J. Physiol. (1977), 264, pp. 63-87 With 9 text-figures Printed in Great Britain

63

AN ESTIMATE OF THE SECONDARY SPINDLE RECEPTOR AFFERENT CONTRIBUTION TO THE STRETCH REFLEX IN EXTENSOR MUSCLES OF THE DECEREBRATE CAT

BY K. KANDA AND W. Z. RYMER* From the Laboratory of Neural Control, NINCDS, Bethesda, Maryland 20014, U.S.A. (Received 8 April 1976) SUMMARY

1. Vibration or stretch ofthe medial gastrocnemius muscle in the decerebrate cat each caused a significant increase in the tension of a synergist, the lateral gastrocnemius. 2. Simultaneous vibration and stretch of the medial gastrocnemius resulted in a substantial increase of lateral gastrocnemius tension which was greater than that produced by medial gastrocnemius vibration alone. The size of this force increase was proportional to the amplitude of medial gastrocnemius stretch, for the limited range of amplitudes examined. 3. Since the discharge of the medial gastrocnemius I a afferent fibres was held constant by vibration, the additional tension in lateral gastrocnemius provoked by medial gastrocnemius stretch must have resulted from the activation of an excitatory pathway separate from the I a afferent system. The secondary spindle afferent pathway was considered to be the most likely candidate. 4. The contributions of the Ia afferents and the additional stretch induced excitation to the stretch reflex were compared. The Ia potency was calculated from the ratio of tonic vibration reflex force and the vibration frequency. The total I a contribution to the stretch reflex, which was estimated from the product of this ratio and the primary ending stretch sensitivity, seemed modest, and was consistently smaller than the proposed secondary contribution. 5. The medial gastrocnemius nerve was subjected to anodal blockade at a strength sufficient to eliminate I a transmission. Under these conditions, the lateral gastrocnemius excitation produced by medial gastrocnemius stretch or vibration was largely eliminated. When lateral gastrocnemius Present address: Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A. *

3

P HY 264

K. KANDA AND W. Z. RYMER 64 vibration was superimposed, the excitatory effect of medial gastrocnemius stretch was partly restored suggesting that some central facilitation by group I a afferents may be necessary for group II excitatory effects to be manifested. 6. Although the additional excitatory actions of medial gastrocnemius stretch were examined exclusively in a synergist, it is suggested that similar effects are likely to occur in the homonymous stretch reflex. INTRODUCTION

The functional role of secondary spindle afferent fibres is not well understood. The traditional view holds that secondary afferents, in common with other high threshold afferents from muscle, skin and joints produce widespread inhibition of extensor motoneurones, with concurrent excitation of flexors (Eccles & Lundberg, 1959a, Holmqvist & Lundberg, 1961). An alternative view, proposed by Matthews (1969) is that secondary afferents produce significant excitatory effects on extensor motoneurones. Matthews hypothesis was based on two observations. The first was that the reflex response to vibration of the soleus tendon did not diminish as muscle length was increased. Since the soleus primary endings would have been entrained at the vibration frequency (Brown, Engberg & Matthews, 1967), increasing muscle stretch should have diminished the incremental response to vibration, if the primary endings were a dominant source of excitation in the stretch reflex. Secondly, when the reflex tension response to vibration was expressed as a ratio of force and discharge frequency of primary endings, the calculated values seemed rather small. This lack of occlusion of the vibration reflex together with the apparent modesty of the I a afferent contribution led Matthews to propose an additional source of excitation in the tonic stretch reflex. The secondary spindle afferent pathway was selected as the most likely candidate. These conclusions have been challenged on a number of grounds. Grillner (1970, 1973) has argued that occlusion of the tension response to vibration could have been masked by the increased tension resulting from stretch of the contracting muscle. Other objections have been directed at the adequacy of vibration as a stimulus (Barnes & Pompeiano, 1970a, b). These arguments have been considered in detail in recent publications (Matthews, 1972; McGrath & Matthews, 1973; Grillner, 1973). Additional evidence supporting an excitatory projection from secondary spindle afferents to extensor motoneurones has been provided by Westbury (1972), and more recently by Kirkwood & Sears (1974), although a substantive excitatory synaptic projection remains to be demonstrated. In our present work we have applied stretch and longitudinal tendon

STRENGTH OF SECONDARY AFFERENT EXCITATION 65 vibration to the medial gastrocnemius muscle (MG), and have examined the resultant reflex response in a close synergist, the lateral gastrocnemius (LG) which was held at constant length. This approach permitted us to verify the existence of an excitatory pathway from muscle which was activated by muscle stretch, but which was distinct from the I a afferent system. The secondary spindle receptor could well be the source of this additional excitation, although the characteristics of the excitatory pathway proved to be more complex than we had anticipated. METHODS The experiments were performed on ten cats which were decerebrated by intercollicular transaction. Anaesthetic induction and maintenance were performed with nitrous oxide, oxygen, and fluothane administered in closed circuit. Animal temperature was maintained in the range 36-38° C with a heating pad and infra-red radiation. The blood pressure was recorded via a carotid cannula, and respiration monitored using a Beckman meter, which measured expired carbon dioxide. The triceps surae muscles of the left hind limb were separated, and metal hooks stitched to the MG and LG tendons. Other muscles of the hind limb, hip, and tail were denervated, and cutaneous nerves to the limb were sectioned. A lumbar laminectomy was performed (L4-L7), the animal mounted in a frame, and the left limb immobilized by supporting clamps which were fixed firmly to the lower femur and tibia at the ankle. Mineral oil pools were fashioned using skin of the hind limb and back, and these were kept at 350 C by heating coils submerged within the oil.

Muscle puller8 Two muscle pullers were mounted, as illustrated in Fig. 1. A large electromagnetic puller was used to apply stretch and vibration to the MG muscle. This device incorporated a displacement transducer which provided a signal used to establish feed-back control. This puller was capable of producing vibrations of 200 /tm amplitude, at frequencies up to 300 Hz. The vibration amplitude was monitored using a continuous display of the length transducer output on an oscilloscope. The displacement transducer had a linear response over a wide range of frequencies, exceeding 300 Hz. The stated measurements were accurate to within 5 %, even for small displacements, which were calibrated optically using a microscopic grid. The compliance of this puller was estimated to be 0-06 mm/kg. Force output was estimated indirectly from measurements of the motor coil current. This estimator had limited frequency response characteristics, but proved sufficiently accurate for the measurement of maintained tension. Sinusoidal functions were provided by Wavetec signal generators, and ramp functions by a wave-form generator capable of producing ramp and hold functions of variable slope and duration. The second puller was considerably smaller, consisting of a Pye-Ling vibrator with positional feed-back established using signals provided by a linear voltage displacement transducer (LVDT). This puller had a maximum displacement of 3-8 mm, but was typically used in the mid-position, for LG vibration only. In the initial experiments, muscle tension was estimated from measurements of the vibrator coil current, but this method was eventually replaced by a direct technique, in which four strain gauges were mounted on a thin metal annulus, with each strain gauge connected to the arms of a Tektronix a.c. bridge and amplifier. This annulus was attached to the drive shaft of the puller, in series with the muscle. The compliance of this system was 3 -2

66

K. KANDA AND W. Z. RYMER

considerably larger than that of the major puller (0.20 mmikg). This did not produce

any errors of recorded length or tension, but meant that muscle length was sometimes less than expected, particularly when muscle force was high.

Fig. 1. The MG and LG muscles were separated and their tendons were attached to separate muscle pullers. The larger puller, Pb, was used for vibration and stretch of the MG. The smaller puller, P., was maintained at constant length, and was used both to record LG tension and to vibrate the LG tension. In some experiments, an anodal DC nerve block was applied to the MG nerve via platinum electrodes (B), using current derived from a constant current source (A). A dorsal root filament (C) was used to record the Ia afferent discharge, or to monitor the anodal block. Vibration The amplitude of the tendon vibration was set between 120 and 150 1am for both muscles, and the frequency was usually set between 170 and 200 Hz (typically 180 Hz). In each instance, the amplitude of the vibration was chosen so as to exceed the minimum necessary to produce stable driving of all the primary endings, as evidenced by a constant, maintained tension response to vibration of increasing amplitude. The tension response usually reached a plateau for vibration amplitudes between 80

STRENGTH OF SECONDARY AFFERENT EXCITATION

67

and 100 jsm, consequently amplitudes in excess of 120 jam were considered to provide adequate safety margin. In cases where ramp stretch was combined with tendon vibration, the input signals were summed linearly via an operational amplifier. Primary spindle receptor responses to vibration were optimized by maintaining each muscle near maximum physiological length while experimental observations were performed. Rest shortening between series of trials and restricted periods of vibration with ample rest periods helped maintain peak primary ending responsiveness. Tenmion recording Muscle tension was measured after filtering the tension signal through a low pass filter, set at 100 Hz. This procedure was required to eliminate the tension ripple produced by vibration, but did not influence estimates of mean tension significantly. The tension recorded from each muscle, the length transducer signals and appropriate timing pulses were stored on magnetic tape for later processing with the help of a PDP-12 computer, which sorted, stored, and averaged control trials independently. These calculated averages were stored on digital tape, and later subjected to further analysis with a variety of computer programmes, which calculated mean plateau tensions, variances and displayed or plotted the results as desired. Stimulus tranami88on between MG and LG The muscles were carefully separated in order to limit the transmission of vibration or stretch between them, but the possibility of some mechanical interaction could not be eliminated entirely. For this reason, LG vibration was applied in all trials, to establish control of LG primary ending discharge, which may have been influenced (directly or reflexly) by MG vibration. However, in trials requiring LG vibration and MG stretch alone, the measured LG response may have been inadvertently increased by the reflex effects of MG primary endings activated by vibration transmitted through the animal. In two experiments, the MG response to LG vibration was examined after section of the LG nerve. There was a slight rise of MG tension (maximally 4 g), but the reflex tension increment produced by MG vibration or stretch was not altered significantly. Nerve blockade In five animals we applied a DC anodal block to the MG nerve. The blocking electrodes consisted of two 4 x 3 mm platinum plates, wrapped in cotton thread, and bent to form shallow grooves. Contact between the nerve and plate was improved by moistening the cotton thread with saline. The convex surfaces of the electrodes were insulated with silastic (Dow Corning Medical Grade Adhesive), to prevent current leakage to adjacent muscle or nerves. The current source was a Grass Instruments constant current stimulator, which was driven by a ramp and plateau input signal voltage, producing an output current of similar time course. A slow onset of the blocking current was advantageous in that it reduced stray discharge from the anode when the block was initiated (Zimmerman, 1968; Manfredi, 1970). Some anodal fibre discharge was apparent in dorsal root filament recordings, but it was brief, of small amplitude, and did not appear to effect results adversely. Effectiveness of the block was monitored by differential recording of the compound action potential in a small dorsal root filament teased from L 7. The compound action potential was produced by electrical stimulation of the MG nerve with single shocks, whose voltage was sufficient to produce a maximal Group I and II volley. Total and selective obliteration of the Group I peak was rarely achieved, and in most experiments significant reductions in the amplitude of the Group II peak accompanied Group I

68

K. KANDA AND W. Z. RYMER

reductions. Some Group II reduction was not unexpected, in view of the overlap in the diameters of primary and secondary spindle afferent fibres (Adal & Barker, 1962); but the technique appeared to be less discriminatory than we had hoped. Consequently in many experiments, a small Group I peak was allowed to persist, presumably allowing unrestricted passage of secondary spindle afferent impulses. We were also able to monitor the nerve block by recording the LG tension response to MG vibration with the block activated. The size of the heteronymous tonic vibration reflex proved to be a sensitive indicator of the degree of Ia afferent transmission. Undirectional current passage, even at low current density, probably produces toxic by-products at the electrode fluid interface, damaging the nerve, and also increasing the impedance of the electrode fluid interface. In an attempt to minimize this problem, a 4000 OiF capacitor was mounted in parallel with the stimulus current, and allowed to discharge in the reverse direction in the period intervening between blocking trials. This arrangement produced approximately zero net charge transfer across the electrode interface and appeared to help preserve nerve viability and conduction.

Variability The averages were calculated from series of observations obtained over relatively short periods of time, usually less than 1 hr. The averaging method, which required sequential ordering of test and control trials helped minimize the effects of trends, but other types of variations could have contributed errors. For example, greater variability of stretch-induced responses could have negated the significance of apparent increases of mean tension. However, measurements of LG tension variance showed that there was no substantial increase induced by the MG stretch. RESULTS

Effects of MG stretch on LG tension In the first series of experiments we attempted to verify the existence of an excitatory pathway from extensor muscle which was activated by muscle stretch but which was distinct from the classical primary spindle receptor afferent system. The LG was held at constant length, while the MG was subjected to various combinations of vibration and slow stretch. When MG stretch was superimposed upon MG tendon vibration, the resultant increase in LG tension exceeded that produced by MG vibration alone. The results of a typical experiment are illustrated in Fig. 2. This Figure was derived from superimposed records of two successive trials, differing only in the addition of 1 mm stretch to the MG muscle in one trial. In this instance, the MG extension was responsible for a force increase of 24 g in LG measured 1 sec after the ramp stretch was completed. Since the LG was not stretched, the increased tension was clearly the result of recruitment of new motor units together with a probable increase in firing rate of some already discharging.

STRENGTH OF SECONDARY AFFERENT EXCITATION

69

Relationship between MG stretch amplitude and LG force output When the MG tendon was simultaneously vibrated and stretched, the increase of LG force resulting from MG stretch was roughly proportional to stretch amplitude. The size and time course of this additional force increase varied considerably over the total experiment period, but for a decerebrate with a stable stretch reflex, the observations were often consistent and repeatable over a period of 30-60 min. Fig. 3 demonstrates the additional force produced when the MG was stretched 2 or 4 mm, after the initiation of independent vibration in each muscle. This Figure 8 -

g 1~~~~~~~~~~~100

\

LG tension Vibration

LG 1 mm

MG stretch

1 sec

Fig. 2. Myographic records of LG reflex response to simultaneous MG stretch and vibration. Upper plot: superimposed myographic recordings of LG tension in two successive trials. Each trial consists of an initial period of LG vibration, followed by a 9 see period in which LG and MG are vibrated simultaneously (A). LG vibration was initially responsible for a tonic vibration reflex of 122 g, which was increased to 254 g by added MG vibration. The second trial (B) has an additional 1 0 mm stretch of MG superimposed, which results in a further tension increment of 24-0 g, in excess of the reflex response produced by simultaneous vibration. The tension record has been smoothed with a low pass filter, with cut-off set at 100 Hz. Measurements were made 1 sec after completion of the ramp stretch. The middle and lower sections are diagrammatic illustrations of the time course of tendon vibration and MG extension. Initial force in LG was 96 g, and LG length was - 6*0 mm (i.e. 6*0 mm shorter than physiological maximum length). Vibration was applied to each muscle at 180 Hz, and 150 prm, peak to peak amplitude.

consists of superimposed ensemble averages, calculated from ten trials for each type of stimulus. Here the incremental stretches were responsible for mean LG tension increases of 52 + 1-6 g (S.E. of mean) and 98 + 2*7 g (s.E. of mean) measured 1 sec after the ramp portion of the stretch was completed. Since the primary endings have a fixed discharge rate, the force

70 K. KANDA AND W. Z. RYMER increment in LG could have resulted from increased secondary ending discharge, as described previously. This LG force increment may be quantified as an LG-force to MG-length ratio, which has the dimensions of

stiffness, allowing comparison with stiffness estimates of the homonymous reflex. In this series, the steady-state stiffness attributable to secondary ending discharge was 24 g LG/mm MG.

1100g LG tension Vibration LG

110 M MG

-

MG stretch

-4mm -8 mm

1 sec

Fig. 3. Averaged myographic records, showing the effects of combined tendon vibration and muscle stretch on LG tension. Upper record: traces A, B, and C are averaged tension responses (10 trials per average) recorded from the LG myograph. A is the response to LG and MG vibration combined. Vibration was applied to each tendon at 130 usm peak to peak amplitude, and at a frequency of 180 Hz. MG vibration produced an increase in LG force of approximately 140 g measured one second after ramp completion. B resulted from the addition of 2-0 mm stretches to the vibrated MG, and illustrates a mean tension increase of 52 g in excess of A, measured at point B. C resulted from the addition of 4-0 mm stretches to the MG, which has produced 98 g additional tensions measured at point C. The lower traces are diagrammatic illustrations of the vibration periods and of MG stretch amplitude. Control and test trials were alternated, allowing trace A to act as an effective base line for the measurement of stretch related effects.

Primary and secondary spindle afferent contributions to the tonic stretch reflex Estimates of the primary and secondary spindle receptor contributionsto the tonic stretch reflex were made by intermingling trials using MG stretch and vibration with trials using MG stretch alone. Fig. 4 illustrates the averaged responses calculated for such a stimulus series. In this series, the steady-state stiffness of the heteronymous stretch reflex (MG stretch, LG tension change) was 35l2 g LG/mm MG. The additional force (A minus B) produced by combined MG stretch and vibration was 72 g, or 36 g LG/mm

\_~ -6m

STRENGTH OF SECONDARY AFFERENT EXCITATION 71 MG. This latter value is not significantly different from the total stiffness of the heteronymous stretch reflex (as calculated from C). A more thorough assessment of the possible primary and secondary spindle afferent contributions can be made from graphs relating LG force and MG extension over a range of MG lengths. Fig. 5A illustrates the results of such a series, calculated from a preparation different from that of Fig. 4. Graph a is a plot of the LG force increase produced by MG stretch A

]#r

S_% \ ] 100 g

LG tension

78g Vibration LG

MG MG stretch

\

1 sec

Fig. 4. Comparison of the heteronymous stretch and vibration reflexes. The upper record are averaged myographic responses recorded from LG (ten trials per average). The MG stretch and vibration were initiated simultaneously. C illustrates the response of LG to a series of 2-0 mm MG stretches, superimposed upon a LG tonic vibration reflex. There is no associated MG vibration, thus the tension increase occurring in addition to the LG vibration response (70 g) is a measure of the heteronymous stretch reflex. B is a record of the response to simultaneous vibration of MG and LG. MG vibration produced an LG tension increment of 190 g. A demonstrates the additional effect of superimposed 2-0 mm stretches of the MG in the presence of simultaneous vibration of LG and MG tendons. The lower traces are diagrammatic illustrations of the duration of tendon vibration for each muscle, and the time course of MG extension. Vibration was at 130 ,tm for MG, 150 ,um for LG at a frequency of 180 Hz in each case.

and its slope is a measure of the stiffness of the heteronymous stretch reflex, here 48 1 g LG/mm MG. Graph b is the change in tension of LG resulting from length increases of MG in the presence of simultaneous MG and LG tendon vibration. The slope of this graph, which is a measure of possible secondary ending effects, is 18-3 g LG/mm MG. The dashed line C is calculated from a graph of LG force and MG vibration frequency, as

72 K. KANDA AND W. Z. RYMER illustrated in Fig. 5B. Here, the MG tendon was vibrated over a wide range of frequencies, at constant amplitude, allowing a more precise estimate of the force frequency ratio - 2*6 g LG/Hz MG. Estimates of A

150

-

1-

C

W

E 100 u U C

C

0 C

qX

b

50-

-J

(100)

-

I

(-6) 400 -

I

1 2 MG extension increment (mm)

B

3

i 0

0 .

.--

W

w VI

I

300-

I

S

W C (an

C

0

Wr

I

200~ I

-j

I

,/Hz

I

(-6 mm, 84 g)

100 200 300 MG vibration frequency (Hz)

Fig. 5A, B. For legend

see

facing

page.

primary ending length sensitivity in the steady state suggest a maximum of 10 impulses/sec. mm (Eldred, Granit and Merton, 1953; Jansen & Matthews 1962), although other observations suggest much smaller values (2.2-5.0 impulses/sec. mm; Houk, Harris, & Hasan, 1973). Assuming an inter-

STRENGTH OF SECONDARY AFFERENT EXCITATION 73 mediate value, say 5 impulses/sec. mm, the MG Ia afferent contribution to the heteronymous stretch reflex was calculated to be 13 g LG/mm MG. This value is the slope of the dashed line in Fig. 5A. The estimated primary ending contribution is substantially less than the total stiffness of the heteronymous stretch reflex, and is clearly no greater than the apparent secondary ending effect. The primary spindle afferents appear to contribute less than half of the excitatory drive to the stretch reflex (Fig. 5A), although such estimates must be treated with some caution, because the sum of the primary (13.0 g/mm) and secondary (18.3 g/mm) contributions did not reach the total stiffness of the heteronymous stretch reflex (48.1 g/mm). Some discrepancies were not unexpected since the stretch and vibration reflexes were not measured from the same trial records, but it is also possible that the primary ending contribution may have been underestimated by our methods. This matter will also be further evaluated in the Discussion. Fig. 6 summarizes the results obtained from a different preparation. Fig. 5A, a comparison of the possible primary and secondary spindle afferent contributions to the heteronymous stretch reflex. The tension values illustrated in this Figure are derived from experimental LG myographic records, similar to those presented in Fig. 4. The points represent mean values, calculated from ensemble averages (ten trials per average) and the bars the corresponding standard errors. Tension was estimated one second after ramp completion. a, the observed heteronymous stretch reflex tension induced in LG by MG extension. As in previous Figures, this tension response was superimposed upon a LG tonic vibration reflex. The line represents the least-squares regression fit through the means, and its slope is 48-1 g LG/mm MG (correlation 0.945). b, the additional tension produced by MG stretch when superimposed upon MG vibration. LG vibration is concurrent. The slope is 18-3 g LG/mm MG (correlation 0.938). This line is an estimate of the possible secondary spindle afferent contribution to stretch reflex tension under these conditions. c, the estimated primary contribution, based on the slope of the reflex LG response to MG vibration, over a range of vibration frequencies, as illustrated in Fig. 6B. The slope has the value 13 g LGImm MG. Tendon vibration was at 180 Hz, at a peak-topeak amplitude of 130 jum in each muscle. LG was kept at - 6-0 mm, and MG initial length was - 6-0 mm, B is the relation between LG reflex force, and MG vibration frequency. Data is from the same preparation as Fig. 4. The heteronymous tonic MG vibration reflex was superimposed upon the LG vibration response as illustrated previously. The tension points are mean values, derived from ensemble averages (ten trials per average), in which the tension was measured 3 sec after the commencement of MG vibration. The tension rises linearly, with slope 2-6 g LG/Hz MG, until 160-200 Hz, where it appears to saturate. The initial slope was calculated by least squares regression (correlation 0.97). The vibration amplitude was 150 ,um, peak to peak for tendon. LG vibration was applied at 180 Hz and the muscle was extended to 6-0 mm for all tension measurements.

74 K. KANDA AND W. Z. RYMER Here, the heteronymous stretch reflex (graph A) had a stiffness of 26-2 g LG/mm MG, which was substantially less than that shown in the preparation of Fig. 5. However, the increment of LG force produced by MG 125

A

100

C

E

B

/

() 75

C

C C 0)

-

25

I

(74) (-8)

1 2 3 MG extension increment (mm)

4

Fig. 6. A comparison of the possible primary and secondary spindle afferent contributions to the heteronymous stretch reflex. The tension values are again ensemble averages (ten trials per average), derived in a manner similar to the points illustrated in Figs. 5A and B. The bars represent standard error of the mean. A is the estimated heteronymous stretch reflex tension produced in LG by MG extension, in the presence of ongoing LG vibration. The line has a slope of 26-2 g LG/mmMG (correlation 0 962). B is the additional tension produced by MG stretch superimposed upon MG vibration. The slope is 21-9 g LG/mm MG (correlation 0-831). As in Fig. 4, this line is an estimate of the posible secondary afferent contribution. C is an estimate of the primary ending contribution, based on the response of LG to MG vibration (1 .1 g LGfmm MG) and a stretch sensitivity of 5 impulses/ mm. The slope of C is 5-5 g/mm. Tendon vibration was at 180 Hz, with a peak to peak amplitude of 150 Elm in each muscle. LG was kept at - 6 0 mm, and MG initial length was - 8-0 mm.

stretch in the presence of MG vibration was still considerable at 21-9 g LGfmm MG (graph B). Graph C is again an estimate of the primary ending contribution to the heteronymous stretch reflex. There is a large discrepancy between B, which is an estimate of the possible secondary spindle receptor afferent contribution, and C, the calculated primary contribution, which suggests that the secondary afferent contribution may have been predominant in this series of observations.

STRENGTH OF SECONDARY AFFERENT EXCITATION 75 Table 1 lists the calculated hypothetical contributions of primary and secondary endings to the heteronymous stretch reflex for our series of ten cats. These values represent the slopes of the various graphs as exemplified in Fig. 5. The potential primary ending contribution in this series was apparently quite modest, and in no case exceeded the estimated TABLE 1. Estimates of the primary and possible secondary spindle afferent contributions to the heteronymous stretch reflex The stretch and vibration reflexes were measured in LG, superimposed upon LG vibration. The values in the Table are calculated slopes, derived from graphs of LG tension response to MG stretch and vibration, as exemplified in Figs. 6 and 7. The slopes were least-squares estimates, based upon the mean tension response to a series of MG stretches ranging from 0-5 to 4-0 mm in amplitude. Both the range and mean of the heteronymous tonic vibration reflex tension suggest a modest Ia contribution, with an over-all value of 1-79 g LG/Hz MG. The values in parentheses are the estimated total MG I a contribution to the heteronymous stretch reflex, assuming a sensitivity of 5 impulses/mm. The suggested secondary contribution had a mean value of 24-0 g LG/mm MG. The homonymous MG stretch reflexes are included to illustrate the substantial difference between homonymous and heteronymous stretch reflexes, although the homonymous reflex includes a significant length tension contribution. Estimated MG stretch Heteronymous Estimated primary secondary reflex contribution contribution stretch reflex Expt. no. (g LG/mm MG) (g LG/Hz MG) (g LG/mm MG) (gMG/mmMG) 32-0 1-8 (9.0) 1 28-4 21-9 1-1 (5-5) 2 26-2 118 19-0 17-9 0-8 (4.0) 3 129 18-5 1-1 (5-5) 4 25-6 161 28-5 52-0 1-9 (9.5) 5 141 19-4 2-2 (12.0) 34-0 6 170 18-3 2-6 (13-0) 7 48-1 144 24-1 1-9 (9-5) 29-1 8 116 32-6 2-8 (14.0) 9 40-1 102 26-0 1-7 (8.5) 35-2 10

Mean

33-7

S.D. S.E.

10-1

1-79 0-62

3-2

0-19

24-0 5-24 1-65

135-11 21-71 7*71

secondary ending contribution. The range of measured values was considerable, but for a given preparation the calculated slopes were appropriate in that stretch reflex stiffness usually exceeded the hypothetical secondary effect. In Expt. 1 and 3, however, the calculated secondary effect was greater than the total heteronymous stretch reflex, which may reflect spontaneous or vibration induced changes in spinal neurone excitability. Such changes could influence the mean estimates independently

K. KANDA AND W. Z. RYMER because the MG heteronymous stretch reflex and the 'additional' stretch mediated excitation were examined in different trials. A comparison of heteronymous and homonymous stretch reflex values shows that the mean heteronymous stiffness values were substantially smaller, by a factor of four (33.7 vs. 135*1 g/mm). Some difference between the MG homonymous and heteronymous reflexes was expected in view of the additional length-tension contribution to the homonymous reflex, and to known differences in Ia synaptic projection (Eccles, Eccles & Lundberg, 1957; Burke, 1968b; Mendell & Henneman, 1971), although it is difficult to predict reflex tension differences from the characteristics of monoynaptic e.p.s.p.s alone, since the distribution of relevant polysynaptic pathways may be quite different. We also measured the homonymous effects of MG vibration in preparations 3-10, and obtained a mean estimate of 1*82 g MG/Hz MG. The size of the homonymous and heteronymous vibration reflexes was frequently comparable, whereas the monosynaptic e.p.s.p. sizes are reportedly quite different (e.g. Burke, 1968 b). The size of the heteronymous TVR is increased in part because of the central facilitation produced by LG vibration, but it is unlikely that this would overcome a fivefold difference in monosynaptic e.p.s.p magnitude. These observations suggest that the tonic vibration reflex requires substantial additional polysynaptic activation, perhaps via interneurones receiving strong Ia excitation. 76

The calculations illustrated in Figs. 5, 6 and Table 1 have not taken into account possible central effects of tendon organ discharge. Assuming the I b afferent excitation results in inhibition, then the lb discharge provoked by tension increases and by vibration could have caused us to underestimate the excitation produced by either spindle receptor. Such inhibition is reported to be modest in the decerebrated cat (Eccles & Lundberg, 1959b), but it could have caused some errors. However, it is unlikely to have influenced our basic observation that MG stretch activated an additional excitatory pathway.

Effects of initial LG active tension on the response to MG stretch and vibration As we have described, the response to a given MG stretch proved to be quite variable both for a single preparation in the course of an experiment and between different preparations. This variability took two forms. In many cases the muscular rigidity of the preparation would decline, often transiently, in which event responses to stretch or vibration would diminish proportionately, as would the response to stretch and vibration combined. In two preparations, we observed an inverse correlation between initial vibration-induced LG tension and MG stretch induced excitation of LG. Fig. 7 illustrates two series of observations made 3 hr apart from one of these preparations. The increase of vibration-induced force in LG was

STRENGTH OF SECONDARY AFFERENT EXCITATION 77 accompanied by a decline in the incremental response to a constant amplitude MG stretch. This apparent reduction of the effectiveness of the proposed secondary afferent pathway suggests broadly the possibility of some force-related inhibitory feed-back, although other explanations are tenable. These observations also suggest that the system under investigation may have some non-linear properties, since a constant MG length change produced a smaller excitatory effect on the LG when the initial force was high. 200 bO I-

C

100 C

CII

-J)50I (-6(-6)

~1

~

2 MG extension increment (mm)

3

Fig. 7. The effects of initial LG tension upon the LG incremental response to MG stretch are shown, The MG stretch was 2-0 mm in each trial. The Figure illustrates two series of LG tension responses, recorded from the same preparation, three hours apart. The upper series (filled circles) were recorded earlier, superimposed upon an LG vibration reflex of 150 g, whereas the lower series (open circles) were measured in the presence of approximately 300 g initial vibration reflex force in LG. Each point is the mean of ten observations, with the bar denoting the standard error of the mean. Vibration amplitude was 130 jam in MG, 150 jsm in LG, at a frequency of 180 Hz in each muscle.

The constancy of primary ending response to simultaneous muscle stretch and vibration If the reported increases of LG tension are to be safely attributed to the secondary ending, then it is necessary that we verify constancy of primary ending discharge from the MG muscle under comparable experimental conditions. The relevant evidence is partly direct, but largely indirect. The direct evidence was obtained by recording from primary spindle afferent fibres included in small filaments teased from the L7 or SI dorsal roots, leaving the remaining dorsal roots intact. We examined the behaviour of five primary endings in three preparations. It was observed that stable driving of primary endings was guaranteed with vibration amplitudes

78 K. KANDA AND W. Z. RYMER greater than 100 Wom, at frequencies of 180-200 Hz, particularly if the muscle was extended to a length close to physiological maximum. Fig. 8 illustrates the vibration response of a primary ending in MG which clearly maintained constant discharge during a 2 mm stretch and release. There was a distinct increase in LG tension output without change in the constancy of the phase locked primary ending discharge. LG tension

/

]

1~~~~~~~2 mm

_

MG vibration and stretch

gm

1 sec

MGspindle primary afferent

4J4K

V 10 msec

Fig. 8. The constancy of primary spindle receptor discharge with simultaneous tendon vibration and muscle stretch. Upper trace: LG myographic record of a single trial, with successive initiation of LG vibration, MG vibration, and MG stretch, as in Fig. 2. There is a clear increase of LG tension corresponding to the additional MG extension. Middle trace: MG displacement transducer record. The thick line results from vibration. Lower trace: segment of neural recording of Ia afferent fibre, from a primary spindle receptor in MG. Portion illustrated is taken from record immediately prior to and during the early phase of the ramp stretch. Small additional spikes are from a spontaneously discharging unit not situated in the gastrocnemius muscles. The larger unit maintained constant phase locked discharge during MG stretch and release and could not have contributed to any tension change in the LG. Vibration is at 150 ,tm, peak-to-peak amplitude for each tendon, at a frequency of 180 Hz. Spikes have been retouched by hand. The Ia afferent fibre was identified by its conduction velocity (96 m/sec), the cessation of its discharge during a muscle twitch, and its response to MG vibration.

The recording of I a afferent activity in the course of MG stretch and vibration is of considerable value, but it is difficult to estimate how well the individual receptor response approximates the behaviour of the total receptor pool. Several types of error remain possible. McGrath and Matthews (1973) demonstrated that under similar conditions, vibration of a muscle could sometimes induce a primary ending to discharge two or more impulses per vibration cycle. However, the contribution of any

STRENGTH OF SECONDARY AFFERENT EXCITATION

79

additional discharge would appear to be minimal in our experiments because the tension increase was not confined to the ramp period, and was maintained for many seconds after the MG had reached the plateau position. A potentially greater source of error arose from possible variations in the security of primary entrainment at different muscle lengths. This potential recruitment error was minimized in several ways. At the beginning of each experiment, the tonic vibration reflex in each muscle was examined and a vibration amplitude chosen such that a 25 % change in amplitude did not influence the size of the TVR. This indicated that all primary endings were securely driven, and probably insensitive to small changes in muscle state. In addition, the imposed length changes were of small velocity and amplitude, the largest being 4 0 mm in 2 sec.

Limitation of maximum LG force output In some instances, the LG tension appeared to reach a limit, which was independent of the method of reflex activation, i.e. MG stretch, vibration, or combined vibration and MG stretch. This limit was usually in the range of 400-600 g, but it was not a result of dampening of the vibration amplitude. Several factors could have contributed. As muscle tension increased, the vibration effect would have been progressively more dissipated into the series elastic elements, which included the tendon and its silk thread attachment to the hook. Such an attachment may have had a larger compliance than the tendon itself. We have monitored the movement of a point on the surface of LG, and observed that the vibratory excursion of the muscle belly clearly diminished as the muscle tension rose, although our observations were not quantitative. (Similar observations were reported by Brown et al. (1967) for the soleus muscle.) There is, therefore, a strong probability that the amplitude of movement transmitted to the primary receptor declined progressively with increasing muscle tension. On the other hand, the stiffness of the muscle would have increased with increasing muscular contraction, allowing the muscle to act as a more effective transmitter of the vibration wave. This enhancement of wave propagation would help to offset the reduction of stimulus amplitude at the primary spindle receptor. The latter effect appeared to dominate, because we were able to produce driving of all five primary endings which we examined, over a wide range of MG forces. However, since we did not survey primary ending discharge in many preparations, it remains possible that secure driving was not maintained for all primaries at tensions approaching 500-600 g. For this reason, the apparent force saturation of the LG could have resulted from a reduction of primary ending input, and it could not safely be attributed to some central interaction of force and length regulatory pathways.

Nerve blockade We investigated the independent effects of stretch induced secondary receptor activation by attempting selective anodal blockade of the MG nerve, using an experimental paradigm similar to that described previously. Fig. 9 is an oscillographic record of two successive trials, differing only in the presence of MG stretch in one trial. The LG tension trace demonstrates a modest LG tension response to MG vibration (which verifies the effectiveness of the Group I block), and a small additional response to MG stretch (26 g). This possible secondary contribution was only 13 g LG/mm MG, but the reduction of force amplitude was smaIlr than that sustained

K. KANDA AND W. Z.RYMER 80 by the heteronymous vibration reflex, which produced only 0 3 g LG/mm MG. We did not observe any increase of LG tension in response to MG stretch when LG vibration was eliminated. The disproportionate effects of electrical blockade on the MG-induced TVR in LG and on the excitatory action of slow MG stretch on LG force suggests that these effects are supported by different peripheral nervous

LG tension

1500 g

J

MG tension ~~~~~\~~ ~

]mm

MG extension

1\] 40 #A

MG nerve o blocking current 1 sec

Fig. 9. The effects of MG nerve blockade upon the LG response to MG stretch are illustrated using myographic records of LG response in two successive trials, with a 2 0 mm MG stretch present in the second trial. MG and LG vibration were applied as in previous experiments. A ramp anodal blocking current was applied to the MG nerve in each trial. This current was of sufficient amplitude to eliminate both motor efferent and group I transmission to the MG (second trace), since there was no MG vibration response. The MG tension change illustrated in the second trace is a passive response to muscle stretch. The uppermost trace demonstrates a modest initial LG vibration response which is substantially reduced by MG nerve blockade. The reflex response to MG vibration was also small (58 g), but there is a clear tension increment corresponding to the MG stretch. MG and LG vibration were applied at an amplitude of 150 4um (peak to peak) and a frequency of 180 Hz. The third trace is composed of superimposed length transducer records from MG, showing the period of MG vibration as a thickening of the length trace, together with the amplitude and time course of the MG extension. The period of MG vibration is demarcated by the bar underneath the MG extension record. The lowermost trace is a diagrammatic representation of the anodal blocking current (positive upwards). The undershoot following current plateau results from the discharge of a capacitor which was included to minimize electrode polarization and nerve damage.

pathways. However, the MG stretch induced excitation proved difficult to reproduce, was inconsistent from trial to trial, and was seen clearly in only three of the five preparations in which nerve blockade was attempted. One source of inconsistency in our results was variation in the efficacy of

STRENGTH OF SECONDARY AFFERENT EXCITATION 81 the nerve block, such that a consistently applied current strength would allow the passage of different levels of primary spindle afferent activity in successive trials. This was presumably a result of variable current leakage to ground, and thus variable current flow through the nerve itself. A further problem was the relatively small amplitude of the excitation produced by MG stretch; typically half the tension values observed in the absence of nerve block. These observations suggest that the amount of secondary excitation produced may be dependent upon the state of spinal excitation, which would clearly be greater in the presence of simultaneous transmitted Ia afferent activity from both muscles. DISCUSSION

The experimental observations described have demonstrated that, in the decerebrate cat, extension of the vibrated MG muscle appears to produce an increase in the tension of the synergist LG, which is greater than that induced by MG vibration alone. This increase of LG tension is proportional to the amplitude of MG stretch, apparently requires facilitation by Ia afferent discharge, is dependent on LG tension, and may show saturation. We presume that this excitatory effect results from activation of secondary spindle afferents. Matthews (1969) has argued that secondary spindle receptor afferent fibres are responsible for muscle excitation in experiments utilizing combined muscle strength and vibration, on the grounds that the secondary ending is the only receptor available with appropriate properties, the primary ending discharge being clamped by vibration, and Golgi tendon organ afferents producing inhibition of homonymous motoneurones. Arguments excluding potential contributions from other muscle afferents have also been presented by Matthews (1969) and will not be repeated here. The most serious potential flaw in our experimental method is the possibility of incomplete MG primary spindle receptor entrainment at some muscle lengths. We have detailed the methods used to minimize this possibility, but since a survey of I a afferent population behaviour was not feasible, the evidence remains largely indirect. Additional evidence has been provided by intracellular recordings from MG motoneurones, in a related series of experiments (Rymer & Walsh, 1974). In these, repeated electrical stimulation of the LG-soleus (LGS) nerve at Group I strength produced e.p.s.p.s whose amplitude increased during extension of the vibrated MG. This facilitator effect of MG stretch occurred in the presence of a fixed LGS stimulus and a constant MG Ia afferent input, which indicates that

K. KANDA AND W. Z. RYMER 82 excitation of MG motoneurones is possible without increased la afferent discharge from either muscle. A further experimental problem is the known sensitivity of other muscle receptors to tendon vibration. There is little doubt that Golgi tendon organ discharge may have been significantly affected in our experiments Qs many tendon organs may have had their discharge phase locked or forced into a subharmonic of the vibration with increasing tension (Brown et al. 1967). If we assume an inhibitory role for tendon organs, this disharge could have caused an unduly low estimate of the excitatory effect, and may have contributed to the observed saturation. We believe Ib effects to have been small because of limited efficacy of the Ib pathway in the decerebrate preparation (Eccles & Lundberg, 1959b), but tendon organ influences could clearly not be excluded.

Evaluation of excitatory effect The magnitude of the excitatory secondary effect must be judged in relation to the size of the heteronymous stretch reflex. The average steadystate stiffness of this reflex was 33-7 g LG/mm MG. In most of our experiments, the estimated slope of the 'secondary' induced tension change in LG was 20-30 g LG/mm MG, whereas the calculated contribution of the Ia afferents was maximally 14 0 g LG/mm MG (assuming 5 impulses/mm sensitivity), and wasoften less. Over-all, the estimated stiffness contributed by the secondary spindle afferents was usually at least equal to that calculated for the primary ending, and there appeared to be a few exceptions. This evidence suggests that the primary and secondary spindle afferent contributions may be of comparable significance in the tonic stretch reflex. Several problems exist with these calculations. Polysynaptic pathways are probably important in the homonymous vibration reflex (De Gail, Lance & Neilson, 1966; Kanda, 1972), and we have here suggested that similar paths may also be active in the heteronymous tonic vibration reflex. Thus, the possible existence of a pool of interneurones receiving convergent and intense facilitation from I a and II afferent fibres suggests that estimates of the individual contributions of the receptors should be interpreted with caution, since a constant receptor input may have different effects according to the state of facilitation of the relevant interneurones. In addition, the analogy between vibration and stretch mediated primary ending discharge remains unclear. For example, it remains possible that vibration induced primary ending discharge may be associated with extensive presynaptic inhibition (Barnes & Pompeiano, 1970a, b), which would diminish the effect of each neural impulse in comparison with

STRENGTH OF SECONDARY AFFERENT EXCITATION 83 the effects of activation frequencies prevailing in the stretch reflex. This inhibition could arise from excitation of muscle afferents (Eccles, Magni & Willis, 1962; Devanandan, Eccles & Yokata, 1965) or perhaps from Pacinian corpuscles excited at a distance by vibration (Schmidt, 1971). There also is a possibility that La e.p.s.p.s, when generated at high frequency, are less effective than those produced at frequencies closer to the physiological range (Curtis & Eccles, 1960). The method used to measure I a synaptic strength in Fig. 5 presumes that estimates of I a effectiveness in the tonic vibration reflex are relevant to the analysis of I a effects in the stretch reflex, where much smaller steady-state frequencies almost certainly prevail (Houk et al. 1973). Verification of Ia potency by alternative techniques is necessary, but not presently possible. In view of these uncertainties, our intent is primarily to point out that secondary effects are substantial, and may be comparable in size to those produced by Ia fibres. Tension dependence and force saturation We have described the existence of a force-related effect in which the size of the LG tension increment produced by vibration or muscle stretch appeared to depend upon existing tension in the muscle. This force level was typically less than 600 g, a tension far smaller than the maximal output from LG, which may reach 10 kg for a fused tetanus (Eccles & Sherrington 1930). If vibration were to recruit from within a sharply defined segment of the motoneuronal population, then maximal recruitment of these neurones would be associated with maximal force output, once motoneurones were discharging at peak frequency. At present, there is no evidence of such synaptically distinct populations of motoneurones within the MG (Henneman, Somjen & Carpenter, 1965a, b; Burke, 1967, 1968a, b). There appears to be a smooth gradation of monosynaptic e.p.s.p. amplitude across neurones innervating fast and slow twitch motor units, with no clearly defined discontinuities. It remains possible that polysynaptic projections are less uniform as exemplified by sural nerve projections to fast and slow twitch type motoneurones of the MG (Burke, Jankowska & ten Bruggencate, 1970), but no comparable discontinuities have been demonstrated for muscle afferent projections. The characteristics of the proposed secondary spindle afferent excitation can be used to construct some hypotheses regarding possible mechanisms of central Group II action. A number of potential explanations warrant consideration.

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Post-synaptic excitation (a) Monosynaptic projection. Group II muscle afferents have been reported to produce disynaptic or polysynaptic e.p.s.p.s in flexor motoneurones (Eccles & Lundberg, 1959a; Holmqvist & Lundberg, 1961; Lundberg, Malmgren & Schomberg, 1975), and occasionally in extensor motoneurones (Wilson & Kato, 1965; Lundberg et al. 1975). Recently Kirkwood & Sears (1974) have used the technique of spike triggered averaging to show that the discharge of identified secondary spindle afferent fibres produced small monosynaptic e.p.s.p.s in triceps and thoracic motoneurones. The data provided do not allow an assessment of the relative strengths of primary and secondary afferent monosynaptic projections; however, the demonstrated secondary connexion could certainly have contributed to our observed excitation. (b) Polysynaptic projection. Interneurones have been described which receive monosynaptic or polysynaptic excitation from electrically activated group II afferents (Willis & Willis, 1969) although the only functionally identified category projects to flexor motoneurones (Lundberg et al 1975). Recently, Fukushima & Kato (1975) have reported the existence of interneurones receiving identified group II excitatory projections, but their over-all synaptic input and projection remain unidentified. Nevertheless, the existence of an interneuronal population receiving convergent input from I a and group II afferents, and projecting in turn to extensor motoneurones remains an attractive possibility. Such a neurone population could also remain undetected in the absence of appropriate facilitation, which could explain the lack of significant group II excitation with muscle nerve block of group I fibres (Cook & Duncan, 1971; Cangiano & Lutzemberger, 1972; Emonet-Denand, Jami, Joffroy & Laporte, 1972), or in anaesthetized preparations. Furthermore, such interneurones could be strongly activated by vibration, thus heightening group II central effects to levels which may be greater than those normally encountered in the tonic stretch reflex. The results of the nerve block experiments could be explained if these hypothetical interneurones were to receive predominant projection from muscle afferents of one muscle.

Disinhibition (a) Post-synaptic. An alternative general mechanism, described by Matthews (1970) requires that secondary muscle spindle afferents mediate their effects by disinhibition of extensor motoneurones or appropriate interneurones. Available post-synaptic inhibitory pathways include Ia inhibitory interneurones, Ib interneurones and Renshaw cells. Our results

STRENGTH OF SECONDARY AFFERENT EXCITATION 85 are consistent with this hypothesis, but provide no specific experimental support for a post-synaptic site of action. (b) Presynaptic inhibition. Modulation of the level of presynaptic inhibition is an alternative possibility which has some experimental support. Lund, Lundberg & Vyklicky (1965) showed that the levels of primary afferent depolarization could be reduced by electrical stimulation of muscle afferents at Group II strength. Westbury (1972; see Fig. 8) demonstrated that the amplitude of vibration induced Ia e.p.s.p.s increased at greater muscle lengths, an observation which was confirmed by Rymer & Walsh (1973b). The available range of e.p.s.p. amplitude increase would clearly be limited by the level of primary afferent depolarization, producing a saturation-like effect which primary afferent depolarization was completely suppressed. Of the two general types of possible secondary spindle afferent excitation described, post-synaptic excitation or disinhibition, direct polysynaptic excitation is the more appealing because of its simplicity, and its lack of dependence on ongoing inhibition. A major question remains. Why has secondary spindle afferent excitation proved to be so difficult to verify directly? This difficulty may result from the absence of appropriate interneuronal facilitation in anaesthetized or spinal preparations, in which spinal synaptic physiology has been largely developed. Such dependence upon interneuronal facilitation also allows greater possible flexibility of response to muscle afferent input, ranging from extensor excitation in the decerebrate with vibrated extensor muscle, to inhibition of extensor motoneurones in the spinal preparations of Eccles & Lundberg (1959a) and Holmqvist & Lundberg (1961). The advice and support of Dr R. E. Burke are gratefully acknowledged. Valuable comment has been provided by Drs J. C. Houk and P. E. Crago. Both K. Kanda and W. Z. Rymer were supported as visiting fellows of the Fogarty International

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