TWITCH FORCE AND ACTION POTENTIALS OF

ACTA NEUROBIOL. EXP. 1988. 48: 11-81

TWITCH FORCE AND ACTION POTENTIALS OF SINGLE MOTOR UNITS IN MEDIAL GASTROCNEMIUS MUSCLE OF'THE RAT Kazimierz GROTTEL, Jan CELICHOWSKI and KRZYSZTOF KOWALSKI Department of Neurobiology, Academy of Physical Education in Poznah Bema 10, 61-555 Poznan, Poland

Key words: motor unit, twitch force, action potentials

Abstract. On the basis of a "sag" test in unfused tetanus we classified fast ana slow motor units (MUs) in medial gastrocnemius muscle of the rat. The following parameters of motor units were investigated: single twitch force, duration of the action potentials, maximum-minimum amplitude time (M-MAT), action potentials amplitude and their latencies after stimulus (to the onset of potentials) and the diameter of MU territory in muscle medio-lateral direction. Differences were found between fast and slow MUs in all of the properties. A correlation was found between MU twitch forces and the diameters of territories and amplitudes of action potentials in The whole investigated MU population, as well as between the duration and the M-MAT of the action potentials. INTRODUCTION

Previous experiments have shown that fast and slow muscle units differ with respect to their twitch force and parameters of their action potentials (3, 14, 14-17, 23, 24). The experiments were mainly carried out on cats 1, 3-7, 14-16) and concerned particularly the medial gastrocnemius muscle (4, 6, 7). In rats, the properties of this muscle are not well known, although there are some publications on motor units (MUs) of other muscles (8, 9, 12, 17). The experiments to be described extend our previous study (13) on the motor unit action potentials of the rat

medial gastrocnemius muscle by investigating the relation between MU twitch force and properties 'of action potentials. The classification of motor units as fast or slow has been based on presence or absence of "sag" in unfused tetanus, using criteria of Burke et al. (6). METHODS

The experiments were carried out on 25 adult Wistar rats, weighing 280-480 g, under general pentobarbital anesthesia (Vetbutal 30 mglkg

Fig. 1. Records of MU action potentials and twitch force of two MUs stimulated a t the same time. A, records of action potentials of MUs labeled I and 11 at successive depths, those of the unit I being more delayed than the potentials of the unit 11. The figures to the left of each record indicate the depth of the electrode from the surface of the muscle as shown in B. The action potentials recorded from 4.0 ta 5.5 mm are polyphasic. B, points of depth recording from the left medial gastrocnemius muscle in the medio-lateral direction. The dashed line shows the place of junction with the lateral gastrocnemius. C , record of tension developed by the two MUs stimulated simultaneously. The contribution of units I and II is clearly evident. D, the unfused tetanus of those units at stimulation of 40 Hz.

i.p., supplemented during the experiments with 5 mglkglh). The distal part of the medial gastrocnemius muscle was dissected free in order to connect its tendon to the force transducer but great care was taken to

:?-\

2 G l ass

LEFT MG

ass

'---Fig. 2. Records of action potentials, twitch force and unfused tetanus of a slow MU. A, records of action potentials at successive depths. B, the MU constant twitch force in successive records. C, the unfused tetanus recorded with stimuli at 20 and 40 Hz. No "sag". D, sites of records as in Fig. 1B.

Fig. 3. Records of action potentials, twitch force and unfused tetanus of a fast MU. Records from the same muscle and in the same plane as those from the slow MU, illustrated in Fig. 2. A, records of action potentials. B, decrease in the twitch force during successive records. C, the mfused tetanus at 40 Hz stimulation. The "sag" can be noticed. The figures indicating the depth of t h e electrode refer to Fig. 20.

leave the blood supply of the muscle intact. All other muscles of the hind limb, except the pelvic muscles, which were inaccessible during the experiments, were denervated and the most distal nerve branch innervating the investigated muscle was cut. The knee of the operated limb was fixed in a steel clamp and the thigh bone was cut before the muscle was attached to the force transducer. The laminectomy was performed at the level of L1 to S1 vertebrae and animals were spinalized at L1-L2 level. The exposed spinal roots and the medial gastrocnemius muscle were covered with warm paraffin oil. The body temperature was maintained at 37 1°C. In order to obtain twitches of single MUs, small filaments of ventral roots L5 or L4 were stimulated electrically: they were split until electromyographic and twitch responses recorded from successive points across the muscle showed that only a single motor units was active. When more than one motor unit was stimulated, the muscle action potentials showed notches due to different latencies of activation of individual motor units, as noted in records from the depth of 4-5.5 mm (see Fig. 1A). A contribution of more than one MU could also be seen in twitch records, as illustrated in Fig. 1C. Examples of records from single units are shown in Figs. 2 and 3. Single stimuli and trains of stimuli (0.3 ms duration, 0,2-1.0 V) were used. The trains frequency was 20 to 40 Hz: the train duration was up to 0.5 s. The tensions developed during single twitches and tetanus contractions were recorded with inductive force transducer (with compliance 50 pm/25 g). Prior to recording, the muscle was stretched to passive tension of about 7 g, to allow recording of maximal force of single MU twitch. Muscle action potentials were recorded bipolarly, using double silver electrode, described in the previous paper (13). The electrode was moved in medio-lateral direction, as illustrated in Figs. 1B and 20. The electrodes had recording surfaces of 150 X 75 pm. The distance between their centers was 160 pm. The recording surfaces were arranged successively along the muscle fibers: the proximal recording surface was positive and the distal one was negative. In the bipolar registration method the recording area is narrow (11). The electrode was inserted 0.5 cm distally to the medial side of the muscle and was moved about 0.5 mrn at a time. The successsively ~ecordedaction potentials were photographed at the screen of an oscilloscope, with three records superimposed. The twitch or tetanus forces were recorded simultaneously from the second oscilloscope screen. The animals were grounded via a wire inserted in the contralateral limb. We analyzed 7 motor unit properties: 1 - the presence or absence

+

of the "sag" in unfused tetanus, 2 - twitch force, 3 - maximal amplitude of the action potentials, 4 - duration of the action potentials, 5 maximum-minimum amplitude time (M-MAT), 6 - latency of onset of the action potential, 7 - diameter of MU territory determined with EMG records. The maximum-minimum amplitude time labeled M-MAT in text was measured between points indicating peak-to-peak amplitude of recorded spiks potentials. On the basis of the "sag" in unfused tetanus, the fast motor units were distinguished from the slow ones (4, 6, 14, 16). Means and standard deviations @D) were computed for all properties (on the basis of the mean results for each motor unit), separately for fast (n = 23) and slow (n = 9) motor units. Significance of differences between properties of fast and slow units and correlations between all investigated MU properties were computed. t-Student test was used. RESULTS

Amplitudes and latencies of action potentials of single motor units. The recorded action potentials were biphasic or triphasic, usually of the same configuration along the electrode track, as shown in Figs. 2A and 3A. The peak-to-peak amplitude of these potentials did not exceed 60 pV just outside the slow MU territory and 100 pV in the case of fast MUs. During insertion of the recording electrode between muscle fibres of the investigated MU, the potentials of higher amplitude appeared. The highest reached 720 pV (Table I). When the electrode was moved across the MU .territory, the amplitudes of the potentials first increased and then decreased. After having left the unit territory, the electrode again recorded only potentials of small amplitude. The latter were sometimes monophasic. When the recording electrode was inserted between muscle fibers of the investigated unit, duration of the action potential became also clearly shorter. The mean amplitude of action potential of fast MUs was 385 It 145 pV (mean 2 SD) while it was 187 f 160 pV (mean 2 SD) for slow units. The difference was statistically significant (P = 0.01, Table I). However, there was a considerable degree of variability, especially for fast units and the maximal amplitudes of potentials of some slow MUs were higher than those of some fast MUs (Table I). Also latencies of action potentials of single motor units varied. Sometimes they increased while in other cases they decreased in one track. For the fast MU group they ranged between 1.9 rns and 3.8 m while the latencies of slow units potentials ranged from 2.2 to 3.4 ms. Ther greatest differences between the latencies of the successively recorded action potentials amounted to 0.8 ms for the fast and to '0.4 ms for the slow motor units.

TABLEI Means of six properties of MUs, their standard deviations (SD) range of variability and results of t-Student test. The duration, the M-MAT and latency of action potentials are based on mean values obtained in several records from the indivdual MUs Twitch force

(GI

Max. amplitude of action potentials (PV)

MU territory Duration of diameter action potentials (mm) (ms) 2.03*0.92 1.75*0.33 1.20-2.34 1.0-4.0 n = 15 n = 15

M-MAT (ms)

Latency of action potentials (ms)

0.43&0.09 0.28-0.58 n = 15

2.53f0.39 1.95-3.53 n = 23

5 E

1.6*0.9 0.2-3.6 n = 23

385f145 180-720 n = 16

5E

mean *SD * range 2 number

0.49&0.45 0.14-1.40 n=9

187& 160' 72-530 n=7

0.93 40.45 0.5-1.5 n =7

2.17*0.50 1.30-2.70 n=7

0.50&0.06 0.43-0.60 n=7

2.77*0.36 2.23- 3.40 n=8

Significance of difference

0.002

0.01

0.01

0.05

0.10

0.20

mean f SD range ,$ number w

m

--

Mean latencies were 2.53 k 0.39 ms and 2.77 k 0.36 ms for fast and slow MUs, respectively. The difference between them was significant at P = 0.2 level. No correlation was found between the latencies and other properties of the investigated units. Duration and M - M A T of t h e action potentials. Table I shows that both the duration and the M-MAT of the fast MUs action potentials were shorter than those of slow MUs (significant differences, P = 0.05 and P = = 0.1, respectively). The two properties were correlated for the whole sample (Table 11) with correlation coefficient of 0.701 (n = 22, P = 0.01) and for fast MU group, with correlation coefficient of 0.673 (n;= 15, P = 0.01), but not for slow MUs. Sometimes changes in one of the parameters failed to be paralleled by changes of the other one when action potentials were recorded at different sites within the muscle. Similar results were obtained when the shortest values of the iduration and the M-MAT of action potentials of motor units were compared for fast and slow units. The mean values for fast MUs were 1.56 and 0.35 ms but for slow ones 1.91 and 0.43 ms, respectively. The differences between them were significant (P = 0.01 in both cases). TABLE I1 Correlation coefficients of investigated MUs properties Twitch Max. ampli- MU territory Duration of M-MAT force tude of action diameter action potenpotentials tials Max. amplitude of action potentials 0.499* 0.571** MU territory diameter 0.553** -0.237 Duration of action potentials -0.229 -0.145 M-MAT -0.133 Latency of action potentials -0.343 - 0.198 * P = 0.05; ** P = 0.01. --

-

-

-

-

- 0.200 -0.126 - 0.255

0.701** 0.344

0.161 -

The values of the M-MAT differed not only between different MUs but also within a series of records in the same MU. The differences amounted to 0.5 ms between different MUs but were less than 0.3 ms (mean 0.172 ms) for single MUs. Similar variability occurred in the duration of action potentials. The differences were 1.6 ms and 1.1 ms (mean 0.466 ms), respectively. Usually, potentials with the shortest MMAT occurred in the middle part of a MU territory. The same potentials exhibited usually the largest amplitudes and the shortest duration. Only values of the duration and the M-MAT of potentials recorded within MU territory were analysed. The potentials recorded outside the MU terri-

tory showed higher values of the parameters. No correlations were found between the M-MAT and the duration of action potentials on the on'e hand and other MU properties on the other. Twitch force and "sag" in unfused tetanus. MU twitch force was recorded simultaneously with action potentials. Means and ranges of values for slow and fast MU twitch forces are shown in Table I. Mean twitch forces of fast and slow MUs differred significantly from each other (P = 0.002), although the highest values of slow MUs and the lowesk values of fast MUs overlapped. Twitch force was determined in several registrations. A decrease of twitch force amplitude occurred sometimes in a series of records of twitch force, developed by some fast MUs. It amounted to 8-24010 of initial values. All slow MUs were resistant to repeated single stimuli. Figure 3B illustrates the decrease in a fast unit twitch force and Fig. 2B shows the stable twitch force of a slow unit. The correlation between twitch force on the one hand and maximal amplitudes of action potentials and diameters of MUs territory on the other are shown in Table 11. The correlation coefficient were 0.499 (n, = = 24, P = 0.05) and 0.553 (n = 31, P = 0.01), respectively. Usually 2 or 3 unfused tetani were registrated after recording action potentials and twitch force. The stimulation frequency optimal for evoking unfused tetanus was 40 Hz for fast and 20 Hz for slow units. Figure 3C shows a record of unfused tetanus of a fast unit and Fig. 2C shows such a record for a slow unit. Diameter of MU territory in a-medio-lateral direction. As mentioned above, the amplitude of action potentials of single motor units increased sharply when the electrode was inserted between MU muscle fibres and decreased when it left the MU territory. The diameters of MU territories defined in this way ranged from 0.5 to 1.5 mm for slow units and from 1.0 to 4.0 mm for fast ones (Table I). Statistically, they differred significantly (P = 0.01). The MU diameters correlated with MU twitch forces and with maximal amplitudes of action potentials (r = 0.571, n = 23, P = 0.01). No correlations with other MU properties were found. DISCUSSION

The MU action potential constitutes a sum of action potentials of all its muscle fibers. In some situations, e.g. when the electrode features small recording surfaces, the potentials can be recorded from a small group of muscle fibers or even from a single muscle fiber (10, 12, 21). Therefore, by following changes in potentials after changing position of the recording electrode we can draw conclusions about the extent of the MUs and their components (12, 21). Position of muscle fibers with regard

to the electrodes is of special importance (10, 12, 19, 21) and is reflected by changes in the amplitude and duration of the potentials which, in turn, allow to localise and determine the diameter of the investigated MU territory in the direction of measurement. However, configuration of these potentials depends on the position of the electrodes with respect to the muscle fiber end-plates (19). Since a greater variability has appeared among slow MUs, lower density of muscle fibers in these units can be suggested (4, 15), as previously concluded by Buchthal et al. (2) with respect to human muscles. Greater amplitudes of potentials of fast MUs may be due to larger amplitudes of potentials of single muscle fibers of these units, associated with their greater thickness (4, 15, 23) but also with a higher density in the transverse plane and a larger number of these fibers. The great variability in amplitudes of action potentials of MUs of the same type map be attributed to recording from different parts of MUs territories. The correlation between amplitudes of action potentials and twitch force of individual units is in keeping with the dependence of the two parameters upon the numberrnand the type of muscle fibers. Moreover, the correlation between the force and the diameter of MU territory suggests that the larger is the diameter of MU territory the greater is the number of muscle fibers it contains. The correlation between the duration and the M-MAT of action potentials may be explained by dependence of both these parameters upon the muscle fiber type (11, 19). Besides, the duration of action potentials would depend upon muscle fiber end-plate disposition (in muscle endplate zone) and upon a distance of MU muscle fibers (the potentials of which are recorded from) to the recording electrode (10, 11, 20, 21). StAlberg and Ekstedt (22) reported higher conductance velocity for the muscle fibers of a larger diameter while experiments of Wallinga-de Jonge et al. (24, 25) indicated shorter duration of action potentials of these fibers. However, the differences have been rather small. The results presented here confirm these observations and show that the difference between fast and slow MUs are significant at the level of only 0.05 or 0.10. We have not found any correlation between the duration or the M-MAT of action potentials and other properties of MUs. Lack of such a correlation may be due to a much smaller variability of time parameters when compared to a greater variability in amplitude of potentials, in twitch force and in diameters of MUs territories. Furthermore, the amplitude of the potential may depend primarily on the number of muscle fibers in the close vicinity of the recording electrode while the

force depends upon the total number and type of muscle fibers. Different conduction velocities of muscle fibers of different types are reflected by different latencies of responses of fast and slow MUs. Accordingly, action potentials of slow MU muscle fibres are more delayed. Certainly, this is a l s ~an effect of lower conduction velocity in d m MU motor axons (1, 6, 14, 18). The observkd difference between twitch force of fast and slow units is well known (4, 6, 14, 15). The observed decrease in the twitch force of some fast MUs after successive stimuli indicates that the rat medial gastrocnemius muscle contains both fatiguable and more fatigue resistant fast MUs. This investigation was partly supported by the Project CPBR 11.9. REFERENCES 1. BAGUST, J., KNOTT, S., LEWIS, D. M., LUCK, J . C. and WESTERMAN, R. A.,

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13. GROTTEL, K., CELICHOWSKI, J . and KOWALSKI, K. 1986. Spatial analysis of motor unit potentials of the rat medial gastrocnemius. Acta Physiol. Pol. 37: 219-227. 14. JAMI, L., MURTHY, K. S. K., PETIT, J. and ZYTNICKI, D. 1982. Distribution of physiological types of motor units i n the rat peroneus tertius muscle. Exp. Brain. Res. 48: 177-184. 15. KERNELL, D., DUCATI, A. and SJOHOLM, H. 1975. Properties of motor units in the first deep lumbrical muscle of the cat's foot. Brain Res. 98: 37-55. 16. KERNELL, D., EERBEEK, 0. and VERHEY, B. A. 1983. Motor unit categorization on basis of contractile properties: an experimental analysis of the composition of the cat's m. peroneus longus. Exp. Brain Res. 50: 211-219. 17. KUGELBERG, E. 1973. 1-Iistochemical composition, contraction speed and fatiguability of r a t soleus motor units. J. Neurol. Sci. 20: 177-198. 18. LEWIS, D. M., PARRY, A. J. and ROWLERSON, A. 1982. Isometric contractions of motor units and immunochemistry of mouse soleus muscle. J. Physiol. 325: 393-401. 19. MILLER-LARSSON, A. 1985. Analysis of distribution of field of motor unit action potential in skeletal muscle; a theoretical model and experimental verification (in Polish). Ph. D. thesis, Nencki Inst. Exp. Biol., Warsaw, 301 p. 20. STALBERG, E. 1980. Macro EMG, a new recording technique. J . Neurol. Neurosurg. Psychiatr. 43: 475-482. 21. STALBERG, E. and ANTONI, L. 1980. Electrophysiological cross section of the motor unit. J. Neurol. Neurosurg. Psychiatr. 43: 469-474. 22. STALBERG, E. and EKSTEDT, J. 1973. Single fibre EMG and microphysiology of the motor unit in normal and diseased human muscle. New Develop. Clin. Neurophysiol., Desmedt, p. 113-129. 23. STALBERG, E. and THIELE, B. 1975. Motor unit fibre density in the extensor digitorurn communis muscle. Single fibre electromyographic study in normal. subjects at different ages. J. Neurol. Neurosurg. Psychiatr. 38: 874-880. 24. WALLINGA-de JONGE, W., GIELEN, F. L. H., BROENINK, J., d e JONG, P., van der MEER, E. and WIRTZ, P. 1984. Electrical activity of slow and fast fibres i n rat skeletal muscle. Proc. of a workshop held a t "De Hooge Vuursche", Baarn-Holland, 24-27 February 1984. 25. WALLINGA-de JONGE, W., GIELEN, F. L. H., WIRTZ, P., de JONG, P. and BROENINK, J. 1985. The different intracellular action potentials of fast and slow skeletal fibres. Electroencephalogr. Clin. Neurophysiol. 60: 539-547. Accepted 25 November 1987

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