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.
REFERENCES
1. Fladby T, Jensen JKS. Development of homogeneous fast
and slow motor units in the neonatal mouse soleus muscle.
Development 1990; 109: 723-732.
2. Staron RS, Johnson P. Myosin polymorphism and
differential expression in adult human skeletal muscle.
Comp BiochemPhysiol B 1993; 106: 463-475.
3. Pette D, Staron RS. Myosin isoforms, muscle fiber types,
and transitions. Microsc Res Tech 2000; 50: 500-509.
4. di Maso NA, Caiozzo VJ, Baldwin KM. Single-fiber myosin
heavy chain polymorphism during postnatal development:
modulation by hypothyroidism. Am J Physiol Regul Integr
Comp Physiol 2000; 278: R1099-R1106.
5. Andruchov O, Andruchova O, Wang Y, Galler S. Kinetic
properties of myosin heavy chain isoforms in mouse skeletal
muscle: comparison with rat, rabbit, and human and
correlation with amino acid sequence. Am J Physiol Cell
Physiol 2004; 287: C1725-C1732.
6. Neunhauserer D, Zebedin M, Obermoser M, et al. Human
skeletal muscle: transition between fast and slow fibre types.
Pflugers Arch 2011; 461: 537-543.
7. d'Albis A, Lenfant-Guyot M, Janmot C, Chanoine C,
Weinman J, Gallien CL. Regulation by thyroid hormones of
terminal differentiation in the skeletal dorsal muscle. I.
Neonate mouse. Dev Biol 1987; 123: 25-32.
8. Gardahaut MF, Fontaine-Perus J, Rouaud T, Bandman E,
Ferrand R. Developmental modulation of myosin expression
by thyroid hormone in avian skeletal muscle. Development
1992; 115: 1121-1131.
9. d'Albis A, Chanoine C, Janmot C, Mira JC, Conteaux R.
Muscle-specific response to thyroid hormone of myosin
isoform transition during rat postnatal development. Eur J
Biochem 1990; 193: 155-161.
10. Adams GR, Haddad F, McCue SA, et al. Effects of
spaceflight and thyroid deficiency on rat hindlimb
development. II. Expression of MHC isoforms. J Appl
Physiol 2000; 88: 904-916.
11. Baldwin KM, Haddad F. Effects of different activity and
inactivity paradigms on myosin heavy chain gene expression
in striated muscle. J Appl Physiol 2001; 90: 345-357.
12. Agbulut O, Noirez P, Beaumont F, Butler-Browne G. Myosin
heavy chain isoforms in postnatal muscle development of
mice. Biol Cell 2003; 95: 399-406.
13. Redfern PA. Neuromuscular transmission in new-born rats.
J Physiol 1970; 209: 701-709.
14. Bennett MR, Pettigrew AG. The formation of synapses in
striated muscle during development. J Physiol 1974; 241:
515-545.
15. Brown MC, Jansen JK, Van Essen D. Polyneuronal
innervation of skeletal muscle in new-born rats and its
elimination during maturation. J Physiol 1976; 261: 387422.
16. Betz WJ, Caldwell JH, Ribchester RR. The size of motor
units during postnatal development of rat lumbrical muscle.
J Physiol 1979; 297: 463-478.
17. Bennett MR, Ho S, Lavidis NA. Competition between
segmental nerves at end-plates in rat gastrocnemius muscle
149
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
during loss of polyneuronal innervations. J Physiol 1986;
381: 351-376.
Balice-Gordon RJ, Thompson WJ. Synaptic rearrangements
and alterations in motor units in the neonatal rat extensor
digitorum longus muscle. J Physiol 1988; 398: 191-210.
Laskowski MB, High JA. Expression of nerve-muscle
topography during development. J Neurosci 1989; 9: 175-182.
Ridge RM. Motor unit organization in developing muscle.
Comp Biochem Physiol A Comp Physiol 1989; 93A: 199-210.
Bennett MR, Lavidis NA. Development of the topographical
projection of motor neurons to a rat muscle accompanies loss
of polyneuronal innervation. J Neurosci 1984; 4: 2204-2212.
Kugelberg E. Adaptive transformation of rat soleus motor
units during growth. J Neurol Sci 1976; 27: 269-289.
Shepherd GM. Neurobiology. Oxford University Press, 1988.
Kanda K, Hashizume K. Factors causing difference in force
output among motor units in the rat medial gastrocnemius
muscle. J Physiol 1992; 448: 677-695.
Celichowski J, Drzymala H. Differences between properties
of male and female motor units in the rat medial
gastrocnemius muscle. J Physiol Pharmacol 2006; 57: 83-91.
Mrowczynski W, Celichowski J, Krutki P. Interspecies
differences in the force-frequency relationship of the medial
gastrocnemius motor units. J Physiol Pharmacol 2006; 57:
491-501.
Lochynski D, Krutki P, Celichowski J. Effect of ageing on
the regulation of motor unit force in rat medial
gastrocnemius muscle. Exp Gerontol 2008; 43: 218-228.
Webber SC, Porter MM, Gardiner PF. Modelling age-related
neuromuscular changes in humans. Appl Physiol Nutr Metab
2009; 34: 732-744.
Hammarberg C, Kellerth JO. The postnatal development of
some twitch and fatigue properties of single motor units in
the ankle muscles of the kitten. Acta Physiol Scand 1975; 95:
243-257.
Close R. Properties of motor units in fast and slow skeletal
muscles of the rat. J Physiol 1967; 193: 45-55.
Bagust J, Lewis DM, Westerman RA. The properties of
motor units in a fast and slow twitch muscle during postnatal development in the kitten. J Physiol 1974; 237: 75-90.
Lannerstrand G, Hanson J. The postnatal development of the
inferior oblique muscle of the cat. Acta Physiol Scand 1978;
103: 132-143.
Krutki P, Ciechanowicz I, Celichowski J, CywinskaWasilewska G. Comparative analysis of motor unit action
potentials of the medial gastrocnemius muscle in cats and
rats. J Electromyogr Kinesiol 2008; 18: 732-740.
Flisinski M, Brymora A, Elminowska-Wenda G, Bogucka J,
et al. Morphometric analysis of muscle fibre types in rat
locomotor and postural skeletal muscles in different stages
of chronic kidney disease. J Physiol Pharmacol 2014; 65:
567-576.
Gwag T, Park K, Park J, Lee J-H, Nikawa T, Choi I.
Celastrol overcomes HSP72 gene silencing-mediated muscle
atrophy and induces myofiber preservation. J Physiol
Pharmacol 2015; 66: 273-283.
Mast C, Joly C, Savary-Auzeloux I, Remond D, Dardevet D,
Papet I. Skeletal muscle wasting occurs in adult rats under
chronic treatment with paracetamol when glutathionedependent detoxification is highly activated. J Physiol
Pharmacol 2014; 65, 5: 623-631.
Burke RE, Levine DN, Tsairis P, Zajac FE. Physiological
types and histochemical profiles in motor units of the cat
gastrocnemius. Science 1973; 174: 709-712.
Garnett RA, O'Donovan MJ, Stephens JA, Taylor A. Motor
unit organization of human medial gastrocnemius. J Physiol
1979; 287: 33-43.
39. Grottel K, Celichowski J. Division of motor units in medial
gastrocnemius muscle of the rat in the light of variability of
their principal properties. Acta Neurobiol Exp 1990; 50:
571-588.
40. Celichowski J, Grottel K. The dependence of the twitch course
of medial gastrocnemius muscle of the rat and its motor units
on stretching of the muscle. Arch Ital Biol 1992; 130: 315-324.
41. Kernell D, Eerbeek O, Verhey BA. Relation between
isometric force and stimulus rate in cat's hindlimb motor
units of different twitch contraction time. Exp Brain Res
1983; 50: 220-227.
42. Kernell D, Eerbeek O, Verhey BA. Motor unit categorization
on basis of contractile properties: an experimental analysis
of the composition of the cat's muscle peroneus longus. Exp
Brain Res 1983; 50: 211-219.
43. Stalberg E, Andreassen S, Falck B, Lang H, Rosenfalck A,
Trojaborg W. Quantitative analysis of individual motor
unit potentials: a proposition for standardized terminology
and criteria for measurement J Clin Neurophysiol 1986; 3:
313-348.
44. Celichowski J, Drzymala-Celichowska H. The number of
motor units in the medial gastrocnemius muscle of male and
female rats. J Physiol Pharmacol 2007; 58: 821-828.
45. Deoni SC, Mercure E, Blasi A, et al. Mapping infant brain
myelination with magnetic resonance imaging. J Neurosci
2011; 31: 784-791.
46. Pogrzebna M, Celichowski J. Changes of properties of
motor units in rat medial gastrocnemius muscle after the
treadmill training. Acta Physiol 2008; 193: 367-379.
47. Mrowczynski W, Celichowski J, Krutki P, Gorska T,
Majczynski H, Slawinska U. Time-related changes of motor
unit properties in the rat medial gastrocnemius muscle after
the spinal cord injury. I. Effect of total spinal cord
transection. J Electromyogr Kinesiol 2010; 20: 523-531.
48. Celichowski J, Grottel K. Changes in tension-frequency
relationship of motor units induced by their activity in rat
muscle. Arch Ital Biol 1997; 135: 229-37.
49. Mrowczynski W, Celichowski J, Krutki P, Cabaj A, Slawinska
U, Majczynski H. Changes of the force-frequency relationship
in the rat medial gastrocnemius muscle after total transection
and hemisection of the spinal cord. J Neurophysiol 2011; 105:
2943-2950.
50. Kernell D. Rhythmic properties of motoneurones innervating
muscle fibres of different speed in m. gastrocnemius medialis
of the cat. Brain Res 1979; 160: 159-162.
51. Villarroel, Sakmann B. Calcium permeability increase of
endplate channels in rat muscle during postnatal
development. J Physiol 1996; 496: 331-8.
52. Zuurbier CJ, Huijing PA. Influence of muscle geometry on
shortening speed of fibre, aponeurosis and muscle. J Biomech
1992; 25: 1017-26.
53. Ettema GJ. Elastic and length-force characteristics of the
gastrocnemius of the hopping mouse (Notomysalexis) and
the rat (Rattusnorvegicus). J Exp Biol 1996; 199: 1277-85.
54. Liber RL. Skeletal muscle structure, function and plasticity.
The Physiological Basis of Rehabilitation. Lippincott
Williams & Wilkins, 2002
55. Gallo M, Gordon T, Tyreman N, Shu Y, Putman CT.
Reliability of isolated isometric function measures in rat
muscles composed of different fibre types. Exp Physiol
2004; 89: 583-592.
56. Kadhiresean VA, Hasett CA, Faulkner JA. Properties of
single motor units in medial gastrocnemius muscles of adult
and old rats. J Physiol 1996; 493: 543-552.
57. Gath I, Stalberg E. Frequency and time domain
characteristics of single muscle fibre action potentials.
Electroencephalogr Clin Neurophysiol 1975; 39: 371-376.
150
58. Dumitru D, King JC, Zwarts MJ. Determinants of motor unit
action potential duration. Clin Neurophysiol 1999; 110:
1876-1882.
59. van Eijden TM, Turkawski SJ. Action potentials and twitch
forces of rabbit masseter motor units at optimum jaw angle.
Arch Oral Biol 2002; 47: 607-612.
R e c e i v e d : June 23, 2015
A c c e p t e d : December 17, 2015
Authors' address: Zuzanna Dobrzynska, Department of
Neurobiology, University School of Physical Education, 27/39
Krolowej Jadwigi Street, 61-871 Poznan, Poland
E-mail: [email protected]