Summation of motor unit forces in rat medial gastrocnemius muscle

Journal of Electromyography and Kinesiology 20 (2010) 599–607

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Summation of motor unit forces in rat medial gastrocnemius muscle H. Drzymała-Celichowska *, P. Krutki, J. Celichowski ´ , Poland University School of Physical Education, Department of Neurobiology, 55 Grunwaldzka St., 60-352 Poznan

a r t i c l e

i n f o

Article history: Received 28 September 2009 Received in revised form 23 December 2009 Accepted 25 January 2010

Keywords: Evoked contraction Force summation Medial gastrocnemius Motor unit Rat

a b s t r a c t The summation of contractile forces of motor units (MUs) was analyzed by comparing the recorded force during parallel stimulation of two and four individual MUs or four groups of MUs to the algebraic sum of their individual forces. Contractions of functionally-isolated single MUs of the medial gastrocnemius muscle were evoked by electrical stimulation of thin filaments of the split L5 or L4 ventral roots of spinal nerves. Additionally, contractions of large groups of MUs were evoked by stimuli delivered to four parts of the divided L5 ventral root. Single twitches, 40 Hz unfused tetani, and 150 Hz fused maximum tetani were recorded. In these experimental situations the summation was more effective for unfused tetani than for twitches or maximum tetani. The results obtained for pairs of MUs were highly variable (moreor less-than-linear summation), but coactivation of more units led to progressively weaker effects of summation, which were usually less-than-linear in comparison to the algebraic sums of the individual forces. The variability of the results highlights the importance of the structure of the muscle and the architecture of its MUs. Moreover, the simultaneous activity of fast and slow MUs was considerably more effective than that of two fast units. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The force of muscle contractions is controlled by the central nervous system in two fundamental ways: by the recruitment and decruitment of motor units (MUs) and by the regulation of the motoneuronal firing rate. The summation of the forces of individual fibers and MUs during voluntary contractions have been fairly well studied, but there remain numerous discrepancies in the literature. In the first study dealing with this topic it was observed that the tetanic force recorded during simultaneous contractions of several slow motor units was always considerably higher than the sum of the forces produced by each unit stimulated separately (Emonet-Dénand et al., 1987). The measured forces were larger than the cumulative forces, being related to the number of activated MUs (more-than-linear summation). Clamann and Schelhorn (1988) stimulated pairs of motor units in medial gastrocnemius or soleus muscles, first individually, then together, at the same constant frequencies. On average, the combined tetanic forces of two MUs exceeded the algebraic sum of their respective forces by 12% in the medial gastrocnemius and 5% in the soleus muscles. These authors concluded that MUs produce more force when interacting than when working in isolation. Moreover, they observed that after cessation of the stimulation of one of these units, the remaining unit produced more force than when stimulated alone. In another series of experiments the force developed * Corresponding author. Tel.: +48 61 8355435; fax: +48 61 8355444. E-mail address: [email protected] (H. Drzymała-Celichowska). 1050-6411/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jelekin.2010.01.006

by the combined stimulation of two to four MUs in the cat tibialis posterior muscle was compared to the algebraic sum of the forces produced by each respective MU (Powers and Binder, 1991). The measured force was, on average, 20% greater than the algebraic sum of the individual unit forces. On the other hand, less-than-linear summation of MU forces was demonstrated in the studies of Sandercock (2000, 2003), who divided ventral roots of spinal nerves into two bundles of axons, each innervating about half of the soleus or tibialis anterior muscles. Summation nonlinearities were also observed during interactions of 10 individual MUs and 4 groups of MUs, each containing approximately 10 units (Perreault et al., 2003). These authors hypothesized that more-than-linear summation only occurs when a few motor units are active, whereas less-than-linear summation prevails when more units are active and their force exceeds around 10% of the total muscle force. Troiani et al. (1999) examined the linearity of summation of forces produced by the stimulation of various combinations of type-identified MUs in the cat peroneus longus muscle. In general, slow (S) and fast resistant to fatigue (FR) MUs showed more-than-linear summation, fast fatigable (FF) MUs produced either more- or less-than-linear summation, whereas when FF units were recruited after S or FR units, the summation was always less-than-linear. The divergent results of the studies outlined above might be because the analyses were performed on different muscles (with respect to their size, composition, position of fibers or biomechanical function) and/or with incomparable methods. Therefore, the main purpose of the present study, performed on the heterogeneous rat

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medial gastrocnemius (MG) muscle, was to systematically examine the effects of summation of the contractile forces of two or four type-identified MUs, as well as four large groups of units during parallel stimulation of axons innervating these units. In addition, this study was designed to supplement and expand the results of previous investigations focusing on the MG by comparing the effects of summation of MU forces for twitch contractions evoked by single stimuli as well as for unfused and fused tetanic contractions evoked by 40 Hz and 150 Hz trains of stimuli, respectively.

2. Methods Experiments were performed on 15 adult male Wistar rats, aged 5–6 months and weighing 430–590 g. The animals were housed two per cage, in the same room with the temperature maintained at 20 ± 1 °C. All experimental procedures followed European Union guidelines on animal care as well as the principles of Polish Law on The Protection of Animals and were approved by the Local Bioethics Committee. During all experimental procedures the rats were kept under pentobarbital anesthesia (Morbital, Biowet Puławy, initial dose 60 mg/kg i.p. supplemented after 2 h with additional doses of approximately 10 mg/kg/h). The depth of anesthesia was controlled by monitoring limb withdrawal reflexes. After the experiments, the animals were euthanized with an overdose of pentobarbital (180 mg/kg). 2.1. Surgery The surgical preparation of the rats was performed in two phases. In the first, the MG muscle was partially exposed, carefully isolated from surrounding tissues and separated from other muscles. All the collateral branches of the sciatic nerve leading to the limb muscles were cut, except that innervating the MG. Laminectomy was performed over the lumbar and sacral segments of the spinal cord. In the exposed area, the dura mater was cut and removed. The dorsal and ventral roots of spinal nerves were cut proximally to the spinal cord. The animals were positioned on a warm aluminum plate and immobilized with steel clamps on the tibia, and the Th13 and S2 vertebrae. The operated hind limb and the exposed areas of the spinal cord were covered with paraffin oil which was maintained at 37 ± 1 °C by an automatic heating system. The MG muscle was connected via the Achilles tendon to an inductive force transducer (model FT-510, BIO-SYS-TECH, sensitivity of 100 lm/100 mN).

converter (RTI-800) at sampling rates of 1 kHz for the force records and 10 kHz for the action potentials. 2.3. Experimental protocol The effects of motor unit force summation were examined in three separate series of experiments: (1) two isolated MUs were stimulated separately and then together; (2) four isolated MUs were stimulated separately and together; and (3) four groups of units were stimulated separately and together. The contractions of groups of MUs were examined by splitting the L5 ventral root into four bundles of axons and then applying stimulation (each bundle was most likely composed of >10 units) (Hashizume et al., 1988; Celichowski and Drzymała-Celichowska, 2007). During the trials with combined stimulation of two MUs the following protocol was applied: (i) the averaged single twitch of the first unit (five stimuli at 1 Hz); (ii) the averaged single twitch of the second unit; (iii) the averaged single twitch of the first and the second unit during their simultaneous stimulation; (iv) the unfused tetanus of the first unit (500 ms train of pulses at 40 Hz); (v) the unfused tetanus of the second unit; (vi) the unfused tetanus of the first and the second unit during their simultaneous stimulation; (vii) the maximum tetanus force of the first unit (200 ms train of pulses at 150 Hz); (viii) the maximum tetanus force of the second unit; and (ix) the maximum tetanus force of first and the second unit during their simultaneous stimulation. All trains of stimuli were separated by 5 s intervals. During recordings, the MG muscle was stretched to a passive tension of 100 mN, which ensured measurement of the highest contractile force of the MUs under isometric conditions (Celichowski and Grottel, 1992). The stimulation protocols for the summation of forces of four motor units and of four groups of units consisted of similarly organized stimulation patterns, but the number of steps was multiplied. The force evoked during simultaneous stimulation of four groups of units was also recorded under isometric conditions, but during these trials the MG muscle was stretched to a passive tension of 400 mN, which ensured measurement of the highest twitch force of the muscle (Celichowski and Grottel, 1992). Finally, the resistance to fatigue was tested for all studied motor units using the standard fatigue test (40 Hz trains of stimuli lasting 330 ms and repeated every second for 4 min) (Burke et al., 1973). The forces produced by the combined stimulation of two MUs, four MUs, and four groups of units of the rat MG muscle were compared to the algebraic sum of the forces produced by the respective MUs. 2.4. Data analysis

2.2. Stimulation and recording Functional isolation of MUs was achieved by splitting the L5 or L4 ventral roots of the spinal nerves into thin filaments, which were electrically stimulated with suprathreshold rectangular pulses (amplitude 60.5 V, duration 0.1 ms) generated by a dual channel square pulse stimulator (Grass Instrument Company, model S88) and delivered via four channel silver wire electrodes (0.5 mm diameter) to evoke contractile activity of MUs. Activity of an isolated MU was indicated by the ‘all-or-none’ appearance of both a twitch contraction and a motor unit action potential in response to stimuli of increasing amplitude (Kuffler et al., 1951; Celichowski, 1992). The MU action potentials were recorded using a bipolar silver electrode inserted into the muscle perpendicularly to the muscle fibers, with a distance between electrodes of 5 mm, and amplified using a low-noise multi-channel preamplifier (World Precision Instruments, model ISO-DAM8-A). A ground electrode was inserted into the muscles of the opposite hind limb. The recorded data were stored on a PC using an analog-to-digital 12-bit

All investigated MUs were classified as fast or slow on the basis of sag appearance, which is absent in slow MUs (Burke et al., 1973; Grottel and Celichowski, 1990). Fast MUs, with a sag, were divided into FF and FR types on the basis of the fatigue index, which was <0.5 for the FF and >0.5 for the FR (Kernell et al., 1975; Grottel and Celichowski, 1990). For each averaged MU twitch, a number of parameters were analyzed: the twitch force (TwF, measured from the baseline to the peak), the contraction time (CT, from the onset of the force increase to the peak) and the half-relaxation time (HRT, measured from the peak force to half of its maximum). In each case, the unfused tetanus force (40 Hz) was measured for the final contraction following the last of the stimuli within a train to avoid dynamic changes of force at the beginning of a tetanus including the sag phenomenon observed in fast MUs (compare the records presented in Figs. 1C and 2B). The amplitude of force oscillation was also measured for the final contraction of the unfused 40 Hz tetanus as the amplitude of force increase following the last stimulus. The maximum tetanic

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Fig. 1. Records of the contractile forces of twitches (A and B), 40 Hz unfused tetani (C) and 150 Hz fused tetani (D) evoked by separate activation of two MUs (left-hand side of each panel) and by their simultaneous stimulation (right-hand side of each panel). Two opposite summation effects are illustrated: more-than-linear summation (B and C, upward arrows) or less-than-linear summation (A and D, downward arrows). The physiological types of MUs are indicated: FF – fast fatigable; FR – fast resistant to fatigue; S – slow. Dashed horizontal lines denote the highest force level in each case: either the sum of contractions of two MUs (A and D) or the peak force during the coactivation of two MUs (B and C).

All of the above parameters were analyzed using identical methods from force records evoked following activation of each single MU or group of units, as well as coactivation of two MUs, four MUs or four groups of units. For statistical evaluation of the presented results the non-parametric tests were used: the ANOVA rank Kruskal–Wallis test for comparisons of the force summation effects with respect to the number of active MUs; the ANOVA Friedman test for comparisons between the forces of single twitches, 40 Hz tetani and maximal tetani; the Mann–Whitney U-test for comparisons between the force summation effects with respect to the MU type. 3. Results A total of 128 MUs were investigated in this study: 51 FF, 50 FR and 27 S MUs. Force summation of 2 MUs was analyzed in 40 pairs of units, while force summation of four MUs was studied in 12 cases. Additionally, in 5 separately performed experiments, summation of the forces of four groups of MUs was studied. 3.1. Effects of the summation of forces with respect to the number of active MUs

Fig. 2. Records of the contractile forces of twitches (A), 40 Hz unfused tetani (B) and 150 Hz fused tetani (C) evoked by separate activation of four MUs (left-hand side of each panel) and by their simultaneous stimulation (right-hand side of each panel). Approximately 5% less-than-linear summation effects found for twitch contractions and fused tetani are illustrated (A and C), while for unfused tetani, only slightly lessthan-linear summation was observed (B). MU type and other features as in Fig. 1.

force (TetF) was measured for the fused tetanus (150 Hz) (Celichowski and Grottel, 1995; Hennig and Lømo, 1985).

Considerable diversity was found in the results of trials examining force summation of two MUs, especially for the twitch and the unfused tetanic contractions (Table 1). Some of the observed effects were evidently more-than-linear (in one case the force of two simultaneously active MUs was 35.4% greater than the algebraic sum of separately active MUs for twitches, and 45% higher for 40 Hz unfused tetani; see Table 1), whereas in other cases they were less-than-linear (up to 25.6% or 21.8%, for the twitch and 40 Hz tetanic contractions, respectively) (Fig. 1). For twitches, more-than-linear and less-than-linear effects of force summation were observed in 24 and 16 pairs of units, respectively. However, despite the similar range of values reflecting force summation effects for the unfused tetani, more-than-linear summation predominated for this type of contraction, being found in 31 of the 40 cases

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Table 1 Differences between the force recorded during simultaneous activation of 2 MUs, 4 MUs or 4 groups of MUs, and the algebraic sum of their separately recorded forces.

Data are mean values ± standard deviations, and variability ranges are given below in parentheses. The negative values are effects of less-than-linear summation whereas positive values are effects of more-than-linear summation. The statistically significant differences between effects of force summation for 2 MUs, 4 MUs and 4 groups of MUs are indicated by asterisks, ** – the difference significant at p < 0.01, * – the difference significant at p < 0.05 (ANOVA Kruskal–Wallis test). The statistical significance of differences between force summation effects of twitches, unfused or fused tetani are presented in the right part of the table, ## – the difference significant at p < 0.01, # – the difference significant at p < 0.05, NS – the difference non-significant, p > 0.05 (ANOVA Friedman test).

(see the example in Fig. 1C). For the maximum tetani, slightly smaller variability of the differences between the force of a pair of coactivated MUs and the algebraic sum of the same MUs activated separately was observed (Table 1). More-than-linear or less-than-linear summation effects were recorded, with the latter being predominant (15 and 25 cases, respectively), as presented in Fig. 1D. Less diverse results were obtained in the trials examining the forces of four MUs stimulated simultaneously or individually. In most cases, simultaneous stimulation led to the development of contractile forces that were lower than the algebraic sum of the forces of separately activated units (Table 1, Fig. 2). For 12 analyzed twitch contractions, less-than-linear (10 cases, up to 27%) or morethan-linear effects (2 cases, up to 7.5%) were observed. For the unfused tetani, the differences between the forces of four coactivated MUs and the sum of the forces of the individual MUs were usually minimal, but the less-than-linear summation pattern again predominated (10 of 12 cases). When the summation of forces of maximum tetani of four MUs was examined, only less-than-linear effects were observed. When the effects of coactivation of four groups of MUs were tested, only less-than-linear summation was observed, and the range of variability of force values was the smallest (Table 1). As presented in Fig. 3, the forces of simultaneously contracting MUs activated by axons in all four parts of the L5 ventral root were considerably smaller than the algebraic sum of forces measured when each part of this ventral root was stimulated separately. The histograms presented in Fig. 4 summarize the relative differences between recorded and mathematically added forces for different numbers of MUs. They show the changes in the effectiveness of force summation as the number of coactivated MUs increased, from high variability and frequently more-than-linear effects recorded for two MUs, towards the more regular occurrence of less-than-linear summation for 4 MUs, and the uniformly lessthan-linear summation for large groups of MUs. It is also apparent from Fig. 4 that irrespective of the number of coactive motor units, the forces of 40 Hz unfused tetani summated more effectively than the forces of twitches or the 150 Hz maximum tetani. 3.2. Effects of summation of forces with respect to the type of active MUs The recordings in trials examining the summation of forces of two MUs were further analyzed according to the types of isolated MUs in each pair. Stimulation of two fast MUs (FF and FF, FF and FR or FR and FR) was studied in 20 cases, of one fast and one slow MU in 19 cases, and of two slow MUs in just one case. It should be noted that although the MG muscle is heterogeneous,

Fig. 3. Records of the contractile forces of twitches (A), 40 Hz unfused tetani (B) and 150 Hz fused tetani (C) evoked by separate activation of four groups of MUs, each most likely composed of >10 units (left-hand side of each panel), and by their simultaneous stimulation (right-hand side of each panel). Note the clearly lessthan-linear force summation effect observed in each case. Features as in Fig. 1.

its composition with respect to MU type is uneven, with FF, FR and S MUs comprising 51%, 37% and 12% of MUs in male rats, respectively (Celichowski and Drzymała, 2006). Therefore, the simultaneous isolation of two S MUs would appear to be rather exceptional. As shown by the histograms presented in Fig. 5, the differences between the forces of two simultaneously activated MUs and the sum of the separately recorded forces of the same units varied depending on the types of MUs in each pair. When the forces of two fast MUs were examined, the mean values (±SD) of the summation effects were 2.77 ± 12.59%, 10.12 ± 16.43%, and 3.05 ± 6.36%, for single twitches, 40 Hz unfused tetani and 150 Hz maximum tetani, respectively. However, for the twitch and maximum

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difference (%) Fig. 4. Comparison of the effects of force summation for twitches (A), 40 Hz unfused tetani (B), and 150 Hz maximum tetani (C), for all MUs tested. Histograms present the differences (as a percentage) between the contractile forces recorded during simultaneous activation and the algebraic sum of separately recorded forces for two MUs (black bars), four MUs (gray bars) and four groups of MUs (white bars). Note that coactivation of groups of MUs was always less effective than the sum of forces evoked by their separate stimulation, and that for each type of contraction, clearly positive effects of coactivation (>10% of more-than-linear summation) were observed only for pairs of MUs.

tetanic contractions, less-than-linear effects prevailed (12 and 14 out of 20 cases, respectively), while for the unfused tetani, morethan-linear effects predominated (16 out of 20 pairs). The corresponding calculations for a pair composed of one fast and one slow MU resulted in mean summation effect values of 7.42 ± 11.72%, 5.83 ± 9.31% and 1.94 ± 6.17%, for the twitch, unfused and maximum tetani, respectively. For the twitch and unfused tetanic contractions, a more-than-linear summation of forces was observed in 15 of the 19 pairs of MUs. Differences between effects obtained for two fast and fast-slow MUs were statistically significant at p < 0.01 for twitches, and p < 0.05 fused tetani (the Mann–Whitney test). However, for the maximum tetani, the numbers of more- and less-than-linear effects were nearly equal: 9 and 10 cases, respectively. The contraction times of various MUs differ, especially when fast and slow units are compared. In the present study the twitch forces

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difference (%) Fig. 5. Comparison of the effects of force summation, for twitches (A), 40 Hz unfused tetani (B), and 150 Hz maximum tetani (C) with respect to MU type. Histograms present differences (as a percentage) between the contractile forces recorded during simultaneous activation of two MUs and the algebraic sum of separately recorded forces of 20 pairs of fast MUs (black bars), 19 pairs of fast and slow MUs (gray bars) and one pair of slow MUs (white bars). Note that for two fast MUs, more-than-linear effects of force summation prevailed only in unfused tetani (17 out of 20 cases, the right-hand part of the histogram in B), while for twitch contractions (A) and maximum tetani (C), less-than-linear effects predominated. In contrast, the histograms of the differences between the effects of coactivation and separate activation of fast and slow or two slow MUs show a similar distribution of calculated values for all types of contractions.

were measured at the points of the peak amplitudes of original records, which might influence the force summation calculated with the applied method. To reassess the reliability of these results, algebraic sums of twitch force values measured at the same point following the stimulus (exactly at the time of the peak force for a faster contracting MU) were also calculated. This demonstrated that the two methods gave comparable values. The relative differences between the summated values of twitch forces for one fast and one slow MU, calculated with both of the above methods ranged from 0.1% to 7.1% (mean 1.7 ± 2.3%). Moreover, the mathematical summation of the forces of two fast MUs produced

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Fig. 6. The decrease in the amplitude of the last twitch calculated for responses to the last stimuli within the 40 Hz unfused tetani. The last fragments of unfused tetani records marked by a frame (A) for two fast MUs during their separate activation (left) and coactivation (right) are presented enlarged (B). Curly brackets indicate the analyzed amplitudes of the last twitch force (left, a and b) and are copied to the right-hand part of the figure showing the coactivation of the two MUs to indicate the expected amplitude of the twitch force. Note that the recorded twitch force is smaller (upward arrow) than the expected one, although the summation of forces is more-than-linear (downward arrow). MU type indicated as in Fig. 1.

relative differences between the separate calculation methods that were even smaller, varying between 0% and 4.5% (mean 1.4 ± 1.7%). 3.3. The amplitude of the last twitch within the unfused tetani The amplitude of the last twitch (amplitude of the force increase) in response to the last stimulus within the 40 Hz unfused tetanus was also analyzed and compared to value predicted on the basis of the algebraic sum of these amplitudes for individual MUs (Fig. 6). A decrease in the amplitude of the last twitch was usually observed during the coactivation of two and four MUs or four groups of MUs. Upon the coactivation of two MUs, the amplitude was decreased in the majority of cases (36 out of 40 pairs) by a mean value of 15.6 ± 12.9% (range 50.3% to 0%). In the four remaining cases the force of the last twitch was increased by 2.3–10.3%, with a mean value of 5.6 ± 2.9%. When the summation of forces of four MUs was examined, decreased force of the last twitch was observed in 7 of 12 cases, with a mean value of 20.9 ± 10.6% (range 38.0% to 11.1%). In the five remaining cases, the amplitude of the last twitch increased by a mean value of 10.7 ± 9.0% (2.6–24.3%). In five trials examining the summation of forces of four groups of MUs, a considerable decrease in the amplitude of the last twitch was observed in all cases, with a mean value of 29.5 ± 19.5% (range 48.7% to 7.1%). 4. Discussion 4.1. Variability in the effects of summation of motor unit forces One of the principal findings of this study is that forces produced by MUs within one muscle during simultaneous stimulation summate nonlinearly and can be highly variable. The summation of forces may be more-than-linear (by up to 45.0%) or less-thanlinear (by 33.7%). It was found that the summation effects observed for two coactive MUs do not depend on their individual forces but are probably due to biomechanical reasons (Powers and Binder, 1991), particularly differences in the geometry of the units in the pennate medial gastrocnemius muscle (Zuurbier and Huijing, 1992), which influence their force transmission to the tendon (Powers and Binder, 1991). Significant variability of summation effects has been reported previously, although only one dominant observation was made in the separate studies: less-than-linear

summation (Sandercock, 2000; 2003) or the more-than-linear summation (Emonet-Dénand et al., 1987; Clamann and Schelhorn, 1988; Perreault et al., 2003). However, it should to be emphasized that in these studies on MU force summation the experiments were restricted to the analysis of only one type of contraction with a constant number of motor units. The results of the present study revealed that the effect of force summation was not uniform for various types of evoked contractions. In most cases examined (77% of pairs, 75% of sets of four MUs, 100% of groups of MUs), the forces of unfused tetani summated more effectively than those of twitches or maximum fused tetani. Moreover, the higher the number of MUs activated simultaneously, the lower the force recorded in comparison to the algebraic sum of the individual MU forces. This finding, which confirms a suggestion by Perreault et al. (2003), was extended by the observation that force summation was usually less effective during simultaneous stimulation of two fast motor units (of either the FR or FF type) than during the coactivation of fast and slow MUs, especially for twitch and maximum tetanic contractions (compare histograms in Fig. 5). This is consistent with the results of Troiani et al. (1999), obtained in experiments on MUs of the cat peroneus longus muscle. The effectiveness of summation of MU forces in skeletal muscles undoubtedly depends on the structure of the muscles and architecture of their motor units. The rat medial gastrocnemius is a unipennate muscle with an angle of 19.4 ± 1.5° (Zuurbier and Huijing, 1992) or 20.4 ± 5.5° (Gallo et al., 2004) between its fibers and the long axis of the muscle, and is comprised of muscle fibers of 10.7–13.8 mm in length (Zuurbier and Huijing, 1992; Ettema, 1996). The average length of the unstretched muscle is 24.4 ± 1.1 mm (Celichowski et al., 1997). In the human MG muscle, the angle between its fibers and the long axis of the muscle is 16.7 ± 4.4° (Liber, 2002), which is similar to the value in the rat muscle. The muscle geometry changes with alterations in muscle length (Huijing and Woittiez, 1985), whereas the angle of pennation of muscle fibers is likely to contribute to nonlinearities in force summation (Sandercock, 2000). When part of a muscle contracts, the angles of pennation of the remaining muscle fibers are modified, changing the working environment of simultaneously active MUs (Zajac, 1989; Sandercock, 2000). In the present study, moreor less-than-linear force summation was observed, although the latter effect predominated when more than two units were activated. In contrast, the MU force summation effects observed for 3–10 units in the cat peroneus longus muscle were all more-

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than-linear (Emonet-Dénand et al., 1987). However, the structure of muscle fibers within these two muscles is clearly different, with a considerably smaller pennation angle in the peroneus longus in comparison to the medial gastrocnemius (e.g. on average 10.0° vs. 16.7°, for the respective human muscles) (Liber, 2002). Among factors contributing to nonlinear force summation, the ultrastructure of single muscle fibers, which influences force transmission to the tendon, should also be considered. Contractile force generated within sarcomeres is also transmitted to the actin filaments of the sub-sarcolemmal cytoskeleton by structural proteins, such as desmin at the Z-band. Two types of molecules connect the cytoskeleton to two types of transsarcolemmal protein complexes (Berthier and Blaineau, 1997; Huijing and Jaspers, 2005; Rando, 2001). Dystrophin, the sub-sarcolemmal cytoskeleton, is connected to the transsarcolemmal sarcoglycans and this system is referred to as the dystroglycan complex. Talin connects the sub-sarcolemmal cytoskeleton to the integrins at the sarcolemma. The dystroglycan complex and the integrins are connected to laminin of the basal lamina, and laminin is also connected to the collagen IV reinforcement of the basal lamina (Meijer, 2007). It should be stressed that these systems connect the myofibrils of two neighboring muscle fibers to the same collagen fibers in the basal lamina. Therefore, the force generated by one of these two fibers is transmitted to the tendon by the same fragments of basal lamina and in this way influences force transmission from the neighboring muscle fibers. However, at present it is not possible to quantitatively assess the relative contribution of these mechanisms in the summation of forces of high numbers of intermingled muscle fibers belonging to two or more MUs. Since the muscle fibers of a single motor unit, irrespective of the MU type, are dispersed within the muscle (Bodine et al., 1987; Bodine-Fowler et al., 1990), variable effects of summation of two coactive MUs are probably due to their overlapping territories. Many studies have examined the locations of fibers of slow and fast MUs in the muscle cross-section. The territories of slow MUs are located in deeper parts of the muscle, while those of fast units are situated more superficially (De Ruiter et al., 1995a,b, 1996; Lind and Kernell, 1991). Thus, the territories of fast and slow units usually overlap to a smaller degree than the territories of two fast or two slow units. Therefore, the probability that muscle fibers belonging to one fast and one slow MU will be in close proximity during their simultaneous activity is lower than for two fast or two slow MUs. Taking into consideration the reasons discussed above, considerable overlapping of territories of coactive MUs might decrease the effectiveness of the summation of their forces. This would explain why the less-than-linear effects dominated when higher numbers of units were active during simultaneous contraction. It might also be expected that in the case of non-overlapping territories of two contracting units, the activity of one of them would lead to an increase in the stiffness of muscle within its territory (Parmiggiani and Stein, 1981; Galler and Hilber, 1998). In addition, it has been found that slow-twitch fibers are 30% stiffer than fast-twitch fibers (Cui et al., 2007). Under such conditions, the force generated by two non-overlapping MUs is likely to be more effectively transmitted to the tendon and this would explain more-than-linear force summation. In the present study coactive fast and slow units, in particular, displayed this effect. 4.2. Functional consequences for the recruitment of motor units Motor unit recruitment is the most effective mechanism of muscle force regulation. The recruitment order of voluntary contractions is established according to the size principle: first slow MUs are recruited into contraction, followed by FR, and finally FF MUs (Henneman, 1957; Zajac and Faden, 1985; Feiereisen

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et al., 1997). The experimental arrangement of summation of MU forces may be considered as a model of recruitment. Troiani et al. (1999) analyzed the isometric force summation of an increasing number of MUs in the peroneus longus muscle stimulated in the following sequence: slow, fatigue resistant, fast intermediate and finally fast fatigable MUs. Coactivation of S and FR MUs predominantly gave more-than-linear force summation, whereas additional activation of an FF MU yielded either moreor less-than-linear summation, and when the FF unit was activated after the S and FR MUs, less-than-linear summation always occurred. These authors hypothesized that more-than-linear summation occurs at low force levels when few motor units are recruited, whereas less-than-linear summation prevails when muscle tetanic force exceeds 10%. The results of the present study also showed a gradual decrease in summation effects with an increasing number of active MUs, which supports the above hypothesis. We found that simultaneous stimulation of slow and fast MUs often produced more-than-linear summation, whereas the forces of two fast units more often summated less-than-linearly. During the recruitment process when fast units join previously activated slow MUs, the summation of forces is likely to be very effective. With the progress of recruitment, the territories of active MUs overlap more extensively, which probably decreases the effectiveness of force summation, and therefore, the recruitment of additional fast MUs may contribute less effectively to increasing the muscle force. Taken together, the results of this and previous studies suggest that the recruitment of all motor units of a muscle is probably not necessary to attain the maximal force of a voluntary isometric contraction. Our results also unequivocally demonstrated that force summation for 40 Hz unfused tetani was more effective in comparison to single twitches or 150 Hz maximum tetani. Under physiological conditions neither single twitches nor maximum tetani occur during voluntary contractions, but active MUs generate unfused tetani (over a wide range of fusion degree) which are characterized by force oscillations (Grimby et al., 1979; Raikova et al., 2008). The force oscillations appear to be the principal cause of physiological tremor (Allum et al., 1978; Stein and Lee, 1991; Christakos and Lal, 1979). Moreover, it is known that during muscle activity, the synchronization of action potentials generated by motoneurones occurs, and in consequence, subsequent phases of force-increase during tetanic contractions of coactive motor units can add to each other, thus contributing to physiological tremor (Stein and Lee, 1991). In the study of Stein and Lee (1991) it was demonstrated that during simultaneous unfused tetanic contractions of several MUs, the amplitude of force oscillation decreased in the majority of cases analyzed. This observation suggests that the influence of synchronization of motoneuronal firing on the amplitude of tremor during simultaneous activity of several motor units is less significant than previously expected. The present study was focused on MU forces in isometric conditions and little is known about the summation of MU forces in shortening or lengthening muscle. Furthermore, during natural contractions MUs are not activated by constant frequency patterns of stimuli. Therefore, to gain a greater understanding of the process it will be necessary to examine the summation of forces of unfused tetani during asynchronous stimulation of MUs with variable interpulse intervals, as proposed in recent reports by Krutki et al. (2008) and Celichowski et al. (2008). Further experiments, performed under conditions as close as possible to natural movements, are required. Acknowledgements The study was supported by the Grant No. 2 P05D 029 27 from the Polish Ministry of Science and Informatization. This study con-

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stitutes a part of the doctoral thesis submitted by Hanna Drzymała-Celichowska.

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Hanna Drzymała-Celichowska was born in Poland. She received her Master of Biology degree in 2004 from A. Cieszkowski University of Agriculture in Poznan´ (Poland), she received a Ph.D. degree in neurobiology from the Nencki Institute of Experimental Biology in Warsow (2008). Since 2009 she has been an adjunct at the Department of Neurobiology, University School of Physical Education in Poznan´. Her major research interests are contractile properties of motor units and motor control. Presently, she is studying the summation of motor unit forces during their parallel evoked contractions and the dimorphism of motor units in hindlimb skeletal muscles.

H. Drzymała-Celichowska et al. / Journal of Electromyography and Kinesiology 20 (2010) 599–607 Piotr Krutki was born in Poland in 1967. He graduated the Karol Marcinkowski University School of Medical Sciences in Poznan´ (1992), he received a Ph.D. degree (1997) and the habilitation in neurophysiology from the Nencki Institute of Experimental Biology in Warsaw (2001). Since 2003, he has been an Associate Professor at the Department of Neurobiology, University School of Physical Education in Poznan´. His main fields of research are: spinal neuronal networks, mechanisms of motor control, motor units and plasticity of the neuromuscular system.

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Jan Celichowski was born in Poznan´, Poland in 1960. He received an M.Sc degree from A. Cieszkowski University School of Agriculture (1983), a Ph.D. degree (1989) and the habilitation in neurophysiology from Nencki Institute of Experimental Biology in Warsaw (1996). Since 1997 he has been Professor of Neurophysiology, and since 2000 the Head of the Department of Neurobiology at the University School of Physical Education in Poznan´. His main fields of research are: motor units’ contractile properties and action potentials, plasticity of the neuro-muscular system, mechanomyography.