoris muscle fibers are polyneuronally innervated 1~. The degree of polyneuronal innervation in intact muscles can be markedly increased after denervating the opposite muscle. The proportion of muscle fibers innervated by several motor neurons increased to an average value of 39.7 4- 3.2% (S.E.M.) after transecting the motor nerve to the opposite muscle 14. However, the induction of polyneuronal innervation can also occur in the absence of either denervated muscle fibers or degenerating motor axons. In experimental frogs, after the removal of the left muscle, the incidence of polyneuronal innervation in the right intact muscles increased to an average value of 34.2 :k 2.2 (S.E.M.). As already mentioned, during surgery, some of the muscle fibers that comprise the muscle adjacent to the removed muscle were also damaged. The possibility thus exists that the damaged muscle fibers could induce sprouting and synapse formation in the intact muscle. To test this possibility, both left and right cutaneous-pectoris muscles were examined after damaging neighboring muscle fibers on the left side. The injury was performed under a dissecting microscope with a razor blade or forceps. Muscles were examined at various times and up to several months after the operation. The pattern of innervation in either left or right cutaneous-pectoris muscles did not differ from normal. The incidence of polyneuronal innervation in left muscles was 15 -¢- 1 (S.E.M.) and 16 ~ 1 ~ (S.E.M.) in right muscles. These results demonstrate that sprouting of intact motor neurons can occur in response to neuronal lesion in the absence of either denervated muscle fibers or degenerating axons. Thus, the signal for sprouting must arise from another source, most likely the injured nerve cells. The present results are in good agreement with previous data 14 and further extend them. First, it was shown that sprouting was not induced systemically, since it did not occur when motor nerves that arise from distant segments of the spinal cord were injured. Secondly, one can modulate the degree of sprouting and the latency of its occurrence by varying the extent of the lesion to the opposite nerve and its proximity to the spinal cord, in much the same way as other postaxotomy changes in somata of injured neurons 10. These results offer strong support to the hypothesis that the injured nerve cells that comprise the motor nerve to one cutaneous-pectoris muscle may be the source of the signal for sprouting which they communicate in the spinal cord to the intact motor neurons to the opposite muscle.
356 I am grateful to Drs. U. J. M c M a h a n and S. W. Kuffler for m a k i n g this study possible. The w o r k was s u p p o r t e d by grants f r o m the Muscular D y s t r o p h y Association, D y s a u t o n o m i a F o u n d a t i o n and the N I H .
1 Brown, M. C., Jansen, J. K. S. and Van Essen, D., Polyneuronal innervation of skeletal muscle in newborn rat, J. Physiol. (Lond.), 261 (1976) 387-422. 2 Cotman, C. W. and Lynch, G. S., Reactive synaptogenesis in the adult nervous system. In S. H. Barondes (Ed.), Neuronal Recognition, Plenum Press, New York, 1976, pp. 69-104. 3 Edds, M. V., Collateral regeneration of residual motor axons in partially denervated nuscle, J. exp. Zool., 113 (1950) 517-551. 4 Edds, M. V., Collateral nerve regeneration, Quant Rev. Biol., 28 (1953) 260-276. 5 Guth, L., Trophic influence of nerve on muscle, Physiol. Rev., 48 (1968) 645-687. 6 Gutmann, E., Trophic interactions, Ann. Rev. PhysioL, 38 (1976) 177-215. 7 Harris, A. J. Inductive functions of the nervous system, Ann. Rev. Physiol., 36 (1974) 251-305. 8 Hoffman, H., Local reinnervation in partially denervated muscle. A histochemical study, Austral. J. Exp. biol. med. Sci., 28 (1950) 393-397. 9 Hoffman, H. and Springell, P. H., An attempt at the chemical identification of'neurocletin' (the substance evoking axon sprouting), Austral. J. Exp. biol. reed. Sci., 29 (1951) 417-423. 10 Lieberman, A. R., Some factors affecting retrograde neuronal responses to axonal lesions. In Essays on the Nervous System, Blarden Press, Oxford, 1974, pp. 71-105. 11 Purves, D., In G. S. Stent (Ed.), Function and Formatioll of Neural Systems, Dahlem Konferenzen, 1977, pp. 21-49. 12 Redfern, P. A., Neuromuscular transmission in newborn rats, J. Physiol. (Lond.), 209 (1970) 701-709. 13 Rotshenker, S. and McMahan, U. J., Altered patterns of innervation in frog muscle after denervation, J. Neurocytol., 5 (1976) 719-730. 14 Rotshenker, S. Sprouting of motor neurones and synapse formation, J. Physiol. (Lond.), 273 (1977) 74p. 15 Stein, D. G., Rosen, J. J. and Butters, N. (Eds.), Plasticity and Recovery of Function in the Central Nervous System,, Academic Press, New York, 1970. 16 Van Harreveld, A., On the mechanisms of'spontaneous' reinnervation in paretic muscles, Amer. J. Physiol., 130 (1947) 670-676.
Brain Research, 155 (1978) 354--356 ~,!~Elsevier/North-Holland Biomedical Press
Sprouting of intact motor neurons induced by neuronal lesion in the absence of denervated muscle fibers and degenerating axons
SHLOMO ROTSHENKER Dept. of Neurobiology, HarvardMedical School, Boston, Mass. 02115 (U.S.A.) and Dept. of Anatomy and Embryology*, Hebrew University-Hadassah Medical School, Jerusalem (Israel)
(Accepted June 15th, 1978)
Sprouting of intact nerve fibers and synapse formation constitute a response to injury common to both peripheral and central nervous systems (for reviews see refs. 2, 4-7, 11 and 15). For example, in a muscle, following partial denervation, the remaining intact motor axons sprout and form synaptic connections with the denervated muscle fibersS,4, s. However, sprouting and synapse formation are not restricted to the denervated structure only; denervation of one muscle can induce sprouting in another muscle; after crushing the motor nerve to one cutaneous-pectoris muscle of the frog, the motor nerve to the opposite muscle sprouts and forms additional synaptic connections with intact and already innervated muscle fibers 13,14. A question arises regarding the source of the stimulus for this sprouting and synapse formation. The stimulus could arise from the denervated muscle fibers or degenerating axons, as it has been commonly suggested to occur in partially denervated muscles 4,s,9,16. Alternatively, however, the injured nerve cells could be the source of the stimulus for sprouting. The unique situation where denervation and sprouting occur in two distinctly different pairs of nerve and muscle allows one to determine the role that injured nerve cells play in the induction of sprouting and synapse formation. In the present study I examined the dependence of sprouting and synapse formation in one cutaneouspectoris muscle on the removal of the opposite muscle. The removal of a muscle would thus result in the elimination of the postulated peripheral sources (e.g. denervated muscle fibers or degenerating axons) of the stimulus for sprouting, leaving behind a potential central source for sprouting the injured nerve cells. Frogs (Rana pipiens) were anesthetized (0.1 ~ tricaine methanesulphonate, Ayerst) and their left cutaneous-pectoris muscle removed. Surgery was performed under a dissecting microscope without any damage to the opposite muscle and minimal injury to the neighboring muscle on the same side. Cutaneous-pectoris muscles of normal and experimental frogs were removed, curarized (2-4 × l0 -6 g/ml curare) and examined electrophysiologically to determine the proportion of muscle * Present address for correspondence.
355 fibers innervated by more than one motor neuron. Conventional intracellular recording techniques were used to record end-plate potentials evoked by various intensities of stimulation applied to the motor nerve at a distance of 1 cm from the muscle. In muscle fibers innervated by several motor neurons multiple end-plate potentials were observed as the increasing stimulus intensity recruited a greater number of axons innervating them 1,12,13. From each muscle 50-60 muscle fibers (comprising about 10 of the muscle fiber population) were examined and the incidence of polyneuronal innervation determined. These values were used to calculate the average values given in the following paragraphs. It was shown that in normal frogs about 16 % of the cutaneous-pectoris muscle fibers are polyneuronally innervated 1~. The degree of polyneuronal innervation in intact muscles can be markedly increased after denervating the opposite muscle. The proportion of muscle fibers innervated by several motor neurons increased to an average value of 39.7 4- 3.2% (S.E.M.) after transecting the motor nerve to the opposite muscle 14. However, the induction of polyneuronal innervation can also occur in the absence of either denervated muscle fibers or degenerating motor axons. In experimental frogs, after the removal of the left muscle, the incidence of polyneuronal innervation in the right intact muscles increased to an average value of 34.2 :k 2.2 (S.E.M.). As already mentioned, during surgery, some of the muscle fibers that comprise the muscle adjacent to the removed muscle were also damaged. The possibility thus exists that the damaged muscle fibers could induce sprouting and synapse formation in the intact muscle. To test this possibility, both left and right cutaneous-pectoris muscles were examined after damaging neighboring muscle fibers on the left side. The injury was performed under a dissecting microscope with a razor blade or forceps. Muscles were examined at various times and up to several months after the operation. The pattern of innervation in either left or right cutaneous-pectoris muscles did not differ from normal. The incidence of polyneuronal innervation in left muscles was 15 -¢- 1 (S.E.M.) and 16 ~ 1 ~ (S.E.M.) in right muscles. These results demonstrate that sprouting of intact motor neurons can occur in response to neuronal lesion in the absence of either denervated muscle fibers or degenerating axons. Thus, the signal for sprouting must arise from another source, most likely the injured nerve cells. The present results are in good agreement with previous data 14 and further extend them. First, it was shown that sprouting was not induced systemically, since it did not occur when motor nerves that arise from distant segments of the spinal cord were injured. Secondly, one can modulate the degree of sprouting and the latency of its occurrence by varying the extent of the lesion to the opposite nerve and its proximity to the spinal cord, in much the same way as other postaxotomy changes in somata of injured neurons 10. These results offer strong support to the hypothesis that the injured nerve cells that comprise the motor nerve to one cutaneous-pectoris muscle may be the source of the signal for sprouting which they communicate in the spinal cord to the intact motor neurons to the opposite muscle.
356 I am grateful to Drs. U. J. M c M a h a n and S. W. Kuffler for m a k i n g this study possible. The w o r k was s u p p o r t e d by grants f r o m the Muscular D y s t r o p h y Association, D y s a u t o n o m i a F o u n d a t i o n and the N I H .
1 Brown, M. C., Jansen, J. K. S. and Van Essen, D., Polyneuronal innervation of skeletal muscle in newborn rat, J. Physiol. (Lond.), 261 (1976) 387-422. 2 Cotman, C. W. and Lynch, G. S., Reactive synaptogenesis in the adult nervous system. In S. H. Barondes (Ed.), Neuronal Recognition, Plenum Press, New York, 1976, pp. 69-104. 3 Edds, M. V., Collateral regeneration of residual motor axons in partially denervated nuscle, J. exp. Zool., 113 (1950) 517-551. 4 Edds, M. V., Collateral nerve regeneration, Quant Rev. Biol., 28 (1953) 260-276. 5 Guth, L., Trophic influence of nerve on muscle, Physiol. Rev., 48 (1968) 645-687. 6 Gutmann, E., Trophic interactions, Ann. Rev. PhysioL, 38 (1976) 177-215. 7 Harris, A. J. Inductive functions of the nervous system, Ann. Rev. Physiol., 36 (1974) 251-305. 8 Hoffman, H., Local reinnervation in partially denervated muscle. A histochemical study, Austral. J. Exp. biol. med. Sci., 28 (1950) 393-397. 9 Hoffman, H. and Springell, P. H., An attempt at the chemical identification of'neurocletin' (the substance evoking axon sprouting), Austral. J. Exp. biol. reed. Sci., 29 (1951) 417-423. 10 Lieberman, A. R., Some factors affecting retrograde neuronal responses to axonal lesions. In Essays on the Nervous System, Blarden Press, Oxford, 1974, pp. 71-105. 11 Purves, D., In G. S. Stent (Ed.), Function and Formatioll of Neural Systems, Dahlem Konferenzen, 1977, pp. 21-49. 12 Redfern, P. A., Neuromuscular transmission in newborn rats, J. Physiol. (Lond.), 209 (1970) 701-709. 13 Rotshenker, S. and McMahan, U. J., Altered patterns of innervation in frog muscle after denervation, J. Neurocytol., 5 (1976) 719-730. 14 Rotshenker, S. Sprouting of motor neurones and synapse formation, J. Physiol. (Lond.), 273 (1977) 74p. 15 Stein, D. G., Rosen, J. J. and Butters, N. (Eds.), Plasticity and Recovery of Function in the Central Nervous System,, Academic Press, New York, 1970. 16 Van Harreveld, A., On the mechanisms of'spontaneous' reinnervation in paretic muscles, Amer. J. Physiol., 130 (1947) 670-676.
