J. Physiol. (1978), 281, pp. 285-299 With 8 text-figurem Printed in Great Britain
285
A MORPHOLOGICAL STUDY OF THE AXONS AND RECURRENT AXON COLLATERALS OF CAT a-MOTONEURONES SUPPLYING DIFFERENT HIND-LIMB MUSCLES
BY STAFFAN CULLHEIM AND JAN-OLOF KELLERTH From the Department of Anatomy, Karolinska In-stitutet, 104 01 Stockholm 60, Sweden
(Received 23 January 1978) SUMMARY
1. Intracellular injections with horseradish peroxidase were performed in cat a-motoneurones supplying various hind-limb muscles. 2. Ten a-motoneurones from each of the quadriceps, posterior biceps, gastroenemius-soleus and anterior tibial pools as well as from the pool supplying the short plantar muscles were collected for morphological analysis of the intramedullary axonal systems including the recurrent axon collaterals. 3. The diameter of the a-motor axons showed considerable variation within each motoneurone pool, the total range being from 4 6 to 9 0 ,tm. No significant difference in mean axon diameter was obtained between the different pools. 4. All a-motoneurones supplying the short plantar muscles and one single amotoneurone supplying the quadriceps muscle lacked collaterals completely, while the remaining motoneurones gave off one to five collaterals. 5. The number of axon collateral outbulgings, interpreted as synaptic boutons, which originated from a single a-motoneurone showed large variation within each pool that possessed axon collaterals, the total range being from seventeen to 158. The mean number varied from forty-four (quadriceps) to eighty-two (anterior
tibial). 6. The axon collateral outbulgings were distributed not only in the Renshaw cell area ventromedial to the main motor nuclei but also in those parts of the motor nuclei which were located in the vicinity of the parent cell bodies. In the rostrocaudal direction, the outbulgings were distributed within a distance of less than 1 mm around the position of the parent cell bodies. 7. Some physiological implications of the lack of axon collaterals from a-motoneurones supplying the short plantar muscles were discussed in relation to the functional characteristics of plantar muscles and motor units. INTRODUCTION
The physiological effects of activity in the recurrent axon collaterals of cat spinal a-motoneurones have been subjected to extensive investigation. Thus, the recurrent axon collaterals have been shown to excite small interneurones, the socalled Renshaw cells (Renshaw, 1946; Eccles, Fatt & Koketsu, 1954), which in
S. CULLHEIM AND J.-O. KELLERTH turn inhibit a-motoneurones (Eccles et al. 1954), y-motoneurones (Brown, Lawrence & Matthews, 1968; Ellaway, 1968, 1971), Ia-interneurones (Hultborn, Jankowska & Lindstr6m, 1968, 1971 a), other Renshaw cells (Ryall, 1970) and ventral spinocerebellar tract neurones (Lindstr6m & Schomburg, 1973). Although the full physiological implications of these complex reflex actions still remain to be revealed, it is evident that the activity of a-motoneurones is strongly influenced by the recurrent motor axon collaterals. The distribution of this influence among the a-motoneurone pools has been thoroughly examined (Renshaw, 1941; Eccles et al. 1954; Wilson, Talbot & Diecke, 1960; Eccles, Eccles, Iggo & Ito, 1961; Hultborn, Jankowska & Lindstrbm, 1971 b) and the results indicate that the strongest effect is inhibition between synergistic motoneurones. Until recently, morphological investigations on motor axons and their recurrent axon collaterals have been sparse, due to the difficulties in visualizing myelinated axons with standard silver techniques. However, the introduction of the staining technique using intracellular injection of horseradish peroxidase (HRP) in neurones (Snow, Rose & Brown, 1976; Jankowska, Rastad & Westman, 1976; Cullheim & Kellerth, 1976) have made it possible to visualize, both light and electron microscopically, the intramedullary axonal systems of identified a-motoneurones including their axon collaterals and synaptic terminals (Cullheim & Kellerth, 1976; Cullheim, Kellerth & Conradi, 1977; Cullheim & Kellerth, 1978a). Thus, with this technique it has been possible to demonstrate previously unknown direct synaptic interconnexions between synergistic a-motoneurones via the recurrent axon collaterals (Cullheim et al. 1977). In the present paper, we have performed a quantitative study of the axons and recurrent axon collaterals of a-motoneurones of five muscles or muscle groups representing different functions around joints in the hind limb of the cat. Thus, ten a-motoneurones of each of the quadriceps (knee extensor), posterior biceps (knee flexor), gastrocnemius-soleus (ankle extensor), anterior tibial (ankle flexor) muscles and the plantar (toe extensor) muscle group were collected for analysis. 286
METHODS Twelve adult cats (2.3-3.5 kg) were anaesthetized with sodium pentobarbitone (Nembutal, Abbot, 40 mg/kg Ir.p.). Additional anaesthetics could be administered when needed through a catheter in the right femoral vein. Eight of the cats were immobilized with gallamine triethiothide and respired artificially, the remaining cats being allowed to breathe spontaneously. In all cats, the end-tidal C02 and the blood pressure were continuously monitored. The body temperature was maintained around 36-38 0C with infra-red light. After a lumbosacral laminectomy, intracellular recordings and injections of horseradish peroxidase (HRP) were made in a-motoneurones identified by antidromic invasion after stimulation of the nerves supplying the left quadriceps, posterior biceps, gastrocnemius-soleus, anterior tibial muscles as well as the tibial nerve supplying the flexor digitorum brevis and the short plantar muscles. The techniques for HRP injections, tissue fixation, histochemistry and reconstructions of the neurones have been described in detail elsewhere (Cullheim & Kellerth, 1976, 1978a). Briefly, the injections were made by passing a positive current of 20-30 nA for 5-15 min through micro-electrodes filled with 25 % HRP (type VI; Sigma) in 0-1 M-NaOH. After 1-8 hr the cats weie perfused with 5% glutaraldehyde through the descending aorta. The spinal cord was cut transversely in 30 #sm thick sections with a Vibratome (Oxford Lab., U.S.A.), and the sections were processed for HRP according to Graham & Karnovsky (1966). After dehydration in acetone, the sections were embedded between plastic foils (Hollander, 1970) in Vestopal.
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The entire intramedullary axonal systems of ten a-motoneurones from each of the five motoneurone groups were reconstructed (as exemplified in Fig. 1) by use of a Leitz microscope equipped with a drawing tube. The diameters of the stained main motor axons were measured on light micrographs taken
N. **N
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Fig. 1. Transverse reconstruction of the cell body and the intramedullary axonal system of a-motoneurone posterior biceps 6 (see Table 1). Four first order collaterals left the motor axon at places indicated by arrows and gave rise to thirty collateral end branches and seventy-one axon collateral swellings. The dashed line indicates the border between the spinal grey and white matter. Scale = 500 jm.
S. CULLHEIM AND J.-O. KELLERTH
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with an oil immersion objective. The final magnification of the micrographs was 3000 x . The diameters of the main motor axons in the white matter were obtained as the mean of ten measurements along each axon at intervals of 50 /tm. A collateral of the first order was defined as the collateral extending from a node of Ranvier of a main motor axon to the first branching point. The only exception to this was the rare instances when a main collateral divided immediately after leaving the motor axon and before it became surrounded by a myelin sheath. Collaterals of the first order were then defined as the branches originating from this initial unmyelinated collateral. An 'axon collateral swelling' was defined as a discrete outbulging of a collateral axon having at least twice the diameter of adjacent parts of the axon. Such swellings usually represent synaptic boutons as revealed in the electron microscope although the synaptic contacts may not always be associated with outbulgings (see Discussion of Cullheim & Kellerth, 1978a). 4
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Fig. 2. Histograms of axon diameters in the five motoneurone pools studied. The mean value of each distribution is indicated by an arrow. RESULTS
Motor axons. In Fig. 2 is shown the distribution of the mean diameters of the main motor axons in the white matter. When comparing the different motoneurone pools with each other no significant differences were obtained, the mean values of the pools ranging from 6-7 to 7O0am. However, within each pool considerable variations were found, the total range being from 4-6 to 90 jtm. Motor axon collateral. In Table 1, a number of quantitative data from the axon collateral systems studied have been collected. The most striking finding is the complete lack of axon collaterals from the a-motoneurones innervating the short plantar muscles, while all other neurones, except for one quadriceps a-motoneurone,
AXONAL SYSTEMS OF HIND-LIMB a-MOTONEURONES
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TABLE 1. Number of first order axon collaterals, collateral end branches and collateral swellings of ten cells from each of the short plantar, gastrocnemius-soleus, anterior tibial, quadriceps and posterior biceps a-motoneurone pools Total No. of no. of axon No. of swellings No. of collateral collateral end from each a-moto- first order neurone collaterals branches axon collateral tree swellings 0 0 0 0 1-10 Short plantar 101 45 24, 26, 31, 20 1 4 Gastrocnemius soleus 25 12 3 2 3, 14, 8 26 12 2 8, 18 3 65 31 32, 20, 13 4 3 70 3 30 30, 24, 16 5 36 7,29 18 2 6 69 3 27 2, 33, 34 7 73 25, 31, 17 32 3 8 146 2 57 96, 50 9 33 10 4 13 5, 5, 8, 15 64-4 ± 37.9 22*2 ± 18-3 Mean ± S.D. 2.9 + 0.7 27.7 + 14.9 67 1 1 67 28 Anterior tibial 29 13 17, 12 2 2 74 1 74 32 3 88 35 2 4 79, 9 104 1 104 38 5 79 1 79 36 6 117 2 69, 48 36 7 58 2 24 8 9,49 44 44 1 17 9 158 2 97, 61 69 10 81*8 + 37.5 54.5 + 31*4 Mean ±S.D. 15±+0-5 32-8 ± 15*3 65 15 34 1 50, 2 Quadriceps 45 24, 21 21 2 2 20 11 2 3 9, 11 36 36 14 1 4 21 21 10 1 5 70 22, 48 34 2 6 27 27 12 1 7 117 54 2 82, 35 8 43 20 2 16, 27 9 0 0 0 0 10 44-4 ± 331 29-6 ± 18*9 Mean+s.D. 1-5+0-7 21-0±15-7 142 4 52 47, 60, 20, 15 1 Posterior biceps 66 3 20 24, 21, 21 2 31 13 9, 13, 9 3 3 66 66 1 30 4 77 49, 17, 11 3 35 5 71 4 30 25, 15, 22, 9 6 17 17 9 1 7 97 53, 20, 24 3 39 8 68 27 2, 3, 23, 4, 36 5 9 59 21 38, 21 2 10 32-1 69-4 ± 16-9 ± 23-9 MeanS.D. 29±l1-3 276±_12-7 IO
PHY 28i
S. CULLHEIM AND J.-O. KELLERTH 290 gave off one to five first order collaterals. In the present material the quadriceps and anterior tibial a-motoneurones were on an average found to have a smaller number of first order collaterals than the posterior biceps (P < 0.01) and gastrocnemiussoleus (P < 0-001) neurones. Number of axon collateral end branches and swellings. The number of axon collateral end branches and swellings showed very large variations between different a-motoneurones (Table 1). This variation is graphically expressed in Fig. 3 where the a-motoneurones have been grouped according to the total number of axon collateral 10 81
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Fig. 3. Histograms of the number of axon collateral swellings given off by each motoneurone in the five pools studied. The mean value of each distribution is indicated by an arrow.
swellings that each neurone produced. Differences in the number of axon collateral swellings between the plantar a-motoneurones and all other pools (P < 0.001) and between the quadriceps and anterior tibial a-motoneurones (P < 0.05) were obtained. From Table 1 it is also evident that the number of end branches and swellings originating from different axon collateral trees of one and the same neurone showed considerable variation and that the total number of end branches and swellings could be almost identical in two a-motoneurones in spite of a large difference in the number of first order collaterals (see e.g. cells 4 and 9 in the posterior biceps pool). Vice versa, very different numbers of collateral end branches and swellings could be found in neurones with identical numbers of first order collaterals (see e.g. anterior tibial neurones 2 and 10). It was noticed that the a-motoneurones of the anterior tibial pool possessed the highest mean number of collateral end branches
AXONAL SYSTEMS OF HIND-LIMB ci-MOTONEURONES 291 and swellings in spite of having very few first order collaterals. As a consequence, the collateral trees of the anterior tibial pool had on an average more axon collateral swellings than the trees belonging to the posterior biceps and gastrocnemius-soleus pools (P < 0.001) or the quadriceps pool (P < 0.02). When plotting the number of axon collateral swellings of the a-motoneurones against the diameter of their axons in the white matter (Fig. 4) a weak positive correlation (P < 0.05) was obtained. The considerable variation seen is not only due to differences between the various a-motoneurone pools but also to variations within each pool. 160
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Fig. 4. Graph showing the relation between the number of axon collateral swellings and the diameter of the parent a-motor axon (n = 50). The a-motoneurones belonged to the short plantar (@), gastrocnemius-soleus (O), anterior tibial (@), quadriceps (0) and posterior biceps (v) pools. The linear correlation coefficient r = + 0-284 (P < 0.05).
Di8tribution of axon collateral swellings. The transverse distribution of axon collateral swellings in the spinal cord has been summarized in Figs. 5 and 6 where all swellings originating from each motoneurone group have been indicated. It was noticed that the majority of swellings were located ventromedial to the main motor nuclei. However, in all four groups of neurones swellings were also observed within the motor nuclei in the vicinity of the cell bodies of the parent a-motoneurones (see also Cullheim et al. 1977). The rostrocaudal distribution of the axon collateral swellings of each a-motoneurone group is shown in Figs. 7 and 8. In all groups the swellings were located within rather narrow limits around or just caudal to the position of the parent cell bodies. 10-2
S. CULLHEIM AND J.-O. KELLERTH
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AXONAL SYSTEMS OF HIND-LIMB cx-MOTONEURONES A
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B
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Fig. 6. Transverse distribution in the L5 ventral horn of 444 axon collateral swellings originating from ten quadriceps a-motoneurones (A) and in the L7 ventral horn of 694 axon collateral swellings from ten posterior biceps a-motoneurones (B). The dashed lines and the circles are defined as in Fig. 5.
293
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S. CULLHEIM AND J.-O. KELLERTH DISCUSSION
The present results are based on a morphological analysis of c-motoneurones stained intracellularly with HRP and it may be asked to what extent the various types of measurements may have been influenced by errors such as traumatic volume changes of the neuronal processes and incomplete HRP-staining of the axonal A
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systems. For a full methodological discussion of these topics we refer to a previous study (Cullheim & Kellerth, 1978a), from which it may be concluded that the results of the present study should not be significantly influenced by any artifacts inherent in the staining technique used.
295 AXONAL SYSTEMS OF HIND-LIMB c-MOTONEURONES Motor axons. The present results have not revealed any significant differences in axon diameters between the a-motoneurone pools studied while the variation within each pool was considerable. Since a strong positive correlation has been found between the axon diameter in the white matter and the axon conduction velocity of sciatic oc-motoneurones (Cullheim, 1978), the results indicate that no significant A
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Fig. 8. Histograms showing the rostrocaudal distribution of 444 axon collateral swellings originating from ten quadriceps a-motoneurones (A) and 694 axon collateral swellings from ten posterior biceps a-motoneurones (B) in relation to the location of the parent cell bodies.
differences in mean conduction velocity exist between the pools but rather a spectrum of conduction velocities within each pool. This is in agreement with earlier physiological studies on the conduction velocity of ax-motor axons in the hind limb of the cat (Eccles, Eccles & Lundberg, 1958; Kuno, 1959; Eccles et al. 1961; Appelberg & Emonet-D6nand, 1967; Burke, 1967). In a subsequent paper (Cullheim & Kellerth, 1978b) it will be shown that the variation of axon diameters in the gastrocnemiussoleus pool is largely explained by differences between the various L-motoneurone types within the pool. A full morphological discussion on a-motor axons in general has been presented in a previous paper (Cullheim & Kellerth, 1978a).
S. CULLHEIM AND J.-O. KELLERTH 296 Occurrence of motor axon collateral8. The most conspicuous finding in the present material was the fact that all the a-motoneurones supplying the short plantar muscles were completely devoid of axon collaterals. These neurones were collected from two different animals which should reduce the risk for an accidental selection of any particular subgroup of neurones within the plantar pool. Our results therefore suggest that the plantar ac-motoneurones should produce no or only negligible recurrent effects. This is also supported by physiological studies of plantar motoneurones in the monkey (H. Hultborn, E. Jankowska & S. Lindstr6m, personal communication). Unfortunately, there is no similar study of the plantar motoneurones of the cat. With respect to the quadriceps, posterior biceps, anterior tibial and gastrocnemius-soleus motoneurones all the axons studied, except for one quadriceps axon, possessed collaterals, and recurrent effects have also been demonstrated physiologically from all these motoneurone pools (Renshaw, 1941; Eccles et al. 1954; Wilson et al. 1960; Eccles et al. 1961; Hultborn et al. 1971 b). The number of collaterals from each axon varied considerably even between neurones of the same pool but, on an average, there were about twice as many first order collaterals originating from the posterior biceps and gastrocnemius-soleus motor axons as compared to the anterior tibial and quadriceps motor axons. This difference, however, was not necessarily coupled to a difference in the mean total number of collateral swellings originating from the motor axons (see below and Table 1). Number of axon collateral end branches and swellings. The present results have revealed a wide variation in the number of collateral end branches and swellings of the a-motoneurones, also when studying each motoneurone pool separately. In a subsequent paper (Cullheim & Kellerth, 1978b) it will be shown that this variation may be partly explained by differences between various functional types of motor units. However, the reasons for the wide variation in the branching pattern of collateral trees originating from one and the same motor axon is enigmatic. Moreover, it seems puzzling that one neurone would need five first order collaterals to produce the same number of swellings as another neurone with only one axon collateral. Distribution of axon collateral swellings. A previous morphological investigation has demonstrated the existence of direct monosynaptic interconnexions between a-motoneurones via axon collaterals terminating within the motor nuclei (Cullheim et al. 1977). Also in the present material axon collaterals were found to project to the motor nuclei, but this should not be interpreted as if these are the only collaterals to make monosynaptic contacts with c-motoneurones. On the contrary, motor axon collaterals contacting ax-motoneurone dendrites have been found also within the 'Renshaw cell area' (P.-A. LagerbAck & L.-O. Ronnevi, personal communication), which is not entirely surprising when considering the large extensions of the dendritic trees of c-motoneurones (see e.g. Fig. 1 in Cullheim & Kellerth, 1976). Of course, the evaluation of the functional significance of the recurrent motor axon collateral system is impeded by the fact that the direct synaptic interconnexions between a-motoneurones so far lack a physiological correlate. These problems are presently being subject to studies in our laboratory. The rostrocaudal distribution of the axon collateral end branches and swellings of single neurones was invariably found to be very restricted. This is in disagreement with the Golgi study of Scheibel & Scheibel (1971) where a substantial part (10-20 %)
AXONAL SYSTEMS OF HIND-LIMB a-MOTONEURONES 297 of the motor axon collaterals were claimed to run rostrally or caudally for one segment before termination. However, the study of the Scheibels was performed on immature kittens in contrast to the present study on adult animals. Our findings indicate that the post-synaptic neurones should be located rather close to the presynaptic ones and that intersegmental effects of the a-motor axon collaterals (Renshaw, 1941; Wilson et al. 1960; Eccles et al. 1961; Hultborn et al. 1971b; Ryall, Piercey & Polosa, 1971) should not contain any major monosynaptic components. Physiological considerations. The observation that a-motoneurones supplying the short plantar muscles seem to lack axon collaterals deserves some comments on the physiology of these muscles with special reference to theories of the function of the recurrent axon collateral pathway. Reflex studies have indicated that the plantar muscles of the cat should be classified as physiological extensors (Engberg, 1964). The a-motoneurones of particularly the deep plantar muscles receive only little heteronymous excitation from Ia afferents (Engberg, 1964), which contrasts to the more diverse Ia connexions to the motoneurones of the hip, knee and ankle muscles (Eccles, Eccles & Lundberg, 1957; Eccles & Lundberg, 1958). The function of recurrent inhibition of ax-motoneurones has been proposed to be to limit the extent of diverse Ia effects, thus acting as a lateral inhibition (Brooks & Wilson, 1959; Wilson et al. 1960; Granit & Renkin, 1961). According to this hypothesis, there would be less need for recurrent inhibition from the plantar a-motoneurones than from the other a-motoneurone groups investigated here. Recurrent inhibition has been shown to stabilize the output frequency of triceps surae cz-motoneurones during varying degrees of homonymous muscle stretch Meyer-Lohmann & Henatsch, 1966). Thus, the grading of tension should, at least within certain ranges of muscle stretch, be more dependent on the number of activated motor units than on changes in the firing frequency of the individual units. Against this background, the lack of axon collaterals from the plantar amotoneurones should imply that the grading of tension in the plantar muscles is achieved by changes in the number of active motor units as well as by changes in firing frequency of the individual units. This is in fact in accordance with a recent investigation on a small muscle (first deep lumbrical) of the cat's foot (Kernell & Sj6holm, 1975) where an increase in firing rate of already active motor units was found to be of great importance for an increase in muscle tension. This study was supported by grants from the Swedish Medical Research Council (project 02886) and the Karolinska Institutet. We wish to thank Miss Maj Berghman and Mrs Lillebil Stuart for their technical assistance.
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KERNELL, D. & SJOHOLM, H. (1975). Recruitment and firing rate modulation of motor unit tension in a small muscle of the cat's foot. Brain Res. 98, 57-72. KuNo, M. (1959). Excitability following antidromic activation in spinal motoneurones supplying red muscles. J. Physiol. 149, 374-393. LINDSTROM, S. & SCHOMBURG, E. D. (1973). Recurrent inhibition from motor axon collaterals of ventral spinocerebellar tract neurones. Acta physiol. scand. 88, 505-515. MEYER-LOHMANN, J. & HENATSCH, H.-D. (1966). Die Bedeutung der natiirlichen recurrenten Hemmung (NRH) fur die Frequenz tonisch entladender Extensor-a-Motoneurone. Pflugers Arch. 291, R3. RENSHAW, B. (1941). Influence of discharge of motoneurons upon excitation of neighboring motoneurons. J. Neurophysiol. 4, 167-183. RENSHAW, B. (1946). Central effects of centripetal impulses in axons of spinal ventral roots. J. Neurophysiol. 9, 191-204. ROMANES, G. J. (1951). The motor cell columns of the lumbo-sacral spinal cord of the cat. J. comp. Neurol. 94, 313-364. RYALL, R. W. (1970). Renshaw cell mediated inhibition of Renshaw cells: Patterns of excitation and inhibition from impulses in motor axon collaterals. J. Neurophysiol. 33, 257-270. RYALL, R. W., PIERCEY, M. F. & POLOSA, C. (1971). Intersegmental and intrasegmental distribution of mutual inhibition of Renshaw cells. J. Neurophysiol. 34, 700-707. SCHEIBEL, M. E. & SCHEIBEL, A. B. (1971). Inhibition and the Renshaw cell. A structural critique. Brain Behav. & Evol. 4, 53-93. SNOW, P. J., ROSE, P. K. & BROWN, A. G. (1976). Tracing axons and axon collaterals of spinal neurons using intracellular injection of horseradish peroxidase. Science, N. Y'. 191, 312-313. WILsON, V. J., TALBOT, WV. H. & DIECKE, F. P. J. (1960). Distribution of recurrent facilitation and inhibition in cat spinal cord. J. Neurophysiol. 23, 144-153.
285
A MORPHOLOGICAL STUDY OF THE AXONS AND RECURRENT AXON COLLATERALS OF CAT a-MOTONEURONES SUPPLYING DIFFERENT HIND-LIMB MUSCLES
BY STAFFAN CULLHEIM AND JAN-OLOF KELLERTH From the Department of Anatomy, Karolinska In-stitutet, 104 01 Stockholm 60, Sweden
(Received 23 January 1978) SUMMARY
1. Intracellular injections with horseradish peroxidase were performed in cat a-motoneurones supplying various hind-limb muscles. 2. Ten a-motoneurones from each of the quadriceps, posterior biceps, gastroenemius-soleus and anterior tibial pools as well as from the pool supplying the short plantar muscles were collected for morphological analysis of the intramedullary axonal systems including the recurrent axon collaterals. 3. The diameter of the a-motor axons showed considerable variation within each motoneurone pool, the total range being from 4 6 to 9 0 ,tm. No significant difference in mean axon diameter was obtained between the different pools. 4. All a-motoneurones supplying the short plantar muscles and one single amotoneurone supplying the quadriceps muscle lacked collaterals completely, while the remaining motoneurones gave off one to five collaterals. 5. The number of axon collateral outbulgings, interpreted as synaptic boutons, which originated from a single a-motoneurone showed large variation within each pool that possessed axon collaterals, the total range being from seventeen to 158. The mean number varied from forty-four (quadriceps) to eighty-two (anterior
tibial). 6. The axon collateral outbulgings were distributed not only in the Renshaw cell area ventromedial to the main motor nuclei but also in those parts of the motor nuclei which were located in the vicinity of the parent cell bodies. In the rostrocaudal direction, the outbulgings were distributed within a distance of less than 1 mm around the position of the parent cell bodies. 7. Some physiological implications of the lack of axon collaterals from a-motoneurones supplying the short plantar muscles were discussed in relation to the functional characteristics of plantar muscles and motor units. INTRODUCTION
The physiological effects of activity in the recurrent axon collaterals of cat spinal a-motoneurones have been subjected to extensive investigation. Thus, the recurrent axon collaterals have been shown to excite small interneurones, the socalled Renshaw cells (Renshaw, 1946; Eccles, Fatt & Koketsu, 1954), which in
S. CULLHEIM AND J.-O. KELLERTH turn inhibit a-motoneurones (Eccles et al. 1954), y-motoneurones (Brown, Lawrence & Matthews, 1968; Ellaway, 1968, 1971), Ia-interneurones (Hultborn, Jankowska & Lindstr6m, 1968, 1971 a), other Renshaw cells (Ryall, 1970) and ventral spinocerebellar tract neurones (Lindstr6m & Schomburg, 1973). Although the full physiological implications of these complex reflex actions still remain to be revealed, it is evident that the activity of a-motoneurones is strongly influenced by the recurrent motor axon collaterals. The distribution of this influence among the a-motoneurone pools has been thoroughly examined (Renshaw, 1941; Eccles et al. 1954; Wilson, Talbot & Diecke, 1960; Eccles, Eccles, Iggo & Ito, 1961; Hultborn, Jankowska & Lindstrbm, 1971 b) and the results indicate that the strongest effect is inhibition between synergistic motoneurones. Until recently, morphological investigations on motor axons and their recurrent axon collaterals have been sparse, due to the difficulties in visualizing myelinated axons with standard silver techniques. However, the introduction of the staining technique using intracellular injection of horseradish peroxidase (HRP) in neurones (Snow, Rose & Brown, 1976; Jankowska, Rastad & Westman, 1976; Cullheim & Kellerth, 1976) have made it possible to visualize, both light and electron microscopically, the intramedullary axonal systems of identified a-motoneurones including their axon collaterals and synaptic terminals (Cullheim & Kellerth, 1976; Cullheim, Kellerth & Conradi, 1977; Cullheim & Kellerth, 1978a). Thus, with this technique it has been possible to demonstrate previously unknown direct synaptic interconnexions between synergistic a-motoneurones via the recurrent axon collaterals (Cullheim et al. 1977). In the present paper, we have performed a quantitative study of the axons and recurrent axon collaterals of a-motoneurones of five muscles or muscle groups representing different functions around joints in the hind limb of the cat. Thus, ten a-motoneurones of each of the quadriceps (knee extensor), posterior biceps (knee flexor), gastrocnemius-soleus (ankle extensor), anterior tibial (ankle flexor) muscles and the plantar (toe extensor) muscle group were collected for analysis. 286
METHODS Twelve adult cats (2.3-3.5 kg) were anaesthetized with sodium pentobarbitone (Nembutal, Abbot, 40 mg/kg Ir.p.). Additional anaesthetics could be administered when needed through a catheter in the right femoral vein. Eight of the cats were immobilized with gallamine triethiothide and respired artificially, the remaining cats being allowed to breathe spontaneously. In all cats, the end-tidal C02 and the blood pressure were continuously monitored. The body temperature was maintained around 36-38 0C with infra-red light. After a lumbosacral laminectomy, intracellular recordings and injections of horseradish peroxidase (HRP) were made in a-motoneurones identified by antidromic invasion after stimulation of the nerves supplying the left quadriceps, posterior biceps, gastrocnemius-soleus, anterior tibial muscles as well as the tibial nerve supplying the flexor digitorum brevis and the short plantar muscles. The techniques for HRP injections, tissue fixation, histochemistry and reconstructions of the neurones have been described in detail elsewhere (Cullheim & Kellerth, 1976, 1978a). Briefly, the injections were made by passing a positive current of 20-30 nA for 5-15 min through micro-electrodes filled with 25 % HRP (type VI; Sigma) in 0-1 M-NaOH. After 1-8 hr the cats weie perfused with 5% glutaraldehyde through the descending aorta. The spinal cord was cut transversely in 30 #sm thick sections with a Vibratome (Oxford Lab., U.S.A.), and the sections were processed for HRP according to Graham & Karnovsky (1966). After dehydration in acetone, the sections were embedded between plastic foils (Hollander, 1970) in Vestopal.
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The entire intramedullary axonal systems of ten a-motoneurones from each of the five motoneurone groups were reconstructed (as exemplified in Fig. 1) by use of a Leitz microscope equipped with a drawing tube. The diameters of the stained main motor axons were measured on light micrographs taken
N. **N
*N
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I I I I
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Fig. 1. Transverse reconstruction of the cell body and the intramedullary axonal system of a-motoneurone posterior biceps 6 (see Table 1). Four first order collaterals left the motor axon at places indicated by arrows and gave rise to thirty collateral end branches and seventy-one axon collateral swellings. The dashed line indicates the border between the spinal grey and white matter. Scale = 500 jm.
S. CULLHEIM AND J.-O. KELLERTH
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with an oil immersion objective. The final magnification of the micrographs was 3000 x . The diameters of the main motor axons in the white matter were obtained as the mean of ten measurements along each axon at intervals of 50 /tm. A collateral of the first order was defined as the collateral extending from a node of Ranvier of a main motor axon to the first branching point. The only exception to this was the rare instances when a main collateral divided immediately after leaving the motor axon and before it became surrounded by a myelin sheath. Collaterals of the first order were then defined as the branches originating from this initial unmyelinated collateral. An 'axon collateral swelling' was defined as a discrete outbulging of a collateral axon having at least twice the diameter of adjacent parts of the axon. Such swellings usually represent synaptic boutons as revealed in the electron microscope although the synaptic contacts may not always be associated with outbulgings (see Discussion of Cullheim & Kellerth, 1978a). 4
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Fig. 2. Histograms of axon diameters in the five motoneurone pools studied. The mean value of each distribution is indicated by an arrow. RESULTS
Motor axons. In Fig. 2 is shown the distribution of the mean diameters of the main motor axons in the white matter. When comparing the different motoneurone pools with each other no significant differences were obtained, the mean values of the pools ranging from 6-7 to 7O0am. However, within each pool considerable variations were found, the total range being from 4-6 to 90 jtm. Motor axon collateral. In Table 1, a number of quantitative data from the axon collateral systems studied have been collected. The most striking finding is the complete lack of axon collaterals from the a-motoneurones innervating the short plantar muscles, while all other neurones, except for one quadriceps a-motoneurone,
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TABLE 1. Number of first order axon collaterals, collateral end branches and collateral swellings of ten cells from each of the short plantar, gastrocnemius-soleus, anterior tibial, quadriceps and posterior biceps a-motoneurone pools Total No. of no. of axon No. of swellings No. of collateral collateral end from each a-moto- first order neurone collaterals branches axon collateral tree swellings 0 0 0 0 1-10 Short plantar 101 45 24, 26, 31, 20 1 4 Gastrocnemius soleus 25 12 3 2 3, 14, 8 26 12 2 8, 18 3 65 31 32, 20, 13 4 3 70 3 30 30, 24, 16 5 36 7,29 18 2 6 69 3 27 2, 33, 34 7 73 25, 31, 17 32 3 8 146 2 57 96, 50 9 33 10 4 13 5, 5, 8, 15 64-4 ± 37.9 22*2 ± 18-3 Mean ± S.D. 2.9 + 0.7 27.7 + 14.9 67 1 1 67 28 Anterior tibial 29 13 17, 12 2 2 74 1 74 32 3 88 35 2 4 79, 9 104 1 104 38 5 79 1 79 36 6 117 2 69, 48 36 7 58 2 24 8 9,49 44 44 1 17 9 158 2 97, 61 69 10 81*8 + 37.5 54.5 + 31*4 Mean ±S.D. 15±+0-5 32-8 ± 15*3 65 15 34 1 50, 2 Quadriceps 45 24, 21 21 2 2 20 11 2 3 9, 11 36 36 14 1 4 21 21 10 1 5 70 22, 48 34 2 6 27 27 12 1 7 117 54 2 82, 35 8 43 20 2 16, 27 9 0 0 0 0 10 44-4 ± 331 29-6 ± 18*9 Mean+s.D. 1-5+0-7 21-0±15-7 142 4 52 47, 60, 20, 15 1 Posterior biceps 66 3 20 24, 21, 21 2 31 13 9, 13, 9 3 3 66 66 1 30 4 77 49, 17, 11 3 35 5 71 4 30 25, 15, 22, 9 6 17 17 9 1 7 97 53, 20, 24 3 39 8 68 27 2, 3, 23, 4, 36 5 9 59 21 38, 21 2 10 32-1 69-4 ± 16-9 ± 23-9 MeanS.D. 29±l1-3 276±_12-7 IO
PHY 28i
S. CULLHEIM AND J.-O. KELLERTH 290 gave off one to five first order collaterals. In the present material the quadriceps and anterior tibial a-motoneurones were on an average found to have a smaller number of first order collaterals than the posterior biceps (P < 0.01) and gastrocnemiussoleus (P < 0-001) neurones. Number of axon collateral end branches and swellings. The number of axon collateral end branches and swellings showed very large variations between different a-motoneurones (Table 1). This variation is graphically expressed in Fig. 3 where the a-motoneurones have been grouped according to the total number of axon collateral 10 81
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Fig. 3. Histograms of the number of axon collateral swellings given off by each motoneurone in the five pools studied. The mean value of each distribution is indicated by an arrow.
swellings that each neurone produced. Differences in the number of axon collateral swellings between the plantar a-motoneurones and all other pools (P < 0.001) and between the quadriceps and anterior tibial a-motoneurones (P < 0.05) were obtained. From Table 1 it is also evident that the number of end branches and swellings originating from different axon collateral trees of one and the same neurone showed considerable variation and that the total number of end branches and swellings could be almost identical in two a-motoneurones in spite of a large difference in the number of first order collaterals (see e.g. cells 4 and 9 in the posterior biceps pool). Vice versa, very different numbers of collateral end branches and swellings could be found in neurones with identical numbers of first order collaterals (see e.g. anterior tibial neurones 2 and 10). It was noticed that the a-motoneurones of the anterior tibial pool possessed the highest mean number of collateral end branches
AXONAL SYSTEMS OF HIND-LIMB ci-MOTONEURONES 291 and swellings in spite of having very few first order collaterals. As a consequence, the collateral trees of the anterior tibial pool had on an average more axon collateral swellings than the trees belonging to the posterior biceps and gastrocnemius-soleus pools (P < 0.001) or the quadriceps pool (P < 0.02). When plotting the number of axon collateral swellings of the a-motoneurones against the diameter of their axons in the white matter (Fig. 4) a weak positive correlation (P < 0.05) was obtained. The considerable variation seen is not only due to differences between the various a-motoneurone pools but also to variations within each pool. 160
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Fig. 4. Graph showing the relation between the number of axon collateral swellings and the diameter of the parent a-motor axon (n = 50). The a-motoneurones belonged to the short plantar (@), gastrocnemius-soleus (O), anterior tibial (@), quadriceps (0) and posterior biceps (v) pools. The linear correlation coefficient r = + 0-284 (P < 0.05).
Di8tribution of axon collateral swellings. The transverse distribution of axon collateral swellings in the spinal cord has been summarized in Figs. 5 and 6 where all swellings originating from each motoneurone group have been indicated. It was noticed that the majority of swellings were located ventromedial to the main motor nuclei. However, in all four groups of neurones swellings were also observed within the motor nuclei in the vicinity of the cell bodies of the parent a-motoneurones (see also Cullheim et al. 1977). The rostrocaudal distribution of the axon collateral swellings of each a-motoneurone group is shown in Figs. 7 and 8. In all groups the swellings were located within rather narrow limits around or just caudal to the position of the parent cell bodies. 10-2
S. CULLHEIM AND J.-O. KELLERTH
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AXONAL SYSTEMS OF HIND-LIMB cx-MOTONEURONES A
Quadriceps
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B
n
Fig. 6. Transverse distribution in the L5 ventral horn of 444 axon collateral swellings originating from ten quadriceps a-motoneurones (A) and in the L7 ventral horn of 694 axon collateral swellings from ten posterior biceps a-motoneurones (B). The dashed lines and the circles are defined as in Fig. 5.
293
294
S. CULLHEIM AND J.-O. KELLERTH DISCUSSION
The present results are based on a morphological analysis of c-motoneurones stained intracellularly with HRP and it may be asked to what extent the various types of measurements may have been influenced by errors such as traumatic volume changes of the neuronal processes and incomplete HRP-staining of the axonal A
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systems. For a full methodological discussion of these topics we refer to a previous study (Cullheim & Kellerth, 1978a), from which it may be concluded that the results of the present study should not be significantly influenced by any artifacts inherent in the staining technique used.
295 AXONAL SYSTEMS OF HIND-LIMB c-MOTONEURONES Motor axons. The present results have not revealed any significant differences in axon diameters between the a-motoneurone pools studied while the variation within each pool was considerable. Since a strong positive correlation has been found between the axon diameter in the white matter and the axon conduction velocity of sciatic oc-motoneurones (Cullheim, 1978), the results indicate that no significant A
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differences in mean conduction velocity exist between the pools but rather a spectrum of conduction velocities within each pool. This is in agreement with earlier physiological studies on the conduction velocity of ax-motor axons in the hind limb of the cat (Eccles, Eccles & Lundberg, 1958; Kuno, 1959; Eccles et al. 1961; Appelberg & Emonet-D6nand, 1967; Burke, 1967). In a subsequent paper (Cullheim & Kellerth, 1978b) it will be shown that the variation of axon diameters in the gastrocnemiussoleus pool is largely explained by differences between the various L-motoneurone types within the pool. A full morphological discussion on a-motor axons in general has been presented in a previous paper (Cullheim & Kellerth, 1978a).
S. CULLHEIM AND J.-O. KELLERTH 296 Occurrence of motor axon collateral8. The most conspicuous finding in the present material was the fact that all the a-motoneurones supplying the short plantar muscles were completely devoid of axon collaterals. These neurones were collected from two different animals which should reduce the risk for an accidental selection of any particular subgroup of neurones within the plantar pool. Our results therefore suggest that the plantar ac-motoneurones should produce no or only negligible recurrent effects. This is also supported by physiological studies of plantar motoneurones in the monkey (H. Hultborn, E. Jankowska & S. Lindstr6m, personal communication). Unfortunately, there is no similar study of the plantar motoneurones of the cat. With respect to the quadriceps, posterior biceps, anterior tibial and gastrocnemius-soleus motoneurones all the axons studied, except for one quadriceps axon, possessed collaterals, and recurrent effects have also been demonstrated physiologically from all these motoneurone pools (Renshaw, 1941; Eccles et al. 1954; Wilson et al. 1960; Eccles et al. 1961; Hultborn et al. 1971 b). The number of collaterals from each axon varied considerably even between neurones of the same pool but, on an average, there were about twice as many first order collaterals originating from the posterior biceps and gastrocnemius-soleus motor axons as compared to the anterior tibial and quadriceps motor axons. This difference, however, was not necessarily coupled to a difference in the mean total number of collateral swellings originating from the motor axons (see below and Table 1). Number of axon collateral end branches and swellings. The present results have revealed a wide variation in the number of collateral end branches and swellings of the a-motoneurones, also when studying each motoneurone pool separately. In a subsequent paper (Cullheim & Kellerth, 1978b) it will be shown that this variation may be partly explained by differences between various functional types of motor units. However, the reasons for the wide variation in the branching pattern of collateral trees originating from one and the same motor axon is enigmatic. Moreover, it seems puzzling that one neurone would need five first order collaterals to produce the same number of swellings as another neurone with only one axon collateral. Distribution of axon collateral swellings. A previous morphological investigation has demonstrated the existence of direct monosynaptic interconnexions between a-motoneurones via axon collaterals terminating within the motor nuclei (Cullheim et al. 1977). Also in the present material axon collaterals were found to project to the motor nuclei, but this should not be interpreted as if these are the only collaterals to make monosynaptic contacts with c-motoneurones. On the contrary, motor axon collaterals contacting ax-motoneurone dendrites have been found also within the 'Renshaw cell area' (P.-A. LagerbAck & L.-O. Ronnevi, personal communication), which is not entirely surprising when considering the large extensions of the dendritic trees of c-motoneurones (see e.g. Fig. 1 in Cullheim & Kellerth, 1976). Of course, the evaluation of the functional significance of the recurrent motor axon collateral system is impeded by the fact that the direct synaptic interconnexions between a-motoneurones so far lack a physiological correlate. These problems are presently being subject to studies in our laboratory. The rostrocaudal distribution of the axon collateral end branches and swellings of single neurones was invariably found to be very restricted. This is in disagreement with the Golgi study of Scheibel & Scheibel (1971) where a substantial part (10-20 %)
AXONAL SYSTEMS OF HIND-LIMB a-MOTONEURONES 297 of the motor axon collaterals were claimed to run rostrally or caudally for one segment before termination. However, the study of the Scheibels was performed on immature kittens in contrast to the present study on adult animals. Our findings indicate that the post-synaptic neurones should be located rather close to the presynaptic ones and that intersegmental effects of the a-motor axon collaterals (Renshaw, 1941; Wilson et al. 1960; Eccles et al. 1961; Hultborn et al. 1971b; Ryall, Piercey & Polosa, 1971) should not contain any major monosynaptic components. Physiological considerations. The observation that a-motoneurones supplying the short plantar muscles seem to lack axon collaterals deserves some comments on the physiology of these muscles with special reference to theories of the function of the recurrent axon collateral pathway. Reflex studies have indicated that the plantar muscles of the cat should be classified as physiological extensors (Engberg, 1964). The a-motoneurones of particularly the deep plantar muscles receive only little heteronymous excitation from Ia afferents (Engberg, 1964), which contrasts to the more diverse Ia connexions to the motoneurones of the hip, knee and ankle muscles (Eccles, Eccles & Lundberg, 1957; Eccles & Lundberg, 1958). The function of recurrent inhibition of ax-motoneurones has been proposed to be to limit the extent of diverse Ia effects, thus acting as a lateral inhibition (Brooks & Wilson, 1959; Wilson et al. 1960; Granit & Renkin, 1961). According to this hypothesis, there would be less need for recurrent inhibition from the plantar a-motoneurones than from the other a-motoneurone groups investigated here. Recurrent inhibition has been shown to stabilize the output frequency of triceps surae cz-motoneurones during varying degrees of homonymous muscle stretch Meyer-Lohmann & Henatsch, 1966). Thus, the grading of tension should, at least within certain ranges of muscle stretch, be more dependent on the number of activated motor units than on changes in the firing frequency of the individual units. Against this background, the lack of axon collaterals from the plantar amotoneurones should imply that the grading of tension in the plantar muscles is achieved by changes in the number of active motor units as well as by changes in firing frequency of the individual units. This is in fact in accordance with a recent investigation on a small muscle (first deep lumbrical) of the cat's foot (Kernell & Sj6holm, 1975) where an increase in firing rate of already active motor units was found to be of great importance for an increase in muscle tension. This study was supported by grants from the Swedish Medical Research Council (project 02886) and the Karolinska Institutet. We wish to thank Miss Maj Berghman and Mrs Lillebil Stuart for their technical assistance.
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