THE JOURNAL OF COMPARATIVE NEUROLOGY 301:104-113 (1990)

Development of Compartmentalized hervation of the Rat Gluteus Maximus ARTHUR WM.ENGLISH Department of Anatomy and Cell Biology, Emory University, Atlanta, Georgia 30322

ABSTRACT The innervation territories of the nerves to the gluteus maximus muscle were investigated in neonatal rats by using evoked electromyographic mapping techniques. Similar methods were employed to determine the terminal fields of individual motoneurons. Both at birth and after the period of neuromuscular synapse elimination, rat gluteus maximus is partitioned by its two muscle nerves. Each of these partitions is further divided into neuromuscular compartments by the primary branches of the muscle nerves. The terminal fields of individual motoneurons are about as well localized in early neonates as in 26 day-old animals. Intracellular records from gluteal muscle cells reveal that some myotubes in a narrow region near the center of the muscle receive inputs from both of the muscle nerves during the period of polyneuronal innervation. Such cross-compartmental innervation could be demonstrated only until the eighth postnatal day, even though polyneuronally innervated muscle cells were found as late as postnatal day 18. Analysis of the trajectories of individual axons filled with horseradish peroxidase indicates that they branch into only one primary branch of the nerves to gluteus maximus, both in newborn and 14 day-old pups. The innervation of gluteus maximus at birth is essentially restricted to neuromuscular compartments. Synapse elimination probably plays a minor role in establishing neuromuscular compartments. Key words: electrophysiology,synapse elimination, peripheral nervous system

A number of recent studies have shown that mammalian muscles are partitioned with respect to both their motor and sensory innervation. In the ankle extensor muscles of both cats and rats, these partitions are organized about the primary branches of the respective muscle nerves and are known as neuromuscular compartments (English and Letbetter, '82; English et al., '85). Each of the first order branches of a nerve as it enters a muscle innervates fibers in a distinct region of the muscle and contains a unique group of motor axons. That is, branches of individual motor axons course exclusively in only one primary muscle nerve branch (English and Weeks, '84; Janun and English, '86). At the time of birth, muscle fibers receive synaptic inputs from more than one motoneuron. During the immediate postnatal period, all but one of these synapses is eliminated and an adult type of innervation is established. It has been postulated that this postnatal elimination of synaptic inputs could shape the adult pattern of innervation from a less precise fetal pattern (Brown and Booth, '83; Bennett and Lavidis, '84). In a previous report from this laboratory (Donahue and English, '87) using the compartmentalized rat lateral gastrocnemius (LG) as a model, it was shown that the pattern of innervation of neuromuscular compartments at birth is essentially similar to that observed in O

1990 WILEY-LISS, INC.

adult animals. Compartmentalized innervation is not shaped by synapse elimination, although, within a compartment, some shaping may occur (Bennett and Ho, '88). The rat LG is organized into four neuromuscular compartments, each of which contains, as at least part of its boundary, one of the tendons of origin or insertion that divide LG into heads (Donahue and English, '87). Thus it is possible that these tendons might serve as either physical partitions or as axon guidance cues during development, resulting in the restriction of axons to a particular compartment of LG. In a compartmentalized muscle without such an extensive connective tissue framework, a different type of neonatal innervation might be found. The purpose of this study was to determine the neonatal and adult innervation patterns of one such muscle, the rat gluteus maximus, and to compare its pattern of innervation with that known already for LG. The innervation of gluteus maximus was found to be compartment-specific, even in the first postnatal week, suggesting that postnatal synapse elimination plays at best a minor role in establishing compartmental boundaries in gluteus maximus. A preliminary report has been made (English, '86). Accepted July 23,1990

INNERVATION OF GLUTEUS MAxDlUS

METHODS Electrophysiology All experiments were performed on rats of the Fischer 344 strain. Ages are given in postnatal (P) days, with the day of birth being designated as PO. In animals aged < P4 anesthesia was induced by 10-15 minutes of hypothermia (0°C). Older animals were anesthetized with pentobarbital sodium (90 mgkg, IP). Animals were perfused transcardially with oxygenated Ringer's solution (Rees, '781, quickly eviscerated, decapitated, and transferred to a dish of oxygenated Ringer's solution. The left gluteus maximus muscle was then dissected free from its spinal origin and, along with most of the adjacent tensor fasciae latae and a part of the anterior portion of biceps femoris, it was removed from its insertion onto the gluteal tubercle of the femur. In experiments in which the innervation territories of the nerves to gluteus maximus were explored, the inferior gluteal nerve (IGN) was cut as it branched from the sciatic nerve just caudal to the pyriformis muscle and the superior gluteal nerve (SGN) was cut as it passed beneath the inferior and anterior margins of the gluteus medius muscle. The muscle and nerves were then transferred to a plastic culture dish lined with Sylgard (Dow-Corning) and perfused with oxygenated Ringer's solution for the duration of the experiment. The muscle was then flattened, stretched, and pinned over a specially constructed multiple lead electromyographic (EMG) patch electrode. The patch electrode was made from a sheet of thin, Dacron-reinforced Silastic film (Dow-Corning) into which had been sewn the bared ends of 12 Teflon-coated, multi-stranded, stainless steel wires (see English, '84). The 12 leads were spaced in two rows of six and individual wires were separated by approximately 500 pM. One side of the patch was insulated by means of Medical Adhesive A (Dow-Corning). The electrode leads were connected to a switching apparatus such that bipolar differential recordings could be made from six positions along the patch. A diagram of the patch used is shown in Figure 1A. The muscle was always positioned so that the entry point of the IGN lay approximately over the left-most electrode pair. The muscle was stretched such that the entry of the SGN lay approximately over the right-most electrode pair and the direction of the muscle fibers ran parallel to the pairs of electrodes (Fig. 1B). The positions of the electrode leads relative to the entry points of the two nerves was verified by transillumination. In small animals, the muscle had to be stretched extensively to achieve this configuration. In older animals less stretching was employed. In all cases the stretched muscle was pinned extensively, at 8-10 places along its borders, in order that whole muscle activation would produce as little movement as possible. The pattern of branching of the two nerves was drawn onto a standard outline of the muscle, similar to Figure 2B. The IGN and SGN were then drawn into separate suction electrodes and EMG activity was recorded at each of the six sites in

Abbreviations EDL EMG EPP HRP 1GK LG SGN

extensor digitorum longus electromyographic end-plate potential horseradish peroxidase inferior gluteal nerve lateral gastrocnemius superior gluteal nerve

105 response to supramaximal stimulation of the two nerves. Records were obtained by using a laboratory computer system and stored on a magnetic disk for later analysis. Myoelectric signals were recorded with high input impedance (l0"CU differential amplifiers with a high common mode rejection (80 dB at 1,000 Hz) at a gain of 1,000. The signals were band pass filtered (50 Hz-5 kHz). By using signal averaging techniques to acquire the evoked EMG potentials, background noise could be kept very small ( < 10 pV, peak-to-peak), thus enabling detection of very small potentials. Experiments were performed on 14 animals ranging in age from PO through P5 and on nine animals aged P13 to P30.

Analysisofsingleunit innervationterritories In nine pups ages PO-P5 and eight animals aged P13P26, the innervation territories of single motor units were examined. The procedure for anesthesia and initial preparation was the same as described above. A lumbar laminectomy was performed and the left L4 and L5 dorsal roots were cut near their exit from the spinal ganglia. The ventral roots of these segments were cut near their exit from the spinal cord and then removed along with the spinal ganglia and sciatic nerve. The left gluteus maximus was then dissected free, as described above, and the IGN and SGN were dissected free of surrounding tissues with small glass probes, leaving them in continuity with the dissected roots. The roots, nerves, and muscle were mounted in the dish as described above. Individual motor units were isolated from split ventral rootlets and their innervation territories were determined by recording their evoked EMG activity at different positions along the patch. Units were isolated by visual observation of twitching as well as by the waveform of the evoked EMG activity. A unit was considered to be isolated if a clear all-or-none contraction was noted at low stimulus strengths ( < 10 FA), if only one EMG waveform was noted (see below), and if no indication of recruitment of a second motor unit was noted if the stimulus strength was raised to three times that needed to evoke the first visible twitch. If these criteria were not met, the rootlet was split by using fine glass instruments, and the testing procedure was repeated. If the criteria for isolation could not be met in the smallest dissectable rootlet, then that rootlet was not studied further.

IntracellularRecording In 34 animals aged P3-Pl8, muscles were removed and prepared for stimulation of the SGN and IGN as described above. After the threshold for activation of the two nerves had been established, the Ringer's solution in the bath was removed and replaced by a solution of 2 M formamide in oxygenated Ringer's (Herrera, '84).This paralytic agent was left in the bath until the evoked muscle contractions were notably weakened. This routinely took less than 10 minutes. The 2 M formamide solution was then slowly washed from the bath by replacing the normal Ringer's perfusing medium with a more dilute (0.05 M) formamide solution in oxygenated Ringer's. This modification of Herrera's ('84) procedure has been used effectively for intracellular recording experiments in rat LG, where it was shown that more synaptic inputs could be detected than when using curare (Donahue and English, '89a). Muscle cells were then impaled with glass micropipettes containing 3 M KC1. Only cells with initial resting membrane potentials of

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Fig. 1. A: Schematic diagram of the patch-type EMG electrode used in this study. I t is constructed from Dacron-reinforced Silastic film into which 12 fine wires were sewn, in two rows of six. Each of the electrode wires is separated by approximately 500 km. In each experiment, the gluteus maximus muscle is placed over the patch with its fascicles

oriented parallel to the rows of electrode surfaces as shown. Potentials were obtained by recording differentially between the top and bottom wire of each pair. B The position of the gluteus maximus muscle over the patch is shown diagrammatically. The positions of the superior (SGN) and inferior (IGN) gluteal nerves are indicated.

at least 40 mV were considered for analysis. It is likely that stretching of the muscle results in depolarization of its fibers (Laskowski and Sanes, '87). In unstretched muscles, larger membrane potentials were recorded (data not shown). However, such preparations were mechanically unstable and not suitable for the extensive intracellular recording needed for this study. The responses of each cell to graded electrical stimulation of the SGN and the IGN were observed. Nerve stimulation normally resulted in an end-plate potential (EPP)recorded from the muscle cell. In polyinnervated muscle cells, each stepwise increase in EPP amplitude that occurred at a clear stimulus threshold was taken as evidence of an additional input to the cell (Redfern, '70). In the case of each EPP recorded, the nerve stimulated and its strength were noted as well as the latency, rise time, and amplitude of the potential. At least 15 cells were analyzed in each muscle.

In 15 rat pups aged P2-P5 and in five animals aged P13-Pl6, the trajectories of axons in the IGN and SGN were followed by using the anterograde transport of horseradish peroxidase (HRP). After bilateral removal of gluteus maximus and its nerves according to the method described above, the muscles were pinned out in a dish of oxygenated Ringer's solution and the nerves were dissected free of excess connective tissue. Each nerve was packed with silicone grease (Dow-Corning)until it was completely coated except for its cut end. Care was taken to ensure that the entire circumference of the nerve was coated in grease so that the cut stump of the nerve would be isolated from the nearby muscle. The level in the Ringer's bath was then lowered until the stumps of the nerves were exposed. Small Gelfoam (Upjohn) pieces soaked in distilled water were

Analysisof axonaltrajedories

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by using a camera lucida. The drawings from different sections were superimposed, using the direction of the muscle fibers and the individual axons for alignment, such that the trajectory of the axons could be reconstructed.

RESULTS ArchitectureandnervebranchpattermofGM Fibers of gluteus maximus arise from the deep or medially directed surface of an aponeurosis attached to the iliac crest, the sacroiliac joint, and the sacral spinous processes as far caudad as the ischial tuberosity. At its caudal end this aponeurosis also serves as part of the origin of fibers of the anterior portion of biceps femoris. At its cranial end the aponeurosis serves as origin of the fibers of the tensor fasciae latae muscle, which blends imperceptibly into gluteus maximus. From this origin, fibers course distally to attach to the superficial or laterally directed surface of a tendon of insertion which is attached to the gluteal tubercle of the femur. Some of the more cranial fibers of gluteus maximus and all of tensor fasciae latae attach to the B superficial layer of deep fascia of the thigh, the fascia lata. Thus gluteus maximus contains no internal tendons and only short connective tissue sources of origin and insertion. The innervation of gluteus maximus can best be visualN ized in vitro after removing the muscle with its nerves and pinning it out with its deep or internal surface uppermost (Fig. 2B). The IGN enters the muscle at its caudal extreme, close to where it branches from the sciatic nerve, in the Fig. 2. The anatomy and architecture of the gluteus maximus is company of some large inferior gluteal blood vessels and the shown diagrammatically as the muscle appears in situ (A) when nerves supplying the proximal part of biceps femoris. The removed and placed in vitro (B). The pattern of branching of the pattern of branching of IGN is highly stereotyped from superior (SGN) and inferior (IGN) gluteal nerves is shown in B. animal to animal. Its two most caudal primary branches (Fig. 2B: a,b) course distally after the IGN enters the muscle, each running parallel to the caudal border of the placed on the exposed nerve stumps. After 10 minutes of muscle. The remaining branches (Fig. 2B: c-e) are found as soaking in water, the Gelfoam pieces were replaced with either distally or proximally directed branches of a main similar pieces soaked in a 50% solution of HRP in a 16 nerve trunk which runs almost directly craniad from its pg/ml solution of hyaluronidase in normal saline. The point of entry to the muscle. The SGN enters the muscle at its cranial end. Two hyaluronidase is thought to enhance the uptake of HRP by damaged axons (Weeksand English, '85). The HRP soaking distinct branches of the SGN were always noted: one was continued for at least 2 hours while the bath was coursing caudad to innervate the gluteus maximus and the replenished with oxygenated Ringer's solution such that other coursing rostrad to innervate the tensor fasciae latae the muscle and nerves were covered up to the level of the muscle. The branches of the part of the SGN innervating silicone grease. The Gelfoam was then removed and the gluteus maximus were also found to be highly stereotyped. tissue was left in the bath for another 12 hours. It was then One branch courses parallel to the cranial margin of the fixed in a solution of 4% paraformaldehyde and 0.1% muscle (Fig. 2B: i) and the remainder take either proximal glutaraldehyde in phosphate buffered saline (PBS) for 1 or distal paths from the caudal-directed course of the main hour at 4°C. The muscle was then transferred to a solution trunk of the SGN (Fig. 2B: f-h). of 20% sucrose in PBS, also at 4°C. The next day the tissue Innervation patterns of GM was mounted on a cryostat chuck and sectioned at 80 pm in a plane parallel to the direction of the muscle fibers (en The results of analysis of EMG potentials evoked from face). This plane was chosen to increase the likelihood of stimulation of the IGN and the SGN on the day of birth encountering long stretches of axons. The sections were (PO) and in an animal aged P14 are shown in Figure 3. It is processed for the demonstration of HRP by using a cobalt clear from this analysis that axons of the IGN innervate the intensified diaminobenzidine reaction, mounted on gelatin- caudal part of the muscle, whereas those of the SGN chrome alum coated slides, and allowed to dry overnight at innervate the cranial part of the muscle. However, the 4°C. In some cases the tissue was then processed for the territories of the two nerves overlap slightly, so that EMG demonstration of acetylcholinesterase (see, e.g., Donahue activity can be evoked in the center of the muscle by et al., '88). In other cases tissue sections were lightly stimulation of either the IGN or the SGN (Fig. 3A). In counterstained with neutral red. A small number (usually recordings made from younger (PO-P5) animals the num< 10)of axons were labelled in each of the nerves so that ber of sites from which activity could be recorded by each could be followed through serial sections for consider- stimulation of either nerve was larger (Fig. 3B) than in able distances. The trajectory of each well-labelledaxon was older animals. Both of the sets of records shown in Figure 3 studied at 500 x magnification and drawn on tracing paper were chosen because they display the maximum amount of

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Fig. 3. Panel A shows raw electromyographic (EMG) records obtained from the patch electrode shown in Figure 1.All records are from the gluteus maximus of a 14 day-old rat pup. The six rows of traces are from the six patch positions and correspond to locations from the caudal (top) to the rostral (bottom) parts of the muscle. The two columns of traces show recordings made from supramaximal stimulation of the inferior gluteal nerve (IGN) and the superior gluteal nerve (SGN). In the graph to the right, the same data are plotted as bars, each representing the value of the rectified and integrated EMG activity at one of the six different patch positions. The bars are scaled as a percentage of the largest EMG intensity recorded. Panel B shows similar dat.a in the same format from a rat pup on the day of birth.

this “overlap” in the areas from which recordings of both IGN and SGN evoked activity could be obtained. In many of the muscles examined, the amount of “overlap” encountered was less.

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Fig. 4. The evoked EMG activity recorded in response to stimulation of single gluteus maximus motor units in neonatal (PO-P4) rats is shown in the same format used in Figure 3. The data shown are typical of 39 units examined in animals aged < P5 and were chosen because they demonstrate the full range of localization of EMG potentials encountered.

Innervation territoriesofsjngle motorunits Representative patterns of EMG activity in gluteus maximus evoked by stimulation of single motor units are shown in Figure 4 for newborn (PO) rats and in Figure 5 for animals aged P26. Analysis of the distribution of evoked potential amplitudes at different patch positions shows that the gluteal muscle fibers innervated by single motoneurons are localized. Note that this localization of EMG potential amplitude is found both in muscles from early neonatal animals and from muscles of older animals.

h ~ l l u l a r r e c o ~ To establish the time course of postnatal synapse elimination in gluteus maximus, and to examine the specificity of

innervation of muscle cells in the area of overlap of the two nerves, intracellular recordings were made during graded stimulation of the SGN and the IGN in muscles from 34 animals aged P3-Pl8. The time course of postnatal synapse elimination in gluteus maximus is shown in Figure 6. Whether expressed as the proportion of recorded cells receiving more than one input or the average number of inputs per muscle cell, postnatal synapse elimination in gluteus maximus continues for at least 2 weeks. Whereas on P3, 90% of GM muscle cells (n = 3) receive more than one synaptic input, by P14 and older, only 8% of the sampled cells were polyneuronally innervated (n = 3). The small number of remaining polyneuronally innervated cells

INNERVATION OF GLUTEUS MAXIMUS

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Fig. 5. The evoked EMG activity recorded in response to stimulation of single gluteus maximus motor units in an adult (P26)rat is shown in the same format used in Figure 4. The data shown are typical of eight units examined in animals aged > P13.

at ages > P14 is not particularly unusual. Taxt et al. (’83) showed that a residual amount of such multiple innervation exists for sometime in rat soleus muscle fibers. This residual polyneuronal innervation is almost certainly not an artefact of the intracellular recording technique, as similar results have been obtained by using anatomical methods (Donahue et al., ’88).Thus the end of the period of polyneuronal innervation of skeletal muscle must be considered to be a gradual process. Although the results of evoked EMG recordings suggested that an area of overlap in innervation territories of the IGN and the SGN is greater in younger animals (see above), this was difficult to verify with intracellular recordings. In all animals studied, the region of gluteus maximus

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in which a single electrode penetration yielded either cells innervated by the SGN or the IGN was extremely narrow. The boundary between the two nerve zones was usually quite distinct. If the electrode was moved a small distance rostra1 or caudal to this zone of overlap, innervation by only one of the nerves was noted. This would suggest that the evoked EMG technique may actually overestimate the size of the innervation territories. Within the zone of “overlap,” some cells receiving inputs from both nerves were found routinely in animals younger than P8. An example of records from such a cell is shown in Figure 7. Since these cells receive inputs from axons

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Fig. 7. An intracellular recording from a single gluteus maximus muscle cell at age P3 shows single end-plate potentials obtained in response to stimulation of either the superior (SGN) or inferior (IGN) gluteal nerves. This cell represents a cross-compartmentally innervated muscle cell.

coursing in primary branches of different muscle nerves they are, by definition, cross-compartmentally innervated. In most experiments the region of gluteus maximus containing the boundary between the innervation territories of the IGN and the SGN was sampled extensively to look for such cells. The proportion of cross-compartmentally innervated cells in the zone varied from 5 to 40%. However, because generally more cells were sampled in the region of overlap than elsewhere, the significance of the proportion of crosscompartmentally innervated cells is questionable. No cells were found which received inputs from both nerves in animals older than P8.

Trajectoriesof axom in the IGN and SGN The results of experiments in which the trajectories of individual HRP-filled axons were re-constructed are illustrated in Figure 8. In nearly all cases, individual axons branched into only one primary muscle nerve branch. At each muscle nerve branch point, an axon either branched in a proximal or distal direction or continued along the course of the main nerve trunk. In only one instance (out of 42 cases studied in animals aged P14.

DISCUSSION Partitioningofrat gluteusmsudmus One of the principal findings of this study was that the rat gluteus maximus is partitioned into elements by the primary branches of its two muscle nerves. The analysis of EMG potentials recorded from different parts of gluteus maximus that were evoked from muscle nerve stimulation shows that its two nerves (IGN and SGN) innervated different territories. These two territories are not to be construed as different neuromuscular compartments, since they are territories innervated by whole nerves, not the primary branches of the nerve. It seems likely that they reflect the development of gluteus maximus from two

distinct anlagen, as has been proposed for human gluteus maximus muscles (Tichf and Grim, ’85). The analysis of the EMG activity evoked from stimulation of individual gluteus maximus motor units, along with the analysis of the trajectories of individual axons which had been filled with HRP, together lead to the conclusion that axons of the IGN and SGN innervate motoneurons with more restricted territories. Since these data are compatible with observations on the pattern of primary branching of the IGN and SGN, I would speculate that gluteus maximus is composed of distinct neuromuscular compartments. Although the precise anatomical boundaries between compartments could not be determined by using the electrophysiological techniques employed here, such a speculation would not require the fundamental organization of gluteus maximus to be different from the compartmentalized plan of other muscles (e.g.,Balice-Gordonand Thompson, ’88;English and Letbetter, ’82; English et al., ’85; Galvas and Gonyea, ’80; Hardman and Brown, ’87; Richmond et al., ’85). This conclusion is also compatible with earlier descriptions of the contraction regions of gluteus maximus motor units made from visual observations in adult animals (Brown and Booth, ’83) and the innervation of different gluteal regions by spatially distinct groups of motoneurons (Hardman and Brown, ’85).It is also similar to the topographic partitioning of the serratus anterior and diaphragm reported by Laskowski and Sanes (’87).

Development of partitionsin @ueuSmaximus A second major finding was that single motor units in gluteus maximus of all neonatal rats are largely restricted to one of its compartments. The same lines of evidence used to support the conclusion that gluteus maximus is compartmentalized in animals older that P15 were used to show that the innervation of neuromuscular compartments in gluteus maximus on PO is compartment-specific. The regions of gluteus maximus from which EMG potentials could be evoked from stimulating single motor units were localized in both young ( < P5) and older (P13-P26) neonates. At first this observation might seem at odds with results obtained by IGN and SGN stimulation. In young animals the regions of gluteus maximus from which EMG potentials could be recorded from both IGN and SGN stimulation were larger than in older animals (Fig. 3). However, the large amount of “overlap” noted with whole nerve stimulation in the smaller muscles may be more a matter of EMG recording artefacts than a true “overlap.” Despite using reasonably selective recording electrodes and high common mode rejection differential amplifiers, the number of sites from which significant EMG potentials can be recorded probably overestimates the extent of an innervation territory, as the results of intracellular recording suggest (see below). Electromyographic potentials can be passively conducted from their source to be recorded at considerable distances (English and Weeks, ’89; Gydikov et al., ’82). This problem of EMG “cross-talk’’ is undeniably greater with whole-nerve stimulation than with stimulation of single units. Further, it might also be a greater problem in smaller muscles, where the volume through which potentials conduct is smaller. Smaller muscles were stretched more extensively over the recording patch than larger muscles, so that motor unit territories might be expected to appear more extensive when analyzed from evoked EMG activity. Such more extensive territories were observed when all gluteus maximus motor units are synchronously active, as

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Fig. 8. A reconstruction of the trajectories of HRP-filled axons in the nerve to gluteus maximus for neonatal (P4) and older (P14) rats. Axons coursing in individual primary muscle nerve branches are designated by lowercase letters corresponding to the patterns shown in Figure 2. In the P4 animal, one axon was found which branched into more than one muscle nerve branch. Although no labelled axons were found coursing into some muscle nerve branches in the P4 animal, those branches were nevertheless present.

during IGN or SGN stimulation, but not during activation of single units. If these results are considered along with the results of intracellular recording experiments and the analysis of the trajectories of HRP-labelled axons, then despite the limitations of the EMG recording technique, one could conclude that the innervation of GM at birth is essentially compartment-specific. At birth, all mammalian muscle fibers receive synaptic inputs from more than one motoneuron (e.g., Redfern, '70). During the next 3 weeks of postnatal life, all but one of these inputs disappears, leaving virtually all muscle fibers in an adult, singly innervated state. One role that might be played by the process of synapse elimination is in the

shaping of the compartmentalized pattern of innervation from some less precise neonatal innervation pattern. If this were the case, then synapses would be lost in a compartmentspecific manner, since they would be eliminated selectively from aberrantly innervated compartments. This notion has received support from studies on gluteus maximus (Brown and Booth, '83) and lateral gastrocnemius (Bennett and Lavidis, '84) which have suggested that individual motoneurons reduce their terminal field sizes during synapse elimination. In a recent study of the developmentof compartmentalized innervation of lateral gastrocnemius (Donahue and English, '871, the existence of only a small number of cross-compartmental connections could be demonstrated.

112 Further, none of these inputs could be demonstrated after P5, even though in LG synapse elimination continues until at least P18. Thus it is unlikely that significant numbers of cross-compartmental projection "errors" are corrected by synapse elimination in lateral gastrocnemius. A similar conclusion has been reached by Balice-Gordon and Thompson ('88) from studies of the extensor digitorum longus muscle (EDL). Rat LG contains an extensive system of internal tendons which might conceivably contribute to compartmentspecific innervation by providing a favorable substrate for axon elongation, or even by acting as a barrier to growth. In gluteus maximus, however, no such tendons are found, suggesting that the neonatal compartment-specific pattern of innervation of gluteus maximus must be established independently of the arrangement of tendons. This conclusion is at odds with that of Brown and Booth ('83), who used the Same muscle, probably because of differences in the techniques used. Those authors isolated single motor units from dissected ventral rootlets and estimated their innervation territory in gluteus maximus by monitoring muscle contraction visually. However, it is impossible to be certain that even the smallest dissected rootlet contains only a single active motor axon, a difficulty the authors acknowledge. In the EMG studies described above, stimulation of very small rootlets often evoked more than one EMG potential or single waveforms that appeared to be two or superimposed potentials, even at very low stimulus strengths. The part of gluteus maximus observed to contract during stimulation of these rootlets was often large. In older ( > pi31 animals, in which myelination of ax on^ is advanced, such multiple unit EMG records were encountered less frequently, though still on occasion. of the large contraction It is possible, therefore, that regions reported by Brown and Booth ('831, but not observed in the present study, may have resulted from stimulation of rootlets which contained more than one gluteal motor axon. Motor units with large innervation territories were reported for about 60% of the sample of neonatal animals by Brown and Booth ('83' Fig' 2)' The innervation territories of the remaining 40% of this sample were within the range they described for older (P13-Pl7) animals. Thus incomplete isolation of only a portion of the units studied could account for the differences observed. It is also possible that gluteus maximus motor units with large innervation territories were not using the EMG techniques This detected Seems unlikely, since the available evidence suggests that the EMG technique Overestimates the regions of muscle contracting during stimulation of a single motor unit. The results of intracellular recording experiments, while the conclusions based On EMG indicate that, at the boundaries between compartments, muscle cells may receive inputs from axons from more than one primary muscle nerve branch. It was not possible to determine the proportion of cells in gluteus maximus which receive such cross-compartmental inputs or to compare their rate of loss to the loss of inputs within a compartment, as has been done for LG (Donahue and English, '87b). Nevertheless, the complete absence of any cross-compartmental inputs in animals older than P8 suggests that they are eliminated selectively. The time course o f synapse elimination in gluteus maximus is roughly similar to that

A.W. ENGLISH reported for LG (Donahue and English, '89a), but it is strikingly different from the later and more rapid appearance of singly innervated cells seen in both rat (Brown et al., '76) and rabbit (Gordon and Van Essen, '83) soleus. By P8, when all of the cross-compartmental inputs have been eliminated, only 70% of the total number of gluteus maximus inputs which will be eliminated have been lost. Fully 30%of the inputs which will be eliminated by P18 have not yet been lost. A different conclusion has been reached by Balice-Gordon and Thompson ('881, based on their studies of EDL. In EDL, examples of cross-compartmental innervation can be found throughout most of the period of synapse elimination. They argued that a random loss of inputs could account for the loss of cross-compartmental inputs at a compartment border. This possibility could be easily tested by slowing the rate of synapse elimination, for example, by tenotomY O r Paralysis- In LG (Donahue and Endish, '89b), this led to an overall slowing of synapse elimination, but did not effect the rate of loss of Cross-ComPartmental COnnections.

Implicationsfor the developmentof compartment specificity The results presented above, along with other findings (Donahue and English, '87; Balice-Gordon and Thompson, '88), are consistent with neuromuscular connectivity being initially more precise than iS generally considered. Studies of neuromuscular specificity have generally focused on the innervation of whole muscles. Thus, even in the presence of modest perturbations of either the motoneurons or their target muscles, spatially distinct groups of motorIe~rons innervate particular muscles, with little evidence for connectional errors (see, e.g., Landmesser, '86). It is likely that the same can be said for the innervation of mammalian neuromuscular compartments, as the results discussed above indicate, so that the concept of neuromuscular specificity must be considered to operate at a finer level of focus, at the level of neuromuscular compartments. Both guidance and selective synapse formation must be considered in any model postulated to explain the basis for the initial accuracy of innervation of muscle compartments. However, of these two processes, axon @idance would Seem to be more important, since support for selective formation of synapses is not strong; gluteus maximus myotubes receive inputs from more than one muscle nerve. On the other hand, the trajectories of HRP-filled axOnS in the gluteal are strikingly similar to those Seen by Tosney and (,85a,b) in the chick hindlimb. Those authors strongly implicated axonal pathfinding in the generation of specific neuromuscular connections. It is thus tempting to speculate that similar mechanisms are operative in the development of neuromuscular compartments and that they result in the precise innervation patterns noted in the present study.

ACKNOWLEDGMENTS This work was supported by grant number NS20545 from the USPHS. Thanks are due to Kimberly Patrick, Melissa Cook, and Gail Schwartz for their technical support, to Wes Thompson for his helpful suggestions on the conduct of the experiments, and to Donald Wigston for his critical reading of the manuscript.

INNERVATION OF GLUTEUS MAXIMUS

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Development of compartmentalized innervation of the rat gluteus maximus muscle.

The innervation territories of the nerves to the gluteus maximus muscle were investigated in neonatal rats by using evoked electromyographic mapping t...
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