Receptor Mechanisms Underlying Heterogenic Reflexes Among the Triceps Surae Muscles of the Cat

T. Richard Nichols

Department of Physiology, Emory University, Atlanta, Georgia 30322


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Nichols, T. Richard Receptor mechanisms underlying heterogenic reflexes among the triceps surae muscles of the cat. The soleus (S), medial gastrocnemius (MG), and lateral gastrocnemius (LG) muscles of the cat are interlinked by rapid spinal reflex pathways. In the decerebrate state, these heterogenic reflexes are either excitatory and length dependent or inhibitory and force dependent. Mechanographic analysis was used to obtain additional evidence that the muscle spindle primary ending and the Golgi tendon organ provide the major contributions to these reflexes, respectively. The tendons of the triceps surae muscles were separated and connected to independent force transducers and servo-controlled torque motors in unanesthetized, decerebrate cats. The muscles were activated as a group using crossed-extension reflexes. Electrical stimulation of the caudal cutaneous sural nerve was used to provide a particularly strong activation of MG and decouple the forces of the triceps surae muscles. During either form of activation, the muscles were stretched either individually or in various combinations to determine the strength and characteristics of autogenic and heterogenic feedback. The corresponding force responses, including both active and passive components, were measured during the changing background tension. During activation of the entire group, the excitatory, heterogenic feedback linking the three muscles was found to be strongest onto LG and weakest onto MG, in agreement with previous results concerning the strengths of heteronymous Ia excitatory postsynaptic potentials among the triceps surae muscles. The inhibition, which is known to affect only the soleus muscle, was dependent on active contractile force and was detected essentially as rapidly as length dependent excitation. The inhibition outlasted the excitation and was blocked by intravenous strychnine. These results indicate that the excitatory and inhibitory effects are dominated by feedback from primary spindle receptors and Golgi tendon organs. The interactions between these two feedback pathways potentially can influence both the mechanical coupling between ankle and knee.


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Evidence has accumulated that heterogenic, or intermuscular, spinal reflexes play an important role in determining the mechanical properties of multisegmented limbs and in coordinating the component joints (Gielen et al. 1988; He et al. 1991; Hogan 1985; Nichols 1994). These reflexes depend on the fundamental variables, length and force. Furthermore they link muscles with similar lines of action at a given joint in the case of length feedback, and they couple antigravity muscles that cross different joints in the case of force feedback (Nichols 1989a, 1994). Because of the dependencies on length and force, it is natural to assume that the underlying pathways arise from muscle spindles in the case of length-dependent reflexes and from Golgi tendon organs in the case of force-dependent reflexes. In view of the uncertain roles of these two receptors in limb coordination, however, it is important to determine the basis of heterogenic reflexes in specific receptors and pathways.

The primary receptors of muscle spindles have been shown to constitute the dominant receptor component of the autogenic stretch reflex (Houk and Rymer 1981; Houk et al. 1981) and the myotatic unit (Bonasera and Nichols 1996; Lloyd 1946; Nichols 1989b; Nichols and Koffler-Smulevitz 1991) based on dynamic characteristics, distribution to antagonists, and latency. Group Ia afferents also provide excitatory feedback to all the triceps surae motoneuron pools (Burke et al. 1976; Eccles et al. 1957a, 1962; Scott and Mendell 1976). Prochazka (1990) has estimated the relative contributions of primary and secondary spindle afferents from all three muscles of the triceps surae to depolarization of motor units of the medial gastrocnemius muscle during locomotion, but insufficient data are available to determine the contributions to lateral gastrocnemius and soleus muscles as well. Therefore the contributions of spindle receptors to heterogenic reflexes in the triceps surae during active contractions remain unknown.

Group Ib afferents from Golgi tendon organs are thought to have a wider distribution among limb muscles than group Ia afferents (Eccles et al. 1957b; Jami 1992; Jankowska 1992; Powers and Binder 1985). The potentially short latencies associated with these receptors make them good candidates for the force-related inhibition that links a variety of load-bearing muscles in the cat hindlimb (Nichols 1994). Step-wise regression analyses have revealed for some muscle combinations that the inhibition strongly depends on the force of the muscle of origin (Bonasera and Nichols 1994). However, the adequate stimulus for the Golgi tendon organ is active contractile force rather than total force (Houk and Henneman 1967). Therefore a relationship between reflex effect and active force would establish more firmly the role of the Golgi tendon organ in the reflex inhibition.

The purpose of the experiments reported here was to obtain further insights into the receptor mechanisms underlying heterogenic reflexes among the triceps surae in the decerebrate cat. It was found that excitatory feedback under conditions of muscle activation was distributed approximately according to heteronymous Ia linkages determined under quiescent conditions. Inhibitory feedback from the gastrocnemius muscles to soleus was found to depend on active contractile force and to act essentially as rapidly as the stretch reflex, supporting the hypothesis that this feedback arises from Golgi tendon organs. New information concerning the duration of the inhibition also was obtained. Finally, the inhibition could be blocked using strychnine, indicating a glycinergic and postsynaptic basis.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

The data presented here were drawn from experiments on 32 adult cats of either sex. The methods have been described in detail elsewhere (Bonasera and Nichols 1994; Nichols 1987), so only a brief description with aspects relevant to this study is provided. All protocols are in complete accordance with the guidelines of both the National Institutes of Health and the Emory Institutional Animal Care and Use Committee.

Surgical preparation

Under halothane or isoflurane anesthesia, the animal underwent tracheotomy, bilateral carotid ligature, and cephalic vein cannulation. Core temperature was maintained at ~37°C using a rectal temperature controller and a heat lamp. Fluids were infused through the venous cannula. Two steel pins welded to small metal blocks were inserted longitudinally into the tibia and femur at the right knee to provide fixation. After intercollicular decerebration, the right caudal cutaneous sural nerve was dissected free and the right triceps surae muscle group dissected from surrounding connective tissue. Thread markers were placed around the fibula and through the tendons of each of the triceps muscles for setting muscle length with respect to a standard length (L90) corresponding to a 90° ankle angle. The knee was set to an angle of ~110°. In some experiments, the lateral (LG) and medial gastrocnemius (MG) muscles were separated and in others they were left intact. The tendons of the gastrocnemius and soleus (S) were separated from their attachments on the calcaneus with a small bone chip on each tendon. The tendons were fitted with small clamps for attachment to the torque motors. The posterior tibial nerve was freed near the ankle in both limbs for stimulation. The limb was immobilized using hip pins attached to the stereotaxic frame, and the bone pins and an ankle clamp fixed to mechanical ground by magnetic bases. At the end of the experiment, the animal was euthanized with an overdose of pentobarbital and a pneumothorax was performed.

Mechanical stimulation and recording

Muscle length was controlled using two rotary servomotors and precision potentiometers. Linear positioning was achieved by connecting the motor pulleys to linear ball slides by stainless steel cables. Elastic bands were used to cancel the tendency for the cables to unwind and exert force against the muscle. Therefore motor-shaft position corresponded to ball slide position, and the potentiometer signal could be used to measure muscle length. Force was measured using strain-gauge myographs mounted on the ball slides through miniature universal joints to ensure alignment between myograph and muscle. The myographs were manufactured in the laboratory using semiconductor strain gauges mounted on U-shaped aluminum beams. Stiffness in tension of the combined myograph and servomotor system exceeded 200 N/mm.

A personal computer with custom software was used to actuate the motors in the appropriate sequence (see next section), provide the ramp and hold input signals, and acquire the length, force and electromyographic (EMG) data. Samples were acquired at the rate of 500 Hz and with a precision of ~0.05 N. Analogue sample/hold modules were used to extract changes in force and initial force from the total force signal prior to sampling. The sampled initial force and subsequent change in force signals were stored during each trial.

Experimental design

Initial lengths and forces were controlled for each muscle. Initial lengths were set electronically through the feedback controller. In most experiments, except where noted, muscles were held at standard initial lengths (L90) corresponding to an ankle angle of 90° and a knee angle of 110°. The duration and amplitude of the ramp and the duration of the hold period were set under software control. Muscle force was modulated by evoking either a crossed-extension reflex through electrical stimulation of the contralateral tibial nerve or by stimulating the ipsilateral, caudal cutaneous sural nerve (Figs. 1-3). The stimulation rate for all three nerves was 50 pps. For both reflexes, force increased rapidly then declined slowly. The sural nerve reflex was included to decouple forces in the medial gastrocnemius muscle (MG) from the force in the soleus muscle (SOL) (Clark et al. 1993; Kanda et al. 1977; Labella et al. 1989). Stimulation of this nerve resulted in two different patterns of response in SOL. This muscle was either inhibited initially followed by a delayed excitation (Fig. 1) or it was activated weakly with the same time course as the strong activation of MG (Fig. 3). In the latter case, therefore, the force in SOL was maintained over a narrower range compared with the force in the gastrocnemius muscles.



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Fig. 1. Force responses of the soleus (S), medial gastrocnemius (MG), and lateral gastrocnemius (LG) muscles during reflex activation and periodic stretch of MG (protocol 1). A: triceps surae muscles were activated using a crossed-extension reflex. Contralateral posterior tibial nerve was stimulated continuously at 50 pps. LG and S were constrained isometrically at lengths corresponding to 2 and 4 mm longer than L90, and MG was subjected to ramp and hold stretches (ramp duration = 50 ms, amplitude = 4 mm, hold period = 250 ms). Note that the background force declined more slowly in LG and S than MG. Force responses were excitatory in LG and mixed in S (see Fig. 2). B: triceps surae muscles were activated by stimulating the ipsilateral caudal cutaneous sural nerve (50 pps). Experimental protocol was otherwise the same as shown in A. Note that LG and S initially were inhibited and then activated after a delay. Force responses were mixed in S and excitatory in LG. Calibration bars correspond to 10 N and 1 s.



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Fig. 2. Force responses of triceps surae muscles during activation by a crossed-extension reflex and periodic stretch of MG (protocol 1). Records were obtained from the experimental run shown in Fig. 1A and superimposed for each muscle. Right: ecords are shown with a more expanded time base. Vertical solid lines indicate the beginning of stretch and the vertical dashed line indicates a 16-ms delay corresponding to the expected latency of the stretch reflex. Note that the responses of the isometrically constrained LG and S are delayed by ~16 ms for both excitatory and inhibitory responses. Responses of MG include an instantaneous component due to the intrinsic mechanical properties of the muscle. Note also that the responses of S at lower initial forces consist of an initial excitatory followed by an inhibitory component.



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Fig. 3. Force responses and length changes of the soleus (S) and gastrocnemius (G) muscles during activation by stimulation of the ipsilateral sural nerve and alternating stretches of S alone and S with G (protocol 2). Note that the force in G is modulated over a larger range than the force in S. Responses of S were inhibited at high forces in G and enhanced at low forces in G. Fs = absolute force in S; Delta Fs = force in S with basline subtracted; Es = electromyogram (EMG) in S; Ls = length of S; FG = absolute force in G; EG = EMG of G; LG = length of G. First 2 force records in trace FG were saturating in the chart recorder.

In a given experimental trial, either or both muscles were stretched by 4 mm at a constant velocity of either 40 or 20 mm/s. The amplitude was chosen to approximate the magnitude of the stretch of SOL during the E2, or yield, phase of the step during trotting (Goslow et al. 1973). The hold period was set at 350 ms. Each experimental trial consisted of a stretch of one or both muscles. Trials were conducted as muscle force slowly decayed to obtain responses at different levels of initial force. In cases in which all three muscles of the triceps surae were involved, one muscle was connected to an isometric myograph so that its force could be measured during stretch of either or both of the other muscles (see Fig. 1).

The stretches were sequenced according to one of two protocols. In protocol 1, a single muscle was stretched in each trial (Fig. 1). Forces in the stretched muscle as well as isometric forces in one or two others were measured. Responses of the stretched muscle consisted of intrinsic and stretch reflex components (Nichols and Houk 1976), and responses of the isometrically constrained muscles consisted of heterogenic reflex components only. Mechanical artifacts due to direct mechanical action of the stretched muscle on those isometrically constrained were indicated by essentially instantaneous latencies or by effects observed after pharmacological block of heterogenic reflexes. The motors were repositioned so as to minimize these artifacts. Trials were rejected if these artifacts exceeded 5% of the total response. In protocol 2, one muscle (1) was stretched on each trial and the other (2) on alternate trials (Fig. 3). Using this protocol, the interaction of intermuscular reflexes from muscle 2 and the stretch reflex from muscle 1 was measured. Because the triceps surae muscles were all stretched during the yield phase of movement (Goslow et al. 1973), protocol 2 better approximates natural conditions. Protocol 1 was employed mainly to observe reflex effects in isolation from the complex intrinsic properties of stretched muscle (Hayward et al. 1988).

In six experiments, <= 0.7 mg strychnine was injected intravenously in graded doses to block inhibitory reflexes. In two of these experiments, the animals were pretreated with the N-methyl-D-aspartate (NMDA) antagonist D(-)-2-amino-7-phosphonoheptanoic acid (DAP7) through the intrathecal route. In one additional experiment, the broad spectrum excitatory amino acid antagonist kynurenic was injected intrathecally. A small laminectomy was performed at a low thoracic level, and an intrathecal cannula inserted caudally under the dura to the L7 level. In the case of DAP7, 1-3 mg dissolved in <= 500 µl of saline, and in the case of kynurenic acid, 1.25 mg in 300 µl of saline, was injected. Control measurements were obtained before and after insertion of the cannula and after infusion of drug. These drugs did not alter either the relationship between the magnitude of the stretch reflex and initial force or the corresponding magnitudes of inhibition, but they did substantially reduce the tendency of the preparations to exhibit seizures (Nichols and Koffler-Smulevitz 1991). This procedure therefore made it possible to achieve near complete block of the inhibition with reduced risk of seizures. In a previous experiment, kynurenic acid in a similar dosage did reduce the gain of the stretch reflex by about 10% (Nichols and Koffler-Smulevitz 1991). Because it is known that kynurenic acid blocks non-NMDA as well as NMDA receptors (Stone and Burton 1988), the small reduction in reflex gain in one case indicates that the dosage used here may have been near the threshold for non-NMDA receptors that mediate synaptic transmission from group Ia afferents to motoneurons (see Jahr and Yoshioka 1986). Because the effects of kynurenic acid on reflex gain were small and variable, we attribute the effects of this drug in reducing seizure activity to its effects on NMDA receptors.

Data analysis

Force responses were collected into groups according to muscle species and protocol. Responses formed a single group for protocol 1 (Fig. 2) and two groups for protocol 2. In the latter case, one group consisted of muscle responses obtained with the stretch of muscle 1 only. The other group consisted of responses taken with stretch of muscles 1 and 2. Latencies of any changes in force were checked to rule out mechanical artifacts. The latency for a mechanical effect in the stretched muscle was essentially instantaneous due to the intrinsic mechanical responses of the active motor units (Fig. 2, MG). Excitatory or inhibitory reflex effects occurred with a latency of ~16 ms (Fig. 2, LG and SOL), which interval is less than or equal to the response time of the earliest components of the stretch reflex in other systems (Bonasera and Nichols 1994; Nichols and Houk 1976). Baselines for each response were estimated using a linear regression of samples obtained during the first and last 50 ms of each record. Measurements of the magnitudes of each response at selected time points then were made from the fitted baselines. Responses were measured at the end of the ramp stretch ("dynamic" response) and at the end of the hold phase ("static" response). Although the force responses included both active and passive components, the initial lengths of the muscles were chosen in part to minimize the passive components. These measurements then were plotted versus the initial forces of either muscle (Figs. 4, 5, 8, and 9). The initial forces were obtained from voltages stored in the sample/hold modules during the experiment. The points for a given group of responses were fitted by a quadratic polynomial and plotted along with the 95% confidence limits. Comparison of the two lines obtained under protocol 2 revealed the sign of the net reflex effect and the dependence of this effect on background force.



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Fig. 4. Enhancement of dynamic responses of MG and LG by heterogenic input from MG, LG, or S (protocol 2). , autogenic responses are indicated; , responses during combined stretch. Responses were measured from the force baseline at the end of the ramp stretch. Fitted lines represent quadratic polynomials determined by the method of least squares. ···, 95% confidence limits. Muscles were activated by crossed-extension reflexes. Initial lengths of MG, LG, and S were 4 mm longer than L90. A: responses of LG to stretch of LG alone (LG+) or LG and MG together (LG + MG+). B: responses of LG with and without stretch of S. C: responses of MG with and without stretch of LG. D: responses of MG with and without stretch of S. Note that the excitation is essentially independent of force. LG received stronger excitation than MG. Insets: pairs of responses matched for background tension to illustrate that the heterogenic excitation remained in effect for the duration of the hold period.



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Fig. 5. Enhancement of static responses of S by heterogenic input from G during a crossed-extension reflex in a preparation with weak heterogenic inhibition. , autogenic responses of S; , responses of S during stretch of both S and G. Inset: pair of responses at the highest forces. Force-independent excitation was apparent for most of the force range in this experiment. Lack of enhancement at the highest background forces may have been due to the appearance of heterogenic inhibition from G or to saturation of the recruitable motor units.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Excitatory reflexes

In four preparations in which MG, LG, and SOL were independently stretched according to protocol 2, it was found that these three muscles were linked by excitatory reflexes (Figs. 4 and 5). These excitatory actions were generally found to be independent of initial force for a substantial range of forces (Figs. 4 and 5). That is, the absolute increase in force due to the heterogenic reflex was approximately constant over the range of initial forces. The heterogenic effect also could be expressed as a percentage increase over the autogenic force response (Figs. 4 and 5). The strongest effects were found consistently to extend from either MG or SOL onto LG (Fig. 4, A and C), and the weakest heterogenic reflexes extended from either SOL or LG onto MG (Fig. 4, B and D). In 15 other preparations in which MG and LG were left intact and stretched as a group to minimize muscle damage, excitation with similar characteristics occurred between SOL and the gastrocnemius muscles (G). In preparations with weak inhibitory reflexes, substantial excitation from the gastrocnemius muscles onto SOL also was observed for a wide range of forces (Fig. 5). Using measurements obtained according to protocol 1, latencies of ~15-20 ms were found for this excitation (Fig. 2).

Inhibitory reflexes

As previously described for a more limited set of data (Nichols 1989a), inhibitory reflexes were observed for reflexes from either LG or MG onto SOL but not in the reverse direction. This inhibition increased with force and cancelled the short-latency excitation at the highest forces (Fig. 2, SOL). Therefore, the latencies of inhibition and excitation appeared to be comparable. These interactions are illustrated further in Figs. 6 and 7 for a case in which LG and MG were left intact and activation was achieved using a crossed-extension reflex. Pairs of records of the stretched G and isometric SOL are shown for a range of initial forces (Fig. 6). The relatively brief excitation at low forces and prolonged inhibition at high forces are apparent from the figure. The brief excitation onto SOL contrasts with the excitation from MG onto LG that mirrors the duration of the stretch (Fig. 2, LG). The transition from net inhibition to excitation as force declined is further illustrated in Fig. 7. For each 10 ms during the experimental record, quadratic polynomials were constructed from the responses of SOL and the corresponding initial forces (see METHODS). These polynomials were concatenated and are shown in Fig. 7 as a surface that relates response magnitude to initial force and time. The gradual emergence of excitation and decrease of inhibition as force declines can be explained provisionally by summation of the two reflexes and force dependence of the inhibitory component. It can be argued that the excitation itself does not decline as force increases, because it remains high over a wide range of forces in preparations in which the inhibition is weak (Fig. 5). The excitatory component has a rapid onset and a dynamic phase, whereas the inhibitory component develops more slowly and outlasts the excitatory component.



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Fig. 6. Heterogenic excitation and inhibition from G onto S during a crossed-extension reflex. Each pair of responses represents the force response of G and S to stretch of G (protocol 1). Right: initial forces during the declining reflex for each pair of responses. Bottom: length traces corresponding to each muscle. As force declined, the inhibitory response decreased and excitatory component increased. Initial lengths of both muscles were 4 mm longer than L90.



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Fig. 7. Time course and integration of heterogenic excitation and inhibition from G to S. Composite surface constructed from responses of S to stretch of G during crossed-extension reflex shown in Fig. 6. Quadratic polynomials of responses at different initial forces were fitted for each 10-ms epoch of the responses. Note that the inhibition nearly cancelled the excitation at high forces. Inhibition exceeded the excitation in duration.

The inhibition from G onto SOL was found to depend mainly on the force in the muscle of origin. This conclusion was reached by comparing the extent of inhibition during activation by crossed-extension and sural nerve reflexes. In the former case, forces in both muscles covaried over a large range (Fig. 1). In the latter case, force in G was modulated widely, whereas the force in SOL varied over a more limited, low range (Fig. 3). Static responses obtained under protocol 2 are shown plotted versus the initial force in G for both forms of activation in Fig. 8A. The responses of SOL were smaller in the case of sural nerve stimulation because the background forces are low for this muscle, but the transitions from net inhibition to net excitation occurred at similar initial forces of G. In the case shown in Fig. 8, the transitions from net inhibition to excitation for the static responses occurred at ~5 N (4.8 and 5.0 N) in G for either reflex. The corresponding forces in SOL, however, were 2.2 N for the crossed-extension reflex and only 1.2 N for the sural nerve reflex. In the case of the dynamic responses, the transitions occurred at ~9 N in G for either reflex. The corresponding forces in SOL, however, were ~8 N for the crossed-extension reflex and only 2 N for the sural nerve reflex. These results are summarized in Fig. 8B in relation to the initial forces in SOL and G for both reflexes. The transition forces that corresponded to the dynamic and static responses are indicated by arrows. The fact that these transitions were less dependent on the force in SOL than on the force in G indicates that the balance between excitation and inhibition was determined primarily by the feedback from G and not by the conditions of activation in SOL. In another experiment, the transition forces for the two reflexes varied by ~10% for the forces of G (13 vs. 12.5 N) and threefold for the forces of SOL (3 vs. 1 N). In addition, the magnitudes of inhibition at corresponding levels of initial force were similar for the two forms of activation and therefore were related primarily to the force in the muscle of origin of the inhibitory reflex. The interpretation of this experiment is complicated somewhat by the fact that MG and LG initially are activated in different proportions during the two reflexes (Fig. 1) (Nichols et al. 1993), so the sources of the inhibition might have differed early during each trial. However, SOL receives strong inhibition from both MG and LG, and the transition forces in the gastrocnemius muscles as a group were quite similar for both reflexes.



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Fig. 8. Dependence of heterogenic inhibition on muscle of origin. Static force responses of S with and without stretch of G (protocol 2) during reflex activation. , autogenic responses; , responses of S during stretch of S and G. A: responses of S are plotted against the background forces in G. Muscles were activated using a crossed-extension reflex (top) and a sural nerve reflex (bottom). Note that the heterogenic input switches from net inhibition to net excitation at a background force of 4-6 N (down-arrow ). B: these transition forces, for both dynamic and static responses, are indicated (down-arrow ) on plots of initial force in S against initial force in G for the crossed-extension (top) and sural nerve reflexes (bottom). Note that these transition forces are less dependent on the initial force in S than the initial force in G. B, bottom, insets: pairs of responses obtained at low (left) and high forces (right). Note the short-latency excitation in the former. Latency of inhibition (right) is difficult to determine because the heterogenic action is composed of a mixture of excitation and inhibition.

Evidence was obtained that the inhibition was related to active contractile force rather than total force during three experiments in which the initial length of G was varied systematically. Records were obtained according to protocol 1 during crossed-extension reflexes (Fig. 9). At each initial length of G, the initial forces of the two muscles varied according to straight-line relationships (Fig. 9B), but these relationships differed according to slope and intercept. The differences in intercept reflect the different passive forces of G at each length. The different slopes presumably reflect the increased force-generating capacity of G at the longer lengths. Active contractile forces of G were obtained by subtracting the passive (intercept) forces from the measured initial forces. When the responses of SOL were plotted against the calculated active force of G, the points corresponding to different initial lengths of G fell on a single line (Fig. 9A). These results indicate that the magnitude of inhibition from G onto SOL was related to the active, initial force of G but not the total force.



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Fig. 9. Dependence of heterogenic inhibition on active contractile force. During crossed-extension reflexes obtained at 3 different initial lengths of G, G was stretched and S constrained isometrically (protocol 1). A: isometric responses of S plotted against active background force for all 3 initial lengths. Active force was calculated by subtraction of passive forces. All data points were used to determine the fitted line. B: absolute initial force in S plotted against initial force of G for each of the 3 initial lengths. Three fitted lines are offset by amounts corresponding to the passive muscle forces obtained at each of the 3 different lengths.

It was demonstrated further in six preparations that the inhibition could be blocked using an intravenous injection of strychnine with or without pretreatment with antagonists of excitatory amino acids (see METHODS). Results from such an experiment using protocol 2 are shown in Fig. 10. In this experiment, the inhibition was particularly strong throughout the range of forces (Fig. 10, top). Control data were obtained before and after insertion of an intrathecal cannula to infuse DAP7 (see METHODS) and after infusion of DAP7 (shown in Fig. 10, top). At an intravenous dose of 100 µg of strychnine, autogenic responses of SOL were unaltered, but the inhibition was reduced significantly (Fig. 10, middle). At a dose of 200 µg, autogenic responses () were slightly elevated and the inhibition was essentially absent (Fig. 10, bottom). The elevated autogenic reflex could indicate that some autogenic inhibition had been present, but the main result was the complete block of heterogenic inhibition from G onto SOL. This finding suggests that the inhibition is postsynaptic and glycinergic (Lodge et al. 1977; Pratt and Jordan 1987) and that it sums with the stretch reflex to produce combinations of excitation and inhibition (Fig. 7) (see Nichols and Koffler-Smulevitz 1991). Similar results were obtained using strychnine alone or using intrathecal kynurenic acid as a pretreatment.



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Fig. 10. Reduction of heterogenic inhibition by strychnine. Each panel shows the static responses of S when stretched alone () or with G (). Preparation was pretreated with intrathecal D(-)-2-amino-7-phosphonoheptanoic acid (DAP7; see METHODS). Inhibition was initially strong (top). After 100 µg strychnine injected intravenously (middle), the autogenic responses were little affected but the inhibition was significantly reduced. - - -, fitted polynomials from the control (top) run. After 200 µg strychnine (bottom), heterogenic inhibition was absent and autogenic responses slightly elevated. Initial lengths were L90 for G and L90 + 4 mm for S.

Although inhibition was observed only from MG, LG, or G onto SOL, the extent of this inhibition varied markedly from preparation to preparation. This variability is apparent from a comparison of the force level at which inhibition supercedes excitation in different preparations. The range of the variability is illustrated by comparing Figs. 5 and 10 (top). The inhibition was expressed over the entire range of initial forces in the latter but was absent over most of the range in the former. In most experiments, the magnitude of the inhibition was intermediate between these two extremes.


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Mechanisms

The data presented in this paper can be explained most simply using known properties of primary receptors of muscle spindles and Golgi tendon organs and their homonymous and heteronymous projections within the triceps surae muscle group. The earliest detectable excitatory effects, both autogenic and heterogenic, occurred at ~16 ms after the initiation of muscle stretch. This number corresponds to that obtained for the long toe flexors (Bonasera and Nichols 1994) and is less than the 23 ms obtained for the stretch reflex of SOL using a different stretching device (Nichols and Houk 1976). Considering that the time interval of 16 ms includes the events between sensory coding of mechanical inputs and force production in the muscle, a rapid spinal pathway is probably responsible for this earliest response. Assuming Lloyd's classification of afferent fibers (Lloyd 1943) and an afferent conduction distance of 150 mm for the feline triceps surae muscles, afferent fibers in the group III range would require 5-25 ms for the afferent limb of the response alone. Therefore afferents in the group II or group I range are implicated in these early excitatory responses. Because the monosynaptic group II inputs from muscle spindles are relatively weak (Prochazka 1990), group Ia afferents from primary muscle spindle receptors are more likely candidates to mediate these rapid responses, although a role for group II afferents cannot be excluded.

An additional clue concerning the afferent pathways involved in the early excitatory responses is the independence of the response on initial force as observed for other combinations of Ia synergists, such as peroneus longus and peroneus brevis, and flexor digitorum longus and flexor hallucis longus (Bonasera and Nichols 1994, 1996). Similar results were obtained for reciprocal inhibition, another system that is thought to be mediated by Ia afferents (peroneus brevis and tibialis posterior: Bonasera and Nichols 1996; tibialis anterior and soleus: Nichols and Koffler-Smulevitz 1991). The fact that the heterogenic excitation was less dependent on initial force than the stretch reflex itself (Hoffer and Andreassen 1981) can be explained provisionally by the absence of intrinsic muscle stiffness in the heterogenic response (Hayward et al. 1988). The stretch reflex is composed of the stiffness of contracting motor units and additional force contributions of recruited motor units (Cordo and Rymer 1982; Nichols and Houk 1976). The progressive increase in the former component with force is thought to account for the increase in autogenic reflex with force. In the case of an heterogenic reflex, however, the force increase results solely from recruitment of new motor units and therefore should depend on force to a lesser extent. Some increase in heterogenic reflex magnitude might be expected based on the recruitment nonlinearity (Houk et al. 1970), but this increase is apparently limited even for the reflex component of the stretch reflex (Hoffer and Andreassen 1981). Saturation of motor unit recruitment or firing rate might account for this limitation.

The hypothesis that heterogenic excitation is due mainly to feedback through Ia afferents is strengthened further by considering the distribution of excitatory effects among LG, MG, and SOL. Figure 11 shows, in histogram form, the fractional heteronymous excitatory postsynaptic potential (EPSP) amplitudes obtained by Eccles et al. (1957a) using animals anesthetized with pentobarbital sodium. Figure 11 also shows the data obtained in Nichols (1989a) of the distribution of excitatory mechanical effects, expressed as a percentage of autogenic response, obtained in decerebrate preparations under quiescent conditions. Both sets of data agree substantially in that MG receives the weakest relative input and LG the strongest. The data can be explained largely by a combination of Ia projection frequencies (Scott and Mendell 1976) and spindle numbers (Chin et al. 1962). The autogenic response of LG is increased substantially by stretch of MG and SOL because MG and SOL contain approximately twice as many muscle spindles as LG. The relatively small responses of MG can be related to the lower heteronymous projection frequency from LG and SOL and the smaller number of muscle spindles in LG.



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Fig. 11. Comparison of the distribution of excitatory reflexes obtained by electrophysiological and mechanographic methods. Top: strengths of group Ia excitatory postsynaptic potentials measured by Eccles et al. (1957) in animals anesthetized with pentobarbital sodium; bottom: distribution of stretch-evoked excitation in the decerebrate cat. Note the similarity in these 2 distributions.

These trends are also observed during activation of the triceps surae (Figs. 4 and 5), supporting the major contribution of the monosynaptic Ia afferents to heterogenic excitation under these conditions. No longer latency effects, which might have been attributed to group II spindle receptors, were noted, although such effects might not be distinguishable due to the filtering properties of muscle. Given the longer latencies of these pathways and the smaller size of group II EPSPs (Prochazka 1990), any such effects might not have been discernible in the present experiments.

Arguments based on time course, distribution, and force dependence also can be used to support the hypothesis that the heterogenic inhibition from MG or LG to S includes feedback from Golgi tendon organs. Despite the delays associated with oligosynaptic transmission (Eccles et al. 1957b), the inhibitory action occurred early enough to progressively diminish what is presumed to be monosynaptic excitation as initial force increased (Fig. 7). The inhibition also outlasted the excitation (Figs. 2 and 7). The longer duration of the inhibitory component is consistent with the longer duration and dispersion of latencies of inhibitory postsynaptic potentials compared with Ia EPSPs (Eccles et al. 1957b; Fetz et al. 1979; Hamm et al. 1987; Watt et al. 1976). Additional temporal spread in inhibitory action could come about because of asynchronous recruitment of inhibitory interneurons. This temporal spread would be expected to apply less to the monosynaptic pathways for the excitatory component. An additional factor that could account for the relatively shorter duration of the excitatory response is the brevity of the initial burst in the response of the primary ending of the muscle spindle (Houk et al. 1981).

The finding that the inhibition is long lasting is in apparent conflict with recent data concerning inhibitory fading. In cats anesthetized using either chloralose or pentobarbital, Zytnicki et al. (1990) showed that electrical stimulation of motor units produces inhibitory effects in motor units that fade within 100 ms even in the presence of ongoing stimulation and receptor discharge. These authors suggested that the effect could be due to presynaptic inhibition of Ib afferent fibers onto other Ib afferents. They also suggested additional hypotheses including a decrease in synchrony of Ib interneurons or changes in conductance of motoneuronal membranes. Subsequent work suggested that pathways from group II or even group III afferents might be involved (LaFleur et al. 1993) and that the transient nature of the inhibition occurs in other inhibitory systems (Heckman et al. 1994). The reason why the inhibition did not subside more rapidly in the studies described here is not clear. However, in the preceding quoted studies that showed rapid adaptation of the inhibition, the inhibitory responses were evoked by electrical stimulation of peripheral nerves or synchronous activation of motor units. In the studies reported here as well as previously (Bonasera and Nichols 1994, 1996), inhibitory effects were evoked by muscle stretch and lasted for the several hundred milliseconds during which the muscle was held at the longer length. In a study from another laboratory (Hayward et al. 1988), stretch-evoked inhibition was maintained for >2 s. Therefore the manner in which the inhibitory reflexes were evoked appears to influence the time course of the reflex effect. The apparent discrepancy in results could be explained in a number of ways, such as a difference in the synchrony with which the afferent fibers were activated or the composition of the population of stimulated afferents. In any case, the present results as well as the previous results using muscle stretch suggest that there are important inhibitory reflexes that are not limited to transient conditions (cf. Heckman et al. 1994).

When the triceps surae muscles are stretched as a group, the autogenic response of SOL will be altered by the excitatory and inhibitory heterogenic components from MG and LG. The extent of the alteration will depend on the strength of the inhibition, but the dynamic phase of the response generally will be enhanced due to the addition of autogenic and heterogenic excitation. In contrast, the static phase will tend to be diminished due to the longer-lasting inhibitory component from MG and LG. The assumption that the various sources of feedback sum in the recipient motoneurons is supported by the finding that the inhibition can be blocked by strychnine, a potent blocker of gluycinergic, postsynaptic inhibition (Lodge et al. 1977).

Electrophysiological studies (Eccles et al. 1957b; Powers and Binder 1985) have suggested that extensor muscles at several joints are interconnected by inhibitory feedback from Ib afferents. Therefore the occurrence of some inhibitory feedback among the triceps surae was expected, although the inhibition is not expressed for all combinations of these muscles (Nichols 1989a, 1994). Data from the 32 experiments described here confirmed that no short latency, inhibitory feedback onto either MG or LG is detectable using these methods. Therefore the net reflex responses of LG and MG during stretch of the triceps surae group are composed primarily of the autogenic responses and heterogenic excitation from the other two members of the triceps surae group.

Although several studies have documented the relationships between heterogenic inhibition and muscle force (Bonasera and Nichols 1994, 1996; Nichols 1989b), the results presented here extend the previous work by further defining the adequate stimulus of the inhibition. The use of the sural (caudal cutaneous sural) nerve and crossed-extension reflexes made it possible to decouple the activation patterns of the triceps surae to determine whether the inhibition required activation in all three muscles or only the presumed muscles of origin. The sural nerve reflex primarily activates MG (Hagbarth 1952; Labella et al. 1989) initially, and then may activate other motor units with some delay (Fig. 1). This pattern of activation is preparation specific because SOL is activated simultaneously with MG, in some cases even though the forces are much smaller (Fig. 3). In contrast to either pattern of activation through sural nerve stimulation, the crossed-extension reflex activates all three muscles (Fig. 1). A complication of this method is that the source of the inhibition probably did change to some extent from MG during the sural nerve reflex to a combination of MG and LG during the crossed-extension reflex. This problem is mitigated, however, by the fact that both MG and LG are important sources of inhibitory feedback to SOL (Nichols 1989a). Another problem could have arisen if the two reflexes activated different ensembles of motor units within MG such that different numbers of Golgi tendon organs had been stimulated. It is assumed that the population of activated tendon organs is the same for a given force during either reflex because size-ordered motor unit recruitment is preserved in both cases (Clark et al. 1993; Cope and Pinter 1995).

Besides the short latency of the inhibition, the other major evidence for the participation of Golgi tendon organs was the finding that the inhibition is related to active contractile force rather than total force. Houk and Henneman (1967) established that tendon organs have low thresholds to active forces and that the adequate stimulus is contractile force exerted by the muscle fibers directly in series with the receptors. It has been shown that the discharge frequency of tendon organs in the cat soleus muscle is a good predictor of active muscle force (Crago et al. 1982; Stauffer and Stephens 1977; but see Horcholle-Bossavit et al. 1990). In view of this evidence, it is parsimonious to conclude that the inhibition results from pathways arising from Golgi tendon organs in MG and LG. However, it cannot be excluded on the basis of this data that the inhibition could result from recurrent inhibition (Hamm 1990). This possibility is unlikely in view of the evidence that heterogenic inhibition is markedly reduced in the soleus muscles of animals in which LG and S had been self-reinnervated and consequently have reduced proprioceptive feedback emanating from those muscles (Cope et al. 1994).

Functional considerations

The asymmetrical distribution of excitation among the triceps surae has biomechanical consequences. In association with the smaller numbers of spindle receptors in LG compared with MG and SOL (Chin et al. 1962), the autogenic reflexes in LG are smaller than those on MG (Figs. 4 and 5). The larger fractional EPSPs from MG onto LG (Eccles et al. 1957a) apparently lead to the larger heterogenic excitation in that direction (Fig. 4 and 5). The net result is that when the triceps surae are lengthened, the net reflex responses in MG and LG tend toward the same magnitude. Therefore the line of action of the response of the muscle group will be some average of the force vectors of the two muscles (Lawrence et al. 1993; Nichols et al. 1993). When MG is activated individually with the suppression of LG and S, as occurs during a sural nerve reflex, the direction of pull should be dominated by that of MG because this muscle receives most of its excitatory feedback autogenically and because reflex responses of LG should be suppressed. This evidence supports the hypothesis that the mechanical actions of MG on the ankle are unique among the triceps surae because this muscle provides a significantly larger component of abduction torque than either SOL or LG.

It has been argued elsewhere that the inhibitory reflexes from MG and LG to SOL are associated with the differences in action of these muscles at the knee rather than their different motor unit compositions (Dacko et al. 1996; Nichols 1989a). The data presented here provide further details concerning the dynamics of the resulting reflex activity in the triceps surae muscles. Early during the response to stretch of the triceps surae, mixed inhibition and excitation from MG and LG onto SOL preserves or enhances the dynamic phase of the stretch reflex of SOL so that the response of the triceps surae as a whole is dominated by shared excitation. After the excitatory effect from LG and MG has declined, the remaining inhibition reduces the magnitude of the tonic stretch reflex in SOL. This later phase of the response therefore is dominated by the stiffness of MG and LG. Because these muscles span both ankle and knee joints, the extent of coupling of the two joints is increased with a consequent increase in stability.

This interpretation of the role of inhibition in interjoint coupling and limb stability is subject to the expression of positive force feedback during some motor behaviors (Pearson 1995). Excitatory, group I feedback can help regulate the pattern of locomotion as well as provide a loading reflex to enhance the activity of antigravity muscles (Dietz et al. 1989). If inhibitory force feedback was replaced entirely by excitatory feedback during a given behavior, then the preceding arguments concerning the stabilizing effects of force feedback would not apply during that behavior. However, little information is available concerning the expression and distribution of these two types of force feedback during natural behaviors. Recent evidence using intramuscular stimulation in the standing cat, for example, suggested that both types of feedback can coexist with different distributions (Pratt 1995). Although instances of positive force feedback were shown during standing, stimulation of LG still led to inhibition of SOL (Pratt 1995). The force feedback system appears to be subject to considerable flexibility, as indicated by studies of locomotion and standing as well as the variability of inhibition observed among preparations reported here.


    ACKNOWLEDGMENTS

The author thanks Dr. Timothy C. Cope for valuable comments on the manuscript, two anonymous reviewers for valuable feedback, and M. Witten and D. Koffler-Smulevitz for programming assistance.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-20855.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 February 1998; accepted in final form 19 October 1998.


    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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