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Specificity of innervation among Xenopus twitch muscle fibers
Brain Research, 330 (1985) 353-357 Elsevier
353
BRE 20689
Specificity of innervation among Xenopustwitch muscle fibers BRUCE NUDELL and ALAN D. GRINNELL
Jerry Lewis Neuromuscular Research Center, UCLA, Los Angeles, CA 90024 (U.S.A.) (Accepted October 30th, 1984)
Key words: synapse - - specificity - - motoneuron - - motor unit - - neuromuscular - - Xenopus
More than 50% of the Xenopus pectoralis twitch muscle fibers with two distant endplates are innervated at both sites by the same neuron. This study indicates that there are three separable twitch motor unit types in this muscle which show very little overlap in innervation. This study also shows that each motor unit is topographically localized and that similar type units are shifted relative to one another. It is concluded that these two factors may contribute importantly to the observed high incidence of mononeuronal innervation. In an earlier paper 13 we reported that over 50% of the Xenopus pectoralis muscle fibers with two distant endplates were innervated at both sites by branches of the same motoneuron (mononeuronal innervation). It seemed unlikely that random innervation of muscle fibers would result in so much mononeuronal innervation, since it has been reported that 50 motoneurons innervate this muscle of approximately 1000 fibers 8. Feeling that some selective influences must underly this ordered pattern, we have extended our study to examine carefully the innervation pattern of individual motoneurons within the pectoralis muscle. We find that the muscle fibers receiving suprathreshold innervation from a given m o t o n e u r o n are of similar size (input resistance), and that three classes of motor units can be distinguished on this basis. We also find that motor units occur in spatially restricted regions of the muscle. While each motor unit class is represented throughout the entire field of the posterior pectoralis nerve (see below), motor units of the same class are topographically shifted relative to one another with overlap occurring primarily at their common borders. These studies suggest that the spatial segregation of the synaptic projection fields of similar type motoneurons and the lack of overlap of innervation between neurons innervating different size fibers both contribute importantly to the observed high incidence of mononeuronal innervation.
The Xenopus pectoralis is a triangular, fiat muscle lying between the sternum and the shoulder, innervated by two nerves which enter the muscle at its anterior and posterior bordersS. The posterior nerve usually innervates about two thirds of the surface fibers in this muscle. For that reason we restricted our studies to axons contained in this nerve. In young adult animals (1.75-2 in. in body length), approximately 90% of the fibers are innervated at two sites separated by 2 0 - 4 0 % of the fiber's length 13. In the present experiments, individual motor axons were mechanically teased from the main nerve trunk and stimulated using a small suction pipette. (Criteria for single motor axon isolation were visual appearance at 100 × magnification in the light microscope and the all-or-none recruitment of motor unit tension upon reaching a threshold stimulation intensity.) Action potentials or endplate potentials (EPPs) were monitored in the muscle fibers, using methods employed in our earlier study 13. T h e superficial fibers of this muscle vary widely in size - - showing a 10-12-fold range in input resistance. However, all can generate action potentials and therefore are classifiable as twitch fibers. Early in these experiments, it became apparent that any given motor axon provides suprathreshold innervation selectively to fibers of similar size, covering only a fraction of the full range of fiber sizes. On the basis
Correspondence: B. Nudell, Jerry Lewis Neuromuscular Research Center, UCLA, Los Angeles, CA 90024, U.S.A. 0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Diagrammatic representations of the fields of isolated single motor units in different pectoralis muscle preparations of similar size which were bathed in normal Ringer solution. Adjacent superficial muscle fibers were impaled with microelectrodes to measure their input resistance and to see whether an action potential (+), EPP (©), or no response (O) was elicited upon single stimulation of the isolated axon. If an action potential did not occur in a fiber, but if evidence for a subthreshold synapse was obtained, a recording electrode was moved to the site which showed the largest EPP and that value is given below. As preliminary recordings were generally made just central to the medial endplate site, it is possible that some very weak lateral junctions which produced small, subthreshold EPPs escaped our detection. In all the experiments presented in this figure, no other surface fibers were activated (showed action potentials) in response to stimulation of the isolated axon. The top panels of this figure show data from three muscles of similar size and the bottom panels present data from three smaller muscles of equivalent size. A 1 and A2: two large motor units as defined by the fact that they consisted of the largest muscle fibers in their muscles. (Thirty fibers represent about 25% of the muscle surface.) B 1 and B2: two intermediate units. In B 1 fibers number 10 and 15 showed 20 and 15 mV EPPs, respectively. In B 2 fiber number 11 showed a 20 mV EPP. C l and C2: two small units. In C2, two fibers belonging to the isolated unit lay at an appreciable distance (separated by 12 fibers) from the nearest other member of the unit and were well within the territory controlled by the anterior pectoralis nerve. Input resistance measurements were not made for the fibers lying between fiber number 29 and the two members of the unit in question. Fiber number 42 showed a 10 mV EPP.
of comparison of isolated motor unit properties in similar sized muscles, such as those illustrated in Fig. 1, we feel that three largely non-overlapping motor unit classes could account for the full range of fiber sizes exhibited in each muscle. For the purpose of this paper therefore, we will refer to motor units as 'large', 'intermediate' or 'small', here defined by the size of the muscle fibers innervated (exhibiting either
EPPs or action potentials). Although we cannot rigorously exclude the possibility that there is a continuum in motor unit types, these data suggest a correlation with the finding by L~innergren and Smith 11 of three distinct twitch fiber types in the Xenopus iliofibularis muscle, characterizable on the basis of their size, biochemistry and physiological performance. Our findings thus support the prediction of Smith and
355 Ovalle 16 that there are three distinct twitch fiber motor unit types in Xenopus. A precise determination of the amount of synaptic overlap between the different classes of motor unit can probably only be achieved by simultaneously examining different type units in the same regio n (see below) of a single muscle. Nevertheless, we feel that the data presented above strongly suggest that neurons innervating fibers of a given size generallydo not innervate fibers of a distinctly different size category in a suprathreshold manner. Occasionally, however, we did find evidence of neurons projecting synaptic inputs to 'inappropriate' sized muscle fibers. These inputs were often subthreshold. In Fig. 1B 1 for instance, fibers number 3 and 15, both of which were innervated by the isolated intermediate motor unit neuron, were large enough to also be part of a large motor unit (see Fig. 1A1). Similarly, in Fig. 1B 2 and 1C2, the isolated intermediate and small motor unit neurons formed subthreshold synapses on relatively large fibers (fibers number 11 and 42, respectively) which probably also belonged to motor units of different size classes. This degree of uniformity in muscle fiber size within a given motor unit might be expected in view of the reported similarity of fibers innervated by individual motoneurons in mammalian muscle 3. However, it has not been shown in other anuran muscles, and in fact is not the case in the frog cutaneous pectoris, where single motor units may include fibers varying by up to 5-fold in input resistance 19. Motor units also displayed a degree of spatial consolidation not previously reported in anuran muscle. This was shown by identifying those fibers in unblocked preparations which exhibited an action potential or EPP in response to the stimulation of a single motor axon. In each of these experiments all of the surface muscle fibers were sampled. The pattern of innervation shown in Fig. 1A was typical of that exhibited by the eight large motor units that we studied in this manner. Within a limited region (25-35%) of the muscle surface a single large motor unit neuron activates nearly all of the large muscle fibers (those fibers in the lowest input resistance category). Outside this region are fibers of similar size which are not members of the isolated unit but rather presumably belong to other large motor units. Virtually all of the fibers driven by an individual large motor unit axon
were found close to one another and were not separated by intervening large fibers driven exclusively by other large motor unit neurons. Occasionally, however, we found one or two fibers in a unit that were located apart from the other members of the unit and which were well within the domain of an adjacent large motor unit. It was not possible to ascertain the degree to which the large units we studied were restricted to the superficial layer of the muscle. We did find some evidence, however, for the spatial segregation of large units within the vertical plane of the muscle. We often isolated what appeared to be large motor unit axons which generated a strong twitch in the muscle but which did not activate any surface fibers. Comparable localization of projections has been shown in mammalian intercostal muscles 4, and regional localization of different segmental nerve fields has been shown in the frog gluteal muscle 1. However, the sharply restricted localization of single motor units that we find has not been reported in other anuran muscle, and is not the case in the frog cutaneous pectoris TMor sartorius muscles (Trussell, unpublished observations). (A cautionary note should be interjected here, namely that our techniques only allowed us to study the pattern of motor unit organization on the superficial face of the muscle. Thus we cannot definitely state that the topographic consolidation of motor units which is evident on the muscle surface is also characteristic of the underlying fiber layers.) Motor units comprised of small and intermediate sized fibers also appear to be spatially localized. Fig. 1B and C show examples of the five units of each type studied. From these figures it can also be seen that the small and intermediate sized motor units studied contained fewer as well as smaller muscle fibers than the large units. In order to get a more precise picture of the innervation pattern of individual motoneurons, we also examined as many of the synaptic inputs as possible in surface fibers in the region innervated by isolated motor axons. After isolating the axon, the muscle was curarized until twitching was suppressed, using concentrations ranging from 2/~M for small motor units to 8 ~M for large units. The relative resistance of large units to curare block reflects the fact that their synapses have higher safety factors than do smaller unitsT, 19.
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Fig. 2. Summarizes diagrammatically the innervation source and EPP amplitude (in mV) of all of the accessible endplates on adjacent fibers lying within the field of an isolated large motor unit neuron in a single preparation. The muscle was bathed in a Ringer solution containing 8 pM curare. The EPPs generated by the isolated neuron are labeled I, and those generated by unidentified neurones are labeled U. Some fibers lay beneath the superficial plane of the muscle for part of their length, and for that reason, one of their endplate regions (denoted by []) could not be studied. Several very small fibers (denoted by []) failed to show any synaptic activity; their junction(s) (about 20% of the small motor unit fibers have only one endplate (Nudell, unpublished data)) appeared to be totally blocked by the curare. Fibers belonging to the isolated unit and from which data were obtained for both endplates were either mononeuronally (mono) or heteroneuronally (hetero) innervated. Fiber number 23 had 3 endplates, the lateral-most of which was not accessible to local recording.
Fig. 2 presents characteristic data from 30 adjacent muscle fibers which spanned the field of an isolated large motor unit neuron. In this experimental protocol, additional stimulating electrodes were placed on the posterior nerve trunk at a point distal to the site of motor axon isolation and also on the anterior pectoralis nerve. By activating the intact nerves we could monitor the synapses formed by the nonisolated motor axons and also assess the frequency with which the isolated motor axon contributed to polyneuronally innervated junctions. It was sometimes impossible to record from one of the endplates on a fiber, however, since some fibers lay beneath other fibers for part of their lengths. The large motor unit's innervation pattern depicted here illustrates the tendency of a large motor unit neuron to contact its fibers at both endplate sites over much of its field, while often contacting only one of each fiber's available endplates at the edges of its field. In this experiment, for instance, the fibers on the right side of the field were only contacted by the isolated neuron at
heir lateral endplate sites. Fig. 2 also shows, however, that even in the center of the large unit's field some fibers were heteroneuronally innervated or had one polyneuronally innervated junction. Overall, the incidence of m o n o n e u r o n a l innervation of distant endplates among large motor unit fibers was 66% ; 36 of the 57 fibers (in the five preparations) studied were contacted at both endplate sites by the isolated axon. (While we did observe instances of m o n o n e u ronal innervation in smaller units, too few small (n = 3) and intermediate (n = 2) motor units and fibers were studied to assess reliably the incidence of m o n o n e u r o n a l innervation among the smaller units.) Two factors that are probably important in generating the observed pattern of m o n o n e u r o n a l innervation in large units (Fig. 2) are the limited a m o u n t of overlap between the different motor unit classes and the topographical shifting of large units relative to one another. Taken together, these factors help explain the high incidence of m o n o n e u r o n a l innervation in the center of large motor unit fields and its
357 lowered incidence at the b o r d e r s of large units; i.e. in regions of the muscle where two large units adjoin. The pattern of synaptic connections that we observed may yield insights into the functional organization of the pectoralis muscle. The spatial localization of m o t o r units and the high incidence of mononeuronal innervation of muscle fibers imply that activation of each m o t o r unit generates a different vector of force. F u r t h e r m o r e , the regular intercalation of different sized m o t o r units allows for the gradual recruitment of tension in each section of the muscle 10. O u r findings raise the question of w h e t h e r there exists an analogous anatomical organization of m o t o neurons in the premuscular nerve trunk and/or the ventral horn of the spinal cord. Questions relating to the origins of hetero- and m o n o n e u r o n a l innervation can p r o b a b l y be best ans-, wered in a d e v e l o p m e n t a l context. H o w e v e r , it is of interest to briefly consider what mechanisms may be responsible for the segregation of the different m o t o r
unit classes. Some evidence2,13 suggests that there is a
1 Bennett, M. R. and Lavidis, N., Development of the topographical projections of motoneurones to an amphibian muscle accompanies motoneurone death, Develop. Brain Res., 2 (1981) 448-452. 2 Bennett, M. R. and Pettigrew, A. G., The formation of synapses in amphibian striated muscle during development, J. Physiol. (Lond.), 252 (1975) 203-239. 3 Burke, R. E. and Edgerton, V. R., Motor unit properties and selective involvement in movement, Exercises and Sport Sciences Rev., 3 (1975) 31-81. 4 Dennis, M. J. and Harris, A. J., Transient inability of neonatal rat motoneurons to reinnervate muscle, Develop. Biol., 74 (1980) 173-183. 5 Feng, T. P., Zhou, C. and Zhu, P., Transformation of ultrastructural type of fast-twitch muscle fibres after cross-innervation by tetrodotoxin-blocked slow muscle nerve, Scientia Sinica B, 25 (1982) 935-960. 6 Gordon, H., Postnatal Development of Motor Units in Rabbit and Rat Soleus Muscles, Ph. D. Thesis, California Institute of Technology, 1984. 7 Grinnell, A. D. and Trussell, L. O., Synaptic strength as a function of motor unit size in the normal frog sartorius, J. Physiol. (Lond.), 338 (1983) 221-241. 8 Haimann, C., Mallart, A., Tomas I Ferre, J. and Zilber Gachelin, N. F., Patterns of motor innervation in the pectoral muscle of adult Xenopus laevis: evidence for possible synaptic remodelling, J. Physiol. (Lond.), 310 (1981) 241-256. 9 Hebb, D. O., Organization of Behaviour, John Wiley and Sons, New York, 1949. 10 Henneman, E. and Olson, C. B., Relations between struc-
ture and function in the design of skeletal muscles, J. Neurophysiol., 28 (1965) 581-598. 11 L~innergren, J. and Smith, R. S., Types of muscle fibres in toad skeletal muscle, Acta physiol, scand., 68 (1966) 263-274. 12 Lichtman, J. W. and Wilkinson, R. S., Repeating pattern of three fiber types in a single-fiber-thick muscle of the garter snake, Soc. Neurosci. Abstr., 9 (1983) 1059. 13 Nudell, B. M. and Grinnell, A. D., Regulation of synaptic position, size and strength in anuran skeletal muscle, J. Neurosci., 3 (1983) 161-176. 14 Phillips, W. D. and Bennett, M. R., Development of primary myotube types and their topographical distribution in wing muscles is independent of motor nerves, submitted. 15 Sayers, H. and Tonge, D. A., Persistence of extra-junctional sensitivity to acetylcholine after reinnervation by a foreign nerve in frog skeletal muscle, J. Physiol. (Lond.), 335 (1983) 569-575. 16 Smith, R. S. and Ovalle, W. K., Varieties of fast and slow extrafusal muscle fibres in amphibian hind limb muscles, J. Anat., 116 (1973) 1-24. 17 Thompson, W. J., Sutton, L. A. and Riley, D. A., Fibre type composition of single motor units during synapse elimination in neonatal rat soleus muscle, Nature (Lond.), 309 (1984) 709-711. 18 Trussell, L. O., The Regulation of Synaptic Strength in Motor Units of the Frog, Ph.D. Thesis, University of California at Los Angeles, 1983. 19 Trussell, L. O. and Grinnell, A. D., The regulation of synaptic strength within motor units of the frog cutaneous pectoris muscle, J. Neurosci., in press (1984).
sequential addition of endplates on amphibian twitch fibers as they increase in size. It could be that the neuron which gains control of the initial endplate site determines the subsequent activity or trophic demands of the fiber, and that neurons with different activity patterns 9 or trophic characteristics 5,15 would be at a relative disadvantage in establishing themselves at a second, distant e n d p l a t e site. Alternatively, embryonic muscle fibers m a y differ intrinsically in their properties6,12,14,17, and only accept m o t o n e u rons of a suitable type. W e would like to thank Drs. I. Chow, A. J. D ' A l o n zo, S. Hagiwara, A . H e r r e r a , S. H u t t n e r , F. Knight, S. Spector, and L. Trussell for p r o v i d i n g helpful criti-. cisms of this manuscript. W e also thank Frances Knight for expert technical assistance. This w o r k was supported by grants from N I H (NS06232) and the National Science F o u n d a t i o n (BNS83-2566).
353
BRE 20689
Specificity of innervation among Xenopustwitch muscle fibers BRUCE NUDELL and ALAN D. GRINNELL
Jerry Lewis Neuromuscular Research Center, UCLA, Los Angeles, CA 90024 (U.S.A.) (Accepted October 30th, 1984)
Key words: synapse - - specificity - - motoneuron - - motor unit - - neuromuscular - - Xenopus
More than 50% of the Xenopus pectoralis twitch muscle fibers with two distant endplates are innervated at both sites by the same neuron. This study indicates that there are three separable twitch motor unit types in this muscle which show very little overlap in innervation. This study also shows that each motor unit is topographically localized and that similar type units are shifted relative to one another. It is concluded that these two factors may contribute importantly to the observed high incidence of mononeuronal innervation. In an earlier paper 13 we reported that over 50% of the Xenopus pectoralis muscle fibers with two distant endplates were innervated at both sites by branches of the same motoneuron (mononeuronal innervation). It seemed unlikely that random innervation of muscle fibers would result in so much mononeuronal innervation, since it has been reported that 50 motoneurons innervate this muscle of approximately 1000 fibers 8. Feeling that some selective influences must underly this ordered pattern, we have extended our study to examine carefully the innervation pattern of individual motoneurons within the pectoralis muscle. We find that the muscle fibers receiving suprathreshold innervation from a given m o t o n e u r o n are of similar size (input resistance), and that three classes of motor units can be distinguished on this basis. We also find that motor units occur in spatially restricted regions of the muscle. While each motor unit class is represented throughout the entire field of the posterior pectoralis nerve (see below), motor units of the same class are topographically shifted relative to one another with overlap occurring primarily at their common borders. These studies suggest that the spatial segregation of the synaptic projection fields of similar type motoneurons and the lack of overlap of innervation between neurons innervating different size fibers both contribute importantly to the observed high incidence of mononeuronal innervation.
The Xenopus pectoralis is a triangular, fiat muscle lying between the sternum and the shoulder, innervated by two nerves which enter the muscle at its anterior and posterior bordersS. The posterior nerve usually innervates about two thirds of the surface fibers in this muscle. For that reason we restricted our studies to axons contained in this nerve. In young adult animals (1.75-2 in. in body length), approximately 90% of the fibers are innervated at two sites separated by 2 0 - 4 0 % of the fiber's length 13. In the present experiments, individual motor axons were mechanically teased from the main nerve trunk and stimulated using a small suction pipette. (Criteria for single motor axon isolation were visual appearance at 100 × magnification in the light microscope and the all-or-none recruitment of motor unit tension upon reaching a threshold stimulation intensity.) Action potentials or endplate potentials (EPPs) were monitored in the muscle fibers, using methods employed in our earlier study 13. T h e superficial fibers of this muscle vary widely in size - - showing a 10-12-fold range in input resistance. However, all can generate action potentials and therefore are classifiable as twitch fibers. Early in these experiments, it became apparent that any given motor axon provides suprathreshold innervation selectively to fibers of similar size, covering only a fraction of the full range of fiber sizes. On the basis
Correspondence: B. Nudell, Jerry Lewis Neuromuscular Research Center, UCLA, Los Angeles, CA 90024, U.S.A. 0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Diagrammatic representations of the fields of isolated single motor units in different pectoralis muscle preparations of similar size which were bathed in normal Ringer solution. Adjacent superficial muscle fibers were impaled with microelectrodes to measure their input resistance and to see whether an action potential (+), EPP (©), or no response (O) was elicited upon single stimulation of the isolated axon. If an action potential did not occur in a fiber, but if evidence for a subthreshold synapse was obtained, a recording electrode was moved to the site which showed the largest EPP and that value is given below. As preliminary recordings were generally made just central to the medial endplate site, it is possible that some very weak lateral junctions which produced small, subthreshold EPPs escaped our detection. In all the experiments presented in this figure, no other surface fibers were activated (showed action potentials) in response to stimulation of the isolated axon. The top panels of this figure show data from three muscles of similar size and the bottom panels present data from three smaller muscles of equivalent size. A 1 and A2: two large motor units as defined by the fact that they consisted of the largest muscle fibers in their muscles. (Thirty fibers represent about 25% of the muscle surface.) B 1 and B2: two intermediate units. In B 1 fibers number 10 and 15 showed 20 and 15 mV EPPs, respectively. In B 2 fiber number 11 showed a 20 mV EPP. C l and C2: two small units. In C2, two fibers belonging to the isolated unit lay at an appreciable distance (separated by 12 fibers) from the nearest other member of the unit and were well within the territory controlled by the anterior pectoralis nerve. Input resistance measurements were not made for the fibers lying between fiber number 29 and the two members of the unit in question. Fiber number 42 showed a 10 mV EPP.
of comparison of isolated motor unit properties in similar sized muscles, such as those illustrated in Fig. 1, we feel that three largely non-overlapping motor unit classes could account for the full range of fiber sizes exhibited in each muscle. For the purpose of this paper therefore, we will refer to motor units as 'large', 'intermediate' or 'small', here defined by the size of the muscle fibers innervated (exhibiting either
EPPs or action potentials). Although we cannot rigorously exclude the possibility that there is a continuum in motor unit types, these data suggest a correlation with the finding by L~innergren and Smith 11 of three distinct twitch fiber types in the Xenopus iliofibularis muscle, characterizable on the basis of their size, biochemistry and physiological performance. Our findings thus support the prediction of Smith and
355 Ovalle 16 that there are three distinct twitch fiber motor unit types in Xenopus. A precise determination of the amount of synaptic overlap between the different classes of motor unit can probably only be achieved by simultaneously examining different type units in the same regio n (see below) of a single muscle. Nevertheless, we feel that the data presented above strongly suggest that neurons innervating fibers of a given size generallydo not innervate fibers of a distinctly different size category in a suprathreshold manner. Occasionally, however, we did find evidence of neurons projecting synaptic inputs to 'inappropriate' sized muscle fibers. These inputs were often subthreshold. In Fig. 1B 1 for instance, fibers number 3 and 15, both of which were innervated by the isolated intermediate motor unit neuron, were large enough to also be part of a large motor unit (see Fig. 1A1). Similarly, in Fig. 1B 2 and 1C2, the isolated intermediate and small motor unit neurons formed subthreshold synapses on relatively large fibers (fibers number 11 and 42, respectively) which probably also belonged to motor units of different size classes. This degree of uniformity in muscle fiber size within a given motor unit might be expected in view of the reported similarity of fibers innervated by individual motoneurons in mammalian muscle 3. However, it has not been shown in other anuran muscles, and in fact is not the case in the frog cutaneous pectoris, where single motor units may include fibers varying by up to 5-fold in input resistance 19. Motor units also displayed a degree of spatial consolidation not previously reported in anuran muscle. This was shown by identifying those fibers in unblocked preparations which exhibited an action potential or EPP in response to the stimulation of a single motor axon. In each of these experiments all of the surface muscle fibers were sampled. The pattern of innervation shown in Fig. 1A was typical of that exhibited by the eight large motor units that we studied in this manner. Within a limited region (25-35%) of the muscle surface a single large motor unit neuron activates nearly all of the large muscle fibers (those fibers in the lowest input resistance category). Outside this region are fibers of similar size which are not members of the isolated unit but rather presumably belong to other large motor units. Virtually all of the fibers driven by an individual large motor unit axon
were found close to one another and were not separated by intervening large fibers driven exclusively by other large motor unit neurons. Occasionally, however, we found one or two fibers in a unit that were located apart from the other members of the unit and which were well within the domain of an adjacent large motor unit. It was not possible to ascertain the degree to which the large units we studied were restricted to the superficial layer of the muscle. We did find some evidence, however, for the spatial segregation of large units within the vertical plane of the muscle. We often isolated what appeared to be large motor unit axons which generated a strong twitch in the muscle but which did not activate any surface fibers. Comparable localization of projections has been shown in mammalian intercostal muscles 4, and regional localization of different segmental nerve fields has been shown in the frog gluteal muscle 1. However, the sharply restricted localization of single motor units that we find has not been reported in other anuran muscle, and is not the case in the frog cutaneous pectoris TMor sartorius muscles (Trussell, unpublished observations). (A cautionary note should be interjected here, namely that our techniques only allowed us to study the pattern of motor unit organization on the superficial face of the muscle. Thus we cannot definitely state that the topographic consolidation of motor units which is evident on the muscle surface is also characteristic of the underlying fiber layers.) Motor units comprised of small and intermediate sized fibers also appear to be spatially localized. Fig. 1B and C show examples of the five units of each type studied. From these figures it can also be seen that the small and intermediate sized motor units studied contained fewer as well as smaller muscle fibers than the large units. In order to get a more precise picture of the innervation pattern of individual motoneurons, we also examined as many of the synaptic inputs as possible in surface fibers in the region innervated by isolated motor axons. After isolating the axon, the muscle was curarized until twitching was suppressed, using concentrations ranging from 2/~M for small motor units to 8 ~M for large units. The relative resistance of large units to curare block reflects the fact that their synapses have higher safety factors than do smaller unitsT, 19.
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22 2324 25 26 27 28 29 30 MEDIAL
FIBER
Fig. 2. Summarizes diagrammatically the innervation source and EPP amplitude (in mV) of all of the accessible endplates on adjacent fibers lying within the field of an isolated large motor unit neuron in a single preparation. The muscle was bathed in a Ringer solution containing 8 pM curare. The EPPs generated by the isolated neuron are labeled I, and those generated by unidentified neurones are labeled U. Some fibers lay beneath the superficial plane of the muscle for part of their length, and for that reason, one of their endplate regions (denoted by []) could not be studied. Several very small fibers (denoted by []) failed to show any synaptic activity; their junction(s) (about 20% of the small motor unit fibers have only one endplate (Nudell, unpublished data)) appeared to be totally blocked by the curare. Fibers belonging to the isolated unit and from which data were obtained for both endplates were either mononeuronally (mono) or heteroneuronally (hetero) innervated. Fiber number 23 had 3 endplates, the lateral-most of which was not accessible to local recording.
Fig. 2 presents characteristic data from 30 adjacent muscle fibers which spanned the field of an isolated large motor unit neuron. In this experimental protocol, additional stimulating electrodes were placed on the posterior nerve trunk at a point distal to the site of motor axon isolation and also on the anterior pectoralis nerve. By activating the intact nerves we could monitor the synapses formed by the nonisolated motor axons and also assess the frequency with which the isolated motor axon contributed to polyneuronally innervated junctions. It was sometimes impossible to record from one of the endplates on a fiber, however, since some fibers lay beneath other fibers for part of their lengths. The large motor unit's innervation pattern depicted here illustrates the tendency of a large motor unit neuron to contact its fibers at both endplate sites over much of its field, while often contacting only one of each fiber's available endplates at the edges of its field. In this experiment, for instance, the fibers on the right side of the field were only contacted by the isolated neuron at
heir lateral endplate sites. Fig. 2 also shows, however, that even in the center of the large unit's field some fibers were heteroneuronally innervated or had one polyneuronally innervated junction. Overall, the incidence of m o n o n e u r o n a l innervation of distant endplates among large motor unit fibers was 66% ; 36 of the 57 fibers (in the five preparations) studied were contacted at both endplate sites by the isolated axon. (While we did observe instances of m o n o n e u ronal innervation in smaller units, too few small (n = 3) and intermediate (n = 2) motor units and fibers were studied to assess reliably the incidence of m o n o n e u r o n a l innervation among the smaller units.) Two factors that are probably important in generating the observed pattern of m o n o n e u r o n a l innervation in large units (Fig. 2) are the limited a m o u n t of overlap between the different motor unit classes and the topographical shifting of large units relative to one another. Taken together, these factors help explain the high incidence of m o n o n e u r o n a l innervation in the center of large motor unit fields and its
357 lowered incidence at the b o r d e r s of large units; i.e. in regions of the muscle where two large units adjoin. The pattern of synaptic connections that we observed may yield insights into the functional organization of the pectoralis muscle. The spatial localization of m o t o r units and the high incidence of mononeuronal innervation of muscle fibers imply that activation of each m o t o r unit generates a different vector of force. F u r t h e r m o r e , the regular intercalation of different sized m o t o r units allows for the gradual recruitment of tension in each section of the muscle 10. O u r findings raise the question of w h e t h e r there exists an analogous anatomical organization of m o t o neurons in the premuscular nerve trunk and/or the ventral horn of the spinal cord. Questions relating to the origins of hetero- and m o n o n e u r o n a l innervation can p r o b a b l y be best ans-, wered in a d e v e l o p m e n t a l context. H o w e v e r , it is of interest to briefly consider what mechanisms may be responsible for the segregation of the different m o t o r
unit classes. Some evidence2,13 suggests that there is a
1 Bennett, M. R. and Lavidis, N., Development of the topographical projections of motoneurones to an amphibian muscle accompanies motoneurone death, Develop. Brain Res., 2 (1981) 448-452. 2 Bennett, M. R. and Pettigrew, A. G., The formation of synapses in amphibian striated muscle during development, J. Physiol. (Lond.), 252 (1975) 203-239. 3 Burke, R. E. and Edgerton, V. R., Motor unit properties and selective involvement in movement, Exercises and Sport Sciences Rev., 3 (1975) 31-81. 4 Dennis, M. J. and Harris, A. J., Transient inability of neonatal rat motoneurons to reinnervate muscle, Develop. Biol., 74 (1980) 173-183. 5 Feng, T. P., Zhou, C. and Zhu, P., Transformation of ultrastructural type of fast-twitch muscle fibres after cross-innervation by tetrodotoxin-blocked slow muscle nerve, Scientia Sinica B, 25 (1982) 935-960. 6 Gordon, H., Postnatal Development of Motor Units in Rabbit and Rat Soleus Muscles, Ph. D. Thesis, California Institute of Technology, 1984. 7 Grinnell, A. D. and Trussell, L. O., Synaptic strength as a function of motor unit size in the normal frog sartorius, J. Physiol. (Lond.), 338 (1983) 221-241. 8 Haimann, C., Mallart, A., Tomas I Ferre, J. and Zilber Gachelin, N. F., Patterns of motor innervation in the pectoral muscle of adult Xenopus laevis: evidence for possible synaptic remodelling, J. Physiol. (Lond.), 310 (1981) 241-256. 9 Hebb, D. O., Organization of Behaviour, John Wiley and Sons, New York, 1949. 10 Henneman, E. and Olson, C. B., Relations between struc-
ture and function in the design of skeletal muscles, J. Neurophysiol., 28 (1965) 581-598. 11 L~innergren, J. and Smith, R. S., Types of muscle fibres in toad skeletal muscle, Acta physiol, scand., 68 (1966) 263-274. 12 Lichtman, J. W. and Wilkinson, R. S., Repeating pattern of three fiber types in a single-fiber-thick muscle of the garter snake, Soc. Neurosci. Abstr., 9 (1983) 1059. 13 Nudell, B. M. and Grinnell, A. D., Regulation of synaptic position, size and strength in anuran skeletal muscle, J. Neurosci., 3 (1983) 161-176. 14 Phillips, W. D. and Bennett, M. R., Development of primary myotube types and their topographical distribution in wing muscles is independent of motor nerves, submitted. 15 Sayers, H. and Tonge, D. A., Persistence of extra-junctional sensitivity to acetylcholine after reinnervation by a foreign nerve in frog skeletal muscle, J. Physiol. (Lond.), 335 (1983) 569-575. 16 Smith, R. S. and Ovalle, W. K., Varieties of fast and slow extrafusal muscle fibres in amphibian hind limb muscles, J. Anat., 116 (1973) 1-24. 17 Thompson, W. J., Sutton, L. A. and Riley, D. A., Fibre type composition of single motor units during synapse elimination in neonatal rat soleus muscle, Nature (Lond.), 309 (1984) 709-711. 18 Trussell, L. O., The Regulation of Synaptic Strength in Motor Units of the Frog, Ph.D. Thesis, University of California at Los Angeles, 1983. 19 Trussell, L. O. and Grinnell, A. D., The regulation of synaptic strength within motor units of the frog cutaneous pectoris muscle, J. Neurosci., in press (1984).
sequential addition of endplates on amphibian twitch fibers as they increase in size. It could be that the neuron which gains control of the initial endplate site determines the subsequent activity or trophic demands of the fiber, and that neurons with different activity patterns 9 or trophic characteristics 5,15 would be at a relative disadvantage in establishing themselves at a second, distant e n d p l a t e site. Alternatively, embryonic muscle fibers m a y differ intrinsically in their properties6,12,14,17, and only accept m o t o n e u rons of a suitable type. W e would like to thank Drs. I. Chow, A. J. D ' A l o n zo, S. Hagiwara, A . H e r r e r a , S. H u t t n e r , F. Knight, S. Spector, and L. Trussell for p r o v i d i n g helpful criti-. cisms of this manuscript. W e also thank Frances Knight for expert technical assistance. This w o r k was supported by grants from N I H (NS06232) and the National Science F o u n d a t i o n (BNS83-2566).