Activity of Hindlimb Motor Units During Locomotion in the Conscious Rat

Monica Gorassini,1 Torsten Eken,2 David J. Bennett,1 Ole Kiehn,1 and Hans Hultborn1

 1Department of Medical Physiology, Section of Neurophysiology, University of Copenhagen, 2200 Copenhagen N, Denmark; and  2Institute of Neurophysiology, University of Oslo, N-0317 Oslo, Norway


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Gorassini, Monica, Torsten Eken, David J. Bennett, Ole Kiehn, and Hans Hultborn. Activity of Hindlimb Motor Units During Locomotion in the Conscious Rat. J. Neurophysiol. 83: 2002-2011, 2000. This paper compares the activity of hindlimb motor units from muscles mainly composed of fast-twitch muscle fibers (medial and lateral gastrocnemius: MG/LG, tibialis anterior: TA) to motor units from a muscle mainly composed of slow-twitch muscle fibers (soleus: SOL) during unrestrained walking in the conscious rat. Several differences in the activation profiles of motor units from these two groups of muscles were observed. For example, motor units from fast muscles (e.g., MG/LG and TA) fired at very high mean frequencies of discharge, ranging from 60 to 100 Hz, and almost always were recruited with initial doublets or triplets, i.e., initial frequencies >= 100 Hz. In contrast, the majority of SOL units fired at much lower mean rates of discharge, approx 30 Hz, and had initial frequencies of only 30-60 Hz (i.e., there were no initial doublets/triplets >= 100 Hz). Thus the presence of initial doublet or triplets was dependent on the intrinsic properties of the motor unit, i.e., faster units were recruited with a doublet/triplet more often than slower units. Moreover, in contrast to units from the slow SOL muscle, the activity of single motor units from the fast MG/LG muscle, especially units recruited midway or near the end of a locomotor burst, was unrelated to the activity of the remainder of the motoneuron pool, as measured by the corresponding gross-electromyographic (EMG) signal. This dissociation of activity was suggested to arise from a compartmentalized recruitment of the MG/LG motoneuron pool by the rhythm-generating networks of the spinal cord. In contrast, when comparing the rate modulation of simultaneously recorded motor units within a single LG muscle compartment, the frequency profiles of unit pairs were modulated in a parallel fashion. This suggested that the parent motoneurons were responsive to changes in synaptic inputs during unrestrained walking, unlike the poor rate modulation that occurs during locomotion induced from brain stem stimulation. In summary, data from this study provide evidence that the firing behavior of motor units during unrestrained walking is influenced by both the intrinsic properties of the parent motoneuron and by synaptic inputs from the locomotor networks of the spinal cord. In addition, it also provides the first extensive description of motor-unit activity from different muscles during unrestrained walking in the conscious rat.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

One way to study the central processes involved in the production of locomotion is to record the activity of single motoneurons during walking. Traditionally this has been done by examining locomotor-like behavior induced by stimulation of the mescencephalic locomotor region (MLR) in the decerebrate cat (Brownstone et al. 1992; Orsal et al. 1986; Severin et al. 1967; Tansey and Botterman 1996; Zajac and Young 1980). Single-motor-unit activity recorded in unrestrained, conscious preparations during self-initiated walking is rare because of the technical difficulties in obtaining such data (cat, Hoffer et al. 1987; rat, Eken 1998; human, DeSerres et al. 1995; Grimby 1984; Jakobsson et al. 1988; Ogawa et al. 1991). Because of this, detailed information from only a limited sample of motor-unit types is available, including the anterior thigh muscles in the intact cat and the tibialis anterior and the short toe extensor muscles in the human.

One aim of the present study therefore was to record from various types of motor units in the conscious animal that differ in both action (extensors vs. flexors) and motor-unit type (slow vs. fast). Single-motor -unit activity was recorded in the medial and lateral gastrocnemius (MG and LG), soleus (SOL), and tibialis anterior (TA) muscles of the rat hindlimb. We used the rat because of the increasing number of laboratories using the neonatal rat preparation to study locomotion (Kiehn and Kjaerulff 1998) and the need to establish what the normal adult motoneuron patterns are during unrestrained, self-initiated walking.

When comparing motoneuron behavior during MLR-induced activity and self-initiated walking, several interesting discrepancies arise. For example, during MLR-induced locomotion, there is little rate modulation of motoneuron activity during a locomotor burst, i.e., the firing frequency profiles are flat. In addition, firing rates are affected only slightly by changes in either synaptic input (Severin et al. 1967; Zajac and Young 1980) or injected current (Brownstone et al. 1992). In contrast during self-initiated walking, motor-unit frequency profiles are more rounded, and they follow the envelope of their corresponding gross electromyograms (EMGs) (DeSerres et al. 1995; Eken 1998; Hoffer et al. 1987). Likewise, mean firing rates tend to increase as the speed of locomotion increases (Grimby 1984; Hoffer et al. 1987; Ogawa et al. 1991). In addition, during MLR-induced locomotion, there tends to be a greater percentage of motoneurons that fire with initial doublets (Zajac and Young 1980, although cf. Brownstone et al. 1992) in comparison to self-initiated walking (Hoffer et al. 1987; Ogawa et al. 1991) [an initial doublet is defined as the occurrence of a short interspike interval (i.e., <= 10 ms) between the first two motor-unit action potentials (MUAPs) in a burst (Zajac and Young 1980)].

The poor rate modulation and high occurrence of initial doublets may be due to stronger and more abrupt synaptic activation of motoneurons by MLR input than during self-initiated walking. However, it is also possible that motoneurons with different intrinsic properties (i.e., fast vs. slow) differ in their recruitment and firing behavior. This latter possibility has not been excluded because the motoneurons studied in the MLR preparations often were not identified with respect to motoneuron type or muscle they supplied and the data from self-initiated walking come from only a few muscle types. We therefore compared the firing patterns of motor units in muscles with predominantly fast twitch muscle fibers (approx 90%; LG, MG, and TA) with those of motor units in a muscle with 90% or more slow twitch muscle fibers (SOL) (Gillespie et al. 1987; Tötösy de Zepetnek 1992) to examine if there were systematic differences in the recruitment and rate modulation patterns of the different motor-unit populations. Parts of this paper have been presented in abstract form (Gorassini et al. 1995).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Single-motor-unit recordings were obtained from 18 (of 24) adult male Wistar rats weighing between 270 and 390 g. Intramuscular micro-EMG electrodes were implanted in the MG, LG, and TA muscles to record single MUAPs. The firing behavior of five TA, three MG, and nine LG motor units during unrestrained walking are described (the activity of 3 of these units during imposed muscle stretch is described in Gorassini et al. 1999). In all rats, gross (i.e., whole muscle) EMG electrodes were implanted in both flexor (TA) and extensor (MG or LG) muscles. In a separate series of experiments performed at the University of Oslo by Torsten Eken, data from the SOL muscle were obtained from four male adult Møll-Wistar rats (n = 5 units). Experimental details and a qualitative description of these SOL motor units during tonic activity and walking can be found in Eken (1998). Experiments were approved by the local ethics committees in Copenhagen and Oslo, respectively.

Implant procedures and data recording

Details of the construction and implantation of the micro- and gross-EMG electrodes can be found in Eken (1998) and Gorassini et al. (1999). Note that the gross-EMG electrodes were inserted on either side of the corresponding micro-EMG electrodes, separated by approx 1 cm, to record from the same population of motor units.

All walking data were obtained from over-ground locomotion. Animals walked freely in a 1.0 × 0.8 m glass aquarium with the walking surface covered by a rubber mat. In this space, the rat could take approx 10 consecutive steps along the length of the aquarium. Gross-EMG activity from TA and LG or MG was used to assess the quality of walking. The swing and stance phases of the step cycle were defined as occurring during the periods of TA and MG/LG gross-EMG activity, respectively. Unit activity was analyzed only when good alternation between flexor and extensor muscles was present and during sequences of at least four steps. For the SOL units, flexor activity was not recorded but quality of walking was assessed from video recordings taken during the walking sequences. Details of the recording set-up and single-unit discrimination techniques can be found in Eken (1998) and Gorassini et al. (1999).

Discrimination of all MUAP waveforms in an entire locomotor burst was possible in only approx 10% of the steps recorded due to movement of the electrodes or from superimposition of other units. These steps were distributed randomly in the recordings, i.e., there were no particular speeds or gross-EMG profiles in which single MUAP waveforms could be discriminated more easily. In steps where only a portion of the MUAPs could be discriminated, the firing frequency profiles appeared very similar to the fully analyzed steps.

Data analysis

MEAN FREQUENCIES. For each unit, the mean interspike interval in a single locomotor burst was calculated (interspike intervals <= 10 ms and >= 200 ms were omitted) using Linux-based custom software and SigmaPlot 3.0. The individual means for each step then were summed and averaged together [each unit had an average of 16 ± 10.5 steps (mean ± SD)]. The resulting mean interspike interval value for a given unit then was used to calculate the mean firing-frequency value. Table 1 displays the group averages for units in the various muscle and motor-unit groups. The mean firing-frequency values for the initial doublet/triplets also were calculated from the mean interspike interval values and expressed as means ± SD.


                              
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Table 1. Summary of motor-unit activity during stepping

CORRELATION BETWEEN ACTIVITY OF SINGLE MOTOR UNITS AND CORRESPONDING GROSS-EMG. In 12 units (5 LG, 5 SOL, 2 TA), the amplitude of the rectified and smoothed gross-EMG signal (30 Hz low-pass digitally filtered, 0°-phase shift) was plotted against the firing frequency of the single unit (Hoffer et al. 1987). This was done to test if the firing frequency of a single motor unit was modulated in a similar manner to the activity of the entire motoneuron pool, as represented by the amplitude of the gross-EMG signal. The equation Rate(t) beta *EMG(t) + b (where t = time, beta  = the slope of the regression line, and b = the horizontal offset) was fitted to the data, using both Matlab custom-written software and Sigma Plot 3.0 (note that we did not force the regression line through the origin) (cf. Hoffer et al. 1987). The coefficient of determination, r2 (r = correlation coefficient) was calculated to obtain a measure of the total variance in motor-unit discharge that could be accounted for by fluctuations in the gross-EMG profile (Hoffer et al. 1987). The mean absolute error (MAE) with respect to the regression line also was calculated to determine the error in predicting frequency from the preceding linear EMG relation. Initial doublets/triplets and other frequencies >200 Hz and <5 Hz were omitted to remove outlying points for the regression analysis.

CORRELATION BETWEEN ACTIVITY OF SIMULTANEOUSLY RECORDED MOTOR-UNIT PAIRS. The firing rate profiles of motor units that were discriminated from a single micro-EMG record also were compared to examine if the activity of motor units located close to one another were modulated in a similar manner. Three motor-unit pairs located in the superficial layer of the LG muscle were analyzed. The firing rate profiles of each unit in a pair were compared at similar points in time by fitting a fifth-order polynomial to the individual frequency profiles (Sigma Plot 3.0). Frequency values from the two polynomials were sampled every 25 ms and plotted against one another (see Fig. 8). Finally, a straight line was fitted to these data points and r2 values calculated, as described in the preceding text.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In total, the activation profiles of 21 motor units were analyzed during locomotion (see Table 1 for summary). Two main groups of motor units were found in the extensor muscles MG/LG and SOL. One group was recruited at the beginning of a locomotor burst and was classified as "early-stance phase-activated units" and the second group was recruited later in the locomotor burst and was defined as "mid-late-stance phase-activated units." Two groups of TA motor units also were recorded, one type that fired only two or three MUAPs in a single locomotor burst (doublet/triplet only) and a second type that fired longer trains of MUAPs.

"Fast" mid-late-stance phase-activated MG/LG units

The majority of MG and LG units (10/12) that were discriminable from the raw micro-EMG records were recruited either mid-way through or at the end of the stance phase as the hindlimb pushed on the ground to propel the rat forward. These units will be referred to as mid-late-stance phase units. An example is shown in Fig. 1 where a large amplitude unit in the single-unit recording (micro-EMG; 3rd trace) was recruited halfway through the locomotor burst. The firing frequency profile of the unit is shown in the bottom trace. A striking feature of the mid-late-stance phase MG/LG units was the high mean firing rates reached during walking. In the two steps in Fig. 1, the mean rate was approx 100 Hz (means for all steps, excluding doublets, ranged from 54 to 86 Hz, Table 1). For comparison, the firing rates of neonatal rat motoneurons during transmitter-induced locomotion only reach 5-10 Hz (Bertrand et al. 1998; Hochman and Schmidt 1998; MacLean et al. 1997). The mid-late-stance phase units fired briefly during a locomotor burst, with an average of 16 spikes per step (see 1st 5 MG/LG units in Fig. 5A for individual values). The number of MUAPs fired related poorly to the duration of the corresponding gross-EMG burst (Fig. 5B), with r2 values ranging from 0.12 to 0.31. 



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Fig. 1. Mid-late-stance phase lateral gastrocnemius (LG) motor unit. In the single-unit electromyogram (EMG; micro-EMG record; 3rd trace), an LG unit was activated midway through the locomotor burst. Bottom trace: instantaneous firing-frequency profile of the unit. Note the high firing rates reached and the presence of initial doublets/triplets, even during the reactivation of the unit in the first step. Rectified and smoothed gross-EMG from tibialis anterior (TA) and LG are shown in traces 1 and 2, respectively. Superimposed motor-unit action potentials (MUAPs) of the single unit are displayed to the right of the frequency graph (as in subsequent figures).

An unexpected finding, as demonstrated in Fig. 1, was the high occurrence of very short interspike intervals (<= 10 ms) of the first two or three MUAPs. These initial doublets or triplets occurred in all mid-late-stance phase recruited MG/LG units and in 82% of all steps analyzed (87/106 steps, see Table 1 for MG and LG group values). Initial doublets/triplets were present at all speeds of walking, and thus data from the various walking speeds were grouped together. Even when a unit fired two discrete bursts in a single step (3/12 units), each burst often would start with an initial doublet (e.g., step 1, Fig. 1). Discrete bursts were defined as discharges separated by >= 200 ms (Hennig and Lømo 1985).

The mid-late-stance phase units were most likely fast twitch motor units as judged by their high mean firing rates and phasic discharge pattern (Hennig and Lømo 1985). In addition, the sample of MG and LG units probably was biased toward large, fast units considering >90% of MG/LG units are fast-twitch (Gillespie et al. 1987) and tend to have large-amplitude signals that are easier to discriminate throughout the locomotor burst.

"Fast" early-stance phase-activated LG units

The unit in Fig. 2 was one of two LG units that were recruited at the beginning of the stance phase (referred to as early-stance phase units). These units displayed activation properties that were intermediate to the mid-late-stance phase MG/LG units and the SOL units described in the following text. As seen in Fig. 2, the early stance phase units also commenced firing with initial doublets (in 46 and 70% of all steps, Table 1) and fired more MUAPs per step cycle than other LG units (see * for these 2 units, Fig. 5A) despite their lower frequency of discharge (see Table 1). Note that in some of the step cycles in Fig. 2, changes in rate by as much as 50% occurred within a single locomotor burst (e.g., last 2 step cycles). The second early-stance phase unit that was recorded (not shown) was distinct from all other MG/LG units recorded in that the number of MUAPs fired increased linearly as a function of the duration of the corresponding gross-EMG activity (see unit black-triangle, Fig. 5B).



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Fig. 2. Early-stance phase-activated LG motor unit. In this 4-step sequence, the unit was recruited with either an initial doublet or triplet (instantaneous frequency, bottom trace: y axis is clipped to increase vertical resolution). An acceleration in firing occurred midway through the stance phase in some steps (steps 1, 3, and 4) whereas in others (step 2), the firing rate remained constant. Rectified and smoothed EMG activity from a second nonselective micro-EMG electrode (middle trace) displayed a different activation profile, decreasing in amplitude mid-way through the locomotor burst. Rectified and smoothed gross-EMG activity for the TA muscle is shown in the top trace.

"Slow" early-stance phase-activated SOL units

Motor-unit activity recorded from the "slow" soleus muscle showed very different activation patterns during walking as compared with the MG/LG motor units. In general, the frequency profiles of the SOL units were more rounded in shape, and their activity was more related to the corresponding gross-EMG signal (Fig. 3) (see also Fig. 9 in Eken 1998). Even though the firing rates were the lowest of all extensor motor units recorded (25-31 Hz, Table 1), the SOL units fired more MUAPs per locomotor burst than the mid-late-stance phase MG/LG units (26 on average, see Fig. 5A for individual values), for step cycles of similar duration. The SOL units also differed from the MG/LG units in that the number of MUAPs increased linearly with the duration of the corresponding gross-EMG activity (Fig. 5C), with r2 values ranging from 0.72 to 0.91. 



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Fig. 3. Activation profile of 2 slow early-stance phase-activated soleus (SOL) units. A: firing-frequency profile of a SOL unit with high initial firing frequencies (i.e., the interspike interval of the 1st 2 MUAPs was 1 of the shortest in the locomotor burst). In step 1, the first interspike interval was classified as a doublet because the frequency was >= 2 times higher than the mean frequency in the burst. B: example of a different SOL unit with a more gradual recruitment pattern (e.g., steps 1 and 3). In both A and B, the rectified and smoothed gross-EMG are shown in the 1st trace. Flexor activity was not recorded but quality of walking was assessed from video records. Note the rounded frequency profiles in both units.

None of the slow soleus units had initial interspike intervals that were <10 ms, but in two units the initial frequencies were, on occasion, twice that of the mean frequency in the locomotor burst (e.g., step 1, Fig. 3A). We also have classified this overshoot in frequency as an initial doublet, and it occurred in only 18% of the steps cycles for these two units (8/44 steps). In a large number of steps (50%), however, SOL units were recruited with frequencies above or close to the peak rate in the locomotor burst (e.g., steps 2 and 3 in Fig. 3A). A more gradual recruitment pattern was seen in the remainder of the steps (32% e.g., steps 1 and 3 for a different unit in Fig. 3B); however, initial frequencies rarely fell below 20 Hz.

These SOL units were considered to be slow-twitch, fatigue-resistant (S) due to the high percentage of slow twitch muscle fibers in the SOL muscle (approx 90%) (Gillespie et al. 1987) and because of their low mean firing rates and ability to fire tonically for several minutes (see Eken 1998 for recordings of these units during static posture). The remaining SOL units have been shown to be fast, fatigue-resistant (FR) (Chamberlain and Lewis 1989), and the SOL unit described in the next section may fall into this category.

"Fast" SOL unit

There was one SOL unit that had activation properties similar to the early-stance phase LG units described in the preceding text. This unit was recruited at high levels of gross-EMG activity and had a higher mean firing rate (45 Hz, Table 1) and a greater interval to interval variability in firing rate than the other "slow" SOL units. Unlike the slow SOL units, this unit fired initial doublets with interspike intervals that were <= 10 ms. The proportion of initial doublets (46% of all steps) and relationship between the number of MUAPs to the gross-EMG duration (see unit , Fig. 5C) were similar to the early stance phase LG unit described above (unit black-triangle in Fig. 5B). This characteristic firing profile suggested that the recorded SOL unit was of the fast, FR type (Eken 1998; Hennig and Lømo 1985).

"Fast" TA flexor units

In contrast to the extensor units, the flexor motor units (n = 5) fired very briefly when activated with an average of 4.3 MUAPs per step cycle (see Fig. 5A for individual unit means). In almost every step (99%, Table 1), the TA units were recruited with a doublet or triplet (Fig. 4). In one unit, two to three very rapid MUAPs were fired per step with an average initial interspike interval of only 2.4 ± 1.1 ms (approx 417 Hz, n = 47 steps). The mean firing rates of the TA motor units were the highest of all units investigated (see Table 1), ranging from 82 to 109 Hz (excluding initial doublets). The firing rates within a locomotor burst were quite variable (Fig. 4), but a relative undershoot in firing was often seen after the doublet/triplet (Fig. 6Bvii). The TA units recorded during walking were most likely fast due to the firing properties described in the preceding text and because 94% of unit types in the TA muscle are fast-twitch (Tötösy de Zepetnek et al. 1992). In addition, the micro-EMG electrodes were implanted into the superficial layers of the TA muscle where there is a preponderance of FF unit types (Tötösy de Zepetnek et al. 1992).



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Fig. 4. Activation profile of fast TA motor unit. Each step was initiated with a doublet or triplet. First interspike interval for all steps (n = 28) in this unit averaged 3.9 ms (256 Hz), and the mean firing rate after the doublet/triplet was 100 Hz. Only 5-6 MUAPs were fired in each locomotor burst. Rectified and smoothed gross-EMG of LG and TA are shown in the top and middle traces, respectively. Reprinted from Fig. 2B in Gorassini et al. (1999).



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Fig. 5. Average number of MUAPs per step cycle and its relationship to duration of the gross-EMG burst for TA, medial gastrocnemius (MG)/LG, and SOL units. A: individual averages (±SD) for units where data were available for >= 5 steps (number of steps shown at bottom). Group mean for the TA units was 4.3 MUAPs/step cycle, MG/LG units, 16.0, and SOL units, 26.4. Early-stance phase LG units (*) had significantly more MUAPs/step cycle, (33.5, P < 0.01). B: relationship between the number of MUAPs and the duration of the gross-EMG burst for MG/LG units. Same symbol for each unit is used in both A and B. Coefficient of determination (r2) and slope of the regression line (beta ) are given in the top left-hand corner. Units with negative beta  values (marked with "-" in A) are not shown for clarity. Note that 1 of the early-stance phase-recruited units (black-triangle) displayed a fairly strong linear relationship. C: same plot as in B but for SOL units. Unit marked () is the fast SOL unit.

Initial doublets and triplets

When comparing the initial activation patterns of motor units from the various muscles, it turned out that there was a positive relationship between the mean firing rate of the first two MUAPs and the mean firing rate during the sustained phase of a locomotor burst. For example, the group mean firing rates (i.e., the average of the individual unit means for a particular muscle or motor-unit group) for the first two MUAPs were TA 286 Hz > MG/LG 238 Hz > fast SOL 161 Hz > slow SOL 68 Hz (see Fig. 6A for individual values). Correspondingly, the group mean firing rates were TA 97 Hz > MG/LG 69 Hz > fast SOL 45 Hz > slow SOL 28 Hz (Table 1). An r2 value of 0.6 was obtained from the linear regression when the individual mean firing rates of 13 units were plotted against the corresponding mean initial firing rates (not shown).



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Fig. 6. Summary of initial doublet and triplet firing patterns. A: average interspike interval values (±SD, left y axis) and corresponding firing frequencies (right y axis) for the 1st 2 MUAPs for steps with initial doublets/triplets (the number of steps averaged are shown at the bottom of each bar). TA units had the highest group mean frequency for the 1st interspike interval (286 Hz), compared with the MG/LG (238 Hz), fast SOL (161 Hz), and slow SOL (68 Hz) units. B: representative step cycles from various units illustrating the 2 doublet and triplet firing patterns recorded (2 step cycles for each pattern are shown). A common feature is the undershoot in firing frequency after the initial doublet/triplet that is followed by an overshoot in firing (steps ii, iii, iv, vi, vii, and viii, down-arrow ). Note the different time resolution in the individual graphs. All ticks on time axis are 100 ms, and ticks on frequency axis are 100 Hz.

A variety of initial doublet and triplet firing patterns were recorded in the various units. Figure 6B gives examples of the four general patterns observed. A common occurrence, however, was that the interspike interval after the initial doublet/triplet was the longest in the spike train, resulting in a frequency that "undershot" all the others in the train (Fig. 6B, iii, iv, vi, and viii). A "rebound" in firing often would occur one or two intervals after the frequency undershoot (arrows, Fig. 6B, ii, iii, iv, vi, vii, and viii). Two patterns of triplet firing were observed, one in which the second interspike interval was longer than the first, but still under 10 ms (see triplet decrement: 6B, v and vi) and the second in which the second interspike interval was even shorter than the first, resulting in an initial increase in firing frequency ("triplet increment", 6B, vii and viii). Motor units with the highest mean firing rates (see preceding text) exhibited the highest percentage of initial triplets, i.e., TA units 30% (33/111 steps) > MG/LG units 18% (25/136 steps) > fast SOL unit 0.03% (1/33 steps) > slow SOL units 0% (0/65 steps), with an r2 value of 0.93 for this linear relation.

Relationship between single motor-unit firing frequency and rectified gross-EMG amplitude

Similar to that shown for cat motor units (Hoffer et al. 1987), the firing profiles of the slow rat SOL units roughly followed the envelope of their corresponding rectified and smoothed gross-EMG signals. This suggested that the firing frequency of a single unit was modulated in a similar manner to the activity of the remaining motoneuron pool as represented by the amplitude of the gross-EMG signal. Figure 7A, left, demonstrates this for one of the slow SOL units where the firing frequency profile of the unit was superimposed over the corresponding rectified and smoothed gross-EMG profile. When these two parameters were plotted against each other and a straight line was fit through the data (Fig. 7A, right), a relatively positive correlation emerged in comparison with the MG/LG units described below. The r2 values obtained from the fitted lines for the slow SOL units ranged from 0.14 to 0.35 (n = 4 units). The MAE with respect to the fitted line also was calculated for the slow SOL units to determine the average error in predicting frequency from the linear EMG relation (see METHODS). The MAE values ranged from 6.3 to 7.1 Hz, approx 16-18% of the modulation depth of the units during stepping, which was 40 Hz on average (modulation depth = maximum rate - minimum rate, excluding initial doublet/triplets).



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Fig. 7. A: relationship between the firing frequency of a single slow SOL motor unit and the amplitude of the corresponding rectified and smoothed gross-EMG. Right: scatter plots of frequency vs. gross-EMG amplitude for all steps (n = 22); left: 3 representative step cycles from the group data with the firing frequency superimposed on the rectified and smoothed gross-EMG record. B: same graph as in A but for a late-stance phase LG unit (n = 29 steps). Steps in the left panel are taken from 3 different walking sequences.

In comparison, the firing profiles of the mid-late-stance phase MG/LG units related poorly to the corresponding gross-EMG signal, with r2 values ranging from 0.0015 to 0.010 and MAEs from 20.2 to 31.0 Hz (see representative example of an LG unit in Fig. 7B). The interval-to-interval variability in the discharge rates of these units was greater than the slow SOL units, and this may account for the poorer correlation to the gross-EMG signals. Another contributing factor may be that because the MG/LG muscles are compartmentalized (DeRuiter et al. 1996; English and Letbetter 1982), the gross-EMG signals probably were not good indicators of overall motoneuron pool activity, i.e., different compartments of the MG/LG motoneuron pools may have received different drives from the locomotor networks. Because the gross-EMG electrodes most likely were recording activity from these different compartments, the correlation to a single motor unit in one compartment may have been poor, unlike that for the more homogeneous SOL motoneuron pool (Chamberlain and Lewis 1989; Gillespie et al. 1987).

To compare the rate modulation of motor units within a single LG muscle compartment, the activation profiles of pairs of motor units discriminated from a single micro-EMG record were examined. We wanted to investigate if the firing rates of simultaneously activated units were modulated in a parallel fashion and thus responding in a similar manner to a common locomotor drive, unlike that seen for motoneurons during MLR-induced walking, which display flat frequency profiles (Brownstone et al. 1992). Activity from a pair of LG motor units discriminated from a single micro-EMG record (and thus probably from the same muscle compartment) is shown in Fig. 8. A representative step cycle is shown in Fig. 8A, which clearly demonstrates that the firing profiles of both units were similarly modulated. The two rate profiles were fitted with a fifth-order polynomial to obtain a smoothed rate profile (see Fig. 8A, , unit A and , unit B). When the smoothed rate of one unit was plotted against the smoothed rate of the other at similar points in time, a positive linear relationship emerged (see Fig. 8B, r2 value of regression line = 0.62, n = 6 steps). A similar parallel rate modulation was seen in two other motor-unit pairs that were discriminated from the same micro-EMG electrodes, with r2 values of 0.53 and 0.71 from the linear regression.



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Fig. 8. Relationship between modulation pattern of 2 simultaneously recorded LG motor units. A: frequency profiles of unit A () and B () for a single step were fitted with a fifth-order polynomial to obtain a smoothed rate profile. B: data points from the fitted lines for unit A and B were taken every 25 ms and plotted against one another to test if the frequency profiles of both units were modulated in a similar fashion (n = 6 steps). ---, R2 = 0.6.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Differences in firing properties between fast and slow motor units during locomotion

A consistent finding from this study was that motor units often would jump to a relatively high-frequency of discharge when recruited. Only the fast motor units (i.e., MG/LG, fast SOL and TA units), however, initiated firing with a doublet or triplet. These fast motor units therefore displayed similar recruitment patterns to the hindlimb motor units recorded during MLR-induced locomotion (Brownstone et al. 1992; Tansey and Botterman 1996; Zajac and Young 1980). The slow SOL units, on the other hand, did not initiate firing with a doublet or triplet but had frequencies that were above or close to the mean rate in a locomotor burst, similar to the anterior thigh motor units recorded during locomotion in the intact cat (Hoffer et al. 1987). From our data, therefore it appears that the firing of initial doublets/triplets was dependent on the type of motor unit recorded (i.e., fast vs. slow) rather than from the type of locomotion studied (i.e., self-initiated vs. MLR-induced).

Likewise, the presence or lack of doublets/triplets was probably not a result of different locomotor drives to the different muscles (i.e., an abrupt synaptic drive to MG/LG and TA motoneuron pools compared with a more gradually increasing drive to the SOL motoneuron pool). This was supported by the fact that during gradual muscle stretch (Gorassini et al. 1999), only units with very high mean rates of discharge (>40 Hz and thus probably fast motor units) (Hennig and Lømo 1985) would initiate firing with a doublet, whereas units with mean firing rates of <40 Hz (and thus probably slow motor units) did not. It is not known why motor units jump to such high frequencies of discharge at recruitment. The activation of intrinsic conductances at recruitment may be one possibility and this is discussed in Initial doublets/triplets.

Another difference between the slow and fast motor units observed in this study was that the slow motor units (i.e., early stance phase SOL) were activated as a more homogeneous group during locomotion in contrast to the MG/LG units. This was evident in the linear relationship that existed between the number of MUAPs fired and the duration of the corresponding gross-EMG for the SOL but not the mid-late-stance phase MG/LG motor units. In addition, only the firing rate profiles of the slow SOL units followed the envelope of their gross-EMGs, indicating that these units were being modulated in a similar manner by a common locomotor drive.

The dissociation of activity of the mid-late-stance phase MG/LG units from the gross-EMG profiles may have resulted from a compartmentalized recruitment of the MG/LG motoneuron pool. For example, anatomic and physiological compartmentalization of the MG/LG muscle has been shown in both cats and rats with FR unit types occupying deeper parts of the muscle and FF unit types in the more superficial layers (DeRuiter et al. 1996; English and Letbetter 1982; Vanden Noven et al. 1994). The majority of MG/LG units recorded in this study were most likely from the superficial compartment because the micro-EMG electrodes were inserted very close to the surface of the muscle belly. These potentially large and fast units were activated preferentially midway through or toward the end of the stance phase and may be activated especially to provide the force needed to propel the animal forward at the end of stance. The delayed recruitment of these motor units may have resulted from a separate drive from the rhythm-generating networks in the spinal cord (rather than from an orderly recruitment in response to a gradually increasing central drive) because the recruitment of the mid-late-stance phase MG/LG units could occur as the corresponding gross-EMG activity (recorded in deeper muscle compartments) was declining (e.g., Figs. 2 and 7B).

Rate modulation during locomotion

The parallel modulation between the firing rate profiles of the SOL units and their corresponding gross-EMG activity also provides further evidence that the firing rates of motor units can be modulated during a locomotor burst in contrast to the poor rate modulation of hindlimb motor units that occurs during MLR-induced locomotion in the cat (Brownstone et al. 1992; Severin et al. 1967; Zajac and Young 1980). Further evidence was provided by the parallel rate modulation of pairs of simultaneously recorded motor units within a single LG muscle compartment. Perhaps the lack of rate modulation of motoneuron activity that occurs during MLR stimulation in the cat results from a rate limiting of motoneuron discharge due to maximal synaptic excitation (Binder et al. 1996). Alternatively, changes in intrinsic properties that increase motoneuron excitability and repetitive firing (Krawitz et al. 1997) may also contribute given that firing frequencies tend to be higher during MLR locomotion (40-50 Hz) (Brownstone et al. 1992) compared with intact walking (25-35 Hz) (Hoffer et al. 1987). In conclusion, it appears from this and other studies (see also DeSerres et al. 1995; Eken 1998; Hoffer et al. 1987) that rate modulation of motor-unit activity occurs during natural, self-initiated walking.

Initial doublets/triplets

The recruitment of the mid-late-stance phase MG/LG motor units with doublets/triplets most likely was a result of intrinsic conductances activated at recruitment, rather than from an abrupt increase in excitatory synaptic drive. Evidence to support this comes from observations in which initial doublets/triplets have been shown to occur without abrupt increases in the corresponding gross-EMG activity or in the firing rates of other concurrently active motor units (Gorassini et al. 1999). One possible intrinsic mechanism may be the activation of voltage-dependent, noninactivating plateau potentials considering it recently has been shown that when plateau potentials are activated near the threshold for action potential generation, the plateau can produce an abrupt increase in the rate of rise of the membrane potential at the time of recruitment, resulting in high initial firing rates (Bennett et al. 1998).

The added depolarization produced by the plateau at recruitment may have acted like an abrupt current injection through a microelectrode, which is known to cause doublets and triplets (Granit et al. 1963a). Furthermore in cat motoneurons it was shown that when sodium spikes were inactivated with QX314, an overshoot in membrane potential that lasted for approx 80 ms emerged when a plateau initially was activated (Bennett et al. 1998; Brownstone et al. 1994). Thus two or three MUAPs could have been fired on the crest of this overshoot, resulting in the initial doublets/triplets, provided that the motor units could fire fast enough. Taking this latter point into consideration, it is interesting that initial doublets (and especially initial triplets) were more prevalent in units that were able to fire two to three MUAPs within this short time period (e.g., TA and late-stance phase MG/LG units), i.e., their firing rates were high enough to "sample" the underlying membrane potential. The SOL units on the other hand only reached maximal initial rates of ~60 Hz, and perhaps they were unable to follow the fast transients in membrane potential. Alternatively, the doublets/triplets may have been produced from a postinhibitory rebound (Bertrand and Cazalets 1998), from the activation of a low-threshold Ca2+ spike (Llinás and Yarom 1981; Russo and Hounsgaard 1996), or from extra spikes arising out of an afterdepolarization (Fulton and Walton 1986: Granit et al. 1963b; see also discussion in Eken 1998), which has been shown to be enhanced during plateau potential activation (Bennett et al. 1998).

Tension-frequency relation of single motor units during locomotion

Regardless of the mechanism(s) involved, a high initial firing rate of a motoneuron has been shown to produce a more rapid onset in motor unit force (i.e., catch property) (Burke et al. 1970). A rapid onset in force is essential during walking, especially for flexor muscles in the rat. For example, during steady walking (1 s/step), a rat must dorsiflex its ankle within <= 50 ms to clear the foot at the beginning of the swing phase due to the very flexed posture of both the hip and knee joints (Gruner et al. 1980). Thus it is not surprising that a large number of motor units are recruited with initial frequencies that are two to five times greater than the mean rate of discharge in a locomotor burst. In addition, these doublets/triplets often are followed by an undershoot in firing frequency, and this pattern of discharge has been shown to optimize the speed at which maximum force is reached in a motor unit (Stein and Parmiggiani 1979). The gross-EMG patterns of a variety of hindlimb muscles that show an initial peak followed by a rapid decline may result from this pattern of motor-unit recruitment (Gorassini et al. 1994; Gruner et al. 1980).

After recruitment, the mean firing rates of the various units during a locomotor burst fell close to the top of their corresponding tension-frequency curves [the tension-frequency relation was taken from Fig. 3 in Henning and Lømo (1985) for the slow SOL and fast extensor digitorum longus (EDL) muscles]. For example, in this study, when the nerve to the SOL muscle was stimulated at a rate of 30 Hz (i.e., the mean rate at which SOL units fired during walking, see Table 1), the muscle reached nearly 90% of its maximum tetanic force at its optimal length. Similarly, in the EDL muscle, which has a similar fast motor-unit type composition as the TA muscle, stimulation of the nerve at 100 Hz (i.e., mean rate of TA units during walking, see Table 1) also produced 90% of the maximum tetantic force. Stimulation rates of 60-80 Hz (i.e., firing rates of MG/LG units during walking) produced 60-80% maximum tetanic force in the EDL muscle and probably would produce even higher forces in the LG/MG muscle because it contains more FR unit types than EDL (DeRuiter et al. 1996).

From these indirect estimates it appears that during walking, motor units (on average) fire at discharge rates that produce forces that are close to their maximum levels. These estimates were taken from experiments in which the muscle lengths were set to produce maximum force. Although motor units may not always be at their optimal lengths throughout a locomotor burst, this may be compensated for by the high rates at recruitment, as discussed in the preceding text.

Summary

The data from this study provide the first extensive description of single motor-unit activity from different muscles during unrestrained walking in the conscious adult rat. It is important to establish this considering the increasing number of studies looking at the control of motoneuron activity during transmitter-induced locomotion in the neonatal rat. For example, neonatal motoneurons do not initiate firing with initial doublet/triplets, and they fire at frequencies that are 5-10 times slower than adult motoneurons during unrestrained walking (Bertrand et al. 1998; Hochman and Schmidt 1998; MacLean et al. 1997).

In summary, evidence was provided to suggest that the activation patterns of single motor units during unrestrained locomotion in the rat are shaped both by the intrinsic properties of the parent motoneuron and from synaptic inputs from the rhythm-generating networks of the spinal cord. The integration of these two effects on motor-unit recruitment and firing aids in the shaping of the final force output of the respective muscle fibers to produce the appropriate activation patterns required for locomotion.


    ACKNOWLEDGMENTS

The authors acknowledge the excellent technical assistance of I. Kjaer for construction of the micro-EMG wires, L. Grøndahl for help in animal care, and E. Gudbrandsen for building electrical equipment.

This research was supported by the Danish Medical Research Council and the Novo Nordisk Foundation. D. J. Bennett was supported by the Alberta Heritage Foundation for Medical Research and M. Gorassini by the Danish Research Academy. T. Eken was supported by the Norwegian Research Council for Science and the Humanities. O. Kiehn is a Hallas Møller Associate Professor supported by the Novo Foundation.

Present address of D. J. Bennett: Division of Neuroscience, Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, Alberta T6G 2G4, Canada


    FOOTNOTES

Present address and address for reprint requests: M. Gorassini, 3-48 Corbett Hall, Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, Alberta T6G 2G4, Canada.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 July 1999; accepted in final form 8 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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