College of Medicine, Salt Lake City 12, Utah, U.S.A.

Transcription

1 43 J. Phy8iol. (1962), 164, pp With 9 text-figurea Printed in Great Britain A COMPARZISON OF MONOSYNAPTIC AND POLYSYNAPTIC REFLEX RESPONSES FROM INDIVIDUAL FLEXOR MOTONEURONES BY E. R. PERL From the Department of Physiology, University of Utah College of Medicine, Salt Lake City 12, Utah, U.S.A. (Received 11 May 1962) Evidence from a number of types of experiments has shown that gradation of muscular contraction is accomplished by changes both in the number of motoneurones active and in the frequency of discharge of individual motoneurones (Creed, Denny-Brown, Eccles, Liddell & Sherrington, 1932). It is also well known that different spinal reflexes have different abilities to excite fractions of the motoneurone pool supplying a particular muscle. On the other hand, there appears to be little information on one important feature of the functional organization of motoneurones particularly relevant to quantitative control of skeletal muscle contraction, namely, the degree of influence various reflex pathways have on a particular motoneurone in comparison to other motoneurones of the same pool. The present study examines the effectiveness of the monosynaptic and several polysynaptic reflex arcs in discharging individual knee flexor motoneurones. These experiments should be useful in indicating whether a given motoneurone responds equally well to different reflex pathways or whether variation in excitation throughout a particular pool is dependent upon the nature of the reflex process. Information on such a point would be valuable in helping to ascertain the manner in which different mechanisms combine to determine the participation of motoneurones in activity. For instance, it has been postulated that a significant control of motoneurone excitability is exerted via the fusimotor (gamma) effect upon the afferent limb on the monosynaptic reflex, the Group I fibres from the muscle spindle (Eldred, Granit & Merton, 1953). Fitting the motor control of the spindle into the framework of mechanisms concerned with the functional organization of motoneurones would require knowledge of how the effectiveness of the monosynaptic drive varies among members of a pool in comparison to other excitatory pathways. The motoneurones studied were taken from the pool monosynaptically excited by Group I volleys from the peripheral nerves supplying the

2 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 431 synergistic knee flexor muscles, biceps femoris posterior and semitendinosus. Under conditions comparable to the present experiment this population would innervate one or another of these synergistic muscles (Lloyd, 1943b; Lloyd, Hunt & McIntyre, 1955). The monosynaptic response evoked from a motoneurone by volleys of different size from the muscle nerves supplying the knee flexors was studied, and then the response of the same motoneurone was examined following volleys graded in size from nerves evoking generalized polysynaptic flexor reflexes. Usually, the relative responsiveness of motoneurones was similar in the several reflex tests; however, interesting exceptions did exist. METHODS Adult cats were used after being made spinal under ether anaesthesia by a transection at C 1. The brain anterior to the transection was killed by the occlusion of the carotid and vertebral arteries, and after death of the head anaesthesia was discontinued. The preparations were artificially ventilated and end-tidal CO2 monitored by an infra-red detector (Liston-Becker LB-1). Expired CO2 was held constant during any series of observations by adjustment of the respirator. Rectal temperature was maintained in the range between 37 and 39 C by use of external heat, and was not allowed to vary more than + 3 C during the recording period. Changes in motoneurone response are reported to occur as the result of relatively small temperature changes (Lloyd et al. 1955), a fact confirmed in the course of the present observations. Gallamine triethiodide (Flaxedil, American Cyanamide) was given intravenously to paralyse the preparation. The lumbar spinal cord was exposed and the ventral roots from L 7 to S 2 were cut; occasionally ventral root L6 was also divided. All branches of the sciatic nerve were cut. The sural nerve (S) and the major nerve branches innervating triceps surae (TS) were dissected free from other nerves for at least 3 cm to minimize possibility of stimulus escape. The nerves to the semitendinosus and biceps posterior femoris were traced into the respective muscles and separated from other hamstring nerve branches. Nerves innervating only these knee flexor muscles ordinarily number three, a short and a long branch to semitendinosus and one branch to posterior biceps. Occasionally, stimulation indicated that a second branch to biceps contained motor fibres only to the posterior biceps muscle. The branches to semitendinosus and biceps posterior were examined for mutual monosynaptic facilitation and the divisions showing such facilitation were stimulated together as the BST nerve. Interelectrode distances of the stimulating leads was 6-12 mm. Nerve volleys were evoked by rectangular pulses from generators whose output could be re-set to - 1 %. Exposed nerves and spinal cord were covered with mineral oil equilibrated with 95 % 2 and 5 % CO2. Oil pool temperatures ranged from -5 to 1-5 C below rectal temperature. This difference was constant for a given experiment. Two recordings were regularly displayed on the oscilloscope; one was a volume lead from the dorsal-root-spinal-cord junction at the segmental level showing largest potentials, and the other indicated the discharge of motoneurones in a ventral root filament. Unitary potentials of motoneurone axons of the pool discharging monosynaptically to stimulation of BST nerve were obtained by splitting ventral root (usually S1) filaments selected at random with the aid of a dissecting microscope. The unitary nature of the response was then checked for all-or-none appearance of an impulse: (1) to near threshold stimuli, (2) after tetanus of BST (5/sec) at Group I maximal intensity and (3) after stimulating BST, S and TS at 5 x threshold value. If several units were encountered in one filament, an attempt was made to isolate and study eaeh. This was not always successful. In some instances two moto-

3 432 E. R. PERL neurones were studied simultaneously when the recorded action potentials were sufficiently different to allow ready identification and the criteria for 'isolation' were otherwise met. In most cases, including all cases of two units simultaneously studied, the evoked activity appearing in both the dorsal-root and ventral-root leads was displayed on the oscilloscope and photographed from a time base of or 5 msec duration. The firing index or FI (Lloyd & McIntyre, 1955), (number of responses x 1)/(number of test stimuli), was determined for every motoneurone at each intensity of stimulus to each of the three nerves. Many of the motoneurones discharged repetitively to single large volleys (Perl, 1962). Only the first discharge was used to calculate FI. Usually 5 tests were made at every stimulus intensity, but in many cases this was expanded to 1 or more. Collection of data from each motoneurone took from 45 min to 2 hr. Accordingly, the number of units which could be studied in each animal was limited, the experiment being stopped whenever indication of deterioration of the preparation was noted. To judge such deterioration, both C2 production and the nature of spinal-cord circulation observed through the dissecting microscope were used. Two to seven motoneurones were studied in each of 24 experiments. RESULTS The afferent input The afferent input was graded by adjusting the size of single afferent volleys evoked by 5,usec pulses. The volleys employed varied from a few low-threshold, large-diameter fibres to all the myelinated afferent fibres in each nerve. The large range of afferent fibres made accurate description of the volleys difficult. Despite some obvious limitations in the technique, the volleys from each nerve will be described by the stimulus intensity used to evoke them, expressed as multiples of the intensity required to produce a threshold response at the dorsal-root-spinal-cord junction. The threshold stimulus for dorsal-root response was determined by examining the dorsal-root recording at high amplification and was checked several times during each series of stimuli to a particular nerve. Usually threshold values stayed reasonably stable for 1-2 hr. In most of the experiments increments of stimulus intensity were made at certain multiples of the threshold (T) value: l 1, 1-2, 1-3, 1-5, 2, 5, 1, 5 for BST and S nerves and 1-5, 2-, 5-, 1 and 5 for TS nerve. Volleys initiated by stimuli equal to specific multiples of threshold for a particular nerve cannot be expected to excite the same number of fibres or fibres with the same range of diameters in different preparations. Comparison of the size of volleys in different animals evoked by stimuli representing a given multiple of threshold did show that some general statements regarding the composition of volleys could be made. Figure 1 illustrates some relationships between volley size and stimulus intensity expressed in this manner. In Fig. 1A the size of the Group I volley evoked at specific multiples of threshold stimulus to BST are plotted for two experiments. As illustrated here, the Group I afferent spike was often of maximal size with stimulus intensities of twice threshold. An

4 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 433 indication of the variation in volley composition for stimuli less than this value is given by the difference between the results from the two preparations. The volume dorsal-root recording used during the course of the experiments frequently showed a variable notch in the BST Group I fibre component (Bradley & Eccles, 1953; Laporte & Bessou, 1957). When components with two different latencies within Group I were present they could be first recognized with stimuli between 1-3 and 1-5T. As indicated in Fig. 1 A, a negative deflexion in the dorsal-root lead consistent with the 1 x me td E o6- a-. H 1 x E Is x co E - e,o 'Stimulus' intensity Fig. 1. Relationship between composition of volleys and stimulus intensity. A. Ordinate: Amplitude of the Group I component evoked by BST stimulation relative to maximal size; recorded from S 1 dorsal root. Abscissa: stimulus intensity relative to Group I threshold, log. scale * (-23) volume lead (monophasic lead from cut dorsal root followed a similar course): (-9) volume lead. Threshold and maximal points for more slowly conducting components (Groups II and III) are indicated for these two experiments. B. Ordinate: Amplitude of am component (left) and of 8 component (right) evoked by sural stimulation. Abscissa: stimulus intensity relative to oab threshold. *, o,b potential recorded from sural; x = a component recorded from sural (-25)., oca component recorded from dorsal root in another experiment (-23).

5 434 E. B. PERL conduction velocity for Group II fibres (Hunt, 1954), was ordinarily discernable for BST (or TS) volleys somewhere between 1-5 and 2T. It always appeared to reach a maximal amplitude at lot. While difficult to determine accurately, the dorsal-root response changed with BST or TS stimuli between 5 and lot, presumably owing to the addition of smaller Group III fibres. This low-amplitude irregular component reached maximal size somewhere between and 5T stimuli to BST and TS. Figure 1 B gives the approximate relationship between sural volley composition and stimulus intensity. The filled circles show the growth of the sural volley in a particular experiment, as recorded from the sural nerve itself. In this case the alpha-beta spike (Gasser, 196) was complete at 5T. The open circles in Fig. LB are from another experiment in which the sural response was recorded from the distal end of a cut dorsal root. It is possible that at least part of the difference between the two curves for the weaker stimuli may represent differences in threshold value, since considerably greater shunting occurred in the case of the dorsal-root recording. Fibres conducting in the delta range of velocities could first be detected with stimuli of 5T to the sural nerve. Figure LB shows changes in the delta component as recorded from the sural nerve as stimulus intensity was increased. Thus for sural nerve it could be reasonably expected that stimuli between 2 and 5T would complete the alpha-beta spike and that significant numbers of delta fibres would be active in volleys evoked by stimuli of lot or more. In some experiments an attempt was made to evaluate whether strong stimulation of the peripheral nerves (-5T) evoked repetitive discharge from some of the primary afferent fibres. Recording from whole dorsal roots or filaments dissected from them at the end of an experiment indicated that repetitive firing of primary afferent fibres did not occur under conditions existing in these experiments. General characteristics of the population studied A total of 11 motoneurones were studied in some detail. The data collected for 99 of these were complete enough to allow comparison of monosynaptic and polysynaptic responses. The range of monosynaptic responsiveness of this population of cells is illustrated in Fig. 2, which shows the firing indices (FI) for 95 neurones at several stimulation intensities to BST. The figure was constructed by ranking motoneurones in order of increasing FI for each of three stimulus intensities, and then plotting this rank against FI. The shape of the curve formed in this manner was sigmoid at all stimulus strengths because some neurones did not respond (FI = ) and some responded to every test (FI = 1) and a number had intermediate FI (between and 1). With increase of stimulus intensity

6 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 435 and volley size the curve shifted to the left, but the number of neurones with intermediate Fl stayed approximately the same, the change occurrmng owing to a decrease of units which did not respond and a concomitant increase in those which discharged to every volley a: so n Rank Fig. 2. Monosynaptic firing indices (FI) of the motoneurones studied for three sizes of BST (biceps-semitendinosus nerve) volleys. Volleys are described in terms of multiples of threshold for this and all other figures. Six cells were excluded from this figure because stimulus intensities other than those illustrated had been used. The cells were ranked according to firing index (FI), lowest rank given to those with the lowest FI. The distribution of monosynaptic FI illustrated in Fig. 2 is quite similar to that described by Lloyd & McIntyre (1955) for the monosynaptic responses of triceps surae motoneurones in spinal cat. One important difference in the range and variety of FI for the two populations is that more of the neurones in the present experiments responded than did the extensor motoneurones studied by Lloyd & McIntyre. Several reasons for

7 436 E. R. PERL this may be given. First, the monosynaptic drive for a given motoneurone in the present experiments was derived from both the muscle it supplied (homonomous) and the immediate synergist (heteronomous), BST being the combination of nerves to two muscles. Secondly, limb flexor motoneurones in the spinal preparation probably have a greater impingement of 'background' excitatory impulses than do extensor motoneurones, the reflex behaviour of this preparation being strongly pitched towards flexion. Thirdly, the combination of long spinal facilitation and posttetanic potentiation of the afferent nerves used by Lloyd & McIntyre to select their units may have brought a type of extensor motoneurones into the discharge zone that had no counterpart in the present flexor population. In agreement with the results of Lloyd & McIntyre, it would appear that the monosynaptic reflex recorded from the whole ventral root would be composed of the discharge of a number of neurones to every incoming volley and the fluctuating participation of a group of cells, those with intermediate Fl. In addition, the change in the size of the monosynaptic reflex with increased afferent input can be expected to result from an increase in the number of neurones that discharge for every test volley, because the number of cells with intermediate FI tends to stay approximately the same. It will be seen from the discussion of the responses of individual cells that recruitment to a FI of 1 comes from those cells with intermediate FI and this is accompanied by a shift of cells with FI of into the range of intermediate FI. The distribution of FI for the same motoneurones (with a few omissions) excited during a polysynaptic reflex (on the basis of latency: Lloyd, 1943a, Perl, 1962) initiated by sural volleys is shown in Fig. 3. The sigmoid shape of the ranked FI for the neurones is similar to that shown by the monosynaptic responses. An increase of volley size produced by increasing the stimulus intensity again resulted in the shift in the curve to the left, with approximately the same number of neurones remaining in the intermediate range of FI. As was the case for the ranked monosynaptic responses, the change in curves occurred from a decrease of cells with a FI of and an increase of those with a FI of 1. The rankings given in Fig. 3 demonstrate that the afferent fibres of the sural nerve are powerfully excitatory for these flexor motoneurones, since a large fraction of the population was discharged by stimuli exciting only the larger myelinated fibres (see Fig. 1). With volleys containing more afferent fibres than those illustrated in Fig. 3, the number of neurones responding with FI of 1 sharply increased, and with maximal volleys only 7 out of 99 neurones could not be discharged at all. The reflex linkage between afferent fibres from TS nerve and the knee flexor motoneurones was not as effective as that evoked by stimulation

8 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 437 of BST or sural nerves. Again, with latency as a criteria, responses following a TS volley were mediated by a polysynaptic reflex (Lloyd, 1943b; Eccles & Lundberg, 1959a; Perl, 1962). Figure 4 gives the distribution of FI for three sizes of TS volley for those cells for which such data were available. It can be seen that volleys containing most of Group I and T Z Rank Fig. 3. Polysynaptic firing indices of the motoneurones illustrated in Fig. 1 to two sizes of S (sural nerve) volleys. Two units at 1*5T and one at 2T of those shown in Fig. 1 were omitted because stimulus intensities other than those shown had been used. Rank determined as in Fig. 1. some Group II fibres (2T) elicited responses from only some 25 % of the cells. Even volleys of 5T could discharge only 43 % of the population and very few cells had FI of 1. With the largest volley illustrated (lot), which included all Group II as well as some smaller fibres, nearly half the motoneurones failed to respond. Only a few more cells were discharged by the maximal volleys employed (5T). It should be noted that the number

9 438 E. R. PERL of cells with intermediate Fl after TS volleys was considerably fewer than that in the intermediate range for S or BST volleys. One possible explanation for this finding is that the internuncial path involved in the pathway between TS afferent fibres and the motoneurone has some form of threshold which varies over the population TS IOT 6 TS it 5 2T Rank Fig. 4. Polysynaptic firing indices of the motoneurones illustrated in Fig. 1 to three sizes of TS (triceps surae nerves) volleys. Five units of those shown in Fig. 1 were omitted because stimulus intensities other than those shown had been used. Rank determined as in Fig. 1. Comparison of monosynaptic and polysynaptic response for individual motoneurones Responsiveness of individual neurones was judged by the relationship between FI and volley size from each afferent source. The differences in polysynaptic reflexes from one preparation to another made it clear that comparison of units from different experiments was of limited value. Cells

10 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 439 studied in a particular preparation varied in their response to polysynaptic drive, but this variation was in keeping with the level of such activity for the preparation. For this reason, and because of the difference in volley composition from animal to animal, the comparison of responsiveness of units to each of the afferent sources was made on an experiment-by-experiment basis. 1 8 : BST _-- s 6 X Z TS 6 4 Unit I Unit 2 Unit 3 Unit F_.i * Multiple of threshold Fig. 5. Firing indices of the four units from one experiment for volleys from each afferent nerve. The horizontal rows are for one afferent source, with the figures on the abscissa referring to multiple of threshold (log. scale). All important data are included, although all volley sizes employed are not shown. Only the monosynaptic discharge to BST and the first discharge for S and TS (during a 5 msec observation period) were used to calculate FI. Units numbered in the order studied. Polysynaptic reflex was small in this experiment The methods and results of these comparisons are illustrated in Figs Figure 5 shows representative firing indices of the four units studied in one experiment. In this animal the polysynaptic reflex evoked by sural stimulation and recorded from the whole ventral root was relatively small. Each horizontal row gives FI from one afferent source. Monosynaptic response to BST volleys is shown in the upper row. The stimulus intensity is indicated below each vertical bar as the multiple of the threshold. Comparison of the monosynaptic FI for the four units shows that they differed considerably. For instance, unit 4 had a FI of approximately 5 for a small volley ( -IT) and a FI of 1 for a 1-2T volley. The other three units of this experiment were judged to have lower monosynaptic re- 29 Physiol. 164

11 44 E. B. PEBL sponsiveness, because near-threshold stimuli were not as effective. For unit 2, a 1llT volley gave a FI of 15, the FI increasing to 9 for a 1-2T volley. Unit 1 first responded to a 1-3T volley, and even with maximal Group I input its FI did not reach 1. Unit 3 rarely discharged monosynaptically even to a large volley. Thus unit 4 was the most responsive, unit 2 next, unit 1 next and unit 3 the least responsive. Similar rating of the response to sural volley is easy, from the data shown in the middle line of Fig. 5, because unit 1 and unit 3 did not respond to maximal volleys and unit 4 was clearly more responsive than unit 2. Thus, other than the inability Unit 1 Unit 2 Unit 3 Unit BT6- A ~ * a: TS *5 25 2*5 25 Multiple of threshold Fig. 6. Firing indices of the four units from one experiment for volleys from each afferent nerve. Figure constructed as Fig. 5. Polysynaptic reflex was prominent in this experiment. -7. to distinguish between units 1 and 3, the relationship between volley size and response was similar for monosynaptic and sural-evoked polysynaptic discharge. Within a context of a less effective polysynaptic linkage, responsiveness to TS volleys varied, as did sural responsiveness, and therefore was parallel to monosynaptic responsiveness. The relationship between afferent input and response for four units from an experiment showing a prominent polysynaptic reflex after sural volleys is shown in Fig. 6. To monosynaptic test, unit 3 was the most responsive, unit 4 next, unit 1 next and unit 2 least responsive. The differences in responsiveness to sural volleys were more subtle, but on close examination the criteria used above would also place unit 3 as the most responsive,

12 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 441 unit 4 next, unit 1 next and unit 2 as the least responsive. Therefore, in the experiment of Fig. 6, as in the previous one, effectiveness of monosynaptic transmission was paralleled by the relative effectiveness of the polysynaptic reflex from sural nerve. Differences in responsiveness to triceps surae volleys in Fig. 6 were so slight that relative responsiveness could not be decided from the available data. Fig Unit 1 Unit 2 Unit 3 BST BT so EC s TS 6 TS SO Multiple of threshold Firing indices of the three units from one experiment for volleys from each afferent nerve. Figure constructed as Fig Figure 7 shows the results of an experiment in which polysynaptic reflexes were of 'average' or intermediate size. In this experiment once again there was a parallelism in monosynaptic and polysynaptic responsiveness. Of the three units, unit 1 was most responsive to both BST and sural volleys, unit 2 being next and unit 3 being the least responsive. The difference in response to TS afferent input is clear and follows the pattern found for BST and sural nerve. The parallel relationship between monosynaptic and polysynaptic responsiveness, judged in the manner described for these three experiments, was found in thirteen preparations. Some minor variations in the relation between relative monosynaptic responsiveness and relative sural polysynaptic responsiveness was observed in the experiments in which five or more cells were studied. Including these latter, the tendency for relative monosynaptic responsiveness to parallel relative polysynaptic responsiveness occurred in 18 of 24 experiments and involved 67 out of

13 442 E. R. PERL motoneurones. On the other hand, marked differences in the response to monosynaptic and polysynaptic reflexes were found in 1 units from six different preparations. In four of the same preparations the response of other units exhibited the more usual parallelism between monosynaptic and sural responsiveness. An experiment in which marked difference existed in the ability of the two different reflexes to drive a unit to discharge are illustrated in Fig. 8. Unit 4 in Fig. 8 was unquestionably the most responsive monosynaptically, because small volleys (1.2T and 1-3T) 1-8- Z BST 6 - Unit 1 Unit 3 Unit 4 Unit , 11l.1i 8, 1 S S TS4-6TS Multiple of threshold Fig. 8. Firing indices of 4 of the 5 units studied in one experiment for volleys from each afferent nerve. Figure constructed as Fig. 5. Unit 2 not shown because data incomplete. -1. were associated with a higher FI than for the other cells and a Fl of 1 was reached at 1-5T. In contrast, the relationship between FI and polysynaptic response indicated that unit 4 was the least responsive. A siumilar difference in the response to the two test reflexes appeared in the case of unit 5, which was rated third in monosynaptic responsiveness but the most responsive of the four as indicated by the relation between polysynaptic FI and sural stimulus intensity. Another instance of marked difference in the effectiveness of monosynaptic and polysynaptic mechanisms in driving motoneurones is shown in Fig. 9, which describes responses for three out of seven units from one experiment. Of the three, unit A would appear the most responsive monosynaptically and polysynaptically but unit C, which was the least re-

14 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 443 sponsive to BST volleys, was more effectively excited by a polysynaptic reflex than unit B. Units B and C illustrate another interesting point. For unit B stimuli of 1-5T did not evoke a monosynaptic response, yet a stimulus of 2T gave rise to a relatively high FI, an unusually steep increase in responsiveness with increments in volley. In contrast, unit C responded infrequently to small volleys, 1-3 and 1-5T, but the Fl then did not increase as more Group I fibres were excited (2 and 5T). Thus for unit C the Group I fibres having monosynaptic connexions concentrated in the low-threshold band and were relatively ineffective, while the converse held for unit B. Fig. 9. A 1, 8- X BST 6:, : 6 FHEH LS III 8 Zlf - 6 ke X TS 4 -IE I B x C AL Multiple of threshold Firing indices of 3 of 7 units studied in one experiment for volleys from each afferent nerve. Figure constructed as Fig Even in those cases in which monosynaptic 'excitability' differed markedly from sural polysynaptic response, as illustrated in Figs. 8 and 9, relative responsiveness to sural and triceps surae volleys tended to be similar. On the other hand, as is illustrated in Fig. 5, good polysynaptic response to sural volleys was not necessarily associated with large reflexes evoked from triceps surae, despite the fact that a degree of parallelism may have existed. Volley composition and monosynaptic discharge The situation of the present experiments differs from the previous studies of monosynaptic reflex discharge for single motoneurones because afferent fibres other than Group I of the homonomous muscle nerve were

15 444 E. R. PERL effectively excitatory to the motoneurones under consideration. Stimuli of 5T to BST nerve regularly completed the afferent spike associated with fibres conducting in excess of 6 m/sec, i.e. Group I. It was somewhat surprising to find that the FI increased or decreased for a number of neurones of low or medium responsiveness with increases of stimulus strength beyond that needed to give a maximal Group I spike (Fig. 5, unit 1; Fig. 6, units 1, 2 and 4; Fig. 7, unit 3; Fig. 8, unit 3; Fig. 9, units B and C). Latency of discharge for these units was too short to permit direct contribution to monosynaptic excitation by slowly-conducting fibres, yet it was recruitment of these latter which appeared to be associated with the changes in monosynaptic responsiveness at stimulation intensities beyond the Group I maximum. While other complex factors may have played a part, an undoubtedly important consideration is that strong stimulation initiated activity in afferent fibres which were capable of producing long-lasting changes in spinal cord activity and this in turn was capable of indirectly influencing monosynaptic response at the testing intervals used (2 sec). A considerable variability in nerves to the knee flexors prompted the use of the combined nerves (BST) as the afferent source for the monosynaptic tests. It was believed that this procedure might give a more regular and reproducible measure of monosynaptic transmission. The FI of a few neurones was separately determined for each of the branches ordinarily combined as the BST nerve. The results so obtained were consistent with those described by Lloyd & McIntyre (1955) and Hunt (1955) for ankle extensor motoneurones. The flexor motoneurones of the present study would respond to one of the nerves used to form BST with a much higher Fl to another than to the others, singly or in combination. DISCUSSION The assumption common to many investigations, that increasing the number of afferent fibres active in an afferent volley increases the excitatory drive for some particular reflex, is inherent in the techniques used in this study. Legitimate objections to this technique can be raised. For example, not only are electrically evoked nerve volleys an artificial combination of afferent activity, but in addition there is evidence to indicate that some but not all of the afferent fibres in each of the afferent sources used are, in fact, excitatory to the knee flexor motoneurones (Eccles & Lundberg, 1959a; Hagbarth, 1953; Laporte & Lloyd, 1952). In the preparations used, however, single volleys from any of the afferent sources were effective in evoking discharge and this effectiveness increased with progressive increments of stimulus. Furthermore, the stimulation conditions were standard from one experiment to another as well as within a given preparation. Thus

16 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 445 the responses from the motoneurones while initiated under 'artificial conditions' have a sound basis for comparison. The finding that the majority of flexor motoneurones have polysynaptic responsiveness in keeping with their monosynaptic excitability is not startling but is relative to several unresolved questions. On the one hand, the population monosynaptic reflex does appear to be an adequate way for measuring excitability changes of motoneurones distributed by internuncial mechanisms concerned with several flexor reflexes. For these flexor reflexes the assumptions underlying the use of the monosynaptic reflex as a test of motoneurone excitability (Lloyd & McIntyre, 1955) seem reasonable and can be supported by the present findings. Adequately arranged monosynaptic tests for the motoneurone population comprising the pool examined in the present experiments would sample the excitability variations associated with either the cutaneous or muscle flexor reflex of all but the few cells with marked polysynaptic responsiveness and low monosynaptic excitability. Further, the influence of the cells included in the testing population but not reached by the polysynaptic flexor effects would be negligible because of their small number, thus masking the reflex excitability change by a passive population would seem unlikely. The findings on relative responsiveness are pertinent to the sequence of motoneurone recruitment during progressively increasing motor activity. It has been known since the classical experiments of Denny-Brown (1929) that certain extensor motoneurones tend to be the first recruited during weak stretch reflexes, with other cells appearing in the course of increasing reflex activity in a specific order. The present experiments suggest that many of the same flexor motoneurones would respond to weak reflex drive, whether it be of monosynaptic origin or the polyneurone flexor mechanism. In addition, at least in reflexes initiated by single volleys recruitment during a progressively increasing flexor reflex would tend to involve additional elements in a manner parallel to recruitment to increasing monosynaptic response. Inasmuch as three ipsilateral reflex connexions to these flexor motoneurones are shown to have a more or less parallel distribution of effects, it is possible to venture the hypothesis that the distribution of motoneurone responsiveness will be similar, regardless of excitatory source. One recently described arrangement, the fusimotor control over muscle-spindle sensitivity and discharge (Kuffler, Hunt & Quilliam, 1951; Eldred et al. 1953), has particular significance in light of this tendency for motoneurones to have polysynaptic responsiveness parallel to the effectiveness of monosynaptic connexions. Those motoneurones most likely to be affected by the muscle-spindle Group I afferent background would be the same as those most sensitive to excitation of a few afferent fibres associated with the flexor pattern. For this reason, both

17 446 E. R. PERL the threshold and gradation ofinternuncially mediated flexor activity would be significantly altered by any change in the muscle-spindle discharge. While parallel variation in reflex responsiveness was usually observed, significant exceptions to this were present. These exceptions may give some insight on processes underlying the variations of motoneurone responsiveness. Some factors contributing to variation of motoneurone responsiveness may reside within the cells themselves, i.e. post-synaptically. Such post-synaptic factors would include the relation between membrane potential and threshold for impulse initiation and differences in adaptation to membrane potential changes (Eccles, 1961; Sasaki & Otani, 1961). Systematic variation in the amount of depolarization necessary to reach threshold for a conducted action potential would be sufficient to explain differences in the responsiveness of elements of a motoneurone pool provided that reflex excitability was always parallel for the various tests. On the other hand, the observation in some motoneurones of a relatively high degree of responsiveness to polysynaptic drive in the presence of a relatively low monosynaptic responsiveness is not consistent with this explanation. A membrane potential close to threshold should be favourable for synaptic transmission from any source. Furthermore, a high degree of monosynaptic responsiveness in the presence of poor polysynaptic responsiveness would also argue against purely post-synaptic determinants of 'excitability', though here adaptation could play a part. From this argument it seems likely that part of the cause of differences in motoneurone responsiveness rests in the presynaptic endings, i.e. the functional effectiveness of synaptic knobs from Group I afferent fibres and from interneurones of the generalized flexor pathway. From the results of the present experiments one could argue equally well that monosynaptic terminals and the terminals of the final interneurone in the polysynaptic flexor reflex are parallel in effectiveness on various motoneurones or that there is no positive correlation between them, because there is no means of measuring the post-synaptic contribution to 'excitability'. It would appear possible to weigh the relative importance of the various factors in functional organization of a motoneurone nucleus only from direct evidence in which the relationship between membrane potential, post-synaptic potentials and discharge was examined in cells excited by different mechanisms. The difference in effectiveness of excitatory connexions of Group II fibres from triceps surae and the myelinated fibres of cutaneous nerve help to explain some variations in the reflex responses which have been described for different experimental preparations. Reflexes initiated by Group II and Group III fibres from triceps surae nerve are absent in decerebrate cats, but appear after spinal transections in the same pre-

18 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 447 parations (Eccles & Lundberg, 1959b; Kuno & Perl, 196). Kuno & Perl (196) demonstrated that such reflexes can be demonstrated in decerebrate cats provided that the muscle afferent volley was preceded by a conditioning volley from cutaneous afferent fibres. These authors suggested that in decerebrate animals the absence of the reflex from Groups II and III fibres of muscle nerve might be related to depression of certain interneurones important for polysynaptic reflex by inhibitory connexions from suprasegmental sources. The present results indicate that the depression of polysynaptic reflexes from a muscle nerve to flexor motoneurones in decerebrate animals is probably also a consequence of the relatively weak functional connexions in this pathway. SIUMMARY 1. One hundred and one flexor motoneurones were studied in twentyfour unanaesthetized, decapitate cats. The cells were those which could be discharged monosynaptically with or without post-tetanic potentiation from biceps posterior and semitendinosus nerves (BST), the responses being recorded in fine ventral root filaments. The reflex excitability of the neurones to single volleys graded in size from BST, sural (S), and triceps surae (TS) nerves was judged by determining firing index from 5 to volleys for each set of conditions. 2. In the absence of facilitation or potentiation, the firing index of the population studied to maximal volleys (for myelinated afferent fibres) from each source ranged from to 1. For several different sized volleys from BST the number of neurones with intermediate monosynaptic firing indices remained roughly the same, progressively larger volleys recruiting units to the 1 level from cells with intermediate firing indices and to intermediate values from units previously not discharging. A similar pattern was demonstrated for polysynaptic response to S volleys. TS volleys were regularly less effective than S volleys in evoking discharge. 3. The motoneurones from each experiment were compared in responsiveness to the three afferent sources on the basis of the maximal firing index achieved for large volleys and the firing index for intermediate sized volleys. Rating the cells of one preparation for responsiveness to each afferent nerve showed that the common and usual feature was for monosynaptic responsiveness (BST volleys) to closely parallel polysynaptic responsiveness (S and TS volleys). A few (1) major exceptions to this occurred in which either relatively high monosynaptic responsiveness was coupled with relatively low polysynaptic responsiveness or the converse, high polysynaptic responsiveness in combination with relatively low monosynaptic responsiveness. 4. It was concluded that (a) the monosynaptic reflex is an adequate

19 448 E. R. PERL criterion of polysynaptic flexor effects; (b) monosynaptic background must have an important part in determining reflex excitability from other sources; (c) many of the same motoneurones activated by weak monosynaptic input would be active in near-threshold generalized flexor reflexes. Further, the exceptions to the general parallelism of monosynaptic and polysynaptic responsiveness led to the deduction that variation in responsiveness of a pool of motoneurones is not solely determined by the characteristics or features of the motoneurones. This work was supported by Grant B from the National Institutes of Health, U.S. Public Health Service. REFERENCES BRADLEY, K. & ECCLES, J. C. (1953). Analysis of the fast afferent impulses from thigh muscles. J. Physiol. 122, CREED, R. S., DENNY-BROWN, D., ECCLES, J. C., LIDDELL, E. G. T. & SHERRINGTON, C. S. (1932). Reflex Activity of the Spinal Cord. London: Oxford University Press. DENNY-BROWN, D. (1929). On the nature of postural reflexes. Proc. Roy. Soc. B, 14, ECCLES, J. C. (1961). The mechanism of synaptic transmission. Ergebn. Physiol. 51, ECCLES, R. M. & LUNDBERG, A. (1959a). Synaptic actions in motoneurones by afferents which may evoke the flexion reflex. Arch. Ital. Biol. 97, ECCLES, R. M. & LU-NDBERG, A. (1959b). Supraspinal control of interneurones mediating spinal reflexes. J. Phy8iol. 147, ELDRED, E., GR.ANIT, R. & MERTON, P. A. (1953). Supraspinal control of muscle spindles and its significance. J. Physiol. 122, GASSER, H. S. (196). Effect of the method of leading on the recording of the nerve fiber spectrum. J. gen. Physiol. 43, HAGBARTH, K.-E. (1953). Excitatory and inhibitory areas for flexor and extensor motoneurones. Acta physiol. scand. 26. Suppl. 94, HUNT, C. C. (1954). Relation of function to diameter in afferent fibres of muscle nerves. J. gen. Physiol. 28, HUNT, C. C. (1955). Monosynaptic reflex response of spinal motoneurones to graded afferent stimulation. J. gen. Physiol. 38, KUFFLER, S. W., HUNT, C. C. & QUILLIAM, J. P. (1951). Function of medullated small-nerve fibers in mammalian ventral roots: afferent muscle spindle innervation. J. Neurophy8iol. 14, KuNo, M. & PERL, E. R. (196). Alteration of spinal reflexes by interaction with suprasegmental and dorsal root activity. J. Physiol. 151, LAPORTE, Y. & BESSOU, P. (1957). ]Ptude des sous-groupes lent et rapide du Groupe I (fibres aff6rentes d'origine musculaire de grand diam6tre) chez le chat. J. Physiol., Paris, 49, LAPORTE, Y. & LLOYD, D. P. C. (1952). Nature and significance of the reflex connections established by large afferent fibers of muscular origin. Amer. J. Physiol. 169, LLOYD, D. P. C. (1943a). Reflex action in relation to pattern and peripheral source of afferent stimulation. J. Neurophysiol. 6, LLOYD, D. P. C. (1953b). Neuron patterns controlling transmission of ipsilateral hindlimb reflexes. in cat. J. Neurophysiol. 6, LLOYD, D. P. C., HUNT, C. C. & MCINTYRE, A. K. (1955). Transmission in fractionated monosynaptic spinal reflex systems. J. gen. Physiol. 38, LLOYD, D. P. C. & MCINTYRE, A. K. (1955). Monosynaptic reflex responses of individual motoneurones. J. gen. Physiol. 38, PERL, E. R. (1962). Observations on the discharge of flexor motoneurones. J. Physiol. 164, SASAKI, K. & OTANI, T. (1961). Accommodation in spinal motoneurones of the cat. Jap. J. Physiol. 11,

20 COMPARISON OF FLEXOR MOTONEURONE RESPONSES 449 Note added in proof. After this paper was submitted, the author became aware of a study by P. G. Kostyuk (1961, Sechenow J. Physiol. 47, ) describing changes of synaptic potentials recorded from flexor motoneurones as their membrane potential was altered. Shift of membrane potential resulted in systematic variation of polysynaptically evoked excitatory synaptic potentials largely similar to the changes described for monosynaptic excitatory potentials. Those differences of behaviour between potentials initiated via the two pathways which were found were attributed to the impossibility of obtaining 'pure' excitation in the polysynaptic reflex. These findings would support the argument in the Discussion which suggested that the level of motoneurone membrane potential should similarly affect the response to monosynaptic and polysynaptic reflex connexions.