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After-potentials and control of repetitive firing in human motoneurones
Electroencephalography and clinical Neurophysiology, 85 (1992)345-353
345
© 1992 Elsevier Scientific Publishers Ireland, Ltd. 0924-980X/92/$05.00
E L M O C O 91627
After-potentials and control of repetitive firing in h u m a n motoneurones L.P. Kudina and N.L. Alexeeva Institute for Problems of Information Transmission, Russian Academy of Sciences, Moscow (Russia) (Accepted for publication: 6 March 1992)
Summary Characteristics of m o t o n e u r o n e after-potentials in m a n were derived from the recovery curve of m o t o n e u r o n e excitability after a single discharge evoked by threshold stimulation of la afferents or by gentle voluntary muscle contraction. The m o t o n e u r o n e excitability was estimated by the firing index of a single motor unit whose potentials were recorded by needle electrodes. The soleus (a slow muscle) and the flexor carpi ulnaris (a fast muscle) were investigated. The duration of m o t o n e u r o n e after-hyperpolarization of the soleus evaluated by this method ranged between 145 and 255 msec; for the flexor carpi ulnaris it was 55-150 msec. In some motoneurones of the fast muscle, an early short-lasting recovery of excitability (within 5 - 2 0 msec after a discharge) was revealed. It was accounted for by delayed depolarization of the motoneurone. The relationship between after-potentials and the characteristics of repetitive firing of motoneurones activated by weak voluntary muscle contraction was analysed. It was observed that the motoneurones with early excitabiity recovery were capable of firing double discharges with a 5-15 msec interspike interval. It was found also that the minimal firing rate of motoneurones (up to 3.1-5.2 i m p / s e c in the soleus and 3.8-9.0 i m p / s e c in the flexor carpi ulnaris) was not correlated with the after-hyperpolarization duration. This differs from the results obtained for cat's motoneurones u n d e r intracellular stimulation. The findings suggest that after-hyperpolarization is not the only leading mechanism controlling the low firing rate of motoneurones u n d e r conditions of their natural activity in man.
Key words: Motor control; Motoneurone repetitive firing; After-potentials
In spinal motoneurones, a somadendritic spike is followed by a long-lasting hyperpolarizing after-potential, the after-hyperpolarization (AHP), caused by an increase in the membrane conductance to potassium ions, which gradually declines after a spike (Coombs et al. 1955). As a result, the motoneurone excitability is low just after a spike and thereafter gradually recovers. The duration of the A H P widely varies among motoneurones and is correlated with their sizes and contraction time of the innervated muscle fibres (Eccles et al. 1958; Burke 1967; Zwaagstra and Kernell 1980). According to Eccles et al. (1958) the A H P duration was more than 130 msec for motoneurones of slow muscles and 50-110 msec for those of fast ones. The A H P is often preceded by a short-lasting depolarizing after-potential, the delayed depolarization (DD), a phase of slower repolarization between the rapid falling phase of the spike and the fast descending limb of the AHP. As supposed, DD is of dendritic origin; in some motoneurones it smoothly decays, in others it has the shape of a " h u m p " (Granit et al. 1963; Kernell 1964; Calvin 1980). D D approaches the mem-
Correspondence to: L.P. Kudina, Institute for Problems of Information Transmission, Russian A c a d e m y of Sciences, Yermolovoy 19, GSP-4, 101447 Moscow (Russia).
brane potential to the threshold level, thereby causing a transitory increase in the motoneurone excitability. After-potentials, especially AHP, are considered an important factor controlling repetitive firing of motoneurones (Kernel 1965; Calvin 1974; Gustafsson 1984). This was deduced from the correlations between motoneurone firing characteristics and their afterpotential properties. DD, especially in a "hump-like" form, was found to be associated with a particular pattern of motoneurone firing, i.e., doublets with a very short intradoublet interval - - up to 11-13 msec (Calvin and Schwindt 1972). Under conditions of intracellular motoneurone depolarization Kernell (1965) showed that the total A H P duration measured up to the peak of late after-depolarization was strongly correlated with the minimal firing rate within the "primary range" which was between 8 and 25 i m p / s e c (usually 15). Since intracellular depolarization and natural synaptic activation are considered interchangeable (Granit 1970), the value of the minimal firing rate of synaptically activated motoneurones is not expected to fall below the mentioned values. However, under conditions of voluntary muscle contraction in man significantly lower rates of motoneurone repetitive firing (from 3-5 to 10-12 i m p / s e c ) were observed (Person and Kudina 1971; Ashby and Zilm 1982; Kudina 1988;
346 Miles et al. 1989b). The mechanisms underlying the control of such low firing rates are difficult to reveal because of the absence of data on after-potential duration in human motoneurones. The aim of the present study was to analyse afterpotentials of human motoneurones in slow and fast muscles with regard to some characteristics of their repetitive firing. Some of these results were published in the form of an abstract (Kudina et al. 1989).
.~lethods Investigation was carried out on 8 healthy subjects aged 22-48 years who volunteered to participate in the study. Each subject was tested in several experiments. The subjects were comfortably seated in arm-chairs and special attention was paid to complete relaxation of a subject during the whole experiment. Motoneurones of the soleus and flexor carpi ulnaris (known to be slow and fast twitch muscles respectively, see Eccles et al. 1958) were examined. A total of 49 motoneurones (27 of soleus and 22 of flexor carpi ulnaris) were tested. In order to analyse the afterpotentials of motoneurones the method of threshold monosynaptic testing (evoking conditioning and test H reflexes) of single motor units (MUs) was used, which made it possible to evaluate the recovery time of motoneurone excitability after a single discharge. To evoke the H reflex of the soleus MUs the tibialis posterior nerve was stimulated by means of a bipolar surface electrode placed in the popliteal fossa. The H reflex of the flexor carpi ulnaris MUs was elicited by stimulating the ulnaris nerve at the elbow. The stimulus strength was threshold for evoking the H reflex, the stimulus duration was 0.5-1.0 msec. The summated H reflex was recorded with surface electrodes placed over the muscles tested. The M U action potentials were picked up by bipolar needle electrodes. The E M G signals were amplified (bandwidth of 20 Hz to 10 kHz) and photographe d for subsequent analysis. The responses of single M U s were evaluated by a firing index (FI), i.e., the relation Of the number of M U responses with H reflex latenci" to the number of applied stimuli (in percentage). In ~ill experiments the stimulation parameters were carefully chosen for each M U tested so as to elicit its threshold response. The threshold responses of MUs were considered to be those with an FI not lower ~than 50%, because the lower FI is known to be very unstable. The FI was calculated from 10 to 2 0 trials. For different MUs the FI ranged from 50 to i 0 0 % ; for a n y M U it could be constant or varied with t h e same limits throughout the experiment.
L.P. KUD1NA, N.L. ALEXEEVA During paired testing the strengths of conditioning and test stimuli were identical and equal to the threshold stimulus strength used at single testing. Test stimuli were applied with a delay gradually increasing from 5 to 300 msec for most of the MUs and up to 1 sec for some of them. The interstimulus step was 3 - 5 and 10-20 msec at short and long delays, respectively. At each step the FI of an MU was calculated in response to conditioning and test stimuli from 10 to 20 trials. The FI to a conditioning stimulus was equal to the FI at single testing and used as a measure of the background excitability of a motoneurone. The recovery of the motoneurone excitability after a single action potential was estimated by the FI of an M U in response to a test stimulus. Gradually increasing the interstimulus delay we were able to find the minimal interstimulus interval at which a motoneurone discharged in response to a test stimulus with the same FI as to a conditioning stimulus. We called it the time of complete recovery of motoneurone excitability and used it as a measure of the A H P duration. The D D duration was evaluated by the time of early recovery of motoneurone excitability at short delays. In some of the experiments, the excitability recovery of motoneurones in both muscles was tested in two ways, after a single reflexive discharge caused by Ia afferent stimulation as described above and after a single voluntary discharge elicited by very gentle voluntary muscle contraction. In the latter case the subject, being relaxed, was trained to recruit one MU (of those within the recording field of the needle electrode) by a single discharge, using visual and audio feedback. Since the potentials of different MUs were unlike one another and could easily be differentiated, the subject had a reliable control of how successful his training was. After 5 - 1 0 min of training a single "voluntary" discharge of the MU tested was used as a trigger to evoke a threshold H reflex of the same M U with gradually increasing delays. Thus, the conditioning discharge of an MU was "voluntary," while the test one, reflectory. In each trial, the peak of an M U response to the test stimulus was calculated with a correction for the H reflex latency, since even in the absence of the shift of a test stimulus (i.e., delay time = 0 msec) the reflex response of an MU appeared with a delay equal to the H reflex latency. This made it impossible to examine the initial period of excitability recovery after a single "voluntary" discharge of an MU, i.e., to decide whether the D D was present or absent. For the evaluation of the A H P duration the minimal interval between single "voluntary" and reflexive discharges of a motoneurone at which the latter responded to the threshold afferent volley with the same FI as at a single testing was determined. The analysis step was from 10 to 20 msec and about 10-20 trials were made for each step value.
MOTONEURONE AFTER-POTENTIALS AND REPETITIVE FIRING
When the after-potential duration of motoneurones in a relaxed muscle was estimated, the subject was instructed to recruit an M U by weak voluntary muscle contraction and keep its steady firing at possibly minimal frequency (fmin)" fmin was calculated as the reciprocal value of the mean interspike interval duration measured over 1 sec and compared with the reciprocal value of the time of a complete excitability recovery determined by paired monosynaptic testing of motoneurones (fAHP)" The occurrence of double discharges in M U rhythmic firing was also examined.
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Threshold stimulation of Ia afferents elicited firing of single MUs with an H reflex latency of 30-44 msec for the soleus muscle and 12-24 msec for the flexor carpi ulnaris (the latency range being within the limits of the summated H reflex duration). For a given M U in both muscles the H reflex latency was constant throughout all trials. During paired stimulation, a m o t o n e u r o n e response to a test stimulus depended on the interstimulus inter-
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INT~P~/dlitl/uU8 ~l~lgR¥/d~, me Fig. 2. The recovery curves of the soleus motoneurone excitability (the firing index, FI, was used as a measure of excitability), a, b: two motoneurones. Abscissa, the interval between conditioning and test stimuli evoking the H reflex of a single motoneurone (msec). Ordinate, the FI in response to a test stimulus, in percentage. The FI in response to a conditioning stimulus was 100%.
val, its probability rising with an increase of the latter. Samples of recorded M U action potentials of the soleus during paired stimulation are presented in Fig. 1. Typical changes in the FI can be seen in Fig. 2. At interstimulus intervals up to 85-90 msec the motoneurones did not fire in response to a test volley ("the inexcitability time"). During further increase of the interstimulus interval, the motoneurones started to respond to test stimuli in some of the trials. The FI gradually increased, reaching the value of the FI to a conditioning stimulus (the latter being equal to 100%) and thus indicating that the motoneurone excitability was completely recovered. The first motoneurone recovered after an interstimulus interval of 190 msec, the second after 160 msec. During subsequent increase in interstimulus delay (up to 1 sec) the FI to a test stimulus remained unchanged, being equal to the FI in response to a conditioning stimulus. In total (21 motoneurones tested), the inexcitability time varied from 90 to 220 msec (the mean + S.D. = 132.6 + 35.9 msec). The complete recovery time ranged between 145 and 255 msec (196.7 + 35.9 msec). In contrast to the soleus motoneurones, those of the flexor carpi ulnaris were sttbdiv~ded into two groups according to the character of their responses to paired stimuli. The MUs of the first group (33% of MUs
348
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Fig, 4. The recovery curves of the flexor carpi ulnaris motoneurone excitability. Legends as for Fig. 2. The FI in response to a conditioning stimulus was 100%.
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Fig. 3, A record sample of the flexor carpi ulnaris MU discharges in response to paired stimulation of the ulnar nerve. At short delays (6-14 msec) the motoneurone responded to a test volley. During an increase of the interstimulus interval up to 50 msec, the motoneurone was silent, beyond this value it retired to a test stimulus. In top right record, the motoneurone fired double discharge in response to a conditioning stimulus. Points mark the stimulation times. Calibration: 100 ~V; 20 msec.
investigated) fired in response to a test stimulus at i n t e r s t i m u l u s intervals as short as 5 - 2 0 msec, with the FI reaching 100% (Figs. 3 a n d 4a), thus showing the early short-lasting excitability recovery. A p a r t from that, the m o t o n e u r o n e s b e l o n g i n g to the first group had the shortest H reflex latency of 12-15 msec a n d were able to fire d o u b l e discharges with interspike intervals of 5 - 1 5 msec in r e s p o n s e to c o n d i t i o n i n g or test stimuli (Fig. 3, top right record). M U s of the second group did not r e s p o n d to a test volley at the intervals m e n t i o n e d (Figs. 4b a n d 5). A t i n t e r s t i m u l u s intervals from 20 to 5 0 - 6 5 msec the FI of the M U s in Figs. 4 a n d 5 was equal to 0%. O n f u r t h e r increase of i n t e r s t i m u l u s intervals, the m o t o n e u r o n e s started to discharge in
response to a test stimulus. As a whole, 20 M U s of the flexor carpi ulnaris recovered completely within a time r a n g i n g from 55 to 150 msec (88.6 + 22.9 msec) a n d the time of m o t o n e u r o n e inexcitability varied from 40 to 90 msec (61.8 + 15.5 msec). In some of the e x p e r i m e n t s the recovery of mot o n e u r o n e excitability was tested after a single discharge evoked by very gentle voluntary muscle contraction. F o r 7 soleus m o t o n e u r o n e s a n d 6 flexor carpi ulnaris m o t o n e u r o n e s the time of complete excitability recovery was 120-225 (151.4 + 32.4) msec a n d 5 5 - 9 0 (75.0 + 13.3) msec respectively, which was within the r a n g e above. Fig. 6 illustrates the results of testing
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Fig. 5. The recovery curve of the flexor carpi ulnaris motoneurone excitability. Abscissa as for Fig. 2. Ordinate, the FI in response to conditioning (open circles) and test (tilled circles) stimuli, in percentage. The FI in response to a conditioning stimulus ranged between 60 and 100%. The arrow marks the time of the complete recovery of motoneurone excitability.
M O T O N E U R O N E AFTER-POTENTIALS AND REPETITIVE FIRING
excitability of a single flexor carpi ulnaris m o t o n e u r o n e after its reflexive and voluntary discharges. As can be seen in Fig. 6a, the early recovery of the motoneurone excitability (up to 20 msec) was revealed by paired testing. The motoneurone excitability had recovered completely at 65 msec of interstimulus interval. While testing the excitability recovery of the same motoneurone after its voluntary discharge it was found that the motoneurone recovered at 55 msec (Fig. 6b). Hence, the recovery times of motoneurone excitability after a single discharge elicited by activation of two different inputs were sufficiently close. The presumed A H P duration determined by the method described above was compared with the minimal firing rate of single MUs during a weak voluntary contraction of the soleus and the flexor carpi ulnaris muscles. A total of 27 MUs of the slow muscle and 22 MUs of the fast one were analysed. For the soleus MUs most of the subjects had no difficulty in maintain-
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Fig. 6. The recovery curves of the flexor carpi ulnaris motoneurone excitability, a, b: the same motoneurone, a: legends as for Fig. 2. b: abscissa, intervals between voluntary and reflexive discharges elicited by weak voluntary muscle contraction and stimulation of la afferents, respectively (msec). Ordinate, the FI in response to an afferent stimulation, in percentage. The F1 in response to a conditioning stimulus (a) and during a single test.(b) was 100%.
ing a low firing rate which ranged from 3.1 to 5.2 (4.5 _+ 0.8) i m p / s e c . However, not all of the subjects were able of maintaining such low firing rates for the MUs of the fast muscle but they improved their ability after some training. The minimal discharge frequency of the flexor carpi ulnaris MUs was 3.8-9.0 (5.2 +_ 1.5) imp/sec. In Fig. 7, the minimal firing rates of MUs were plotted against the reciprocal values of the A H P duration. These latter varied from 3.9 to 8.3 (5.6_+ 1.2) i m p / s e c for MUs of the soleus and from 6.7 to 18.1 (12.0 + 2.6) i m p / s e c for those of the flexor carpi ulnaris. As demonstrated in Fig. 7, the minimal firing rate of MUs in both muscles, especially in the fast one, was significantly lower than the reciprocal value of the A H P duration. In addition to the minimal discharge rate, the pattern of M U repetitive firing during weak voluntary muscle contraction was analysed. It was observed that some of the flexor carpi ulnaris MUs, in contrast to the soleus MUs, fired double discharges, with intradoublet intervals of 5-15 msec, which was far beyond the statistical scatter of other interspike intervals. Double discharges were usually followed by a longer-than-average interval. They were observed during MU recruitment as well as during M U rhythmic firing. The occurrence of double discharges during voluntary muscle contraction was characteristic of those motoneurones for which early excitability recovery was revealed during paired testing.
350 Discussion
In the present study, the method of detecting afterpotentia'ls and estimating their duration in motoneurones was used in order to analyse factors controlling repetitive firing of motoneurones in man. The method was based on the recovery time measurement of motoneurone excitability (evaluated by the firing index) after a single discharge evoked by threshold stimulation of Ia afferents or by voluntary muscle contraction. A similar estimation of after-potentials based on determining the recovery time of motoneurone excitability was first used during extracellular recording in cat (Brooks et al. 1950a, b). Later, the adequacy of such evaluation was confirmed in studies of intracellular records of cat's motoneurone after-potentials (Brock et al. 1952). Thus, the method employed in the present study can provide information about the after-potentials similar to that obtained by intracellular recording. However, other mechanisms capable of decreasing motoneurone excitability after a single discharge elicited by nerve stimulation were not neglected, such as inhibition from group I, II and skin afferents as well as recurrent and presynaptic inhibition. In our experiments, threshold stimulation of Ia afferents was used, thus avoiding the effects of stimulating afferents with a higher threshold (group II and cutaneous). At the same time Ib afferents could be involved because of some overlap in the diameters (and thus the thresholds to electrical stimulation). However, as a result of the overlap in the diameters and the corresponding overlap in conduction velocities, the diand trisynaptic Ib IPSP, occurring in a motoneurone nearly simultaneously with the Ia EPSP, might exert its effect (by hyperpolarization and shunting) on the Ia EPSP, preventing the occurrence of a spike. When an EPSP reaches threshold the generation of a spike extinguishes the remainder of the EPSP (Coombs et al. 1955). Hence, the Ib IPSP could not affect motonehrone excitability after a spike. Furthermore, if a Ib IPSP arrived in a motoneurone later than a Ia EPSP (just after a spike), it would probably be also ineffective, by analogy with the reciprocal and recurrent inhibition. Being evoked by single threshold stimulation these latter were ineffective just after an MU discharge and became very effective at the end of the A H P when the membrane potential approached threshold (Kudina 1980; Kudina and Pantseva 1988). There is no reason, therefore, to implicate Ib afferents in decreasing the motoneurone excitability observed here. Recently, the existence of a Ia afferent inhibitory effect from the gastrocnemius medialis muscle onto human soleus motoneurones was reported by Gritti and Schieppati (1989). The inhibition, which lasted only 8 msec, showed two periods with peaks at about 0 and 5 msec of conditioning-test delay and decreased
L.P. KUD1NA,N.L. ALEXEEVA the test H reflex up to 80-90% of control values. Proceeding from this, the gastrocnemius Ia afferents could not be a contributor to the long-lasting prominent decrease in excitability of soleus motoneurones described. Among the possible mechanisms responsible for the decrease in the motoneurone excitability after a discharge, recurrent inhibition must be mentioned as the most probable contributor (besides the AHP) because it is unalterably involved when motoneurones fire in the conditioning H reflex. Earlier, recurrent inhibition for firing motoneurones during voluntary muscle contraction in humans was found (Kudina and Pantseva 1988; Miles et al. 1989a; Meunier et al. 1990). The effect of recurrent inhibitory volleys (having a latency slightly longer than the H reflex latency) depended on the time of their arrival in the interspike interval, i.e., the time course of the AHP, and thus these volleys were ineffective at the beginning of the A H P (Kudina and Pantseva 1988). This suggests that in the present experiments recurrent inhibition did not contribute to excitability decrease after a discharge. Finally, the participation of presynaptic inhibition in decreasing motoneurone excitability is hardly possible. As has been shown in animal experimentation, in order to evoke presynaptic inhibition capable of decreasing a monosynaptic reflex by 50-80%, repetitive stimulation at the strength maximal for activating Ia afferents is required (Eccles et al. 1962). The presynaptic inhibition caused by a single stimulus (even at the strength maximal for Ia afferents) decreases a monosynaptic response by less than 10% only (Eccles 1964). Thus, the mentioned mechanisms could hardly affect the decrease in motoneurone excitability after single discharges elicited by threshold stimulation of Ia afferents. This point of view is supported by differences in motoneurone excitability decrease durations for the motoneurones of slow and fast muscles. Actually, all other mechanisms, except the AHP, which could be activated by the conditioning stimulus are comparable in the duration of their influence on motoneurones of slow and fast muscles. Finally, the similarity in motoneurone excitability decrease duration after a single motoneurone discharge elicited by both threshold stimulation of Ia afferents and voluntary muscle contraction proves that the decrease in motoneurone excitability is caused by the AHP. In fact, in the case of a single voluntary discharge of a motoneurone the activation of Ib, II and skin afferents, as well as presynaptic inhibition, is excluded due to the absence of conditioning nerve stimulation. The question arises of whether the contraction-induced afferent volleys from Ib and II afferents contribute to the recovery profile of motoneurone excitability after a discharge. Under the conditions explored, the muscle contraction was very gentle since
MOTONEURONE AFTER-POTENTIALS AND REPETITIVE FIRING the (hreshold Ia volleys activated only one or several MUs. Thus, the effects from contracting muscle a p p e a r to have little influence on motoneurone excitability recovery. Taking these considerations into account, we may well suppose that the method employed makes it possible to evaluate the duration of motoneurone A H P in man. Apart from estimating the A H P duration, the method revealed the D D in human motoneurones. According to our data, some motoneurones of fast muscles, in contrast to those of slow ones, revealed early recovery of excitability lasting up to 15-20 msec after a single discharge, which was attributed to the motoneurone DD. Our results on the D D and A H P durations are in good agreement with those obtained for cat's motoneurones (see Introduction). With regard to data on the duration of the motoneurone after-potentials in man, the only hypothesis proposed (Person and Kudina 1971; Khuskivadze 1979; Person 1985) was based on the value of the M U mean interspike interval at which the breakpoint in the plot "standard deviation/interspike interval duration" occurs. For motoneurones of fast muscles - - rectus femoris (Person and Kudina 1971) and biceps brachii (Maton 1977) - - the value was 80-90 msec. For those of slow soleus muscle it was 120-125 msec (Khuskivadze 1979). However, the present data indicate a greater range of the A H P duration in both fast and slow muscles. As reported by Zwaagstra and Kernell (1980), for cat's hind limb motoneurones the A H P duration also had a wide range (from 65 to 255 msec). The reason for the differences in these findings seems to require further analysis. Some information about the human after-potentials can be extracted from the Sabbahi-Sedgwick study (1987) on the H reflex recovery curves of single motoneurones in which early recovering (80-300 msec) and late recovering (up to 3000 msec) motoneurones of the soleus were shown. Our data obtained on the soleus are in keeping with the recovery profile of the early recovering neurones, while late recovering motoneurones were not observed. The reason for such a discrepancy is not clear; it might be attributed to the differences in the details of applying the H reflex methodology (in particular, in the stimulus strength). According to data reported by Sabbahi and Sedgwick, the recovery time of a motoneurone was constant so long as conditions remained the same but was shortened by some factors such as background activity of the muscle under study, mild tension of body, head or arm movements, the late recovering motoneurones showing a considerable recovery time reduction. This is not in contrast with the results presented demonstrating the similarity in the excitability recovery time of a motoneurone after its single reflexive and single volun-
351
tary discharges, since in our experiments the subjects were relaxed completely. So far, the excitability recovery of motoneurones after their single discharges has not been used for investigating the A H P and D D in man. At the same time paired stimulation of Ia afferents (the " H reflex recovery curve") has been widely used to measure the excitability of the whole motoneurone pool. Many authors have emphasized that the H reflex recovery curve is difficult to interpret because of the number of factors involved, though the after-potentials have not been considered very important. Our results demonstrate, however, that the A H P and D D make a significant contribution to the genesis of the H reflex recovery curve for motoneurones of slow and fast muscles. Our data also show that the motoneurones were not affected by inhibition, which confirms the results reported by Sabbahi and Sedgwick (1987). At the same time, the H reflex recovery profile demonstrates the "secondary depression" which is usually attributed to presynaptic inhibition, the latter being abolished by voluntary muscle contraction (Person 1985; Schieppati 1987; Burke et al. 1989). It appears from the present findings that the presynaptic inhibition affects those motoneurones of the pool which have a higher threshold to stimulation but not the low threshold to Ia stimulation as well as voluntary muscle contraction (i.e., the small fatigue resistant ones) tested by us. We believe that these factors should be taken into account when interpreting the H reflex recovery curve of the motoneurone pool in healthy and diseased subjects. Analysis of motoneurone after-potentials with regard to repetitive firing parterns of motoneurones revealed that some of the fast muscle motoneurones, namely, those showing the early excitability recovery, were capable of firing doublets during voluntary muscle contraction. The intradoublet interval duration was within the same range as the D D duration. The findings agree with those reported by Calvin and Schwindt (1972) showing that D D in cat motoneurones is associated with a doublet firing pattern. It has been generally believed that the long-lasting A H P following each cat's motoneurone spike is a factor of great importance as an intrinsic mechanism for the regulation of repetitive firing (Eccles et al. 1958; Kernell 1965). This view has been strongly supported by model studies in which both repetitive firing and voltage trajectories within an interspike interval can be well predicted on the properties of the A H P (Baldissera and Gustafsson 1974; Gustafsson 1974). Moreover, it has been known that motoneurone repetitive firing at the minimal rates is related to the A H P duration (Kernell 1965). Our results obtained with the use of natural activation of MUs in man demonstrated that the minimal
352
firing rate of human motoneurones activated by gentle voluntary contraction of both slow and fast muscles was not correlated with the AHP duration. After some training the human subjects were able to maintain repetitive firing of MUs with interspike intervals longer than the AHP duration. In the previous reports it has been noted that under conditions of motoneurone natural activation the range of firing rates is extended to the field of lower frequencies within which the motoneurone excitability and inhibitability are increased (Ashby and Zilm 1982; Miles and Tiirker 1986; Kudina 1988; Kudina and Pantseva 1988; Miles et al. 1989b). On the basis of these data and the results presented it can be assumed that within the low range of firing frequencies (in contrast to the higher "primary range" investigated in animal experiments) the AHP does not play a leading role in controlling repetitive firing of human motoneurones. This firing range, which can be considered "subprimary," seems to be regulated, in addition to the AHP, by some other mechanisms of spinal and supraspinal control "switched off" under anaesthesia and spinalization used in animal experimentation. The authors are grateful to L. Kravtsova for scrutinizing the English.
References Ashby, P. and Zilm, D. Relationship between EPSP shape and cross-correlation profile explored by computer simulation for studies on human motoneurones. Exp. Brain Res., 1982, 47: 33-40. Baldissera, F. and Gustafsson, P. Afterhyperpolarization conductance time course in lumbar motoneurones of the cat. Acta Physiol. Scand., 1974, 91: 512-527. Brock, L.G., Coombs, J.S. and Eccles, J.C. The recording of potentials from motoneurones with intracellular electrode. J. Physiol. (Lond.), 1952, 117: 431-460. Brooks, C.McC., Downman, B.B. and Eccles, J.C. After-potentials and excitability of spinal motoneurones following antidromic activation. J. Neurophysiol., 1950a, 13: 9-38. Brooks, C.McC., Downman, B.B. and Eccles, J.C. After-potentials and excitability of spinal motoneurones following orthodromic activation. J. Neurophysiol., 1950b, 13: 157-176. Burke, R.E. Motor unit types of cat triceps surae muscle. J. Physiol. (Lond.), 1967, 193: 141-160. Burke, D., Adams, R.W. and Skuse, N.F. The effects of voluntary contraction on the H-reflex of human limb muscles. Brain, 1989, 112: 417-433. Calvin, W.H. Three modes of repetitive firing and the role of threshold time course between spikes. Brain Res., 1974, 69: 341-346. Calvin, W.H. Normal repetitive firing and its pathophysiology. In: I.S. Lockard and A.A. Ward, Jr. (Eds.), Epilepsy: a Window to Brain Mechanisms. Raven Press, New York, 1980: 97-121. Calvin, W.H. and Schwindt, P.C. Steps in production of motoneurone spikes during rhythmic firing. J. Neurophysiol., 1972, 35: 297-310.
L.P. KUDINA, N.L. ALEXEEVA Coombs, J.S., Eccles, J.C. and Fatt, P. The electrical properties of motoneurone membrane. J. Physiol. (Lond.), 1955, 130: 291-325. Eccles, J.C. Presynaptic inhibition in the spinal cord. In: J.C. Eccles and J.P. Schad6 (Eds.), Progr. in Brain Res. Vol. 12. Physiology of Spinal Neurones. Elsevier, Amsterdam, 1964: 65-85. Eccles, J.C., Eccles, R.M. and Lundberg, A. The action potentials of the alpha motoneurones supplying fast and slow muscles. J. Physiol. (Lond.), 1958, 142: 275-291. Eccles, J.C., Schmidt, R.F. and Willes, W.D. Presynaptic inhibition of the spinal monosynaptic reflex pathway. J. Physiol. (Lond.), 1962, 161: 282-297. Granit, R. The Basis of Motor Control. Academic Press, London, 1970:337 pp. Granit, R., Kernell, D. and Smith, R.S. Delayed depolarization and the repetitive response to intracellular stimulation of the mammalian motoneurones. J. Physiol. (Lond.), 1963, 168: 890-910. Gritti, I. and Schieppati, M. Short-latency inhibition of soleus motoneurones by impulses in la afferents from the gastrocnemius muscle in humans. J. Physiol. (Lond.), 1989, 416: 469-484. Gustafsson, B. Afterhyperpolarization and the control of repetitive firing in spinal neurones of the cat. Acta Physiol. Scand., 1974, Suppl. 416:47 pp. Gustafsson, B. Afterpotentials and transduction properties in different types of central neurones. Arch. Ital. Biol., 1984, 122: 17-30. Kcrnell, D. The delayed depolarization in cat and rat motoneurones. In: J.C. Eccles and J.P. Schad6 (Eds.), Progr. Brain Res., Vol. 12. Elsevier, Amsterdam, 1964: 42-52. Kernell, D. The limits of firing frequency in cat lumbosacral motoneurones possessing different time course of afterhyperpolarization. Acta Physiol. Scand., 1965, 65: 87-100. Khuskivadze, T.K. Comparison of motor unit firing in gastrocnemius and soleus muscles. Fiziol. Celoveka (Moscow), 1979, 5: 102-109. Kudina, L.P. Reflex effects of muscle afferent on antagonist studied on single motor units in man. Electroenceph. clin. Neurophysiol., 1980, 50: 214-221. Kudina, L.P. Excitability of firing motoneurones tested by Ia afferent volleys in human triceps surae. Electroenceph. clin. Neurophysiol., 1988, 69: 576-580. Kudina, L.P. and Pantseva, R.E. Recurrent inhibition of firing motoneurones in man. Electroenceph. clin. Neurophysiol., 1988, 69: 179-185. Kudina, L.P., Churikova, L.I. and Alexeeva, N.L. Firing peculiarities of human motor units within the "subprimary range." In: Abstracts of the 6th Int. Symp. on Motor Control, Albena, 1989: 135. Maton, B. Fr6quence et recrutement des unit6s motrices du muscle bicepse brachial au cours du-travail statistique chez l'homme normal. J. Physiol. (Paris), 1977, 73" 177-199. Meunier, S., Penicaud, A., Pierrot-Deseilligny, E. and Rossi, A. Monosynaptic Ia excitation and recurrent inhibition from quadriceps to ankle flexors and extensors in man. J. Physiol. (Lond.), 1990, 423: 661-675. Miles, T.S. and Tiirker, K.S. Does reflex inhibition of motor units follow the "size" principle"? Exp. Brain Res., 1986, 62: 443-445. Miles, T.S., Le, T.H. and Tiirker, K.S. Biphasic inhibitory responses and their IPSPs evoked by tibial nerve stimulation in human soleus motor neurones. Exp. Brain Res., 1989a, 77: 637-645. Miles, T.S., Tiirker, K.S. and Le, T.H. Ia reflexes and EPSPs in human soleus motor neurones. Exp. Brain Res., 1989b, 77: 628636. Person, R.S. Spinal Mechanisms of Muscle Contraction Control. Nauka, Moscow, 1985:185 pp. Person, R.S. and Kudina, L.P. Frequency of motoneurone discharges under voluntary contraction of human muscle. Neurofiziologiya (Kiev), 1971, 3: 200-209. Sabbahi, M. and Sedgwick, E.M. Recovery profile of single motoneu-
MOTONEURQNE AFTER-POTENTIALS AND REPETITIVE FIRING rones after electrical stimuli in man. Brain Res., 1987, 423: 125-134. Schieppati, M. The Hoffmann reflex: a m e a n s of assessing spinal reflex excitability and its descending control in man. Progr. Neurobiol., 1987, 128: 345-376.
353 Zwaagstra, B. and Kernell, D. The duration of afterhyperpolarization in hind limb alpha motoneurones of different sizes in the cat. Neurosci. Lett., 1980, 19: 303-307.
345
© 1992 Elsevier Scientific Publishers Ireland, Ltd. 0924-980X/92/$05.00
E L M O C O 91627
After-potentials and control of repetitive firing in h u m a n motoneurones L.P. Kudina and N.L. Alexeeva Institute for Problems of Information Transmission, Russian Academy of Sciences, Moscow (Russia) (Accepted for publication: 6 March 1992)
Summary Characteristics of m o t o n e u r o n e after-potentials in m a n were derived from the recovery curve of m o t o n e u r o n e excitability after a single discharge evoked by threshold stimulation of la afferents or by gentle voluntary muscle contraction. The m o t o n e u r o n e excitability was estimated by the firing index of a single motor unit whose potentials were recorded by needle electrodes. The soleus (a slow muscle) and the flexor carpi ulnaris (a fast muscle) were investigated. The duration of m o t o n e u r o n e after-hyperpolarization of the soleus evaluated by this method ranged between 145 and 255 msec; for the flexor carpi ulnaris it was 55-150 msec. In some motoneurones of the fast muscle, an early short-lasting recovery of excitability (within 5 - 2 0 msec after a discharge) was revealed. It was accounted for by delayed depolarization of the motoneurone. The relationship between after-potentials and the characteristics of repetitive firing of motoneurones activated by weak voluntary muscle contraction was analysed. It was observed that the motoneurones with early excitabiity recovery were capable of firing double discharges with a 5-15 msec interspike interval. It was found also that the minimal firing rate of motoneurones (up to 3.1-5.2 i m p / s e c in the soleus and 3.8-9.0 i m p / s e c in the flexor carpi ulnaris) was not correlated with the after-hyperpolarization duration. This differs from the results obtained for cat's motoneurones u n d e r intracellular stimulation. The findings suggest that after-hyperpolarization is not the only leading mechanism controlling the low firing rate of motoneurones u n d e r conditions of their natural activity in man.
Key words: Motor control; Motoneurone repetitive firing; After-potentials
In spinal motoneurones, a somadendritic spike is followed by a long-lasting hyperpolarizing after-potential, the after-hyperpolarization (AHP), caused by an increase in the membrane conductance to potassium ions, which gradually declines after a spike (Coombs et al. 1955). As a result, the motoneurone excitability is low just after a spike and thereafter gradually recovers. The duration of the A H P widely varies among motoneurones and is correlated with their sizes and contraction time of the innervated muscle fibres (Eccles et al. 1958; Burke 1967; Zwaagstra and Kernell 1980). According to Eccles et al. (1958) the A H P duration was more than 130 msec for motoneurones of slow muscles and 50-110 msec for those of fast ones. The A H P is often preceded by a short-lasting depolarizing after-potential, the delayed depolarization (DD), a phase of slower repolarization between the rapid falling phase of the spike and the fast descending limb of the AHP. As supposed, DD is of dendritic origin; in some motoneurones it smoothly decays, in others it has the shape of a " h u m p " (Granit et al. 1963; Kernell 1964; Calvin 1980). D D approaches the mem-
Correspondence to: L.P. Kudina, Institute for Problems of Information Transmission, Russian A c a d e m y of Sciences, Yermolovoy 19, GSP-4, 101447 Moscow (Russia).
brane potential to the threshold level, thereby causing a transitory increase in the motoneurone excitability. After-potentials, especially AHP, are considered an important factor controlling repetitive firing of motoneurones (Kernel 1965; Calvin 1974; Gustafsson 1984). This was deduced from the correlations between motoneurone firing characteristics and their afterpotential properties. DD, especially in a "hump-like" form, was found to be associated with a particular pattern of motoneurone firing, i.e., doublets with a very short intradoublet interval - - up to 11-13 msec (Calvin and Schwindt 1972). Under conditions of intracellular motoneurone depolarization Kernell (1965) showed that the total A H P duration measured up to the peak of late after-depolarization was strongly correlated with the minimal firing rate within the "primary range" which was between 8 and 25 i m p / s e c (usually 15). Since intracellular depolarization and natural synaptic activation are considered interchangeable (Granit 1970), the value of the minimal firing rate of synaptically activated motoneurones is not expected to fall below the mentioned values. However, under conditions of voluntary muscle contraction in man significantly lower rates of motoneurone repetitive firing (from 3-5 to 10-12 i m p / s e c ) were observed (Person and Kudina 1971; Ashby and Zilm 1982; Kudina 1988;
346 Miles et al. 1989b). The mechanisms underlying the control of such low firing rates are difficult to reveal because of the absence of data on after-potential duration in human motoneurones. The aim of the present study was to analyse afterpotentials of human motoneurones in slow and fast muscles with regard to some characteristics of their repetitive firing. Some of these results were published in the form of an abstract (Kudina et al. 1989).
.~lethods Investigation was carried out on 8 healthy subjects aged 22-48 years who volunteered to participate in the study. Each subject was tested in several experiments. The subjects were comfortably seated in arm-chairs and special attention was paid to complete relaxation of a subject during the whole experiment. Motoneurones of the soleus and flexor carpi ulnaris (known to be slow and fast twitch muscles respectively, see Eccles et al. 1958) were examined. A total of 49 motoneurones (27 of soleus and 22 of flexor carpi ulnaris) were tested. In order to analyse the afterpotentials of motoneurones the method of threshold monosynaptic testing (evoking conditioning and test H reflexes) of single motor units (MUs) was used, which made it possible to evaluate the recovery time of motoneurone excitability after a single discharge. To evoke the H reflex of the soleus MUs the tibialis posterior nerve was stimulated by means of a bipolar surface electrode placed in the popliteal fossa. The H reflex of the flexor carpi ulnaris MUs was elicited by stimulating the ulnaris nerve at the elbow. The stimulus strength was threshold for evoking the H reflex, the stimulus duration was 0.5-1.0 msec. The summated H reflex was recorded with surface electrodes placed over the muscles tested. The M U action potentials were picked up by bipolar needle electrodes. The E M G signals were amplified (bandwidth of 20 Hz to 10 kHz) and photographe d for subsequent analysis. The responses of single M U s were evaluated by a firing index (FI), i.e., the relation Of the number of M U responses with H reflex latenci" to the number of applied stimuli (in percentage). In ~ill experiments the stimulation parameters were carefully chosen for each M U tested so as to elicit its threshold response. The threshold responses of MUs were considered to be those with an FI not lower ~than 50%, because the lower FI is known to be very unstable. The FI was calculated from 10 to 2 0 trials. For different MUs the FI ranged from 50 to i 0 0 % ; for a n y M U it could be constant or varied with t h e same limits throughout the experiment.
L.P. KUD1NA, N.L. ALEXEEVA During paired testing the strengths of conditioning and test stimuli were identical and equal to the threshold stimulus strength used at single testing. Test stimuli were applied with a delay gradually increasing from 5 to 300 msec for most of the MUs and up to 1 sec for some of them. The interstimulus step was 3 - 5 and 10-20 msec at short and long delays, respectively. At each step the FI of an MU was calculated in response to conditioning and test stimuli from 10 to 20 trials. The FI to a conditioning stimulus was equal to the FI at single testing and used as a measure of the background excitability of a motoneurone. The recovery of the motoneurone excitability after a single action potential was estimated by the FI of an M U in response to a test stimulus. Gradually increasing the interstimulus delay we were able to find the minimal interstimulus interval at which a motoneurone discharged in response to a test stimulus with the same FI as to a conditioning stimulus. We called it the time of complete recovery of motoneurone excitability and used it as a measure of the A H P duration. The D D duration was evaluated by the time of early recovery of motoneurone excitability at short delays. In some of the experiments, the excitability recovery of motoneurones in both muscles was tested in two ways, after a single reflexive discharge caused by Ia afferent stimulation as described above and after a single voluntary discharge elicited by very gentle voluntary muscle contraction. In the latter case the subject, being relaxed, was trained to recruit one MU (of those within the recording field of the needle electrode) by a single discharge, using visual and audio feedback. Since the potentials of different MUs were unlike one another and could easily be differentiated, the subject had a reliable control of how successful his training was. After 5 - 1 0 min of training a single "voluntary" discharge of the MU tested was used as a trigger to evoke a threshold H reflex of the same M U with gradually increasing delays. Thus, the conditioning discharge of an MU was "voluntary," while the test one, reflectory. In each trial, the peak of an M U response to the test stimulus was calculated with a correction for the H reflex latency, since even in the absence of the shift of a test stimulus (i.e., delay time = 0 msec) the reflex response of an MU appeared with a delay equal to the H reflex latency. This made it impossible to examine the initial period of excitability recovery after a single "voluntary" discharge of an MU, i.e., to decide whether the D D was present or absent. For the evaluation of the A H P duration the minimal interval between single "voluntary" and reflexive discharges of a motoneurone at which the latter responded to the threshold afferent volley with the same FI as at a single testing was determined. The analysis step was from 10 to 20 msec and about 10-20 trials were made for each step value.
MOTONEURONE AFTER-POTENTIALS AND REPETITIVE FIRING
When the after-potential duration of motoneurones in a relaxed muscle was estimated, the subject was instructed to recruit an M U by weak voluntary muscle contraction and keep its steady firing at possibly minimal frequency (fmin)" fmin was calculated as the reciprocal value of the mean interspike interval duration measured over 1 sec and compared with the reciprocal value of the time of a complete excitability recovery determined by paired monosynaptic testing of motoneurones (fAHP)" The occurrence of double discharges in M U rhythmic firing was also examined.
347
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Threshold stimulation of Ia afferents elicited firing of single MUs with an H reflex latency of 30-44 msec for the soleus muscle and 12-24 msec for the flexor carpi ulnaris (the latency range being within the limits of the summated H reflex duration). For a given M U in both muscles the H reflex latency was constant throughout all trials. During paired stimulation, a m o t o n e u r o n e response to a test stimulus depended on the interstimulus inter-
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INT~P~/dlitl/uU8 ~l~lgR¥/d~, me Fig. 2. The recovery curves of the soleus motoneurone excitability (the firing index, FI, was used as a measure of excitability), a, b: two motoneurones. Abscissa, the interval between conditioning and test stimuli evoking the H reflex of a single motoneurone (msec). Ordinate, the FI in response to a test stimulus, in percentage. The FI in response to a conditioning stimulus was 100%.
val, its probability rising with an increase of the latter. Samples of recorded M U action potentials of the soleus during paired stimulation are presented in Fig. 1. Typical changes in the FI can be seen in Fig. 2. At interstimulus intervals up to 85-90 msec the motoneurones did not fire in response to a test volley ("the inexcitability time"). During further increase of the interstimulus interval, the motoneurones started to respond to test stimuli in some of the trials. The FI gradually increased, reaching the value of the FI to a conditioning stimulus (the latter being equal to 100%) and thus indicating that the motoneurone excitability was completely recovered. The first motoneurone recovered after an interstimulus interval of 190 msec, the second after 160 msec. During subsequent increase in interstimulus delay (up to 1 sec) the FI to a test stimulus remained unchanged, being equal to the FI in response to a conditioning stimulus. In total (21 motoneurones tested), the inexcitability time varied from 90 to 220 msec (the mean + S.D. = 132.6 + 35.9 msec). The complete recovery time ranged between 145 and 255 msec (196.7 + 35.9 msec). In contrast to the soleus motoneurones, those of the flexor carpi ulnaris were sttbdiv~ded into two groups according to the character of their responses to paired stimuli. The MUs of the first group (33% of MUs
348
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Fig, 4. The recovery curves of the flexor carpi ulnaris motoneurone excitability. Legends as for Fig. 2. The FI in response to a conditioning stimulus was 100%.
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Fig. 3, A record sample of the flexor carpi ulnaris MU discharges in response to paired stimulation of the ulnar nerve. At short delays (6-14 msec) the motoneurone responded to a test volley. During an increase of the interstimulus interval up to 50 msec, the motoneurone was silent, beyond this value it retired to a test stimulus. In top right record, the motoneurone fired double discharge in response to a conditioning stimulus. Points mark the stimulation times. Calibration: 100 ~V; 20 msec.
investigated) fired in response to a test stimulus at i n t e r s t i m u l u s intervals as short as 5 - 2 0 msec, with the FI reaching 100% (Figs. 3 a n d 4a), thus showing the early short-lasting excitability recovery. A p a r t from that, the m o t o n e u r o n e s b e l o n g i n g to the first group had the shortest H reflex latency of 12-15 msec a n d were able to fire d o u b l e discharges with interspike intervals of 5 - 1 5 msec in r e s p o n s e to c o n d i t i o n i n g or test stimuli (Fig. 3, top right record). M U s of the second group did not r e s p o n d to a test volley at the intervals m e n t i o n e d (Figs. 4b a n d 5). A t i n t e r s t i m u l u s intervals from 20 to 5 0 - 6 5 msec the FI of the M U s in Figs. 4 a n d 5 was equal to 0%. O n f u r t h e r increase of i n t e r s t i m u l u s intervals, the m o t o n e u r o n e s started to discharge in
response to a test stimulus. As a whole, 20 M U s of the flexor carpi ulnaris recovered completely within a time r a n g i n g from 55 to 150 msec (88.6 + 22.9 msec) a n d the time of m o t o n e u r o n e inexcitability varied from 40 to 90 msec (61.8 + 15.5 msec). In some of the e x p e r i m e n t s the recovery of mot o n e u r o n e excitability was tested after a single discharge evoked by very gentle voluntary muscle contraction. F o r 7 soleus m o t o n e u r o n e s a n d 6 flexor carpi ulnaris m o t o n e u r o n e s the time of complete excitability recovery was 120-225 (151.4 + 32.4) msec a n d 5 5 - 9 0 (75.0 + 13.3) msec respectively, which was within the r a n g e above. Fig. 6 illustrates the results of testing
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Fig. 5. The recovery curve of the flexor carpi ulnaris motoneurone excitability. Abscissa as for Fig. 2. Ordinate, the FI in response to conditioning (open circles) and test (tilled circles) stimuli, in percentage. The FI in response to a conditioning stimulus ranged between 60 and 100%. The arrow marks the time of the complete recovery of motoneurone excitability.
M O T O N E U R O N E AFTER-POTENTIALS AND REPETITIVE FIRING
excitability of a single flexor carpi ulnaris m o t o n e u r o n e after its reflexive and voluntary discharges. As can be seen in Fig. 6a, the early recovery of the motoneurone excitability (up to 20 msec) was revealed by paired testing. The motoneurone excitability had recovered completely at 65 msec of interstimulus interval. While testing the excitability recovery of the same motoneurone after its voluntary discharge it was found that the motoneurone recovered at 55 msec (Fig. 6b). Hence, the recovery times of motoneurone excitability after a single discharge elicited by activation of two different inputs were sufficiently close. The presumed A H P duration determined by the method described above was compared with the minimal firing rate of single MUs during a weak voluntary contraction of the soleus and the flexor carpi ulnaris muscles. A total of 27 MUs of the slow muscle and 22 MUs of the fast one were analysed. For the soleus MUs most of the subjects had no difficulty in maintain-
349 fmin. ,imp/s
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Fig. 6. The recovery curves of the flexor carpi ulnaris motoneurone excitability, a, b: the same motoneurone, a: legends as for Fig. 2. b: abscissa, intervals between voluntary and reflexive discharges elicited by weak voluntary muscle contraction and stimulation of la afferents, respectively (msec). Ordinate, the FI in response to an afferent stimulation, in percentage. The F1 in response to a conditioning stimulus (a) and during a single test.(b) was 100%.
ing a low firing rate which ranged from 3.1 to 5.2 (4.5 _+ 0.8) i m p / s e c . However, not all of the subjects were able of maintaining such low firing rates for the MUs of the fast muscle but they improved their ability after some training. The minimal discharge frequency of the flexor carpi ulnaris MUs was 3.8-9.0 (5.2 +_ 1.5) imp/sec. In Fig. 7, the minimal firing rates of MUs were plotted against the reciprocal values of the A H P duration. These latter varied from 3.9 to 8.3 (5.6_+ 1.2) i m p / s e c for MUs of the soleus and from 6.7 to 18.1 (12.0 + 2.6) i m p / s e c for those of the flexor carpi ulnaris. As demonstrated in Fig. 7, the minimal firing rate of MUs in both muscles, especially in the fast one, was significantly lower than the reciprocal value of the A H P duration. In addition to the minimal discharge rate, the pattern of M U repetitive firing during weak voluntary muscle contraction was analysed. It was observed that some of the flexor carpi ulnaris MUs, in contrast to the soleus MUs, fired double discharges, with intradoublet intervals of 5-15 msec, which was far beyond the statistical scatter of other interspike intervals. Double discharges were usually followed by a longer-than-average interval. They were observed during MU recruitment as well as during M U rhythmic firing. The occurrence of double discharges during voluntary muscle contraction was characteristic of those motoneurones for which early excitability recovery was revealed during paired testing.
350 Discussion
In the present study, the method of detecting afterpotentia'ls and estimating their duration in motoneurones was used in order to analyse factors controlling repetitive firing of motoneurones in man. The method was based on the recovery time measurement of motoneurone excitability (evaluated by the firing index) after a single discharge evoked by threshold stimulation of Ia afferents or by voluntary muscle contraction. A similar estimation of after-potentials based on determining the recovery time of motoneurone excitability was first used during extracellular recording in cat (Brooks et al. 1950a, b). Later, the adequacy of such evaluation was confirmed in studies of intracellular records of cat's motoneurone after-potentials (Brock et al. 1952). Thus, the method employed in the present study can provide information about the after-potentials similar to that obtained by intracellular recording. However, other mechanisms capable of decreasing motoneurone excitability after a single discharge elicited by nerve stimulation were not neglected, such as inhibition from group I, II and skin afferents as well as recurrent and presynaptic inhibition. In our experiments, threshold stimulation of Ia afferents was used, thus avoiding the effects of stimulating afferents with a higher threshold (group II and cutaneous). At the same time Ib afferents could be involved because of some overlap in the diameters (and thus the thresholds to electrical stimulation). However, as a result of the overlap in the diameters and the corresponding overlap in conduction velocities, the diand trisynaptic Ib IPSP, occurring in a motoneurone nearly simultaneously with the Ia EPSP, might exert its effect (by hyperpolarization and shunting) on the Ia EPSP, preventing the occurrence of a spike. When an EPSP reaches threshold the generation of a spike extinguishes the remainder of the EPSP (Coombs et al. 1955). Hence, the Ib IPSP could not affect motonehrone excitability after a spike. Furthermore, if a Ib IPSP arrived in a motoneurone later than a Ia EPSP (just after a spike), it would probably be also ineffective, by analogy with the reciprocal and recurrent inhibition. Being evoked by single threshold stimulation these latter were ineffective just after an MU discharge and became very effective at the end of the A H P when the membrane potential approached threshold (Kudina 1980; Kudina and Pantseva 1988). There is no reason, therefore, to implicate Ib afferents in decreasing the motoneurone excitability observed here. Recently, the existence of a Ia afferent inhibitory effect from the gastrocnemius medialis muscle onto human soleus motoneurones was reported by Gritti and Schieppati (1989). The inhibition, which lasted only 8 msec, showed two periods with peaks at about 0 and 5 msec of conditioning-test delay and decreased
L.P. KUD1NA,N.L. ALEXEEVA the test H reflex up to 80-90% of control values. Proceeding from this, the gastrocnemius Ia afferents could not be a contributor to the long-lasting prominent decrease in excitability of soleus motoneurones described. Among the possible mechanisms responsible for the decrease in the motoneurone excitability after a discharge, recurrent inhibition must be mentioned as the most probable contributor (besides the AHP) because it is unalterably involved when motoneurones fire in the conditioning H reflex. Earlier, recurrent inhibition for firing motoneurones during voluntary muscle contraction in humans was found (Kudina and Pantseva 1988; Miles et al. 1989a; Meunier et al. 1990). The effect of recurrent inhibitory volleys (having a latency slightly longer than the H reflex latency) depended on the time of their arrival in the interspike interval, i.e., the time course of the AHP, and thus these volleys were ineffective at the beginning of the A H P (Kudina and Pantseva 1988). This suggests that in the present experiments recurrent inhibition did not contribute to excitability decrease after a discharge. Finally, the participation of presynaptic inhibition in decreasing motoneurone excitability is hardly possible. As has been shown in animal experimentation, in order to evoke presynaptic inhibition capable of decreasing a monosynaptic reflex by 50-80%, repetitive stimulation at the strength maximal for activating Ia afferents is required (Eccles et al. 1962). The presynaptic inhibition caused by a single stimulus (even at the strength maximal for Ia afferents) decreases a monosynaptic response by less than 10% only (Eccles 1964). Thus, the mentioned mechanisms could hardly affect the decrease in motoneurone excitability after single discharges elicited by threshold stimulation of Ia afferents. This point of view is supported by differences in motoneurone excitability decrease durations for the motoneurones of slow and fast muscles. Actually, all other mechanisms, except the AHP, which could be activated by the conditioning stimulus are comparable in the duration of their influence on motoneurones of slow and fast muscles. Finally, the similarity in motoneurone excitability decrease duration after a single motoneurone discharge elicited by both threshold stimulation of Ia afferents and voluntary muscle contraction proves that the decrease in motoneurone excitability is caused by the AHP. In fact, in the case of a single voluntary discharge of a motoneurone the activation of Ib, II and skin afferents, as well as presynaptic inhibition, is excluded due to the absence of conditioning nerve stimulation. The question arises of whether the contraction-induced afferent volleys from Ib and II afferents contribute to the recovery profile of motoneurone excitability after a discharge. Under the conditions explored, the muscle contraction was very gentle since
MOTONEURONE AFTER-POTENTIALS AND REPETITIVE FIRING the (hreshold Ia volleys activated only one or several MUs. Thus, the effects from contracting muscle a p p e a r to have little influence on motoneurone excitability recovery. Taking these considerations into account, we may well suppose that the method employed makes it possible to evaluate the duration of motoneurone A H P in man. Apart from estimating the A H P duration, the method revealed the D D in human motoneurones. According to our data, some motoneurones of fast muscles, in contrast to those of slow ones, revealed early recovery of excitability lasting up to 15-20 msec after a single discharge, which was attributed to the motoneurone DD. Our results on the D D and A H P durations are in good agreement with those obtained for cat's motoneurones (see Introduction). With regard to data on the duration of the motoneurone after-potentials in man, the only hypothesis proposed (Person and Kudina 1971; Khuskivadze 1979; Person 1985) was based on the value of the M U mean interspike interval at which the breakpoint in the plot "standard deviation/interspike interval duration" occurs. For motoneurones of fast muscles - - rectus femoris (Person and Kudina 1971) and biceps brachii (Maton 1977) - - the value was 80-90 msec. For those of slow soleus muscle it was 120-125 msec (Khuskivadze 1979). However, the present data indicate a greater range of the A H P duration in both fast and slow muscles. As reported by Zwaagstra and Kernell (1980), for cat's hind limb motoneurones the A H P duration also had a wide range (from 65 to 255 msec). The reason for the differences in these findings seems to require further analysis. Some information about the human after-potentials can be extracted from the Sabbahi-Sedgwick study (1987) on the H reflex recovery curves of single motoneurones in which early recovering (80-300 msec) and late recovering (up to 3000 msec) motoneurones of the soleus were shown. Our data obtained on the soleus are in keeping with the recovery profile of the early recovering neurones, while late recovering motoneurones were not observed. The reason for such a discrepancy is not clear; it might be attributed to the differences in the details of applying the H reflex methodology (in particular, in the stimulus strength). According to data reported by Sabbahi and Sedgwick, the recovery time of a motoneurone was constant so long as conditions remained the same but was shortened by some factors such as background activity of the muscle under study, mild tension of body, head or arm movements, the late recovering motoneurones showing a considerable recovery time reduction. This is not in contrast with the results presented demonstrating the similarity in the excitability recovery time of a motoneurone after its single reflexive and single volun-
351
tary discharges, since in our experiments the subjects were relaxed completely. So far, the excitability recovery of motoneurones after their single discharges has not been used for investigating the A H P and D D in man. At the same time paired stimulation of Ia afferents (the " H reflex recovery curve") has been widely used to measure the excitability of the whole motoneurone pool. Many authors have emphasized that the H reflex recovery curve is difficult to interpret because of the number of factors involved, though the after-potentials have not been considered very important. Our results demonstrate, however, that the A H P and D D make a significant contribution to the genesis of the H reflex recovery curve for motoneurones of slow and fast muscles. Our data also show that the motoneurones were not affected by inhibition, which confirms the results reported by Sabbahi and Sedgwick (1987). At the same time, the H reflex recovery profile demonstrates the "secondary depression" which is usually attributed to presynaptic inhibition, the latter being abolished by voluntary muscle contraction (Person 1985; Schieppati 1987; Burke et al. 1989). It appears from the present findings that the presynaptic inhibition affects those motoneurones of the pool which have a higher threshold to stimulation but not the low threshold to Ia stimulation as well as voluntary muscle contraction (i.e., the small fatigue resistant ones) tested by us. We believe that these factors should be taken into account when interpreting the H reflex recovery curve of the motoneurone pool in healthy and diseased subjects. Analysis of motoneurone after-potentials with regard to repetitive firing parterns of motoneurones revealed that some of the fast muscle motoneurones, namely, those showing the early excitability recovery, were capable of firing doublets during voluntary muscle contraction. The intradoublet interval duration was within the same range as the D D duration. The findings agree with those reported by Calvin and Schwindt (1972) showing that D D in cat motoneurones is associated with a doublet firing pattern. It has been generally believed that the long-lasting A H P following each cat's motoneurone spike is a factor of great importance as an intrinsic mechanism for the regulation of repetitive firing (Eccles et al. 1958; Kernell 1965). This view has been strongly supported by model studies in which both repetitive firing and voltage trajectories within an interspike interval can be well predicted on the properties of the A H P (Baldissera and Gustafsson 1974; Gustafsson 1974). Moreover, it has been known that motoneurone repetitive firing at the minimal rates is related to the A H P duration (Kernell 1965). Our results obtained with the use of natural activation of MUs in man demonstrated that the minimal
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firing rate of human motoneurones activated by gentle voluntary contraction of both slow and fast muscles was not correlated with the AHP duration. After some training the human subjects were able to maintain repetitive firing of MUs with interspike intervals longer than the AHP duration. In the previous reports it has been noted that under conditions of motoneurone natural activation the range of firing rates is extended to the field of lower frequencies within which the motoneurone excitability and inhibitability are increased (Ashby and Zilm 1982; Miles and Tiirker 1986; Kudina 1988; Kudina and Pantseva 1988; Miles et al. 1989b). On the basis of these data and the results presented it can be assumed that within the low range of firing frequencies (in contrast to the higher "primary range" investigated in animal experiments) the AHP does not play a leading role in controlling repetitive firing of human motoneurones. This firing range, which can be considered "subprimary," seems to be regulated, in addition to the AHP, by some other mechanisms of spinal and supraspinal control "switched off" under anaesthesia and spinalization used in animal experimentation. The authors are grateful to L. Kravtsova for scrutinizing the English.
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