changes in contractile properties and action potentials of motor units in

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2016, 67, 1, 139-150 www.jpp.krakow.pl

Z. DOBRZYNSKA, J. CELICHOWSKI

CHANGES IN CONTRACTILE PROPERTIES AND ACTION POTENTIALS OF MOTOR UNITS IN THE RAT MEDIAL GASTROCNEMIUS MUSCLE DURING MATURATION Department of Neurobiology, University School of Physical Education, Poznan, Poland

The early phase of development of muscles stops following the disappearance of embryonic and neonatal myosin and the elimination of polyneuronal innervation of muscle fibres with the formation of motor units (MUs), but later the muscle mass still considerably increases. It is unknown whether the three types are visible among newly formed MUs soon after the early postnatal period and whether their proportion is similar to that in adult muscle. Moreover, the processes responsible for MUforce regulation by changes in motoneuronal firing rate as well as properties of motor unit action potentials (MUAPs) during maturation are unknown. Three groups of Wistar rats were investigated - 1 month old, 2 months old and the adult, 9 months old. The basic contractile properties and action potentials of MUs in the medial gastrocnemius (MG) muscle were analysed. The three types of MUs were distinguishable in all age groups, but higher proportion of slow MUs was noticed in young rats (29%, 18% and 11% in 1, 2 and 9 months rats, respectively). The fatigue index for fast fatigable MUs in 1 month old rats was about 2 times higher than in 9 months old rats. The twitch time parameters of fast MUs were shortened during the maturation; for these units, the force-frequency curves in young rats were shifted towards lower frequencies, which suggested that fast motoneurons of young animals generate lower firing rates. Higher twitch-to-tetanus ratios noted for the three MU types in young rats suggested the smaller role of rate coding in force regulation processes, and the higher role of MU recruitment in young rats. No significant differences in MUAP parameters between two groups of young and adult animals were observed. Concluding, the maturation process evokes deeper changes in fast MUs than in slow ones. K e y w o r d s : motor units, maturation, development, skeletal muscle, gastrocnemius muscle, action potentials, fast motor units, slow motor units, muscle fatigue

INTRODUCTION The properties of skeletal muscles change during development under the influence of hormones, changes in innervation and activity level (1). Most studies on the development of muscles and nerves have concerned the embryonic and neonatal period of development. It has been shown that two developmental isoforms, embryonic (Emb) and neonatal (Neo), are replaced within the first weeks after the birth by adult MHC isoforms, slow type (I) and three fast types (IIA, IIX, IIB) (2, 3); in rodents, these processes are observed within first postnatal weeks. In 40 day old rats, the Neo isoform completely disappears and the muscle has an adult MHC pattern (4). It should be stressed that the content of myosin is one of determinants of the type of muscle fibres (3, 5, 6). It has been shown that thyroid hormone has a crucial role in postnatal MHC transition and is an important factor involved in muscle maturation (7, 8). In rats, until birth, the level of thyroid hormone remains low, then increases to reach a maximum value in the 2nd – 3rd postnatal week and stabilises 4 weeks after birth (9). Developmental MHC isoforms are no longer detectable in fast muscle after the maximum peak of thyroid hormone expression has been achieved (10). Moreover, after birth, in developing skeletal muscle, numerous properties of its fibres also progressively change to adopt changes in neuromuscular activity or the maturation of excitation-contraction coupling.

The appearance of slow muscle fibres is heavily dependent on weight-bearing activity (beginning during the second week after birth) which regulates normal growth and the optimal expression of type I MHC (11). In contrast, the weight-bearing activity is not essential for fast-type muscle fibres achieving the adult fast MHC phenotype. It has been evidenced that intact thyroid state appearance is necessary to down-regulate the neonatal isoform and replace its expression with the IIb isoform (4, 10, 11). Moreover, slow and fast skeletal muscles have different rates of development and transition of MHC into the adult pattern. In slow muscle, the Emb and Neo MHC isoforms are eliminated later than in fast, phasic muscle (12). The motor unit (MU) is the smallest functional neuromuscular unit, being composed of one motoneuron and muscle fibres innervated exclusively by this neuron; the adult structure of motor innervation is developed in parallel to changes in the MHC content. Several authors have evidenced that within the first postnatal days in vertebrates, the polyneuronal innervation of muscle fibres can be observed (13-20). Later, polyneuronal innervation is eliminated; in rat lateral gastrocnemius muscle, this is up to the end of the second week after birth (21). However, when MUs of rat soleus muscle in 5 and 34 week-old rats were studied, no changes in the innervation ratio were noted but the proportion of fast type II muscle fibres decreased from 33 to 10% during growth (22), also suggesting the transformation of MU types in this phase of development. The last stage of

140 maturation of the nervous system is the myelination of axons which usually begins late in embryonic life or just after birth, when polyneuronal innervation is eliminated, and is continued for a long time (in humans, this lasts for several years) (23). The majority of studies concerning the MU contractile properties have been conducted on adult (24-26) or aged animals (27, 28). Several papers have also documented changes of the basic contractile properties of MUs during development. However, the data concerning the twitch time parameters are not consistent. It has been revealed that for MUs in cat soleus, the contraction time first shortened (up to 3 weeks after birth) and then increased up to 10 weeks (29), whereas for rat soleus, the increased contraction time was noted when 5 and 12 week old animals were compared (30). On the other hand, the MU contraction time shortened for the fast gastrocnemius and flexor digitorum longus muscles in cats (29, 31), when 2 – 5 week old animals were compared to adult ones. Lennerstrand and Hanson (32) indicated that in inferior oblique muscle, the total twitch duration decreased during maturation. The results concerning MU force changes in development are more convergent. As expected, approximately three-times lower twitch and tetanus forces have been noted for young cats (31, 32) and rats (30) in relation to adults. Additionally, Lennerstrand and Hanson (32) have calculated the twitch-to-tetanus force ratio, which describes a possible range of force regulation with changes of the motoneuronal firing rate, and documented first an increase (up to the 4th postnatal week) and then a decrease of this parameter. Also, for several cat hindlimb muscles, Hammarberg and Kellerth (29) indicated that the distribution of fatigue resistance was changing. They found a unipolar histogram as well as the higher participation of units with the fatigue index exceeding 0.75 at 1 – 2 weeks of age in relation to adult animals where the bipolar distribution was observed. The axonal conduction velocity was nearly two-times higher in 6-week old kittens in relation to 2-week old ones (31). However, it should be stressed that the above developmental changes of MU properties were not related to the MU types. Therefore, there is still a gap in the knowledge concerning changes of MU contractile properties during postnatal development, beyond the early days of life, after elimination of the developmental MHC isoforms and polyneuronal innervation, when MUs as structural and functional elements of skeletal muscles are formed, but the muscle mass still dynamically increases. First, MUs studied by all mentioned above authors were not classified and therefore it is not known whether the three types of MUs are detectable soon after the postnatal period and whether their proportion is similar to that in adult muscle. Second, there are no data concerning the possibility of a force regulation by changes in the

motoneuronal firing rate in this time period. Third, the properties of MU action potentials (MUAPs) during the early life period of maturation have never been analysed. It is known that MUAPs depends on the MU architecture (33), as the density in muscle fibres on a muscle cross-section and the muscle fibre diameter - the parameter modified by numerous factors (34-36) and dynamically changing during the increase of the muscle mass in a development. The studied rat MG muscle contains three physiological types of MUs, slow (S), fast fatigable (FF) and fast resistant (FR) (24, 37-39). Therefore, the study aimed at determining and comparing the changes of basic contractile properties (the twitch time parameters, force, fatigue resistance, the force-frequency of stimulation relationship as well as parameters of MUAPs) separately for each MU type. The MUs in three groups of male Wistar rats were studied: soon after elimination of Neo and Emb MHC, in 35 day old rats (1 mo), in two month old rat (2 mo), and in adult, 9 month old animals (9 mo).

MATERIAL AND METHODS The animals used in the study were pathogen free, male Wistar rats. Animals were housed with one or two per cage and maintained on a 12:12 light-dark schedule at 21°C. They had free access to a standard laboratory rat food (Labofeed B) and tap water supplied by special bottles. Three groups of rats were investigated: 1 month (n = 5), 2 months (n = 5) and 9 months (n = 4) old (Table 1). During electrophysiological experiments, rats were anaesthetised with sodium pentobarbital (initial dose of 60 mg/kg, i.p., supplemented during investigation as required, with doses of 10 mg/kg). The level of anaesthesia was controlled by the observation of withdrawal reflexes. After the experiments, the animals were killed with an overdose of sodium pentobarbital (180 mg/kg). All procedures were accepted by the Local Ethics Committee and followed the European Union guidelines as well as Polish Law on the Protection of Animals. The surgical preparation for the electrophysiological experiment included the careful separation of muscle from the surrounding tissues. The blood vessels and nerve branches to the MG muscle were left intact, while the remaining collaterals of the sciatic nerve were cut. The Achilles tendon was cut distally and connected to the force transducer (custom made, deflection sensitivity of 100 µm per 100 mN, the measurement range 0 – 1000 mN). To record the highest twitch force of single MUs, in 1 month old animals the studied muscle was stretched up to an optimal passive tension of 50 mN (determined for a representative group of 10 MUs in pilot experiments), whereas of 100 mN in 2 and 9 months old rats (40). The force was recorded

Table 1. Body and muscle characteristics. The mean values, standard deviations and variability ranges for the three age groups. Body mass [g]

Muscle mass [g]

Muscle-tobody mass ratio

1 mo rats

146.9 ± 10.8 135 – 163

0.313±0.04 0.243 – 0.392

0.21±0.02 0.18 – 0.25

21.3±2.2 18 – 24

2 mo rats

275.0 ± 11.3 264 – 296

0.757±0.06 0.700 – 0.840

0.28±0.02 0.25 – 0.31

28.4±2.7 27 – 30

9 mo rats

461.3 ± 10.3 450 – 470

1.246±0.09 1.16 – 1.34

0.27±0.02 0.25 – 0.29

35.3±1.5 34 – 37



Muscle length [mm]

141 under isometric conditions. The MUAPs were recorded with a pair of silver-wire electrodes (not insulated, 150 µm in diameter) inserted into the middle part of muscle belly perpendicularly to its long axis, at a distance of 5 – 7 mm between the two electrodes. With this location of the electrode for 94% of MUs the MUAP amplitude exceeded 0.1 mV, the lowest amplitude of potentials taken into analyses (33). The hind limb was immobilised in a special chamber filled with paraffin oil kept at 37 ± 1°C by an automatic heating system. Laminectomy was performed on the lumbar and sacral segments of the vertebrae (L2-S1). The dura mater over the spinal cord was cut and retracted. L1 vertebra and the sacral bone were hung up by steel clamps. The dorsal and ventral roots of L4-L5 spinal nerves were cut close to the spinal cord, and the ventral roots were split into the thinnest possible filaments until they included only one axon of the studied muscle in a bundle. The filaments were electrically stimulated with a bipolar silver electrode, with electrical rectangular pulses (amplitude up to 0.5 V, duration 0.1 ms) produced by a dual channel square pulse stimulator (model S88, Grass Instrument Company). The "all-or-none" appearance of a twitch contraction and of MUAP during a stimulation with a train of stimuli at 1 Hz and the amplitude around the threshold confirmed the isolation of a single MU. MUAPs were amplified by AC amplifier (WPI, ISO-DAM-8A for MUAP recording the high-pass filter at 0.1 Hz and low-pass filter at 3 kHz) were applied and monitored on an oscilloscope screen. The force and MUAPs were stored on a computer disk using an analog - to digital 12-bit converter (model RTI - 800, the sampling rate 1 kHz for MU force and 10 kHz for MUAP recordings). Each electrophysiological experiment was aimed at the functional isolation and characterisation of as many MUs as possible (a mean of 12 MUs per one experiment). All of the investigated MUs were stimulated according to the following protocol: 1) 5 stimuli at 1 Hz (5 single twitches were recorded

and averaged), 2) train of stimuli at frequency of 40 Hz and duration of 500 ms (the unfused tetanus was evoked), 3) train of stimuli at a frequency of 150 Hz and duration of 300 ms (the fused tetanus was evoked), 4) a series of 500 ms trains of stimuli at frequencies of 10, 20, 30, 40, 50, 60, 75, 100 and 150 Hz, 5) the fatigue test (tetanus evoked by trains of 14 stimuli at 40 Hz frequency, repeated every second within 3 minutes) (32). Here, 10 s time intervals were applied between all of the above elements of the protocol. The recorded force and MUAPs were analysed off-line with the custom-made computer program. For each MU, for the averaged twitch recording (point 1 of the protocol), the twitch force (TwF), the contraction time (CT, measured as a time from the beginning of force recording to the highest amplitude of twitch force), and the half-relaxation time (HRT, measured during the relaxation phase between the highest amplitude of the twitch force and half of this value) were calculated. Then, for the fused tetanus (at 150 Hz stimulation, point 3 of the protocol), the maximum tetanus force (TetF) was measured, and the ratio of twitch-to-tetanus forces (Tw/Tet) was calculated. The fatigue index (FatI) was also determined on the basis of the fatigue test (point 5 of the protocol), as a ratio of the tetanus force generated 2 min after the most potentiated contraction at the beginning of the fatigue test to the highest initial force (41). On the basis of values of forces achieved at the applied stimulation frequencies (point 4 of the protocol), the force-frequency curves were plotted. The forces were expressed as a percent of the maximum tetanic force (100%) measured during stimulation at 150 Hz and presented as a function of stimulation frequency. The sensibility of a motor unit to changes of stimulation frequency was presented as the force increase, expressed in percent, at the increase of stimulation frequency by 1 Hz (slope of the curve). This parameter was calculated for the steepest part of the curve, at around 60% of the maximum force (26), where the force-

Table 2. The contractile properties of motor units in the medial gastrocnemius muscle in three age groups. The mean values (± S.D.) and variability ranges are given. FF, fast fatigable; FR, fast resistant; S, slow motor units. CT, the contraction time; HRT, the halfrelaxation time; TwF, the twitch force; TetF, the maximum tetanus force; Tw/Tet, the twitch-to-tetanus ratio; FatI, the fatigue index. Significance of differences: * difference significant at P < 0.01; * difference significant at P < 0.05 (ANOVA, Kruskal-Wallis test). MU type

CT [ms]





HRT [ms]

TwF [mN]

TetF [mN]

Tw/Tet

FatI

39.82 ± 16.07 5.37 – 66.54

90.49 ± 32.44 20.27 – 135.00

0.43 ± 0.08 0.26 – 0.57

0.29 ± 0.10 0.12 – 0.48

FF 1 mo rats n = 22

15.23 ± 1.93 11 – 18

17.27 ± 4.29 10 – 26

2 mo rats n = 16

16.38 ± 1.78 13 – 19

16.19 ± 5.39 10 – 34

70.88 ± 39.27 7.57 – 149.90

176.48 ± 93.23 35.16 – 369.50

0.39 ± 0.09 0.22 – 0.58

9 mo rats n = 28

14.64 ± 2.18 11 – 19

13.36 ± 3.51 10 – 22

84.66 ± 42.41 12.21 – 172.40

301.17 ± 144.15 66.67 – 591.00

0.29 ± 0.10 0.10 – 0.50

0.15 ± 0.09 0.01 – 0.32

FR 1 mo rats n = 30

15.43 ± 2.21 12 – 19

17.07 ± 4.89 11 – 36

9.39 ± 6.42 3.91 – 32.42

29.43 ± 14.58 13.13 – 73.38

0.31 ± 0.09 0.13 – 0.49

0.78 ± 0.13 0.53 – 1.00

2 mo rats n = 33

15.97 ± 1.74 14 – 19

15.66 ± 2.93 11 – 23

18.41±13.60 3.66 – 47.62

69.70 ± 40.30 13.68 – 172.30

9 mo rats n = 46

14.80 ± 2.19 10 – 19

14.84 ± 4.02 10 – 26

22.45 ± 16.11 3.18 – 66.67

126.09 ± 62.91 24.66 – 257.40

0.16 ± 0.05 0.07 – 0.28

0.82 ± 0.15 0.51 – 1.00

S 1 mo rats n = 21

23.38 ± 3.02 20 – 33

27.57 ± 5.36 22 – 40

2.19 ±0.66 1.22 – 3.54

14.50 ± 2.98 10.38--22.41

0.15 ± 0.04 0.11-0.24

1.00 ± 0.05 0.94-1.08

2 mo rats n = 11

24.45 ± 4.13 20 – 35

31.73 ± 11.93 20 – 58

3.27 ± 1.48 1.22 – 5.68

24.67 ± 6.13 14.41 – 33.46

0.13 ± 0.04 0.06 – 0.20

9 mo rats n=9

22.11 ± 1.76 20 – 26

29.78 ± 6.98 18 – 39

4.04 ± 1.81 1.95 – 6.84

49.08 ± 15.17 35.41 – 80.59

0.08 ± 0.03 0.05 – 0.14

**

* **

* **

**

*

** 0.23 ± 0.12 0.05 – 0.49

*

** **

*

*

**

** 0.25 ± 0.09 0.16 – 0.43

**

*

0.75 ± 0.12 0.55 – 0.95 **

** **

*

** 1.01 ± 0.04 0.93 – 1.06 * 1.00 ± 0.01 0.99 – 1.02

142 frequency relation is nearly linear. Finally, the stimulation frequency necessary to reach 60% of the maximum tetanus force was calculated. For all three age groups of rats, the studied MUs were classified basing exclusively on standard physiological classification criteria (37). First, they were divided on fast or slow. A sag phenomenon (tested within point 2 of the protocol) was visible in unfused tetani evoked in 40 Hz in fast units; in slow MUs, the sag was not observed (37, 39). Distribution of the contraction time confirmed this division. For all age groups MUs revealing the sag had the contraction time up to 19 ms, whereas for MUs with no sag the contraction time was 20 ms or longer (Table 2). The further division of fast MUs was based on the fatigue index, which was under 0.5 for fast fatigable (FF) and over 0.5 for fast resistant (FR) MUs (39, 42).

For each MU the MUAP recorded in parallel with the single twitch (point 1 of the protocol) was analysed. Most of these potentials had two or three phases and the duration of the potential, the peak-to-peak amplitude, the peak-to peak duration, i.e. parameters dependent on several factors, as the muscle fibres diameter and MU structure (43), as well as the latency from the stimulus i.e. parameter depending on a nerve length and axonal conduction velocities, were calculated. Following the recordings of all of the isolated MUs, at the end of the experiment, the length of the sciatic nerve was measured between the position of a stimulating electrode (on the ventral root) and the place of insertion of the nerve branch to the studied MG muscle. Finally, the MG muscle was removed and its length and weight were measured. The statistical comparisons of the mean values between three age groups of MUs were made using the ANOVA KruskalWallis test, whereas differences in the proportion of three MU types in the three studied age groups were tested with the MannWhitney U-test.

RESULTS



Fig. 1. The percentage proportions of the three motor unit types in three age groups. FF, fast fatigable; FR, fast resistant; S, slow motor units.

FF

FR

The mean body weight of 1 mo and 2 mo rats was nearly three and two times lower than that of 9 mo rats, respectively, whereas the muscle mass of 1 mo and 2 mo rats were about four and about two times lower than that of adult animals, respectively (Table 1). Moreover, an increase in the muscle mass to body mass during maturation process was observed (Table 1) and the ratio achieved in 2 mo rats was the same as in adult animals. A total of 216 MUs were studied, 73 in 1 mo, 60 in 2 mo and 83 in 9 mo rats. For each age group, the number of tested MUs exceeded the number of units which are present in the muscle (about 57 MUs for rat MG in males) (44). Significant differences in the proportion of slow MUs between 1 mo rats (28.8%) and 9 mo rats (10.8%) were found (Mann-Whitney U-test, P < 0.01) (Fig. 1). The proportion of slow MUs in 2 mo rats had an intermediate value (18.0%). The participations of two types of

S

90 80

CT + HRT [ms]

70 60

* *

50

*

40 30 20 10 0

1

2

9

1

2

9

Age in months

1

2

9

Fig. 2. The sum of the twitch time parameters (CT, the contraction time and HRT, the half relaxation time) for three types of motor units of three age groups. Circle - median, box middle quartiles, whisker diagram - confidence intervals. Significance of differences: **difference significant at P < 0.01; *difference significant at P < 0.05 (ANOVA, KruskalWallis test).

143 fast MUs in the three age groups were not different (MannWhitney U-test, P > 0.05) and for FF MUs participation ranged from 26.6 – 33.7%, whereas MUs for FR MUs the participation ranged from 41.0 – 55.4%. Shorter values of the twitch time parameters were noted in the adult (9 mo) group in relation to the two young groups (1 mo and 2 mo) although differences in the contraction and the halfrelaxation time in all types of MUs were usually non-significant (Table 2). However, when a sum of the contraction and the halfrelaxation times was calculated, for fast MUs in the adult group the sum was smaller than for young rats (1 mo and 2 mo) (Fig. 2), whereas no statistical differences were found for slow units. On the other hand, considerable differences were noticed in the

MU force. The twitch forces for the three MU types were about two-times lower in 1 mo rats in comparison to adult rats, although the tetanus forces were three-to-four times lower (Table 2). For 2 mo rats, the twitch force of studied MUs achieved intermediate values in relation to two remaining groups, which were not significantly lower in relation to 9 mo rats, whereas the differences in the tetanus forces were stronger and significant for FR MUs. The twitch-to-tetanus ratios for all three types of MUs in 1 mo and 2 mo rats were significantly (1.5 – 2 times) higher than in 9 mo ones (Table 2). The fatigue index for FF MUs of 1 mo rats was significantly higher than for 9 mo rats and progressively decreased in a maturation process. For FR and S MUs, no evident changes in

 

Fig. 3. The distribution of the fatigue index of fast motor units for three age groups. Vertical dashed lines indicate the border value for FF/FR division. The arrows indicate the mean values of the index for the two MU types.

144 the fatigue index during the maturation process were observed. Moreover, the distribution of the fatigue index values was different in the three age groups (Fig. 3); therefore, evident separation of fast MUs into FF and FR types was only achieved in adult animals. Analysis of the force-frequency relationships revealed in 9 mo animals a shift of the steep parts of the curves for fast MUs towards higher frequencies (Fig. 4), which was confirmed by significant differences in the frequency necessary to achieve 60% of the maximum force between two groups of young animals (1 and 2 mo rats) and the 9 mo rats (Fig. 5A). Moreover, for FF MUs, an increase in a slope of the curve during the maturation process was noted (Fig. 5B). For slow MUs, no differences between the force-frequency relationships for 1 mo, 2 mo and 9 mo rats were found. Analysis of the relationship between the contraction time and the frequency at 60% of the

maximum force (Fig. 6) for all age groups revealed correlations between the two parameters, the strongest for the adult group of animals. Representative recordings of MUAPs for FF, FR and S type MUs of the three studied age groups are presented on Fig. 7A. The amplitude and time parameters of MUAPs for the three age groups of animals unexpectedly appeared to be similar (Fig. 7B, 7C and 7E). However, for the three MU types, the latency of MUAP was the longest for 9 mo rats, whereas the shortest values of latency were observed for 2 mo rats. For FF and FR motor units in all of the examined groups, these differences were statistically significant (Fig. 7D). The observation concerning MUAP latency was divergent to the nerve length measurements which progressively increased and amounted to 59.8 ± 0.4 mm, 75.1±2.7 mm, and 96.1±1.0 mm for 1 mo, 2 mo and 9 mo rats, respectively.

 

Fig. 4. The relationships between the relative MU contractile force and the stimulation frequency. Plots are presented for the three types of MUs (FF, FR and S) in 1, 2 and 9 month old rats. The horizontal dashed lines indicate 60% of the maximum force.

145

FR

FF



A

* Frequency at 60% Fmax [Hz]

50

S

** *

**

40 30 20 10 0

Slo pe of the curve [% of F max / 1 Hz]

B 6 5 ** **

4 3 2 1 0

1

2

9

1

2

9

1

Age in months

DISCUSSION The present study is the first analysis of maturation-related changes in contractile properties for three different types of MUs in one muscle. The main observation concerns dissimilarities in maturation processes of fast and slow MUs. The study revealed also that the MUs proportion as well as force-frequency relationship were changed within the studied period of postnatal development, whereas the MUAP properties remained unaltered. For the first examined group, 1 mo animals were taken soon after mature innervations of their skeletal muscles had been formed and the muscles had achieved the adult pattern of MHC (4, 12). It is worth specifying that in the studied period of maturation (1–9 months), the studied muscle mass increased 4 times, whereas body mass was only augmented by 3 times. It was observed that the MU proportion changed during development, and slow MU participation in MG muscle progressively decreased. In 1 mo rats, the lowest ratio of muscle mass to body mass was also observed (Table 1), which suggested the activation of a higher proportion of MUs in weight-bearing activity, which was crucial for the development of slow muscle

2

9

Fig. 5. The properties of force-frequency curves of three types of MUs for the three age groups. Frequency at 60% of the Fmax - stimulation frequency necessary to develop this force level; The slope of the curve - the force increase at the increase of the stimulation frequency by 1 Hz, calculated for the steep part of the forcefrequency relationship around 60% of the maximum force. Circle - median, box - middle quartiles, whisker diagram - confidence intervals. Significance of differences: ** difference significant at P < 0.01; * difference significant at P < 0.05 (ANOVA, Kruskal-Wallis test).

fibres (10, 11). Another possible explanation of this observation is related to the regulation mechanism of thyroid hormone (7, 8). The maximum peak appears 3 weeks after birth, when fast (IIb) muscle fibres start to be formed and the process is probably still no ended in 1 month rats. Moreover, analysis of the distribution of the fatigue index of FF MUs in three age groups during maturation revealed a progressive decrease in their fatigue resistance (Fig. 3), which was similar to the observations presented for cat MUs by Hammarberg and Kellerth (29). In 1 mo rats, there were no extremely low resistant MUs (the fatigue index below 0.1), which could be explained by three possible mechanisms. First, as mentioned above IIb muscle fibres (of FF MUs) were still under a formation process because a proper level of thyroid hormone had barely been reached one-two weeks earlier (7). The second possibility was that young animals (1 mo rats) were more active and performed a larger spectrum of various movements, also activating developing FF MUs and therefore no extremely fatigable MUs were found. The higher degree of activation of FF MUs might also be due to the lower ratio of the muscle mass to the body mass in 1 mo rats (Table 1). The

146

  

Fig. 6. The stimulation frequency at 60% of the maximum force (ordinate) as a function of the contraction time (abscissa). Plots are presented for the MUs of the three types taken together, in the three age groups.

third possible explanation concerned the recruitment order, which was probably not finally organised in the still developing central nervous system of the youngest animals studied (45), and therefore FF units were more frequently recruited into activity. The different levels of activity of MUs could modulate their properties without changing their type. The last expectation is supported by observation of the distribution of the fatigue index, which in fact illustrates the present activity level of individual MUs. It was shown that the distribution was deeply modulated by either the increased activity (in general, increase of the fatigue resistance of fast units and appearance of a group of MUs with very high fatigue index as an effect of treadmill training) (46) or the dramatically decreased activity following injury of the spinal cord (the

decrease and equalisation of the fatigue index for all MUs in the population) (47). The twitch time parameters of fast MUs were shortened during maturation. This tendency was not noticed in relation to slow MUs. Similar data were reported by other authors studying cat muscles in young animals. Bagust et al. (31) observed no significant changes in twitch time for slow MUs of soleus whereas Hammarberg and Kellerth (29), for fast MUs in gastrocnemius, and Bagust et al. (31), for fast MUs in flexor digitorum longus, noted shortening of their contraction time. Interestingly, the borderline contraction time for a division into fast and slow MUs amounted to 19 ms for the adult rat MG muscle (26, 39) and appeared to have the same values for young animals. The observations concerning the twitch time

147

A

1 mo

2 mo

9 mo

FF

FR 0.2 mV

S 20 ms FF

FR

S

FF

C

1.5 1.0

S

5 4 3 2

0.5

1

0

0

E 5

**

Peak-to-peak time [ms]

D ** **

**

4 Latency [ms]

FR

7 6

2.0

Duration [ms]

Amplitude [mV]

B 2.5

3 2 1 0

3.5 3.0 2.5 2.0 1.5 1.0 0.5

1

2

9

1

2

9

1

2

9

0

1

Age in months

parameters suggested that the plots of a relationship between the MU force and the stimulation frequency would have a different course for fast MUs because of a strong correlation between the twitch contraction time and the course of a curve, as described in several studies (26, 41, 48-49). Analysis of the force-frequency curves showed differences between young (1 mo and 2 mo rats) and adult (9 mo) animals, which were significant for FF and FR MUs, and related to the maturation-induced changes in twitch time parameters. Shortening of these parameters is the most likely reason for the rightward shift of the force-frequency relationship. This observation indicates that lower frequencies of activation are required in the muscles of young animals (1 and 2 mo rats) to reach similar relative force levels in comparison to adult ones. Moreover, because the range of stimulation frequencies at the steep part of the curve corresponds to a range of motoneuronal firing rate (50), it can be expected that fast motoneurons of young animals generate firings at lower rates. In all three MU types the tetanus force values were threeto-four times higher in adult rats in comparison to 1 mo rats (Table 2), what confirmed the results of earlier studies. Lennerstrand and Hanson (32) evidenced that the twitch and

2

9

1

2

9

Age in months

1

2

9

Fig. 7. The properties of the MUAPs for three types of MUs for the three age groups. (A) Sample recordings of MUAPs of FF, FR and S MUs from 1 mo, 2 mo and 9 mo group. (B-E) properties of MUAPs: (B) MUAP amplitude, (C) MUAP duration, (D) MUAP latency, (E) MUAP peakto-peak time. Circle median, box - middle quartiles, whisker diagram - confidence intervals. Significance of differences: ** difference significant at P < 0.01; * difference significant at P < 0.05 (ANOVA, Kruskal-Wallis test).

tetanus forces of MUs increased linearly from birth to adulthood. Bagust et al. (31) also showed that in cat the tension generated by the whole muscle increased more than three times during first 4 weeks of life. The present study enlarged also earlier observations concerning in the twitch-to-tetanus ratio in the development. Lennerstrand and Hanson (32) observed the highest twitch-totetanus ratio for 4-week old kittens (but not for younger ones). The obtained data revealed that this parameter was explicitly higher for all three MU types in the youngest studied rats what indicates that MUs in developing organisms have smaller possibilities to regulate their force (within a range between the twitch and maximum tetanus force) by changes of the motoneuronal firing rate. As a consequence, the recruitment of MUs plays a larger role in motor control processes in young individuals. Several physiological and biomechanical reasons for the differences in the twitch-to-tetanus ratio should be taken into account. First, it is possible that the mechanisms of calcium release and reuptake in muscle fibres in young organisms are not matured and a low amount of calcium is released (51). Second, it is possible that the muscle fibres in the unipennate

148 MG (52-55) have a different pennation angle and/or the ratio of muscle fibre length to muscle length in young and adult animals. For all three studied age groups, the MUAP amplitudes were highest for FF and lowest for S type MUs, whereas their time parameters were similar for the three MU types, which confirmed earlier observations of MUAP properties in rat and cat MG muscle (25, 33). The MUAP amplitudes mainly reflect differences in the innervation ratio, which were highest for FF and lowest for S MUs (24, 56). However, surprisingly, there were no differences when the studied MUAP properties were compared between the three age groups. The time parameters of MUAP mainly depend on the length and diameter of muscle fibres (57-59), which are probably considerably increased during an increase of body size and muscle mass (Table 1), which could shorten the studied time parameters. On the other hand, due to a fact that muscle fibres in MU territory are dispersed, their spatial distribution, also influencing MUAP duration, considerably changed due to increasing muscle cross section and increasing territories of MUs during maturation. These processes lead to a decrease in muscle fibre density, which could prolong the time parameters. Probably, parallel effects of all of these changes in the muscle fibre properties and in a structure of MUs were compensated, and finally no differences in time parameters of MUAPs were noted between young and adult rats. The overlapping effects were likewise responsible for a lack of differences in the MUAP amplitudes because this parameter also depends on several factors, changing in the studied period of development, as an increase in the size of motor unit territory and in the diameter of muscle fibres, but a decrease of their density in a transverse plane (57). It should be stressed that within the experiment electrode location, differences in muscle fibres distribution between individual MUs also evoked considerable variability of their MUAP amplitudes. The latency of MUAPs of FF, FR and S units was the shortest for 2 mo animals (Fig. 7D), although the shortest nerve length was observed for the youngest animals. This observation indicates that in 1 mo rats, the axonal conduction velocity and/or the motor plate transmission in a muscle are considerably slower in relation to older, 2 mo rats. The most likely reason for this observation is the long-lasting myelination of axons (23). The most important limitation of the study concerns a fact that motor units were classified according to their contractile properties whereas the metabolic profiles and myosin content of their muscle fibres were not determined. Therefore, a question does changes in MU contractile properties fully reflect expression of metabolic enzymes and myosin isoforms is still open. All three physiological types of MUs were present even in young animals, soon after the elimination of neonatal myosin, although a higher proportion of S MUs was observed in 1 mo animals. FF type units in 1 mo rats had higher fatigue resistance in comparison to adult ones, which was most probably related to the fact that a mature pattern of activity of MUs was not finally established. The twitch time parameters of fast MUs were shortened during the maturation; for these units, the force-frequency curves in young rats were shifted towards lower frequencies, which suggested that fast motoneurons of young animals generate lower firing rates. Moreover, the higher twitch-to-tetanus ratios noted for the three MU types in young rats suggested the smaller role of rate coding in force regulation processes, and the higher role of MU recruitment. List of abbreviations: MU, motor unit; mo, month; MUAP, motor unit action potential; MG, medial gastrocnemius; MHC, myosin heavy chain; Emb, embryonic; Neo, neonatal; CT, contraction time; HRT, half relaxation time; TwF, twitch force;

TetF, tetanus force; Tw/Tet, twitch-to-tetanus ratio; FatI, fatigue index Acknowledgements: This study was supported by a grant from the Polish National Science Centre (N/NZ4/04907). Conflict of interests: None declared.

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