purely monosynaptic e.p.s.p. is a prerequisite for the validity of the method. Experimental

Transcription

1 J. Physiol. (1987), 389, pp With 8 text-figures Printed in Great Britain ASSESSING CHANGES IN PRESYNAPTIC INHIBITION OF I a FIBRES: A STUDY IN MAN AND THE CAT BY H. HULTBORN*, S. MEUNIER, C. MORIN AND E. PIERROT-DESEILLIGNYt From Clinical Neurophysiology, Department of Reeducation, H'pital de la Salpetriere, 47 Bd de l'hmpital, Paris, Cedex 13, France and the * Department of Neurophysiology, The Panum Institute, University of Copenhagen, Denmark (Received 12 May 1986) SUMMARY 1. A method to assess changes in presynaptic inhibition of I a afferent terminals in man is proposed. The soleus H reflex was facilitated by a heteronymous Ia volley from quadriceps and the amount of reflex facilitation was used to estimate the size of the conditioning I a excitatory post-synaptic potential (e.p.s.p.). It is argued that the size of this e.p.s.p. as measured by the resulting amount of reflex facilitation reflects the amount of presynaptic inhibition on the corresponding Ia fibres. A decrease in the reflex facilitation may then be ascribed to an increase in presynaptic inhibition of the Ia fibres mediating the conditioning volley. 2. That the heteronymous Ia facilitation from quadriceps to soleus is caused by a purely monosynaptic e.p.s.p. is a prerequisite for the validity of the method. Experimental evidence is therefore given in an Appendix that in man the earliest part (first 0-5 ms) of this heteronymous I a facilitation is mediated through a monosynaptic pathway. Evidence is also given that this earliest facilitation is not yet contaminated by any polysynaptic effects from Ia or Ib afferents. 3. The validity of the method was established in animal experiments in which presynaptic inhibition of I a fibres and post-synaptic events in motoneurones could be assessed by direct tests. It was found that the amount of test reflex facilitation produced by a conditioning I a volley was decreased when I a fibres were subjected to presynaptic inhibition but remained unchanged when the motoneurone pool in which the test reflex was elicited received pure post-synaptic inhibition. 4. In man, presynaptic inhibition of I a fibres was evoked by a short-lasting (three shocks at 200 Hz) vibration applied to the tibialis anterior tendon, Such a vibratory burst reduced the efficiency of the heteronymous I a volley in facilitating the soleus H reflex. By contrast, during a pure post-synaptic inhibition of soleus motoneurones the efficiency of the conditioning volley in facilitating the test reflex remained unchanged. It is therefore argued that the amount of heteronymous Ia facilitation can indeed be used to assess the amount of ongoing presynaptic inhibition exerted onto heteronymous I a fibres from the quadriceps muscle to soleus motoneurones. t To whom correspondence should be addressed.

2 730 H. HULTBORN AND OTHERS 5. The short-lasting tibialis anterior vibration used here evoked a long-lasting ( ms) depression of soleus and quadriceps H reflexes. Even though several mechanisms contribute to this depression, it is argued that presynaptic inhibition of I a fibres mediating the afferent volley of the reflex is the only mechanism responsible for the reflex depression when the test reflex is evoked ms after the onset of vibration. Within this time interval, therefore, the measurement of the vibratory inhibition of the H reflex assesses the excitability of the interneurones mediating presynaptic inhibition of homonymous I a extensor fibres. INTRODUCTION Frank & Fuortes (1957) described a depression of Ia monosynaptic excitatory post-synaptic potentials (e.p.s.p.s) which occurred without observable change in the post-synaptic cell itself. It is now generally accepted that this depression is caused by a conductance increase (and depolarization) of the primary afferent terminals brought about by axo-axonal synapses and that this conductance increase results in a reduced transmitter release (Eccles, 1964; Schmidt, 1971). Although presynaptic inhibition to muscle spindle I a afferents has been extensively investigated in the cat spinal cord (for references see Schmidt, 1971; Baldissera, Hultborn & Illert, 1981), its functional significance in motor control remains difficult to evaluate. Such an evaluation would be possible from experiments performed during natural movements in man. A method permitting estimation of changes in presynaptic inhibition of Ia fibres in man is proposed. It is based on the following principle: a change in the size of the monosynaptic I a e.p.s.p. evoked by a given stimulation may be ascribed to a change in presynaptic inhibition of the corresponding Ia fibres. The amount of H reflex facilitation produced by a conditioning stimulation of Ia fibres is used to estimate the size of the conditioning I a e.p.s.p. Under these conditions the H reflex facilitation resulting from stimulation of a given number of Ia fibres should be constant unless the presynaptic inhibition exerted on the I a fibres mediating the conditioning volley is changing. Accordingly, a decrease in the H reflex facilitation must be ascribed to an increase in presynaptic inhibition of the Ia fibres. This principle has already been used to provide evidence for presynaptic inhibition of soleus Ia fibres in man while employing a homonymous Ia facilitation of soleus motoneurones (Morin, Pierrot-Deseilligny & Hultborn, 1984). However, it is crucial to the proposed method that the facilitation of the test reflex is caused by a pure monosynaptic Ia excitation: if the conditioning excitation is not entirely mediated through the monosynaptic Ia pathway and/or is contaminated by later (e.g. Ib) effects, changes in facilitation of the test reflex cannot be ascribed unequivocally to changes in the size of the conditioning I a e.p.s.p. The monosynaptic I a excitation has therefore to be assessed during its earliest part when it is not yet contaminated by any polysynaptic effect. When the conditioning and test stimuli are applied to the same fibres (homonymous facilitation; Morin et al. 1984) this cannot be achieved since there must be a minimum 3 ms conditioning-test interval to avoid the refractoriness of I a fibres activated by the conditioning stimulus. By contrast when the conditioning and test stimuli are applied to different fibres (heteronymous facili-

3 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN tation) it is possible to adjust the timing so that the two afferent volleys arrive at the spinal level simultaneously. Thus in the present experiments the soleus H reflex was conditioned by a heteronymous I a volley from the quadriceps muscle (Bergmans, Delwaide & Gadea-Ciria, 1978). Experimental evidence is given that during its first 05 ms this heteronymous facilitation is only mediated through a monosynaptic pathway (see Appendix) and not contaminated by any Ib effect. The validity of the method was established in animal experiments in which presynaptic inhibition of I a fibres and post-synaptic events in motoneurones could be verified by direct tests: it was found that the amount of monosynaptic reflex facilitation was decreased when, and only when, presynaptic inhibition of the I a fibres mediating the conditioning volley was increased. Similarly, in human experiments, the efficiency of the conditioning I a volley in facilitating the test reflex was reduced if the I a fibres mediating this volley were subjected to presynaptic inhibition, but remained unchanged if the motoneurones responsible for the test reflex received postsynaptic inhibition. It will be shown in the companion paper (Hultborn, Meunier, Pierrot-Deseilligny & Shindo, 1987) that the present method can be used to study changes in presynaptic inhibition of I a fibres in man during movement. 731 METHODS Experimental procedure in man The experiments were carried out on seven healthy subjects aged years, all of whom gave informed consent to the experimental procedure. In three subjects the experiments were repeated at least twice to test the reproducibility of the results. The subjects were comfortably seated in an armchair and the examined leg was loosely fixed with the hip semi-flexed (120 deg), the knee slightly flexed (160 deg) and the ankle at 110 deg. Test reflexes Surface electrodes were used for both stimulation and recording. Test stimuli were rectangular pulses of 0'5 ms duration delivered through a unipolar electrode every 4 s. The reflex responses were measured as the peak to peak amplitude of muscle action potentials recorded by two nonpolarizable disk electrodes (0 9 cm diameter) placed 1-5 cm apart on the corresponding muscle. The soleu8 H reflex was used for the basic experiment, i.e. the assessment of the excitatory effects of a heteronymous conditioning I a volley from quadriceps to soleus. The active electrode used to stimulate the posterior tibial nerve was a ball (1-5 cm diameter) in the popliteal fossa. The reference electrode was fixed to the anterior aspect of the thigh, adjacent to the patella. Recording electrodes were placed on the soleus below the gastrocnemii muscles. The quadriceps H reflex was used to study the inhibition caused by a short vibration to the tendon of the tibial anterior muscle. The quadriceps H reflex was obtained by stimulating the femoral nerve with an active electrode (half-ball of 2-5 cm diameter) in the femoral triangle, the reference electrode being on the posterior and upper aspect of the thigh. Recording electrodes were placed on the vasto-crureus (6-8 cm above the patella, anterior aspect of the thigh). Conditioning stimuli Heteronymous facilitation of the soleus H reflex from the femoral nerve. The conditioning stimulus was applied to the femoral nerve and was delivered through the same unipolar electrode used to obtain the quadriceps H reflex. By convention, the timing of the test pulse was referred to that of the conditioning pulse. If the afferent volleys following the conditioning and test stimuli are to arrive at the spinal cord simultaneously, the test pulse has to be delivered before the conditioning pulse as the distance from the site of femoral nerve stimulation to the spinal cord is shorter than the distance from the popliteal fossa to the spinal cord. In such cases the conditioning-test interval is said to be negative. The current delivered by the stimulator was measured by a current probe

4 732 H. HULTBORN AND OTHERS (Tektronix 6021). Since it is very difficult to record ascending afferent volleys in man, stimulus intensity was expressed in multiples of the threshold intensity for the direct motor M-wave (times motor threshold x MT). In order to avoid any possible recurrent inhibition of soleus motoneurones conditioning stimulus intensity was below the threshold of quadriceps motor and H waves. Vibratory stimulus. A short-lasting vibration (10 ms: three shocks, 200 Hz) was applied to the skin above the tendon of the tibialis anterior by a vibrator (Bruiel and Kjaer model 4809) driven by monophasic rectangular pulses of 2 ms duration. Using a power amplifier it was possible to grade the strength of the vibration, the amplitude of which remained below 0-8 mm. Stimulation of the gastrocnemius medialis nerve. This nerve was stimulated by single shocks (0 5 ms duration, 0-9 x MT strength) delivered through a bipolar surface electrode placed at the lower and medial part of the popliteal fossa, 6-10 cm below the electrode eliciting the soleus H reflex (Pierrot-Deseilligny, Katz & Morin, 1979). Organization of the experiment. Analysis of the results Each experiment was started by establishing the earliest conditioning-test interval (using 0 1 ms steps) at which it was possible to elicit the heteronymous facilitation of the soleus H reflex from the femoral nerve. The interval used for the experiment was 0-2-0{4 ms longer so as to obtain a sizeable facilitation yet still ensure that only the uncontaminated monosynaptic I a e.p.s.p. was responsible for the facilitation (see Section II under Results). The amount of heteronymous I a facilitation of the soleus H reflex was compared in two situations: (1) control situation without conditioning inhibitory stimulus and (2) with inhibitory conditioning stimulus (either vibration or gastrocnemius medialis nerve stimulation). It has been shown that the susceptibility of an H reflex to facilitation can change with the size of the reference test reflex (not conditioned by femoral nerve stimulation): at low reflex amplitudes (below 10-15% of the maximum M-wave amplitude) the susceptibility to facilitation increases with the increase in size of the unconditioned reflex (Meinck, 1980; Mazieres, Morin & Pierrot-Deseilligny, 1984). Thus, when the test reflex was reduced by an inhibitory stimulus, the test stimulus intensity was increased so that the size of the reference reflex was the same as in control. This will be referred to as 'compensation' of the reference test reflex during inhibition. The amplitude (peak-to-peak) of the H reflexes was measured 'on line' (Apple II) and results stored on disk for later analysis. Various stimulation paradigms were used (see Results). The size of the conditioned H reflex was expressed as a percentage of its unconditioned value. The heteronymous facilitation obtained during pre- or post-synaptic inhibition was also expressed as a percentage of the control facilitation (i.e. the facilitation without the inhibitory stimulation). Whatever the stimulation paradigm, the different alternatives were randomly presented (Fournier, Katz & Pierrot-Deseilligny, 1984) and at least twenty responses foreach were collected. The statistical significance of the results was examined by an F test. Experimental procedure in the cat Experiments were performed on four cats. Ether anaesthesia was used during dissection, but was withdrawn after anaemic decerebration by ligation of both carotid arteries and the basilar artery (Pollock & Davis, 1930). During recording the cats were immobilized with gallamine triethiodide (Flaxedil) and artificially ventilated. Lumbar spinal segments were exposed by a laminectomy from L4 to L7. The ventral roots L6-S1 were transacted on the left side. The following peripheral nerves in the left limb were dissected and mounted on silver wire electrodes for stimulation: the nerves to posterior biceps and semitendinous (knee flexors), the deep peroneal nerve (supplying ankle flexors), the nerve to medial gastrocnemius, the nerve to lateral gastrocnemius and soleus and the sural nerve. The skin flaps around exposed areas of the cord and the hind limb were sewn up to form pools that were filled with warm paraffin oil. The central portion of L7-S1 ventral roots was mounted on electrodes for recording or stimulation (ventral roots L7 and S1 were often divided, one-half used for stimulation and the other for recording). The cats were spinalized at a low thoracic level. Monosynaptic reflexes from triceps surae were recorded from the central part of sectioned ventral roots and their area was measured. Rectangular wave pulses of 0 1 ms duration were used for nerve stimulation. The strength of stimulation was expressed in multiples of threshold ( x T) for the lowest-threshold afferent fibres. Intracellular recordings from triceps surae motoneurones were obtained using glass capillary micro-electrodes filled with 2 M-potassium citrate solution. The tips of the micro-electrodes were broken to a diameter of about 1-5 /sm. The input

5 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN resistance was measured by injecting small current pulses (2-3 na, about 5 ms duration) through the recording micro-electrode. The device allowed compensation of the voltage drop across the micro-electrode resistance (Eide, Jurna & Lundberg, 1968). The excitability of I a primary afferent terminals from triceps surae was tested by stimulation in the motor nuclei ad modum Wall (1958): single stimuli were delivered to the ventral horn through low-resistance glass capillary electrodes filled with 2 M-NaCl solution, while the antidromic I a volleys were recorded from the peripheral muscle nerve. A computer (Nord Data) was used for averaging intracellular potentials and for an on-line analysis of monosynaptic reflexes and antidromic Ia volleys. 733 RESULTS In the method proposed here the soleus H reflex is facilitated by a conditioning stimulation applied on heteronymous Ia fibres from the quadriceps muscle. The heteronymous Ia e.p.s.p. so evoked in soleus motoneurones is monosynaptic (see Appendix). The earliest part of the resulting facilitation of the test reflex is not contaminated by polysynaptic effects (Section II) and can be used to estimate the size of the conditioning monosynaptic e.p.s.p. A constant conditioning stimulation should elicit an e.p.s.p. of constant size in motoneurones, and thus a constant amount of reflex facilitation, unless the amount of presynaptic inhibition is changing: a decrease in the amount of heteronymous I a facilitation, as compared to that in the control situation, may therefore be ascribed to an increase in presynaptic inhibition of the Ia fibres mediating the conditioning volley. The validity of this method was tested as follows in human experiments: it was first demonstrated that a short train of vibration applied to the tibialis anterior tendon elicits a depression of soleus and quadriceps H reflexes with a time course which implies presynaptic inhibition of the I a fibres mediating these reflexes (Section III). The amount of heteronymous I a facilitation of the test reflex was then compared in the control situation and in situations with a pure presynaptic inhibition of I a fibres (caused by the short vibratory train, Section IV) or a pure post-synaptic inhibition of soleus motoneurones (I b inhibition from medial gastrocnemius, Section V). This aimed at showing that the heteronymous Ia facilitation, and thus the underlying conditioning e.p.s.p. in motoneurones, is decreased when the Ia fibres mediating the conditioning volley are subjected to presynaptic inhibition but remains unchanged when the motoneurones responsible for the test reflex receive postsynaptic inhibition. The latter is essential if one wants the method to be a specific test for presynaptic inhibition. Conditioning stimuli causing post-synaptic or presynaptic inhibition both reduced the test reflex size, as compared to the control situation (vibration producing a presynaptic inhibition which is diffusely distributed to I a fibres mediating both conditioning and test volleys). Since the susceptibility to facilitation of the test reflex depends on the size of the reference (unconditioned) reflex (see Methods), it was necessary to increase the intensity of the test stimulus to compensate for this inhibition: thus reference reflexes were of the same size in the control situation and during presynaptic and post-synaptic inhibition. This restoration of the test reflex size of course does not change the size of the conditioning e.p.s.p. Finally, the validity of the proposed method has been confirmed in parallel animal

6 734 H. HULTBORN AND OTHERS ^xsfc --~ 1 A. I <-<(< Recording - :heterony mouse / Recording group < a homonymous F, -d G B 250 S~~~~~~~50ms 5 ins ) ' a_0 50 nro I I I I.... M ,1,,.", H I J K L heterony :mous F lexor F flexor7 AdRecording O > group ' / *~~~~~~~~~~~3 Cuc 150 4/," Ia homonymous wi/i ~~~~~~~~ N 0 Fig. 1. Facilitation of a monosynaptic reflex elicited by a heteronymous Ia volley and excitability of triceps surae I a terminals during presynaptic inhibition in the acute spinal cat. A, schematic diagram of the experimental paradigm for testing heteronymous Ia facilitation; the gastrocnemius medialis (I a homonymous) monosynaptic reflex (recorded from the sectioned ventral root) is facilitated by a heteronymous I a volley (gastrocnemius lateralis-soleus). Monosynaptic Ia e.p.s.p.s are recorded intracellularly from triceps surae motoneurones. Presynaptic inhibition of both homonymous and heteronymous La fibres is elicited by a train of group I volleys in a flexor nerve. Bars and small filled circles indicate excitatory and inhibitory synapses respectively. B, monosynaptic Ia e.p.s.p. elicited by a volley to the nerve to lateral gastrocnemius and soleus (I15 x T) is compared in the control situation (B1) and during presynaptic inhibition (B2) elicited by a train of six shocks to the posterior biceps-semitendinosus (at 300 Hz, 16 x T, conditioning-test interval 90 ms). The conditioning volleys produce no potential change when applied alone (B3). Sample records (C0-) of the reflex responses and averages of fifty trials (H-L, the size of the test reflex being expressed as a percentage of its reference value in the control situation) show the size of the reflex in the different situations. In C-G, upper traces are ventral root recordings and lower traces show dorsal root incoming volleys; the

7 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN experiments. Because the pre- or post-synaptic nature of the inhibitory stimulus could only be ascertained in such experiments (by using direct tests, such as measurement of the excitability of Ia fibres or intracellular recording of motoneurones), the results of these cat experiments will be presented first (Section I). In particular it was of importance to verify that the peak of the conditioning Ia e.p.s.p. is not significantly modified during post-synaptic inhibition, despite the increase in conductance underlying the inhibitory post-synaptic potential (i.p.s.p.). (I) Verification of a selective decrease in heteronymous Ia facilitation during presynaptic inhibition in animal experiments The experimental paradigm employed in these animal experiments was similar to that used in human experiments (Sections IV and V). The nerve to one part of the triceps surae (either medial gastrocnemius or lateral gastrocnemius and soleus) was used for the test reflex while the conditioning stimulus eliciting the heteronymous I a facilitation was applied to the nerve supplying the other part. The different choice for heteronymous Ia facilitation in man (quadriceps) and in the cat (part of triceps surae) is due to the lack of I a excitation from medial gastrocnemius to soleus in man (Pierrot-Deseilligny et al. 1979) while, in the cat, Ia excitation from quadriceps to soleus is too weak (Eccles, Eccles & Lundberg, 1957) to be convenient for the present purpose. In these experiments intracellular recordings from triceps surae motoneurones made it possible to study the size and shape of the conditioning I a e.p.s.p. as well as changes in the membrane potential and conductance caused by the stimuli which evoked pre- or post-synaptic inhibitions of the triceps surae test reflexes (Figs. 1 B and 2B). The excitability of Ia primary afferent terminals was measured ad modum Wall (1958): microstimulation in the triceps surae motor nuclei activates Ia terminals (and other neural elements) of the region and the early antidromic volley recorded in the peripheral nerve to triceps surae (cf. diagram in Fig. 1 M) is solely due to I a 735 lines below the slow records in the left columns indicate the parts which are shown on an expanded time scale in the right-hand column. C and H, reference monosynaptic reflex (gastrocnemius medialis) in the control situation. D and I, heteronymous Ia facilitation of the reflex in the control situation. E and J, inhibition of the monosynaptic reflex by a train of group I volleys in the posterior biceps-semitendinosus nerve. F and K, following the group I train the size of the reference monosynaptic reflex is restored to its control level by increasing the test stimulus intensity. G and L, following the group I train, heteronymous I a facilitation was reduced, as compared to the control situation (this reduction is shown by the interrupted open column in L). Vertical bars 1 standard error of the mean. M, schematic diagram of the experimental paradigm for recording changes in excitability of triceps surae I a terminals; I a terminals are stimulated by a microelectrode in the triceps surae motoneurone pool and the antidromic volley in the I a afferents is recorded from the nerve to gastrocnemius lateralis and soleus. Presynaptic inhibition of I a terminals is evoked by a train of group I volleys in a flexor nerve. N-O, the size of the antidromic I a volley evoked by microstimulation of I a terminals in the motoneurone pool and recorded from the nerve to gastrocnemius lateralis-soleus. The size is expressed as a percentage of its reference value in the control situation (N); the filled column (0) shows the increase in excitability during presynaptic inhibition (same stimulus parameters to the posterior biceps semitendinosus nerves as in B2, E-G and J-L).

8 736 H. HULTBORN AND OTHERS fibres as they are the only fast-conducting afferents projecting to the motor nuclei. The size of the antidromic I a volley reflects the excitability of the I a terminals which is increased during presynaptic inhibition (Wall, 1958). Presynaptic inhibition. Following Eccles, Schmidt & Willis (1962) presynaptic inhibition of the triceps surae I a fibres was evoked by short trains of two to six shocks (300 Hz, x T) to a nerve supplying a flexor muscle (ankle or knee flexor) with a conditioning-test interval of ms. Intracellular recording from twenty-two triceps surae motoneurones confirmed that these trains reduced the amplitude of I a e.p.s.p.s from triceps surae nerve branches without simultaneous changes in membrane potential or conductance. The long conditioning-test interval was chosen to avoid the early short-lasting post-synaptic inhibition that is usually evoked by such trains of group I volleys. This is exemplified in Fig. 1 B, where each of the traces represents the average of twenty-five responses. Trace 1 of Fig. 1 B shows the control I a e.p.s.p. (lateral gastrocnemius and soleus nerve, 1 15 x T), while trace 2 illustrates the efficient decrease in the peak amplitude when preceded (90 ms) by six pulses to the nerves of the knee flexor muscles (posterior biceps-semitendinosus, 1 6 x T). The conditioning stimulation alone produced neither potential change (trace 3) nor conductance change (not illustrated) at this interval. With the same parameters for the conditioning stimulation as in Fig. 1 B there was always an increase in the antidromic I a volley following microstimulation of I a terminals in the ventral horn (compare Fig. 1 N and 0), i.e. an increased excitability of I a terminals reflecting an increase in presynaptic inhibition. The proposed method for assessing presynaptic inhibition of I a fibres was then tested with the same parameters for the conditioning stimulation as in Fig. 1 B and N-O. The test reflex was evoked by stimulation of the gastrocnemius medialis nerve and the heteronymous facilitation was evoked by a simultaneous stimulation of the nerve to gastrocnemius lateralis and soleus muscles (1I15 x T). Sample records of the reflex responses (Fig. 1 C-G) and averages of fifty trials (Fig. 1 H-L) show the size of the reference and of the facilitated reflexes in the control situation and in the situation with presynaptic inhibition. In the control situation the size of the reference reflex was set as 1000% (Fig. 1 C and H) and the heteronymous facilitation increased it to 2300% (Fig. 1 D and I). When the test was preceded (90 ms) by the same train of stimuli to the nerves of the knee flexor muscles the size of the reflex was reduced to 24% (Fig. 1 E and J). The test stimulus intensity was then increased and the gastrocnemius medialis test reflex was restored to 960% of its original size (Fig. 1F and K). As previously mentioned, it is necessary to perform this 'compensation' as the susceptibility of a test reflex to facilitation depends on its size. With the same stimulation of the heteronymous nerve to gastrocnemius lateralis and soleus the facilitation due to monosynaptic Ia excitation was less pronounced during presynaptic inhibition than in the control situation (compare Fig. 1 G and L to D and I). This difference in facilitation was highly significant (P < 001). Post-synaptic inhibition. Post-synaptic inhibition being secondary to an increase in motoneurone membrane conductance it would be expected that other simultaneous synaptic inputs would be reduced by 'shunting'. The conditioning heteronymous I a e.p.s.p. would then be reduced and less efficient in facilitating the test reflex. It was therefore of particular importance to evaluate the extent to which monosynaptic I a e.p.s.p.s were modified by conditioning post-synaptic inhibition.

9 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN 737 In human studies the lb inhibition from gastrocnemius medialis to soleus motoneurones was used as an example of post-synaptic inhibition, which is possible since there is no monosynaptic I a excitation from gastrocnemius medialis to soleus in man (Pierrot-Deseilligny et al. 1979; see Fig. 7B). This combination is unsuitable for cat experiments, since there is a strong monosynaptic I a excitation between all parts of the triceps surae in the cat (Eccles et al. 1957). Therefore, in the cat, post-synaptic inhibition of triceps surae motoneurones was elicited from several other sources: (1) recurrent inhibition (stimulation of the Si and part of the L7 ventral roots, while recording the test reflex from the remaining part of the L7 ventral root), (2) inhibition by low-threshold cutaneous afferents (sural nerve, 1'2-2 x T) and (3) inhibition from high-threshold muscle afferents (single pulse to afferents from extensors). In all cases the absence of contamination by presynaptic inhibition was shown by the absence of a significant increase in the excitability of the Ia terminals (see Fig. 2N-O and diagram in M). The test reflex in Fig. 2C and H was evoked from the nerve to the gastrocnemius medialis muscle as in Fig. 1. A simultaneous stimulation of the heteronymous nerve to gastrocnemius lateralis and soleus (1 1 x T) increased the test to 163 % (Fig. 2D and I). A conditioning stimulation applied to the sural nerve (single shock, 1P2 x T, 8 ms conditioning-test interval) decreased the test reflex size to 33% of its control value (Fig. 2 E and J), i.e. an inhibition in a range similar to that caused by presynaptic inhibition illustrated in Fig. 1 E and J. When the test stimulus was increased to 'compensate' for the inhibition, the reflex attained 95% of its original size (Fig. 2F and K). The heteronymous facilitation caused the test to increase to 168% (Fig. 20 and L), i.e. the facilitation by the heteronymous monosynaptic Ia excitation was as effective during post-synaptic inhibition as under control conditions. Similar results, i.e. inhibition of the monosynaptic reflex, but no change either in the amount of heteronymous I a facilitation of the test reflex or in the excitability of the corresponding Ia terminals, were obtained with recurrent inhibition and inhibition from high-threshold muscle afferents. Intracellular recording from twenty-five triceps surae motoneurones was used to study the extent to which monosynaptic I a e.p.s.p.s were modified by a conditioning post-synaptic inhibition. In the example of Fig. 2B the Ia e.p.s.p. (trace 1) was evoked from the nerve to lateral gastrocnemius and soleus (1 1 x T) and the conditioning post-synaptic inhibition (recurrent inhibition) by a single shock (supramaximal to a-fibres) to the 51 and part of the L7 ventral roots. When given alone, the latter produced a pure recurrent i.p.s.p. This i.p.s.p. obtained in isolation (trace 3) or together with the I a e.p.s.p. (trace 2) are both aligned to the same base line as the control I a e.p.s.p. alone (trace 1). The 'isolated' I a e.p.s.p. during post-synaptic inhibition would be represented by the difference between traces 2 and 3; the result of this subtraction is shown by trace 4 to allow easy comparison with control I a e.p.s.p. in trace 1. It is seen that the peak is virtually unchanged, while the time constant of the passive decay is shortened. As long as conditioning stimuli elicited a relatively moderate post-synaptic inhibition, i.e. an inhibition which reduced the test reflex down to 90-25% of its reference value, it was found that the peak amplitude of the Ia e.p.s.p. was not significantly decreased (cf. Fig. 2B), despite the increase in motoneurone membrane conductance underlying the i.p.s.p. This increase in conductance (measured but not illustrated) might account for the shortening of the time constant of the passive decay and thus the reduction of the Ia e.p.s.p. during its 24 PHY 389

10 738 H. HULTBORN AND OTHERS A ( f/0<f0 C Recording :heteronymouss D /Recording Sural nerve C -, la homonymous F A_ B 200- G 10 ms 4 1~~~~50-05 ms 0In 05mV.U ner Sirns H I J K L M Sural nerve ch /~muius sti~ heterony- ' 200 mous! 150 Recording 0;'. ~ ~~~~~ t i I a homonymous Wvl C N 0 Fig. 2. Facilitation of a monosynaptic reflex elicited by a heteronymous ia volley and excitability of triceps surae la terminals during post-synaptic inhibition in the acute spinal cat. A, schematic drawing of the experimental paradigm for testing heteronymous tafacilitation; the gastrocnemius medialis (Ila homonymous) monosynaptic reflex (recorded from the sectioned ventral root) is facilitated by a heteronymous Ia volley (gastrocnemius lateralis-soleus). Monosynapticia e.p.s.p.s are recorded intracellularly from triceps sure motoneurones. Post-synaptic inhibition is elicited by volleys in lowthreshold cutaneous fibres (or high-threshold muscle afferents, or antidromic volleys in motor axons, i.e. recurrent inhibition). Bars and small filled circles indicate excitatory and inhibitory synapses respectively. B, monosynaptic I a e.p.s.p. elicited by a volley in the gastrocnemius lateralis-soleus nerve (lī x T) is compared in the control situation (B 1) and together with post-synaptic inhibition (B 2) elicited by a maximum antidromic motor volley in the Si and part of the L7 ventral roots (recurrent i.p.s.p.). The recurrent i.p.s.p. alone is shown in B 3 and the 'isolated' Ia e.p.s.p. during post-synaptic inhibition in B4 (obtained by subtracting in each 0-2 ms bin the value of the recurrent i.p.s.p. shown in B 3 from the combined potential caused by recurrent i.p.s.p. + I a e.p.s.p. in B 2). Sample records of the reflex responses (C-G; upper traces are ventral root recordings, lower traces show dorsal root incoming- volleys) and averages of fifty trials (H-L; the size of the test

11 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN falling phase. The apparent discrepancy between the unchanged peak amplitude and the reduction of the falling phase is explained by fundamental biophysical circumstances (see Discussion). In any event, the unchanged peak amplitude of the Ia e.p.s.p. explains that, in experiments with reflexes, post-synaptic inhibition of the motoneurones did not change the amount of reflex facilitation (Fig. 2H-L): because conditioning and test I a volleys were timed to reach the motoneurones simultaneously, the test e.p.s.p. was added to the top of the conditioning e.p.s.p. However, when the conditioning stimulation was strong enough to cause the test reflex almost completely to disappear, the correspondingly larger i.p.s.p. in motoneurones was accompanied by a pronounced conductance increase, and the peak of the I a e.p.s.p. was also significantly reduced. Consequently, if inhibition of the reflex is very large (whatever its origin) a reduction of the heteronymous Ia facilitation is obtained. Thus, in human experiments only conditioning stimuli which reduced the test reflex by less than half were used so that the peak amplitude of the conditioning Ia e.p.s.p. was not reduced by post-synaptic inhibition. (II) Facilitation of the soleus H reflex by stimulating quadriceps I a fibres in man The use of variations in the amount of heteronymous I a facilitation of a test reflex to estimate changes in presynaptic inhibition of I a fibres requires that the facilitation is mediated through a monosynaptic pathway and is not contaminated by any other effect. The first step was therefore to ensure that these requirements were fulfilled when facilitating the soleus H reflex by femoral nerve stimulation (the experimental paradigm is sketched in Fig. 3A). Fig. 3B and C shows the time course. of the variations in the soleus H reflex when conditioned by a stimulation applied to the femoral nerve. The curves were obtained in two different subjects and the intensity of the femoral nerve stimulation was 0O85 x MT and 095 x MT respectively. Facilitation of the test reflex started at the interval -6-7 (Fig. 3B) and -6-2 (Fig. 3C) ms (this negative delay is due to the more proximal position of the conditioning electrode; the distance between conditioning and test electrodes being 44-2 and 41 cm respectively). In both cases this facilitation reflex being expressed as a percentage of its reference value in the control situation) show the size of the reflex in the different situations. C and H, reference monosynaptic reflex (gastrocnemius medialis) in the control situation. D and I, heteronymous Ia facilitation of the reflex in the control situation. E and J, inhibition of the monosynaptic reflex by a volley in the sural nerve (1-2 x T, conditioning-test interval 8 ms). F and K, following the sural nerve volley the size of the reference monosynaptic reflex is restored to its control level by increasing the test stimulus intensity. G and L, following the sural nerve volley, heteronymous I a facilitation is unchanged, as compared to the control situation. Vertical bars 1 standard error of the mean. M, schematic diagram of the experimental paradigm for recording excitability of triceps surae I a terminals; Ia terminals are stimulated by a micro-electrode in the triceps surae motoneurone pool and the antidromic volley in the I a afferents is recorded from the nerve to gastrocnemius lateralis and soleus. Post-synaptic inhibition of motoneurones is evoked by a sural nerve volley. N and 0, the size of the antidromic I a volley evoked by microstimulation of I a terminals in the motoneurone pool and recorded from the nerve to gastrocnemius lateralis-soleus. The size is expressed as a percentage of its reference value in the control situation (N); the filled column (0) shows that there is no significant increase in excitability during post-synaptic inhibition (same stimulus parameters as in E-G and J-L)

12 740 H. HULTBORN AND OTHERS A lb...a B CD c 0 U :t a) cm 4- c CD I 113 Lo w o- o CD oc...ooei _ Lo to ~ co o). Ce n V) a, N 'CA a, x a, Q') a,- H c t i I.I Conditioning-test interval (ims) 150 F G ^ U cor-.oo_ LOCO. WO)O In6t6eni6y.o 6g tu (x MT) Intensity of conditioning stimulus (x MT) Fig. 3. Effect of femoral nerve stimulation on the soleus H reflex. A, schematic diagram showing the pathways of Ia excitation and Ib inhibition from quadriceps to soleus; bars and small filled circles indicate excitatory and inhibitory synapses respectively. The results obtained in two different subjects (B and D-E; C and F-a) are illustrated. The size of the test reflex, expressed as a percentage of its control value, is plotted against either the conditioning-test interval (B and C) or the conditioning stimulus intensity (D-G). B and C, conditioning stimulus intensity is constant (B, 0-85 x MT; C, 0-95 x MT). D-C, conditioning-test interval is constant (D, - 6-5; E, -5-5; F, -6; -4 ims). Each symbol represents the mean of twenty measurements. Vertical bars 1 standard error of the mean.

13 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN had an abrupt onset since it reached its peak 0 7 ms after it started. At its peak, the test reflex size had increased to 150% of its reference value. Later, the facilitation declined, sometimes being replaced, as illustrated in Fig. 3B, by an inhibition. Fig. 3D-G shows the variations in the test reflex when the conditioning stimulus intensity was varied. The facilitation appeared with a low threshold (0-65 x MT). When the conditioning-test interval was within the 0-5 ms following the onset of the facilitation (i.e and -6 ms in Fig. 3D and F respectively), increasing the conditioning stimulus intensity up to 1 x MT resulted in a continuous increase in the soleus H reflex facilitation. With longer conditioning-test intervals (i.e and -4 ms in Fig. 3E and G respectively), the reflex stopped increasing for conditioning stimulations beyond 0-8 x MT. It has already been shown that with such weak stimuli applied to the femoral nerve the only muscle afferents to be activated are group I afferents (Fournier, Meunier, Pierrot-Deseilligny & Shindo, 1986). A contribution of the cutaneous afferents to the variations of the test reflex was eliminated since a pure cutaneous stimulation applied to the anterior aspect of the thigh (and producing the same sensation as the femoral nerve stimulation) did not modify the test reflex size. Thus it appears very likely that the early facilitation and the following inhibition (Fig. 3B) are respectively Ia and Ib in origin. During its most part, I a facilitation is contaminated by Ib inhibition, whose threshold corresponds to the conditioning stimulus intensity (here 0'8 x MT, Fig. 3E and G) at which reflex facilitation stops increasing (Pierrot-Deseilligny, Morin, Bergego & Tankov, 1981). However, at the earliest conditioning-test intervals (first 0 5 ms) the Ia facilitation continued to increase up to 1 x MT (Fig. 3D and F), which indicates that it is not yet contaminated by any Ib effect (Pierrot-Deseilligny et al. 1981). The question is whether this early I a facilitation is indeed mediated through a monosynaptic pathway. A monosynaptic linkage between quadriceps Ia fibres and soleus motoneurones has been demonstrated both in the cat (Eccles et al. 1957) and in the baboon (Hongo, Lundberg, Phillips & Thompson, 1984). To ascertain that it was the same in man, a separate series of experiments was performed on single soleus motor units, and the latency from femoral nerve stimulation to the peak in the poststimulus time histogram (p.s.t.h.) was used to evaluate the central latency. These experiments clearly demonstrated that the I a projections from the femoral nerve onto soleus motoneurones are indeed monosynaptic (see Appendix). (III) Presynaptic inhibition of soles and quadriceps Ia fibres after tibialis anterior vibration In a previous report (Morin et al. 1984) it was shown that a weak (peak-to-peak: 0 3 mm) and short-lasting (three shocks at 200 Hz) vibration applied to the skin above the tibialis anterior tendon caused an inhibition of the soleus H reflex which lasted for ms. It was demonstrated that at conditioning-test intervals between ms this inhibition was purely presynaptic in origin: as sketched in Fig. 4A, the vibration-induced Ia volley from the tibialis anterior muscle causes a presynaptic inhibition of Ia fibres mediating the afferent volley eliciting the soleus H reflex. At earlier intervals post-synaptic inhibition from muscle group I fibres can contribute to this inhibition (cf. Tanaka, 1974; Crone, Hultborn & Jespersen, 1985), 741

14 742 H. HULTBORN AND OTHERS A vii 150- B 0) s D 0 oj 0,-c ( wo D ~~ ty$ , N CD a) wj cu1 Vw m 1 a - v0 l t C 300 E H 0 _ *- 20- { Conditioning-test interval (ims) Fig. 4. Time course of the variations of the soleus (B and filled symbols in D and E) and quadriceps (C) H reflexes when preceded by a short (three shocks, 200 Hz) vibration to the tibialis anterior tendon. A, presumed pathway of the vibration-induced depression of the reflexes; bars and small filled circles indicate excitatory and inhibitory synapses respectively. Curves with filled symbols in D and E were obtained in the two subjects in whom vibration elicited a late facilitation superimposed on the soleus H reflex depression. This facilitation was compared to the effect of a pure cutaneous stimulation applied to the skin beneath the vibrator (A in D). The facilitation had almost completely disappeared after anaesthesia of the skin (O in E). The size of the reflex, expressed as a percentage of its unconditioned value, is plotted against the conditioning-test interval. Each symbol represents the mean of twenty measurements. Vertical bars 1 standard error of the mean.

15 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN while a cutaneous facilitation is, in some subjects, added at an interval of ms after the vibration onset. In the present study the effect of vibration of the tibialis anterior tendon was tested both on quadriceps and soleus H reflexes (Fig. 4). The vibratory inhibition of the quadriceps H reflex was usually weak: to augment it, the peak-to-peak amplitude of the vibratory stimulus was increased to 0-8 mm. With such a vibration the inhibition of both the soleus (Fig. 4B) and the quadriceps (Fig. 4C) H reflex was usually preceded by an early facilitation (maximum at the 20 ms conditioning-test interval). It is likely that the early facilitation of the soleus H reflex is due to spread of the vibration to soleus muscle spindles causing homonymous Ia e.p.s.p.s in soleus motoneurones. This interpretation is supported by the finding that no early facilitation (but the later presynaptic inhibition) was seen with weaker vibration (Morin et al. 1984). The early facilitation of the quadriceps H reflex was frequently larger (Fig. 4C) than that of the soleus H reflex. Although mechanical spread of the vibration to quadriceps muscle spindles cannot be excluded it seems more likely that the present facilitation is caused by the activation of the pathway mediating the strong group I facilitation from tibialis anterior to quadriceps motoneurones (Pierrot- Deseilligny et al. 1981). Although this facilitation was thought to be lb in origin, it is possible that the tibialis anterior vibration here evokes a facilitation of quadriceps motoneurones by a I a facilitation of an excitatory pathway predominantly activated by Ib afferents (Jankowska, McCrea & Mackel, 1981 b). In any event, taking account of the distance between the sites for conditioning and test stimuli, of the duration of vibration and of the total duration of group I e.p.s.p.s it can be estimated that this facilitation would be finished 35 ms after the onset of vibration. In two subjects, there was a marked facilitation between 70 and 150 ms, with a peak at ms, of both soleus (Fig. 4D and E) and quadriceps (not illustrated) H reflexes. Fig. 4D shows that a pure cutaneous stimulation applied to the skin at the site of the vibratory stimulus produced the same H reflex facilitation (A) as that produced by vibration (A) in the same subject. Inversely, local anaesthesia of the skin and subcutaneous tissues beneath the hammer (1% xylocaine) strongly reduced the vibration-induced facilitation (open circles in Fig. 4E), confirming that it is cutaneous in origin. Subjects who presented such a facilitation were excluded from further experiments. Furthermore, it must be pointed out that in subjects who did not display this cutaneous facilitation, anaesthesia of the skin and subcutaneous tissues beneath the hammer did not change the time course of the effect produced by vibration, indicating that activation of cutaneous afferents did not modify the H reflex. In sum, in subjects without cutaneous facilitation the long-lasting depression of soleus and quadriceps H reflexes following tibialis anterior vibration is due to a decreased efficacy of the test afferent volley at presynaptic level. With the exception of the first 35 ms, this presynaptic mechanism is not contaminated by any significant post-synaptic effects. We have therefore used this vibratory inhibition of the H reflex as an example of presynaptic inhibition in the following evaluation of the method proposed to assess presynaptic inhibition of Ia fibres in man. 743

16 744 H. HULTBORN AND OTHERS A,. inhibition Presynaptic B a / quadriceps / ii Vilbraio Test stimulus La soleus 2 mvl 10 ms E F,o 150 rt, 0 U100 6@ 0~~~~~~~~~~~~~0 acn 50 CDO G H / J K Fig. 5. Variations in the heteronymous I a facilitation from quadriceps to soleus when the soleus H reflex is preceded (40 ms) by a vibration to the tibialis anterior tendon. A, schematic diagram of the experimental paradigm; the soleus H reflex is facilitated by a heteronymous Ia volley from quadriceps and the vibration-induced Ia discharge from tibialis anterior caused presynaptic inhibition of both homonymous and heteronymous I a fibres to soleus motoneurones; bars and small filled circles indicate excitatory and inhibitory synapses respectively. Sample records (B-F) of the reflex responses and averages of forty trials (G-K, the size of the test reflex being expressed as a percentage of its reference value in the control situation) show the size of the reflex in the different situations. B and G, reference H reflex in the control situation. C and H, heteronymous Ia facilitation of the reflex in the control situation. D and I, inhibition of the reflex by vibration. E and J, after vibration the size of the reference H reflex is restored to its control level by increasing the test stimulus intensity. F and K, after vibration, heteronymous Ia facilitation was reduced, as compared to the control situation (this reduction is measured by the interrupted open column in K). Vertical bars 1 standard error of the mean. (IV) Changes in the amount of heteronymous I a facilitation from quadriceps to soleus after tibialis anterior vibration In the present experiments (Fig. 5A) the soleus H reflex was conditioned by a femoral nerve stimulation the intensity of which was 075 x MT. The conditioningtest interval was so set that the heteronymous I a facilitation was assessed 0 3 ms after its onset. The amount of heteronymous I a facilitation was compared in the control situation (without vibration) and when preceded by vibration.

17 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN 745 Sample records of the reflex responses (Fig. 5B-F) and averages of forty trials (Fig. 5 G-K) show the size of the reference and of the facilitated reflexes in the control situation and when preceded (40 ms) by tibialis anterior vibration. In the control situation the reference H reflex was elicited by a 6-4 ma stimulus (Fig. 5 B and G) and femoral nerve stimulation increased the test reflex amplitude to of its reference value (Fig. 5C and H). As seen above, vibration caused presynaptic inhibition of Ia 150 A a c I X P I..I Vibration-test interval (ms) Fig. 6. Time course of the variations in the heteronymous I a facilitation from quadriceps to soleus after vibration. The results obtained in two different subjects (A and B) are compared to the vibratory inhibition of either the soleus (A) or the quadriceps (B) H reflex. Heteronymous Ia facilitation (@) and H reflex size (A, 0) are expressed as a percentage of their value in the control situation and plotted against the vibration-test stimulus interval Each point represents the mean of twenty measurements. Vertical bars 1 standard error of the mean. fibres mediating the afferent volley responsible for the H reflex, and thus markedly reduced the soleus H reflex (Fig. 5D and I). The test stimulus intensity was therefore increased to 7-2 ma to 'compensate' for the vibratory inhibition: then a soleus H reflex of the same size as in the control situation was obtained after vibration (Fig. 5E and J). Fig. 5F and K show that the heteronymous Ia facilitation was markedly reduced (129%) after vibration, as compared to the control situation. This reduction (interrupted open column in Fig. 5K) was highly significant (P < 0 01). Similar results were obtained in all seven subjects. The time course of this reduction in the heteronymous I a facilitation from quadriceps to soleus is illustrated in Fig. 6. As seen above, for each interval, vibration-

18 746 H. HULTBORN AND OTHERS induced inhibition of soleus I a fibres was 'compensated' by increasing the test stimulus intensity. Thus the amount of heteronymous I a facilitation with vibration, expressed as a percentage of the facilitation in the control situation without vibration, is plotted against the time interval between the vibratory train and the test stimulus. Filled circles in Fig. 6A show that there was a significant reduction in heteronymous Ia facilitation at ms intervals. This time course was compared to that of the vibratory inhibition of the soleus H reflex (A). The lower efficiency of the excitatory I a volley from quadriceps to soleus (0) is in close temporal relationship with the reduction of the soleus H reflex (A). In Fig. 6B the time course of the reduction in the heteronymous I a facilitation (0) obtained in another subject is compared to that of the vibratory inhibition of the quadriceps H reflex (0): here also there is a close relationship between the two time courses. This is in agreement with the hypothesis that the reduction of heteronymous Ia facilitation reflects presynaptic inhibition exerted on Ia fibres from quadriceps to soleus by tibialis anterior vibration. (V) Maintained heteronymous Ia facilitation during post-synaptic inhibition For the validity of the method as a specific test for presynaptic inhibition it was necessary to demonstrate that the amount of heteronymous Ia facilitation was not modified by a post-synaptic inhibition of soleus motoneurones. Stimulation of group I fibres from the gastrocnemius medialis nerve was used to produce I b inhibition of soleus motoneurones, since this inhibition is not contaminated by any I a excitation: Ia excitation from gastrocnemius medialis to soleus motoneurones, which is strong in the cat (Eccles et al. 1957), becomes weak in the baboon (Hongo et al. 1984) and has completely disappeared in man (Pierrot-Deseilligny et al. 1979). An example of such an inhibition by gastrocnemius medialis nerve stimulation (0 9 x MT) is shown in Fig. 7B: the inhibition of the soleus H reflex appeared at the 2 ms interval, was maximum at 5 ms and ended at 10 ms. This inhibition can be regarded as postsynaptic: the brief latency and short duration almost excludes any contribution of presynaptic inhibition. The soleus H reflex was facilitated by femoral nerve stimulation (0-75 x MT, conditioning-test interval so adjusted that the facilitation was measured 0 3 ms after its onset). The amount of heteronymous I a facilitation was compared in the control situation and when the test reflex was preceded (5 ms; arrow in Fig. 7B) by the gastrocnemius medialis nerve stimulation (single pulse, 0-9 x MT). In the control situation the reference H reflex (set as 100%, Fig. 7C) was elicited by a 4-5 ma stimulus intensity. Femoral nerve stimulation increased the test reflex amplitude up to 138% (Fig. 7D). lb inhibition evoked by gastrocnemius medialis nerve stimulation reduced the amplitude of the reflex to 67% of its control value (Fig. 7E). The amplitude of the reflex was restored by increasing the test stimulus intensity up to 5*1 ma. Comparison of Fig. 7D and G shows that there is no reduction in the amount of heteronymous Ia facilitation when soleus motoneurones undergo postsynaptic inhibition. Similar results were obtained in all four subjects so explored.

19 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN 747 A yr--- B 03 1-* laquadriceps/ t Se I,. b 1m CL 0 5 Ila soleus / Conditioning-test interval (ins) 'a 100- C D. o 0 0 S CD CD~~~~~~~~~~~~O CL 0~~~~~~~~~~~@ C D E FG Fig. 7. Maintained Ia facilitation during post-synaptic inhibition. A, schematic diagram of the experimental paradigm; the soleus H reflex is facilitated by heteronymous Ia volley from quadriceps and soleus motoneurones receive I b inhibition from gastrocnemius medialis (g.m.); bars and small filled circles indicate excitatory and inhibitory synapses respectively. B, the size of the soleus H reflex (expressed as a percentage of its unconditioned value) is plotted against the time interval between gastrocnemius medialis nerve and test stimuli; the arrow indicates the conditioning-test interval used in E-a. c-c, averages (forty trials) of the soleus H reflex, expressed as a percentage of its reference value in the control situation, showing the size of the reflex in the different situations. Vertical bars 1 standard error of the mean. C, reference H reflex in the control situation. D, heteronymous Ia facilitation of the reflex in the control situation. E, inhibition of the reflex after gastrocnemius medialis nerve stimulation. F, after gastrocnemius medialis nerve stimulation, the size of the test reflex is restored to its control level by increasing the test stimulus intensity. C, heteronymous Ia facilitation of the reflex, is the same after gastrocnemius medialis nerve stimulation as in the control situation. DISCUSSION Changes in the amount of heteronymous I a facilitation of a test reflex can be used to assess presynaptic inhibition of Ia fibres The depression of the monosynaptic I a e.p.s.p. evoked in motoneurones by a given stimulation of I a fibres (and occurring without any observable change in motoneurone membrane potential and conductance) has been the first and most direct evidence of presynaptic inhibition (Frank & Fuortes, 1957), and remains a standard test to ascertain presynaptic inhibition of Ia fibres in animal experiments. Using methods based on an estimation of the size of the monosynaptic I a e.p.s.p., evidence for presynaptic inhibition of I a fibres in man has already been presented. The size of

20 748 H. HULTBORN AND OTHERS the homonymous Ia e.p.s.p. was shown to be efficiently reduced under vibration; in these experiments the homonymous Ia e.p.s.p. was either assessed in individual motor units by the peak of increased firing probability induced at monosynaptic latency by homonymous nerve stimulation (the p.s.t.h. method; Ashby & Verrier, 1980) or estimated from the amount of homonymous Ia facilitation of the H reflex (Morin et al. 1984). However, these methods cannot be used to study the changes in presynaptic inhibition of I a fibres during movement. This is obvious when using the p.s.t.h. method which requires recording of a voluntarily activated motor unit, thus precluding a comparison with the level of presynaptic inhibition at rest. The interpretation of the H reflex facilitation by a preceding volley applied to the homonymous nerve is complicated since the refractoriness of Ia fibres makes it necessary to use conditioning-test intervals of 3-5 ms. Both the autogenetic Ib inhibition, to which I a fibres contribute (Jankowska, McCrea & Mackel, 1981 a) and polysynaptic Ia excitation (Fournier et al. 1986) may then contaminate the monosynaptic Ia excitation. Since during voluntary contraction there are changes in transmission in pathways of autogenetic group I inhibition (Fournier, Katz & Pierrot-Deseilligny, 1983) and of polysynaptic Ia excitation (Hultborn, Meunier, Pierrot-Deseilligny & Shindo, 1986), changes in these non-monosynaptic effects would interfere with the assessment of the monosynaptic Ia e.p.s.p. Thus a prerequisite for a suitable method to study changes in presynaptic inhibition during movement is a conditioning-test interval short enough to permit only monosynaptic Ia effects to manifest themselves. As seen above, this can only be obtained with a heteronymous Ia conditioning volley, and heteronymous Ia facilitation from the quadriceps muscle to soleus motoneurones was used here. Experimental evidence is given (see Appendix) that in man, as in the cat (Eccles et al. 1957) and the baboon (Hongo et al. 1984), this heteronymous Ia facilitation is mediated through a monosynaptic pathway. As could be expected, it was demonstrated that during its first 0'5 ms this monosynaptic I a excitation is not contaminated by any polysynaptic effect. Finally it was shown that the heteronymous Ia facilitation is considerably reduced following a tibialis anterior vibration known to produce presynaptic inhibition of Ia fibres (Ashby & Verrier, 1980). This result was fully confirmed in parallel animal experiments where it was possible to ascertain presynaptic inhibition of Ia fibres by direct tests: presynaptic inhibition was shown significantly to reduce the efficiency of the conditioning Ia volley in facilitating the test reflex. Hence, there is no doubt that presynaptic inhibition of Ia terminals is indeed reflected by a reduced heteronymous Ia facilitation of a test reflex. In order to use the method as a specific test for presynaptic inhibition it was essential to ensure that the amount of heteronymous I a facilitation (i.e. the underlying conditioning Ia e.p.s.p.) remained unchanged when the motoneurones pool in which the test reflex is elicited received post-synaptic inhibition. This could not be taken for granted, given the increase in motoneurone membrane conductance underlying the i.p.s.p. which would be expected to reduce other synaptic inputs by ' shunting'. Parallel animal experiments with intracellular recording of motoneurones were therefore performed to evaluate the extent to which monosynaptic I a e.p.s.p.s were modified by conditioning post-synaptic inhibition. It was found that, when post-synaptic inhibition is strong (i.e. reducing the amplitude of the test

21 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN reflex to less than 20% of its control value), both the peak and the falling phase of the Ia e.p.s.p. are reduced. However, as long as the post-synaptic inhibition is relatively moderate (i.e. reducing the test reflex to 90-25% of its control value) it was found that, even though the falling phase of the e.p.s.p. is often reduced, its peak remains virtually unchanged. This apparent discrepancy is due to biophysical circumstances: the very first voltage deflection caused by a synaptic current flow is mainly due to the capacitive charge which is not dependent on membrane conductance. Accordingly, in motoneurones, which have a time constant of about 5 ms, the voltage deflection within the 0 5 ms following the onset of a current flow (i.e. the rising phase of the monosynaptic Ia e.p.s.p. up to the peak) is very much dominated by the capacitive charge and thus quite resistant to changes in conductance. By contrast, after this brief initial period, the voltage deflection is increasingly due to current flow across the membrane resistance and thus depends on changes in conductance. In our experiments with reflexes, post-synaptic inhibition was moderate (i.e. reduced the test reflex at most to 50% of its control value) and the timing was so adjusted that the conditioning and test volleys arrived simultaneously at the spinal level: the test e.p.s.p. was therefore added on the top of the conditioning e.p.s.p., which explains that the heteronymous Ia facilitation was not reduced despite the increase in conductance underlying the i.p.s.p. To sum up, post-synaptic inhibition of the motoneurone pool in which the test reflex is elicited does not modify the amount of conditioning monosynaptic I a facilitation of the reflex provided that (1) the conditioning and test volleys arrive simultaneously at motoneuronal level and (2) the inhibition is not too strong (i.e. reduces the test reflex by no more than 50% of its control value). Within these limits the proposed method provides specific evidence for presynaptic inhibition of the heteronymous I a fibres from quadriceps to soleus. Vibratory inhibition of the H reflex can be used to assess the excitability of interneurones mediating presynaptic inhibition of I a fibres The amount of depression of a test H reflex following the short-lasting vibration applied to the tibialis anterior tendon can also be used, as an independent method, to study the transmission in the pathway subserving presynaptic inhibition. Vibration-induced presynaptic inhibition was first discussed in relation to the soleus H reflex depression evoked by a tonic vibration applied to the Achilles tendon. It was postulated that this depression is presynaptic in origin since it is seen despite a motor discharge (the tonic vibration reflex) reflecting an increased excitability at motoneuronal level (Hagbarth & Eklund, 1966; De Gail, Lance & Neilson, 1966; Delwaide, 1973). When prolonged vibration is applied, however, presynaptic inhibition of those I a fibres that mediate the H reflex is certainly not the only mechanism which contributes to the H reflex depression. Other contributing mechanisms might include: refractoriness of Ia fibres (Hagbarth, 1973), 'transmitter depletion' at Ia terminals (Curtis & Eccles, 1960), post-synaptic reciprocal Ia inhibition (Tanaka, 1974; Crone et al. 1985) due to spread of vibration and activation of muscle spindles in antagonistic muscles (Ashby, Verrier & Carleton, 1980) and post-synaptic 'nonreciprocal' group I inhibition, since I a fibres converge onto the so-called I b inhibitory interneurones (Jankowska et al a). 749

22 750 H. HULTBORN AND OTHERS With the short-lasting vibration used here the post-synaptic effects have vanished 35 ms after the onset of the vibration. Although the vibration was applied to the tibialis anterior tendon, 'transmitter depletion' at the la terminals mediating the test reflex (Curtis & Eccles, 1960) might contribute to the reflex depression, if there was a mechanical spread of the vibration with activation of muscle spindles in triceps surae or quadriceps muscles. Such 'transmitter depletion' was supposed to be the cause of the strong and long-lasting depression of H reflexes following conditioning stimuli just below the threshold for the H reflex or the tendon jerk (Taborikova' & Sax, 1969; Katz, Morin, Pierrot-Deseilligny & Hibino, 1977). The conditioning stimulus used here was indeed much weaker (0-8 mm vibration applied transversely to the tibialis anterior tendon). As a result, the vibratory inhibition of the reflex was always over before 500 ms (Fig. 4B and C) after the onset of vibration. A significant contribution of 'transmitter depletion' in our case can therefore be eliminated, since the depression due to this mechanism has been shown to last for several seconds both in the cat (Eccles & Rall, 1951) and in man (Taborikova' & Sax, 1969; Katz et al. 1977). It was observed that, in some subjects, the cutaneous stimulation by the vibration caused a large facilitation of the reflex which is superimposed on the dominating depression within the interval of ms. In the other subjects anaesthesia of the skin did not modify the vibration-induced changes of the H reflex. Thus, when using an interval of ms between the vibratory train and the test stimulus, the depression of the H reflex selectively reflects the amount of presynaptic inhibition of the I a fibres mediating the test reflex. The two methods can therefore be used in parallel: (1) measurement of the amount of heteronymous monosynaptic I a facilitation which assesses the amount of ongoing presynaptic inhibition exerted onto heteronymous I a fibres from quadriceps to soleus; and (2) measurement of the vibratory inhibition of the H reflex which tests the excitability of the interneurones fed by tibialis anterior I a afferents and mediating presynaptic inhibition of homonymous extensor I a fibres. As shown in the companion paper (Hultborn et al. 1987), congruent results during voluntary contraction were obtained with these two complementary and independent methods. APPENDIX Experimental evidence for a monosynaptic connection from quadriceps I a afferents to soleus motoneurones in man The method of post-stimulus time histograms (p.s.t.h.s) of the firing probability of individual motor units in quadriceps and soleus following stimulation of afferent fibres from these muscles was used to provide experimental evidence for a monosynaptic connection from quadriceps Ia afferents to soleus motoneurones. Ia fibres from vastus lateralis (quadriceps) were stimulated by unipolar electrodes, the active electrode being placed at two different sites: (1) the upper thigh at the level of the femoral arch, and (2) the mid-thigh, 19 cm below the femoral arch and 2 cm lateral to the mid line. I a fibres from soleus were stimulated by a bipolar electrode placed at two different sites: (1) the popliteal fossa and (2) the upper and posterior aspect of the thigh, 31 cm above the electrode in the popliteal fossa. Stimulation was always set to be subthreshold for the compound H reflex. The latency of the earliest increase

23 PRESYNAPTIC INHIBITION OF la FIBRES IN MAN in firing probability after these different stimulations was assessed. From these assessments it was possible (see below) to calculate the conduction velocity in the fastest I a fibres from quadriceps and soleus and, consequently, to compare the central latency of the earliest response elicited in soleus motor units by stimulation of I a fibres from soleus and quadriceps. The electromyogram (e.m.g.) from soleus and vastus lateralis was recorded with surface electrodes while the subjects performed a very weak and steady contraction of the corresponding muscle. After some training, it was always possible to isolate an individual motor unit either because it was the only one active or because it was significantly larger than the others. The e.m.g. potentials of single motor units were converted into standard pulses by a discriminator with variable trigger levels (correction was made for the trigger delay). The motor unit potential and the trigger pulse were continuously monitored to detect false triggers due to other motor units and to ensure that motor unit shape and size and trigger position remained constant within and between sequences. The stimulation of the nerves was triggered at a fixed delay (D) after the preceding motor unit discharge (Fournier et al. 1986) and the latency of the following motor unit potential was computed. The p.s.t.h.s of the voluntarily activated motor unit discharge were constructed for the period ms following the stimulation using 02 ms bins. When the nerve stimulation is delivered at a fixed interval after the previous discharge, it is possible to choose a delay when the probability for a new discharge is high (with the relatively regular firing frequency during voluntary contraction). This implies that the number of stimulations necessary to reveal obvious peaks in the p.s.t.h. can be much lower than if nerve stimulations are given without reference to the previous motor unit discharge. This is important if the same motor unit has to be subjected to a large number of different tests. Stimulation at a fixed interval after the previous discharge also implies that the probability of discharge in the p.s.t.h. does not depend only on the post-synaptic potentials evoked by the stimulation but also on the motoneurone membrane trajectory during the interspike interval. To take account of the latter a histogram of firing probability was also constructed in a control situation without stimulation and subtracted from the histogram with nerve stimulation. Situations with and without stimulation were randomly alternated (same number of triggers) within a sequence, the equivalent bins in both histograms being obtained at the same moment of the motoneurone membrane trajectory. A x2 test was used to determine whether in each bin the firing probability after stimulation differed from that obtained in the control situation. An increase in firing probability was accepted when the firing probability after stimulation was significantly larger (P < 001) in three or more consecutive bins. Latencies of the increases in firing probability of soleus motoneurones following stimulation of the posterior tibial nerve and of the femoral nerve. Fig. 8 shows the p.s.t.h. obtained from a soleus motor unit, for which the 'spontaneous' firing interval was about 140 ms. The delay D between the previous spike and the trigger was 90 ms. Fig. 8B, obtained without stimulation, reflects the motoneurone membrane trajectory during the interspike interval. The p.s.t.h.s obtained after posterior tibial nerve and femoral nerve stimulation are shown in Fig. 8C and D respectively. In 751

24 752 H. HULTBORN AND OTHERS order to isolate the effects produced by the nerve stimuli the histogram obtained without stimulation (control situation) was subtracted from that obtained after posterior tibial nerve (Fig. 8C) and femoral nerve (Fig. 8D) stimulation; the results are shown in Fig. 8E (posterior tibial nerve) and 8F (femoral nerve). A a quadriceps soleus CD a) am. C- +-o 4] 2] o I. -i.a * * MI Id,. AW*- 14 libis111. III-, B I.. I.., II O hi -4,,,,. Fs E c *-E E0Is.LI.,l,,,i F 4 Ii 1Fiilp1 m-il TT 79-,., T -Z:,1,1 L I I,..1 L L Latency (Ms) L½I II I 1II J Fig. 8. Time histograms of the discharge of a voluntarily activated soleus motor unit in control conditions (B) and in response to posterior tibial (C) and femoral (D) nerve stimulation. The experimental paradigm is sketched in A. The histogram obtained without stimulation was subtracted from that obtained after posterior tibial nerve and femoral nerve stimulation and the results of these subtractions are shown in E and F respectively. The spontaneous firing interval was on average 140 ms, and the delay between the previous spike and the stimulation 90 ins. The number of counts, expressed as a percentage of number of triggers, is plotted against the latency. Number of triggers used: 500 for each alternative. The intensity of the posterior tibial nerve stimulation was adjusted to be just below the threshold of the compound H reflex, i.e. at 0 9 x MT in this experiment. This resulted in an increase in firing probability of the motor unit beginning 35 ms after the stimulation and ending at 37-2 ms (Fig. 8E). In order to match the amplitude of the peak of increased firing probability obtained after homonymous (posterior tibial nerve) and heteronymous (femoral nerve) stimulation, the intensity

25 PRESYNAPTIC INHIBITION OF Ia FIBRES IN MAN of the femoral nerve stimulation was adjusted to 1V5 x MT. The resulting increase in firing probability began 29-6 ms after femoral nerve stimulation and lasted for 1i2 ms (Fig. 8F). In both cases the first bin of the peak of facilitation exhibited a highly significant (P < 0001) increase in firing probability and could be used to locate the latency of the onset of facilitation induced by stimulation. Since the results were obtained with the same motor unit, the efferent conduction time (from the motoneurone in Si spinal level to the muscle) was the same in both situations (posterior tibial nerve and femoral nerve stimulations). Hence differences in the afferent conduction time and/or the central latency must account for the 5-4 ( ) ms difference between the latencies of posterior tibial nerve- and femoral nerve-induced facilitation. The afferent conduction time for the Ia volleys elicited by posterior tibial nerve and femoral nerve electrodes was estimated by calculating the conduction velocity of these Ia fibres and measuring the distances from these electrodes to the soleus motoneurone pool. Thus p.s.t.h.s in the same soleus motor unit were constructed after stimulation of soleus Ia fibres at two sites separated by 31 cm. The difference between the latencies of the onset of facilitation elicited by the electrodes placed at these sites was 4-8 ms, which gives a conduction velocity of 64 m/s for the fastest I a fibres. In the experiment, the results of which are illustrated in Fig. 8, the distance from the posterior tibial nerve electrodes to the SI spinal level (i.e. LI vertebra), was estimated at 66 cm, which gives an afferent conduction time of 10-3 ms (066/64). Similarly, p.s.t.h.s in a vastus lateralis motor unit were constructed after stimulation of vastus lateralis I a fibres at two sites separated by 19 cm. The difference in latencies of the increase in firing probability in these two cases was 3-2 ms, which gives a conduction velocity of 59 m/s for the fastest I a fibres from vastus lateralis. The distance from the femoral nerve electrode to the SI spinal level was estimated at 27 cm in the experiment illustrated in Fig. 8. The distance to the soleus motoneurone pool was, however, somewhat longer, since the I a fibres from quadriceps do not enter the spinal cord in Si but in L4 and then project caudally within the spinal cord to reach S1. This additional distance was measured on two corpses as being about 2 cm. Thus the total distance to the soleus motoneurone pool can be estimated at 29 cm, which gives an afferent conduction time of 4-9 ms (0O29/59). The conduction distances between the posterior tibial nerve stimulation site and the SI spinal segment, and the femoral nerve stimulation site and the SI spinal segment cannot be estimated without a certain error, especially for the 'common' abdominal part. It is essential, however, to note that the difference in distance between femoral nerve and posterior tibial nerve sites (39 cm) was estimated quite accurately. Thus the afferent conduction times from the posterior tibial nerve and femoral nerve electrodes to the soleus motoneurone pool differ by 5-4 ms (10i3-4-9). This corresponds exactly to the difference between the latencies of the posterior tibial nerve- and femoral nerve-induced increases in firing probability. Similar results were found with the ten motor units explored in two subjects: the differences in the latencies of increased firing probability in the p.s.t.h.s following femoral nerve and posterior tibial nerve stimulation did not exceed the differences in measured afferent conduction time by more than 02 ms. As seen above, this difference includes possible differences both in afferent conduction time and in central latency. Since it is entirely 753

26 754 H. HULTBORN AND OTHERS explained by the differences in afferent conduction time, this indicates that the earliest homonymous and heteronymous Ia facilitatory effects have the same segmental latency. Since it has been demonstrated that the earliest part of the soleus H reflex (i.e. the homonymous I a excitatory effect) occurs at monosynaptic latency (Magladery, Porter, Park & Teasdall, 1951), this indicates that in man, as in the cat (Eccles et al. 1957) and the baboon (Hongo et al. 1984), the earliest Ia excitatory heteronymous I a effects from quadriceps to soleus are mediated through a monosynaptic pathway. The argument that conceivable oligosynaptic pathways might affect motoneurone discharge in the H reflex (Burke, Gandevia & McKeon, 1984) is admissible, but does not apply to the present situation in which the earliest facilitation in a single motor unit was investigated. The authors wish to express their gratitude to Drs Lena Jami and Kristina Malmgren for reading and commenting upon the manuscript. Our thanks are also due to Annie Rigaudie and Michele Dodo for their unfailing assistance during the course of these experiments, and David MacGregor for scrutinizing the English. This work was supported by University Paris VI, INSERM ( ), ETP (TW 83/146), the Danish Multiple Sclerosis Society, the Warwara Larsen Foundation and le Fond d'etudes du Corps Medical des Hopitaux de Paris. REFERENCES ASHBY, P. & VERRIER, M. (1980). Human motoneuron responses to group I volleys blocked presynaptically by vibration. Brain Research 183, ASHBY, P., VERRIER, M. & CARLETON, S. (1980). Vibratory inhibition of the monosynaptic reflex. In Spinal and Supraspinal Mechanisms of Voluntary Motor Control and Locomotion, Progress in Clinical Neurophysiology, vol. viii, ed. DEsMEDT, J. E., pp Basel: Karger. BALDISSERA, F., HULTBORN, H. & ILLERT, M. (1981). Integration in spinal neuronal systems. In Handbook of Physiology, section I, The Nervous System, vol. II, Motor Control, ed. BROOKS, V. B., pp Bethesda, MD, U.S.A.: American Physiological Society. BERGMANS, J., DELWAIDE, P. J. & GADEA-CIRIA, M. (1978). Short-latency effects of low-threshold muscular afferent fibres on different motoneuronal pools of the lower limb in man. Experimental Neurology 60, BURKE, D., GANDEVIA, S. C. & McKEON, B. (1984). Monosynaptic and oligosynaptic contributions to human ankle jerk and H-reflex. Journal of Neurophysiology 52, CRONE, C., HULTBORN, H. & JESPERSEN, B. (1985). Reciprocal Ia inhibition from the peroneal nerve to soleus motoneurones with special reference to the size of the test reflex. Experimental Brain Research 59, CURTIS, D. R. & EcCLEs, J. C. (1960). Synaptic action during and after repetitive stimulation. Journal of Physiology 150, DE GAIL, P., LANCE, J. W. & NEILSON, P. D. (1966). Differential effects on tonic and phasic reflex mechanisms produced by vibration of muscles in man. Journal of Neurology, Neurosurgery and Psychiatry 29, DELWAIDE, P. J. (1973). Human monosynaptic reflexes and presynaptic inhibition. An interpretation of spastic hyperreflexia. In New Developments in Electromyography and Clinical Neurophysiology, vol. III, ed. DESMEDT, J. E., pp Basel: Karger. ECCLES, J. C. (1964). The Physiology of Synapses, p Berlin: Springer-Verlag. EcCLEs, J. C., ECCLES, R. M. & LUNDBERG, A. (1957). The convergence of monosynaptic excitatory afferents on to many different species of alpha-motoneurones. Journal of Physiology 137, ECCLES, J. C. & RALL, W. (1951). Effects induced in a monosynaptic reflex path by its activation. Journal of Neurophysiology 14, ECCLES, J. C., SCHMIDT, R. F. & WILLIS, W. D. (1962). Presynaptic inhibition of the spinal monosynaptic reflex pathway. Journal of Physiology 161,

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