Wadsworth Center, New York State Department of Health and State University of New York at Albany, Albany, New York 12201
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ABSTRACT |
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Carp, J. S., P. A. Herchenroder, X. Y. Chen, and J. R. Wolpaw. Sag during unfused tetanic contractions in rat triceps surae motor units. Contractile properties and conduction velocity were studied in 202 single motor units of intact rat triceps surae muscles activated by intra-axonal (or intra-myelin) current injection in L5 or L6 ventral root to assess the factors that determine the expression of sag (i.e., decline in force after initial increase during unfused tetanic stimulation). Sag was consistently detected in motor units with unpotentiated twitch contraction times <20 ms. However, the range of frequencies at which sag was expressed varied among motor units such that there was no single interstimulus interval (ISI), with or without adjusting for twitch contraction time, at which sag could be detected reliably. Further analysis indicated that using the absence of sag as a criterion for identifying slow-twitch motor units requires testing with tetani at several different ISIs. In motor units with sag, the shape of the force profile varied with tetanic frequency and contractile properties. Simple sag force profiles (single maximum reached late in the tetanus followed by monotonic decay) tended to occur at shorter ISIs and were observed more frequently in fatigue-resistant motor units with long half-relaxation times and small twitch amplitudes. Complex sag profiles reached an initial maximum early in the tetanus, tended to occur at longer ISIs, and were more common in fatigue-sensitive motor units with long half-relaxation times and large twitch amplitudes. The differences in frequency dependence and force maximum location suggested that these phenomena represented discrete entities. Successive stimuli elicited near-linear increments in force during tetani in motor units that never exhibited sag. In motor units with at least one tetanus displaying sag, tetanic stimulation elicited large initial force increments followed by rapidly decreasing force increments. That the latter force envelope pattern occurred in these units even in tetani without sag suggested that the factors responsible for sag were expressed in the absence of overt sag. The time-to-peak force (TTP) of the individual contractions during a tetanus decreased in tetani with sag. Differences in the pattern of TTP change during a tetanus were consistent with the differences in force maximum location between tetani exhibiting simple and complex sag. Tetani from motor units that never exhibited sag did not display a net decrease in TTP during successive contractions. These data were consistent with the initial force decrement of sag resulting from a transient reduction in the duration of the contractile state.
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INTRODUCTION |
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Mammalian motor units exhibit considerable
variation in force generation, endurance, and speed of contraction and
relaxation (Burke 1981). A substantial portion of this
diversity can be attributed to the presence of distinct sets of
myofibrillar constituents that define two general classes of muscle
fibers, commonly referred to by their hallmark contractile feature as
fast-twitch (F) and slow-twitch (S) fibers (Pette and Staron
1990
; Stephenson et al. 1998
). In attempting to
identify motor units solely on the basis of contractile properties,
Burke et al. (1973)
proposed a classification scheme in
which the presence or absence of "sag" (i.e., a decline in force
after the initial increase during unfused tetanic stimulation) distinguished F and S motor units, respectively. Originally developed for medial gastrocnemius motor units of the cat, this scheme has been
applied widely in other muscles and species (Burke
1981
).
Studies of rat triceps surae (TS) motor unit contractile properties
have produced qualitatively similar results, but there has not been
general agreement on the specific criteria for type identification. The
presence or absence of sag as a lone predictor of the speed of
contraction has been confirmed in some studies (Chamberlain and
Lewis 1989; Einsiedel and Luff 1993
;
Kanda and Hashizume 1989
) but not in others
(Bakels and Kernell 1993a
,b
; Grottel and
Celichowski 1990
). In these studies, sag was evaluated under
widely varying conditions (e.g., at fixed stimulation frequencies or at
frequencies based on the speed of contraction and with single trains or
with batteries of trains at different stimulation frequencies). Sag has
been demonstrated to be present at some stimulation frequencies in a
given unit, but not all units display the same pattern of frequency
dependence or even the same force envelope shapes (Celichowski 1992a
; Einsiedel and Luff 1993
; Gardiner
1993
; Grottel and Celichowski 1990
). Thus it is
not surprising that a consistent classification scheme for F versus S
units has not emerged for rat motor units.
We are currently developing a model for performing motor unit typing in
the intact triceps surae (TS) of the anesthetized rat as part of this
laboratory's ongoing investigation of adaptive plasticity in the
spinal cord (reviewed in Wolpaw 1997). The goal of the
present study was to identify factors that contribute to variation in
sag conformation to develop criteria for rapid and reproducible motor
unit typing in the rat. Portions of this work has been reported
previously in abstract form (Carp et al. 1997
, 1998
).
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METHODS |
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Animal preparation
Experiments were performed in 24 male Sprague-Dawley rats
weighing 461-554 g. All procedures conformed to National Institutes of
Health Publication 85-23, Guide for the Care and Use of Laboratory Animals and had been reviewed and approved by the Institutional Animal
Care and Use Committee of the Wadsworth Center. Animals were
anesthetized initially with 70 mg/kg ip of pentobarbital and
supplemented with 10 mg/kg iv about every 30 min to suppress corneal
and limb withdrawal reflexes and maintain heart rate between 300 and
350 beats per minute. The trachea and a jugular vein were cannulated. A
mixture of dextran, sodium bicarbonate and glucose was infused
continuously at 1.7-3.4 ml/h (Quintin et al. 1989). End
tidal CO2 was 32-42 mm of Hg during recording sessions.
Rectal temperature was maintained at 37-38°C by feedback-controlled
heat lamps.
Animals were mounted in the prone position in a rigid frame and secured by ear and jaw bars and a pair of hip pins applied 12-15 mm caudal to the front end of the ilium. The right hip and knee were extended and inserted into a latex surgical glove. The anterior surface of the hindlimb from the foot to the middle of the thigh was attached with cyanoacrylate glue to the glove, leaving the posterior side accessible for surgery. The hindlimb was immobilized by a 2-cm-wide clamp that gripped the anterior side of the tibia with its proximal edge 3 mm distal to the knee. The clamp was anchored in this position by inserting No. 62 drill bits through the tibia through two pairs of holes in the clamp located 2 mm from each end. This permitted the clamp to be tightened without slipping or impeding blood flow to the TS muscles.
The TS [i.e., medial and lateral gastrocnemius (MG and LG) and soleus (SOL)] muscles of the right hindlimb were dissected from surrounding tissue. The tendon of the plantaris muscle was dissected from the common tendon of the TS muscles and cut, but its position relative to the TS muscles was not altered. To avoid interruption of perfusion, the TS muscles were not separated from each other, and small vessels along the lateral borders of the muscles were left intact to the extent that permitted unimpeded passive stretch of the muscle. The calcaneus was cut leaving a small bone chip attached to the TS tendon. The bone chip was secured in a clamp, which was connected by a 50 × 0.6 mm steel wire to a force transducer (FT03, Grass Instruments).
Electromyographic activity (EMG) from each of the TS muscles (i.e., SOL, MG, and LG) was recorded with three intramuscular pairs of fine stainless steel wires that had 4 (SOL) or 6 (MG and LG) mm of Teflon insulation removed from each tip. Postmortem inspection confirmed electrode positions to be wholly within the targeted muscle in all but one experiment, where one of the wires intended for SOL was inserted into LG.
Motor unit recordings were performed by impalement of the motoneuron
axon in the ventral root (modification of the method of Cope and
Clark 1991). A dorsal laminectomy was performed from the
L1 to L5 vertebrae and the dura mater was cut.
The cut skin flaps were pulled up to hold a mineral oil pool. The
L5 or L6 ventral root was manipulated with fine
glass hooks onto a pair of stainless steel hook electrodes embedded in
a longitudinally hemisected 10-mm-long piece of Silastic tubing (3 mm
OD × 2 mm ID) 1-3 mm above the surface of the spinal cord. In
addition, the tibial nerve was dissected from surrounding tissue, and
its electroneurographic activity (ENG) was recorded using a cuff
electrode formed from a longitudinally hemisected 4-mm-long piece of
silastic tubing (0.4 mm OD × 0.26 mm ID) fitted with a
semicircular loop of 50-µm OD stainless steel wire at its midpoint.
The cuff was positioned underneath the tibial nerve ~10 mm proximal
to the TS muscles. A small incision was made with fine scissors in the epineurium opposite to the wire loop in the cuff. The tip of a 50-µm
OD stainless steel wire from which the final 0.5 mm of Teflon insulation had been removed was inserted gently just beneath the epineurium. Application of a rapid-cure silicone elastomer (Kwik-Cast, World Precision Instruments) to the cuff stabilized and electrically isolated the electrodes.
The TS muscles and tibial nerve then were submerged in a mineral oil pool formed by lifting the edges of the latex surgical glove that had been glued previously to the anterior skin of the hindlimb. Mineral oil pools in the leg and back were maintained at 36-37 and 37-38°C, respectively, by feedback-controlled heat lamps.
Electrophysiological recording
Ventral root axons were impaled by advancing glass
microelectrodes filled with 3 M potassium acetate (12-25 M) in 1- to 3-µm steps at a 45° angle to the long axis of the nerve with tip
pointing caudally. As the electrode advanced, depolarizing current
pulses (2-4 nA, 1 ms) were applied at 1 Hz until an action potential was elicited. Stimulation frequency was immediately reduced to 0.2 Hz,
and motor units were identified by the presence of reproducible all-or-none EMG and twitch responses to single stimuli over at least a
fourfold range of current intensities. Plantaris axon impalements
sometimes elicited measurable EMG in the TS electrodes. These units
were abandoned after observing movement of its cut tendon and transient
force responses at the onset and offset of tetanic stimulation with
little or no sustained force during the tetanus. Ventral root
impalements produced negative resting potentials and action potential
amplitudes of 37 ± 18 mV (mean ± SD, measured from spike
threshold to peak).
A series of twitch and tetanic responses were elicited by current
injection to characterize the properties of each motor unit. The
following sequence of stimulation and recording protocols was initiated
and continued until it was completed or until the unit was lost. First,
unpotentiated twitch force, EMG, and axon voltage were recorded during
0.2-Hz stimulation and averaged (4-16 responses, with more averaged
for low force units). Second, tetanic and twitch force were recorded
during a 600-ms, 200-Hz train of stimuli followed 2 s later by a
single stimulus. This sequence was repeated at 10-s intervals until the
maximum potentiation of the single twitch was seen (usually 5-10
sequences). Third, tetanic force (and EMG in 26 motor units) was
recorded during trains of 25 stimuli repeated at 10-s intervals to
detect the presence of sag. In most units, the first sag train was
elicited with an interstimulus interval (ISI) equal to the
unpotentiated twitch contraction time (CTtw), estimated
during the first step of this protocol as the time from twitch onset to
maximum force. Longer initial intervals were used if this ISI elicited
fused tetani. Stimulus trains were applied with at least three
different progressively longer ISIs incremented in steps of
0.125-0.25 × CTtw. In all experiments, minimum and
maximum values (in terms of CTtw) of the range of ISIs
tested varied across units due to errors in estimating
CTtw. An additional series of tetani were elicited in 12 motor units to evaluate the effect of repetition rate on the
reproducibility of sag. In these units, two series of six tetani each
were repeated at 10- and 0.5-s intervals for up to four different ISIs.
Fourth, fatigue was assessed by recording tetanic force (and EMG in 36 motor units) during application of 70-Hz trains of 14 stimuli delivered
at 1-s intervals for 2 min. Stimulation at 40 Hz is the de facto
standard of motor unit typing in cats (Burke et al.
1973). However, CTtw of TS motor units is substantially shorter in rats (Bakels and Kernell 1993b
;
Chamberlain and Lewis 1989
; Einsiedel and Luff
1993
; Gardiner 1993
; Grottel and
Celichowski 1990
) than that in cats (Burke 1967
;
Burke et al. 1973
; Proske and Waite 1974
;
Wuerker et al. 1965
). Therefore a higher stimulation
frequency was chosen to maintain a comparable degree of tetanic fusion.
A fifth protocol was employed in which individual action potentials were elicited by injection of 2- to 3-ms depolarizing current pulses at 8-16 Hz, and axon voltage and tibial nerve ENG were recorded. Typically, 400-800 responses were averaged, but as many as 4,000 were averaged when the ENG had a low signal-to-noise ratio and recording stability permitted.
Typical recording sessions lasted 6-9 h. TS whole muscle twitch in response to supramaximal stimulation of the tibial nerve was monitored during all experiments. Experiments were terminated when twitch force fell to <75% of its initial value. Animals were killed at the end of the experiment by intravenous pentobarbital overdose. In experiments with ENG recordings, the conduction distance between the impalement site and the subepineurial electrode was measured with the animal still mounted in the experimental apparatus.
Data collection and analysis
EMG and ENG were band-pass filtered (0.03-3 and 0.003-10 kHz,
respectively) and force and axon voltage were low-pass filtered (0.7 and 10 kHz, respectively). All signals were amplified to maximize
resolution during A/D conversion by computer (sampling rate: 2 kHz
for force,
20 kHz for EMG, ENG, and axon voltage).
Peak force (Ptw), CTtw, and half-relaxation time (HRTtw; i.e., time from maximum force to 50% of maximum force) were calculated for the averaged unpotentiated twitches. Maximum tetanic and potentiated twitch forces were calculated for each of the alternating pairs of tetani and twitches, and from these the responses with the largest amplitude tetanus (Ptet) and potentiated twitch (Ppot) were identified. Potentiated twitch contraction time (CTpot) and half-relaxation time (HRTpot) were also calculated for the largest potentiated twitch.
Preliminary experiments were performed to evaluate the effect of background passive force on twitch and tetanic amplitudes. Over a 50- to 550-mN range of forces produced by varying muscle length, Ptw did not vary with background passive force, but Ptet decreased by 2 mN per 100 mN increase in background passive force (P > 0.5 and <0.001 for regression of Ptw and Ptet, respectively, on background passive force by analysis of covariance with motor unit as variate and background passive force as covariate). The mean background passive force ±SE was 133 ± 14 mN for the entire data pool (range = 101-203 mN, with only 1% of the units tested >163 mN). The error in measuring Ptet introduced by differences in background passive force were estimated to be <2% of Ptet in all units, which corresponded to projected errors of <1 mN in all cases. All tetani that were evaluated for sag were elicited in a background passive force range of 111-163 mN. Within this range, there was no significant correlation between background passive force and the likelihood of detecting sag (P > 0.2 and r2 = 0.01 for linear regression of the percentage of trials tested in which sag was detected on background passive force).
The presence or absence of sag (see Criteria for determination of sag status) was noted for all tetani tested in a given motor unit. Failure to detect sag in any of at least four trains (median number of trains = 7) with ISIs falling in the range of 1.0-3.0 × CTtw (median minimum and maximum ISI/CTtw = 1.20 and 2.69, respectively; mean ± SD of ratio of maximum:minimum ISIs = 2.1 ± 0.3) resulted in classification as a nonsagging unit. The presence of sag in one or more of those tetani resulted in classification as a motor unit with sag.
The pattern of force accumulation in unfused tetani in which sag was sought was quantified in two ways. First, a sequential force index (SFI) was calculated as the difference in amplitude between a given peak and its predecessor during the tetanus (with the SFI for the first peak being the difference between that peak and the pretetanic force level) divided by the maximum tetanic force. Second, the time from contraction onset to maximum force (TTP) was calculated for each peak in the tetanus.
Fatigue was quantified by calculating a fatigue index (FI) defined as the ratio of the maximum force during the 120th tetanus to that of the first tetanus.
Motor units were provisionally type-identified according to the
criteria of Burke et al. (1973). Motor units with or
without sag were classified as F or S, respectively. F motor units were subdivided further according to their fatiguability such that units
with a FI
0.75 were classified as fatigue-resistant (FR), those
with 0.25 < FI < 0.75 as having intermediate fatiguability [F(int)] and those with FI
0.25 as fatiguable (FF).
Orthodromically conducted action potentials evoked by intra-axonal current injection usually were detected as triphasic ENG waveforms. Axonal conduction time was calculated as the latency from the onset of the current-evoked action potential to the onset of the large negative component (usually the second peak) of the ENG waveform. The latter time point was considered to reflect the timing of the nodal action potential threshold (i.e., the time when net current flow at the subepineurial electrode was 0). Axonal conduction velocity (CV) was calculated as the ratio of the conduction distance to the conduction time.
The muscle containing the stimulated motor unit was identified nominally by the electrode pair with the largest evoked peak-to-peak amplitude EMG. No qualitative differences were observed in the force envelope shapes of motor units with or without sag among motor units from the different TS muscles. Thus the data were pooled across muscles.
Comparisons of motor unit properties by type were performed using ANOVA. Differences among treatment groups were assessed by Tukey's honestly significant difference test. Relationships between motor unit properties were assessed by linear regression. Differences with P < 0.01 were considered to be statistically significant.
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RESULTS |
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A total of 202 motor units are included in this study. The number of motor units studied per experiment varied widely, with 15 experiments having 1-7 units and 9 experiments having 9-14 units. Of these motor units, 158 (78%) had at least one occurrence of sag at an ISI in the range of 1.0-3.0 × CTtw.
Force profiles of unfused tetanic contractions
The force envelope formed by the peaks of unfused tetanic
contractions exhibited several characteristic patterns, which are illustrated by data from four motor units in Fig.
1. Unit 1 is representative of
the 44 units in which sag was not detected at any ISI tested, even with
ISIs of up to 3.4 × CTtw. The peak force envelope
rose steadily throughout the stimulus train without detection of local
maxima. Unit 2 is one of seven motor units (5-9 different ISIs tested) with peak force envelopes that increased to maxima (wide
arrowheads) and decreased steadily thereafter. This uncomplicated force
accumulation pattern is identified as "simple" sag
(sagS). Unit 3 is 1 of 63 motor units that
exhibited force envelopes that rose to initial peaks (narrow
arrowheads), decreased for one to three stimuli, and then increased
again. This "complex" sag configuration (sagC) has
been observed in both rats (Bakels and Kernell 1993a; Celichowski 1992a
,b
) and cats (Jami et al.
1983
). The two sag patterns were both observed in each of the
remaining 88 motor units (exemplified by unit 4), where
sagS was observed at some ISIs (ISI 1-2) and
sagC was observed at others (ISIs 4-7). Motor units in
which at least one tetanus with sag was observed often had individual
tetani in which sag was not detected (Fig. 1, ISIs 1-3 from unit
3 and ISI 3 from unit 4). These are referred to as
sagN tetani.
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The shape of the force envelope was also dependent on the time between successive tetanic contractions. Sets of six tetani at one to four different ISIs were repeated every 10 s and then every 2 s in 11 motor units. Repetition every 2 s resulted in qualitative changes in the shape of the force envelope of the second to sixth tetani, either through conversion among different sag patterns or by losing the appearance of sag entirely (16 or 30 of 140 tetani tested, respectively; Fig. 2, top). Stimulation at the same ISIs every 10 s produced little change in force envelope shape (Fig. 2, bottom), such that sag status (i.e., sag vs. no sag) did not vary during repetition of tetani in any motor unit and conversions among sag subtypes were detected in only 3 of 138 tetani tested.
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EMG did not vary substantially during the course of the 207 tetani from 26 motor units in which it was recorded. For example, the peak-to-peak amplitude of the EMG response to the last tetanic stimulus of the train was not significantly different from that of the first EMG response (mean % difference ± SE = 0.7 ± 1.0%; P = 0.53 by t-test). In addition, the ratio of the last:first peak-to-peak EMG amplitudes did not vary significantly with tetanic ISI (slope = 0.03 ± 0.02, r2 = 0.01 and P = 0.12 for regression of EMG amplitude ratio on ISI/CTtw). This relationship predicts only a 6% change in EMG for tetani elicited over the entire twofold range of ISI/CTtw values used in this study. Thus any contribution of neuromuscular transmission failure to sag under the conditions described here was probably minimal.
Criteria for determination of sag status
On the basis of the different patterns of force accumulation
described above, two sets of criteria were established to identify sag
during an unfused tetanic contraction. The initial approach used to
detect sag was to identify the earliest local maximum (and subsequent
local minimum, if present) in the force envelope (1st-order method). As
illustrated in Fig. 3, bottom,
the second peak of the tetanus was identified as the first maximum of
the force envelope because it was larger than at least two subsequent peaks ( values under · · · ) by
1% of the
maximum tetanic amplitude and by 1 SD of the baseline force level
before stimulation. Subsequently, the third peak was defined as the
first local minimum because it was smaller than at least two subsequent
peaks by
1% of the maximum tetanic amplitude and by 1 SD of the
baseline force level before stimulation. A tetanus with both a maximum
and a minimum such as this is classified as having sagC1
(i.e., complex sag identified by the 1st-order method). Had no local
minimum been located after detection of the initial maximum, the
tetanus would have been classified as sagS. Requiring
interpeak differences to meet criteria based on both tetanus magnitude
and background passive force variability provided a stringent approach
to identifying maxima and minima. However, one limitation of these
criteria was that tetani with substantial force decrements in only a
single peak subsequent to the maximum were not detected as
sagC1. Thus in cases where sag was not detected using the
first-order method, an alternate approach based on the detection of an
inflection point in the amplitudes of sequential force peaks was used
(2nd-order method). In this method, second-order differences (
values) were calculated between adjacent differences in peak force (
value, as in the preceding text). Units with long CTtw and
monotonically rising peak force profiles (i.e., presumed S units) never
exhibited
values greater than +1.4 mN. Thus this
value
was used as the minimum criterion for identifying the presence of sag.
This method readily identified tetani with substantial single-peak decrements (as described earlier) and an additional group of tetani with points of inflection that did not exhibit overt force decrements (Fig. 3, middle). All tetani identified with points of
inflection more than +1.4 mN were defined as having a complex sag
configuration as detected by the second-order method
(sagC2). Tetani so identified were considered to have their
first local minima at the middle peak of the three that produced the
largest
value. The peak before the one defined as minimum was
defined as the first local "maximum," even if it was not
numerically higher than the middle peak. Tetani that failed to meet
criteria for both first-and second-order methods were considered to not
exhibit sag (Fig. 3, top).
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Average force profiles in motor units with or without sag
Using the criteria described above, sag was identified in 933 (66%) of 1,406 tetani. The tetani with sag consisted of 310 (22%) with sagS, 545 (39%) with sagC1, and 78 (6%) with sagC2. Of the units with sagC2, 35 (2%) had overt force decrements. Of the remaining 473 tetani without sag, 173 (12%) were from motor units in which sag was elicited in at least one other tetanus with a different ISI (i.e., sagN tetani), and the remaining 300 (21%) were from motor units that failed to display sag at any stimulation frequency.
Figure 4 shows the
average increment in force (normalized to maximum tetanic force)
produced by sequential stimuli in the train (i.e., SFI) for units with
and without sag elicited at ISIs in the range of 1.1-1.3, 1.7-1.9,
and 2.5-2.7 × CTtw (Fig. 4, A-C,
respectively). In motor units that failed to exhibit sag at any ISI
tested (Fig. 4, ×), the force of the first contraction represented a
larger fraction of the maximum force as ISI increased. The subsequent
4-10 contractions added a near-constant fraction of the maximum
tetanic force at any ISI, declining gradually thereafter toward an
asymptote. Motor units that displayed sag during at least one sag train
(Fig. 4, ,
,
,
) had larger force increments in response to
the first stimulus than those that never sagged. The large initial
increments diminished rapidly during the subsequent two to six stimuli.
Significant differences were detected in the SFI between each group of
tetani with sag and those from nonsagging motor units for short (peaks
1-3 and 5-25 for sagS; peaks 1-3 and 5-12 for
sagC1; and peaks 1-2, 4-17, and 21 for
sagC2), intermediate (peaks 1-3 and 6-25 for
sagS; peaks 1-2, 4-16, 18, and 20 for sagC1;
and peaks 1-2, 4-16, and 20 for sagC2), and long (peaks 1-2, 5-15, 17, 19, 22, and 24 for sagS; peaks 1, 4-15,
17, 19, 22, and 24 for sagC1; and peaks 1 and 12-14 for
sagC2) ISIs in Fig. 4, A-C, respectively
(P < 0.01 for all by ANOVA).
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The range of SFI values for motor units without sag overlapped those of
motor units with sag for each stimulus in all ISI ranges. The absence
of sag in a motor unit usually was predicted in a given tetanus by a
stable (or gradually decreasing) pattern of SFI values in the second to
final contractions of the tetanus (the SFI of the 1st contraction of
tetani from motor units without sag varied substantially with ISI). For
example, the sag status of a motor unit was correctly identified in 147 of 152, 164 of 166, and 113 of 115 tetani (97, 99, and 98% agreement,
respectively) with nonsagging units defined by SFI of 0.007-0.18,
0.016-0.13, and 0.006-0.11 in the 2nd-12th contractions for
ISI/CTtw values in the range of 1.1-1.3, 1.7-1.9, and
2.5-2.7, respectively. Slightly stronger relationships between sag and
SFI pattern were observed for tetani limited to absolute ISI ranges.
For example, the sag status of a motor unit was correctly identified in
266 of 268 tetani (99% agreement) when nonsagging units were defined
by a SFI of 0.013-0.16 in the 2nd-12th contractions for ISIs in the range of 22.5-27.5 ms. The >97% covariation of SFI values and motor
unit sag status could not be achieved with any single range of SFI
values for all ISI or ISI/CTtw ranges nor for any other combination of sequential contractions within the tetanus. The striking
difference in the patterns of force accumulation between sagging and
nonsagging motor units is consistent with those reported for 40-Hz
tetani in histochemically defined F and S motor units (Tötösy de Zepetnek et al. 1992).
Differences in force profiles among tetani from motor units with sag
were most pronounced at intermediate duration ISIs (Fig. 4B). Differences among tetani elicited at shorter or longer
ISIs were more subtle, but the overall patterns were qualitatively similar. Tetani displaying sagS decayed gradually,
eventually exhibiting their characteristic force decrements toward the
end of the stimulus train at intermediate ISIs ( at stimuli 13 and 25) or earlier in the stimulus train at long ISIs (
at stimuli 6 and
7). The first force increments of tetani with sagC (
and
) were larger than those with sagS (P < 0.001, ANOVA) and decreased more rapidly thereafter. The SFIs of motor
units with sagC1 (
) were nearly identical to those with
sagC2 (
). No significant differences were detected
between them at any stimulus number (P > 0.05 for all
except for stimulus 3 in A, where P > 0.01 by ANOVA). The sagS groups were significantly different
from the sagC1 and sagC2 groups at several
stimuli (short ISIs: peaks 5-7, 12-23, and 25 and peaks 1 and 3-6,
respectively; intermediate ISIs in B: peaks 1, 3-7, 10-22,
and 24-25 and peaks 1, 3-5, 10-14, and 15-16, respectively; long
ISIs in C: peaks 1, 3-4, 7-8, 10, and 12 and peaks 7-8,
respectively; P < 0.01 for all by ANOVA). Both
sagC groups reached minima by the fourth stimulus, but the sagC1 force increment became negative, while the
sagC2 force increment only reached zero. These patterns
reflect the methods used to identify sagC (i.e., based on
force decrement for sagC1 and point of inflection for
sagC2). The sagN force increment pattern (
) was more similar to that of tetani with sag (lacking only the early and
late negative components of sagC and sagS
tetani, respectively) than to that of units that never sagged.
In addition to differences in the force envelope profiles among sagging
and nonsagging motor units, there were also differences in TTP of the
individual contractions comprising the tetani used to test for sag. For
example, the TTP of tetani elicited in the ISI range of 1.7-1.9 × CTtw decreased for all tetani from motor units with sag
and increased for tetani from motor units that never sagged during
peaks 2-6 (Fig. 5). The decrements in
TTP in motor units with sag are associated with force decrements, while
the increasing TTP of motor units that never sagged are associated with
stable force increments (see SFI from peaks 2-6 of comparable tetani
in Fig. 4B). TTP tended to decrease most rapidly in tetani
with sagC ( and
), less rapidly in sagN
tetani (
), and least rapidly in sagS tetani (
). The
differences in the pattern of change in TTP among tetani from motor
units with sag were subtle (sagS tetani
sagC1 tetani at peaks 2-4, sagS tetani
sagC2 tetani at peak 2, and sagS
tetani
sagN tetani at peak 4; P < 0.01 by ANOVA) compared with differences in TTP between motor units
with and without sag [sagC1, sagC2,
sagN, and sagS
tetani from motor units that
never sagged at peaks 2-6; P < 0.0001 for all (except
P < 0.01 for sagS at peak 2) by ANOVA].
After peak 6, the TTP subsequently decreased in tetani from motor units
that never sagged and in tetani with sagS,
sagN, and sagC2 (but not sagC1).
|
Decreases in TTP are associated with force decrements to varying degrees in motor units displaying different types of sag. Figure 6 shows that the force increment provided by sequential stimuli during a tetanus varies directly with TTP for the first three to seven contractions in tetani with sagC1 (A) and sagC2 (B). Subsequent peaks displayed little dependence of force increment on TTP. Tetani with sagS also displayed a similar phenomenon, but the relationship continued from the 2nd to 12th contractions before dissipating (C). These data suggest that the decrease in TTP during the initial contractions contributed to the development of sag in both subtypes, but other factors determined the pattern of force accumulation during latter contractions of tetani. That tetani from motor units that never sagged also had force increments that varied with TTP during the majority of the tetanus (D) suggests that this relationship is not specific to sag but rather is a common outcome of unfused tetanic stimulation.
|
During tetanic stimulation, motor units displaying sagS reached peak amplitude by a median of 13 stimulations within the train of 25 (middle 90% of units ranged from 5 to 20). Motor units displaying sagC reached an initial force maximum by a median of two stimulations (middle 90% of values between 2 and 4). For both sag subtypes, the peak number of the first amplitude maximum tended to decrease at higher ISI multiples of CTtw (e.g., compare ordinal position of peaks among traces from units 2 and 3 in Fig. 1). Regression analysis indicated that the location of the initial force maximum was related inversely to the ISI with ordinal peak location (Fig. 7). This difference in relative location was also evident in the absolute times of the force maxima for sag subtypes. For example, the mean time to the initial maximum ± SD for all tetani elicited at ISIs = 1.7-1.9 × CTtw was 303 ± 132 and 58 ± 28 ms for sagS and sagC1, respectively (P < 0.0001 by t-test). Differences in peak location and frequency dependence seen in the entire sample also are seen in the 88 units in which both sag subtypes were observed. The mean peak position ± SD of pairs of trains with sagS and pairs of trains with sagC decreased by 1.5 ± 4.1 and 0.5 ± 0.9 stimulations per 0.25 × CTtw change in ISI, respectively. The mean peak position ± SD of sagS-to-sagC decreased by 9.8 ± 8.7 stimulations per 0.25 × CTtw change in ISI. These data suggest that the transition from sagS to sagC does not represent a continuous process with the peak shifting smoothly from later to earlier peaks.
|
Tetani with sagC2 shared many features in common with those expressing sagC1 (e.g., force envelope shape, time course of TTP of sequential peaks, ordinal location of the initial maximum peaks), suggesting that they are manifestations of the same underlying phenomenon. The sagC2 tetani without force decrements, although not ordinarily defined as sag, had qualitatively similar force envelopes to sagC2 tetani with force decrements and to sagC1 tetani. Thus tetani with sagC detected by either method are treated here as a single subtype of sag force profiles.
Frequency dependence of sag
Accurate type identification of motor units requires knowing the
probability of detecting sag at a given frequency. For the 158 motor
units with sag, Fig. 8, top,
shows the total number of motor units tested (indicated by the total
height of each bar) as a function of the train ISI. The number of motor
units with or without sag in each ISI bin is indicated by the size of
the hatched and open part of each bar, respectively. Stimulation at a
given ISI can elicit tetani with very different degrees of fusion in F
or S motor units. Therefore we also evaluated these distributions as a
function of ISI expressed as multiples of CTtw,
CTpot, HRTtw, or HRTpot (Fig. 8,
2nd-5th from top, respectively). For all five distributions, there was a wide range of ISIs in which sag was not
detected during individual trains, as indicated by the distribution of
over several ISI bins. The probability of observing sag during any
single train was highest for long ISIs (raw or normalized). ISI/CTtw, ISI/CTpot, and ISI alone produced
distributions with comparable frequency dependence and maximum
probability of detecting sag (i.e., sag was correctly detected in
95% of the units in the uppermost 4-5 ISI bins).
ISI/HRTtw or ISI/HRTpot produced distributions
with lesser frequency dependence and maximum probability of detecting
sag (i.e., sag was detected
95% of the units in only the upper 2 ISI
bins). However, the advantage of using tetani elicited at long ISIs to
detect sag was counterbalanced by the reduced number of tetani
available for analysis. Tetani elicited at long ISIs were often of low
amplitude with little fusion of consecutive contractions, making the
determination of sag problematic. Tetani elicited at short ISIs had a
lower probability of detecting sag correctly. This was due in large
part to variability in the lowest ISI (raw or normalized) at which sag
could be detected. For example, the lowest ISI/CTtw value
that elicited sag in a given motor unit ranged from 0.68 to 2.72 (median = 1.52, with the middle 90% of units in the range of
0.93-2.14). In addition, some motor units failed to sag at
intermediate ISIs where trains at higher or lower multiples had already
elicited sag (e.g., compare ISI 3 to ISI 2 and ISI 4 from unit
4 in Fig. 1). Thus it is unlikely that stimulation at a single ISI
value (or multiple of CT or HRT) could provide a reliable method of
determining the presence or absence of sag in a motor unit.
|
To evaluate the probability of correctly determining sag status using
different numbers of tetani, a simulation was performed in which
individual tetani with and without sag from all units with at least one
tetanus with sag were sorted into eight bins according to their ISI
(raw or normalized to CT or HRT). Although each motor unit in the
simulation had some form of sag, not all units displayed sag in each of
the ISI bins. The number of motor units that would have been identified
successfully as having sag were noted for each ISI bin alone and for
all possible combinations of those ISI bins to simulate the effect of
testing for sag with different numbers of tetani. For example, tetani
with ISI/CTtw = 1.0-3.0 were divided among eight bins at
0.25 × CTtw intervals. A single test tetanus in any
one of the eight bins resulted in 61-137 of 158 units being correctly
identified as having sag. Tests that sought the presence of sag in
either one of two bins (28 combinations) resulted in 84-152 units
being correctly identified. As more tetani were used, more units were
correctly identified. The mean number of units correctly identified
(expressed as a percentage of the total number of units tested) for up
to eight tetani is shown in Fig. 9 ( and
). On average, motor units with sag in at least one tetanus were
correctly identified 95% of the time with three or more tetani and
99% of the time with five or more tetani. Similar results were
obtained using ISI alone or normalized ISIs. Each method of normalizing
ISI had slightly different abilities to distinguish sag correctly
according to the following order: ISI/CTtw>
ISI/CTpot > ISI > ISI/HRTtw > ISI/HRTpot.
|
Responses to individual tetani also were evaluated for the presence of
sag with two different patterns of force decrement (i.e.,
sagS and sagC). For the 158 units tested, 108 exhibited sagC and 77 exhibited sagS. Figure
10 shows the probability of detecting
each type of sag subtypes when distributed according to raw or
normalized tetanic ISI. The frequency dependence of the different sag
subtypes is clear, regardless of ISI treatment. SagS ()
occurred at all ISIs tested, but was more prevalent at short ISIs,
while sagC (
) occurred preferentially at higher ISIs. For example, the median minimum CTtw multiples at which
sagS and sagC were observed were 1.37 (mid-90%
of ISIs between 0.91 and 2.34 × CTtw) and 1.96 (mid-90% of ISIs between 1.28 and 2.95 × CTtw),
respectively. The frequency dependence of sag configuration also was
seen within units where both subtypes were elicited, i.e.,
sagS occurred in tetani with shorter ISIs than
sagC in 78 of 98 units.
|
Relationship between sag and other motor unit properties
The presence of sag was distributed preferentially among motor
units having short contraction and relaxation measurements (Fig.
11). This effect was shown most clearly
for separation by CTtw (Fig. 11A): all units
with CTtw < 20 ms displayed sag in at least one tetanus
(), whereas all those with CTtw > 20 did not exhibit
sag in any tetanus (
). Similar results were obtained, but with a
greater degree of overlap between sagging and nonsagging units, by
basing the distributions of sag on HRTtw,
CTpot, and HRTpot (Fig. 11, B-D,
respectively).
|
Sag, force accumulation pattern, and measurements of contractile speed
each defined two populations of motor units that largely covaried,
presumably reflecting the intrinsic differences between F and S motor
units. We have applied the four-part scheme of Burke et al.
(1973) to the 176 motor units in which both sag and fatigue tests were performed. Motor units with sag (presumed F units) displayed
a wide range of fatigue sensitivity (FI = 0.01-1.12). Of the
motor units that never sagged (presumed S units), most were fatigue
resistant (FI = 0.93-1.11 in 39 of 42 units), but three motor
units displayed a modest degree of fatigue sensitivity (FI = 0.85, 0.84, and 0.72). The latter FI value is below the commonly used
criterion value of 0.75 for defining fatigue resistance. However, FI
values from units with small tetanic force at the onset of the fatigue
test (15, 33, and 26 mN, for these 3 units, respectively) may not be
entirely reliable indicators of fatigue sensitivity due to random
variation in maximum force during repeated tetanization. The long
CTtw values of these three motor units (28, 33, and 29 ms,
respectively) led to their inclusion with other motor units that never sagged.
The properties of 49 FF, 43 F(int), 42 FR, and 42 S motor units are
summarized in Table 1. Results are
generally consistent with those of previous studies in rats.
Potentiated and unpotentiated twitch and tetanic maximum amplitude
followed the expected sequence of FF > F(int) > FR > S
type units. S units contracted and relaxed more slowly than F motor
units. No differences were found among F units in CT, but FR and F(int)
motor units had longer HRTtw values than FF units.
Ptw:Ptet was higher in FF than in S, FR, or FI
motor units. Previous studies found similar differences between FF and
S motor units in twitch:tetanus ratio, but all F units had similar
values (Celichowski and Grottel 1993; Einsiedel and Luff 1993
). A greater degree of twitch potentiation (i.e., lower Ptw:Ppot ratio) was seen in F than in S
motor units; this was consistent with previous results in cats
(Stephens and Stuart 1975
). CV was slower in S than in F
motor units with no differences found among FF, F(int), and FR motor
units.
|
Although sag was, by definition, present in all F motor units, the sag subtypes were not equally distributed among units of different fatigue sensitivity. The relative frequencies of sag subtypes were quantified by calculating the number of occurrences of sagS and sagC for each motor unit as a percentage of the total number of trains applied (%sagS or %sagC, respectively). %SagS was significantly lower in FF and F(int) units than in FR units, whereas the reverse was true for %sagC (P < 0.01 by ANOVA; see Table 1). The differences in sag subtype distribution by motor unit type could not be attributed to differences in background passive force at which tetani were elicited in that no significant correlations were found between background force and %sagS or %sagC (r2 < 0.03 and P > 0.05 for both by linear regression). This suggested the possibility that motor unit contractile properties might be distributed differentially among motor units with different sag subtype prevalences. Several significant correlations were found between the probability of detecting sag subtypes and motor unit contractile properties related to size, speed, and fatigue (linear regression analyses summarized in Table 2). Most notably, the frequency of occurrence of sagC varied inversely with FI (Fig. 12A), HRTtw (Fig. 12C), and HRTpot and directly with Ptw (Fig. 12B), Ppot, and Ptet.
|
|
The FI is linearly related to several of the other contractile
properties that vary significantly with %sagS and
%sagC (r = 0.55, 0.57, and
0.56;
P < 0.0001 for linear regressions of Ptw,
HRTtw, and Ppot on FI). Thus the many
relationships between contractile properties and sag subtype prevalence
probably arise at least in part from covariation in contractile
properties according to motor unit type. This was confirmed by further
analysis of the relationship between %sagC and
Ptw, HRTtw, and FI, the three variables that
alone accounted for the greatest amount of variation in
%sagC (r2 = 0.25, 0.31, and 0.40, respectively, by linear regression). Multiple regression of
%sagC on all three of these variables together only
accounted for 47% of the total variation in %sagC
(P < 0.0001 overall). This indicated that much of the
variation in %sagC accounted for by each variable alone
was common to that accounted for by the other variables (i.e.,
r2 unique to Ptw, HRTtw,
and FI alone was 0.02, 0.04, and 0.09 at P < 0.02, 0.003, and 0.0001, respectively; r2 common to
all three variables was 0.32).
A consequence of using a relatively high-frequency tetanus for fatigue
testing (70 Hz vs. the more commonly used 40 Hz) is an increase in the
contribution of neuromuscular transmission failure to reduced force
production (Krnjevic and Miledi 1958; Kugelberg
1973
). Of the 33 motor units with sag in which EMG was recorded
during fatigue testing, 8 exhibited marked EMG decrements. In these
units, the maximum EMG values during the 120th tetanus of the fatigue
test was 11-64% of that during the 1st tetanus, and FI values were
0.01-0.43 (
in Fig. 12A). All of these motor units had
%sagC values >0.86. However, motor units frequently expressing sagC were not uniquely sensitive to EMG failure
during repeated tetanic stimulation. Several of the 25 motor units with little or no EMG decrement (maximum EMG in 120th tetanus >87% of that
during the 1st tetanus;
in Fig. 12A) were fatiguable and
tended to express sagC (note the 3 motor units with FI
values <0.25 and with %sagC = 0.71-0.88 in Fig.
12A). Thus there was no indication of selective
susceptibility to neuromuscular fatigue among motor units with a high
likelihood of expressing sagC.
Relationship between body weight and sag
The distributions of motor unit histochemical and contractile
properties change with age in rats (Edström and Larsson
1987; Kugelberg 1976
). In the present study,
male animals of different weights (and, presumably, age) contributed
different numbers of motor units to the data set. Differences in animal
age (estimated from body weight to be 4-6 mo) could have confounded
the present results. However, no linear relationships were found
between body weight and %sag, %sagC, %sagS,
Ptw, Ppot, Ptet, CTtw,
CTpot, HRTtw, HRTpot, or FI
(P > 0.15 for all linear regressions). Thus the modest
differences in animal ages are likely to have had little effect on the
findings described in the preceding text.
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DISCUSSION |
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The F and S classifications identify motor units with different
ranges of properties related to contractile speed and tetanic force
accumulation. The different distributions of properties are presumably
the manifestation of their distinct histochemical profiles
(Burke et al. 1973, 1974
; Edström and
Kugelberg 1968
; Kugelberg 1973
; Larsson
et al. 1991
). Histochemical or immunohistochemical classification is not feasible in studies requiring sampling from even
modest numbers of motor units. Thus type-identification of motor units
often has been performed according to criteria based on contractile
properties alone.
Sag and other correlates of contractile speed
Three approaches have been employed to distinguish physiologically
S and F motor units. The first and most direct approach is to base
criteria on the distributions of CT or HRT. Histochemically defined F
and S units tend to have short and long CT and HRT measurements, respectively, but the degree of overlap varies among muscles and species (Burke et al. 1973; Chamberlain and Lewis
1989
; Kugelberg 1973
; Kwa et al.
1995
; Sieck et al. 1996
;
Tötösy de Zepetnek et al. 1992
). Thus use of
these measurements as the sole means of classifying large samples of F
and S motor units is straightforward when their distributions are
clearly bimodal (Close 1967
), but is problematic when
their distributions are continuous (Kernell et al. 1983
;
Reinking et al. 1975
). Placement of a contraction time
criterion at the minimum between apparent clusters of samples may be
questionable in cases of limited sample size. This issue has been
addressed by using the longest contraction time found in motor units of
intermediate-to-high fatiguability as the criterion value for dividing
fatigue-resistant F from S units (Bakels and Kernell
1993b
; Grottel and Celichowski 1990
;
Reinking et al. 1975
). In either case, the validity of
this approach relies on the assumption of nonoverlapping distributions
of CTtw in F and S motor units.
The second approach relies on differences in the pattern of force
accumulation during unfused tetanic contractions to distinguish F and S
motor units (Gardiner 1993; Gillespie et al.
1986
). Motor units with near-linear force increments in
response to successive tetanic stimuli have physiological properties
similar to those of histochemically defined S units, whereas those with
large initial force increments followed by rapidly decreasing
increments to subsequent stimuli have properties consistent with
histochemically defined F units (Tötösy de Zepetnek
et al. 1992
).
The third approach to distinguish F and S motor units by physiological
testing is based on the expression of sag during unfused tetanic
contractions. In the first study to apply the sag phenomenon in this
way (Burke et al. 1973), the presence or absence of sag divided their motor unit sample into F and S units with short and long
CTpot, respectively. The two-part separation by this contractile property was consistent with histochemical differences between these types. Several subsequent studies based on histochemical analyses of modest numbers of motor units have supported the separation of F and S units by sag (Chamberlain and Lewis 1989
;
Dum and Kennedy 1980
; Edström and Larsson
1987
; Kwa et al. 1995
; Martin et al. 1988
; Sieck et al. 1996
). This approach is
attractive because it provides a binary result once criteria for
identifying sag have been defined. It has been observed in different
mammalian skeletal muscles from different species, e.g., cat and rat
gastrocnemius (Burke et al. 1973
; Gardiner
1993
; Gillespie et al. 1986
; Proske and
Waite 1974
), cat extensor and flexor digitorum longus
(Dum and Kennedy 1980
; Dum et al. 1985
), cat and rat
tibialis anterior (Hamm et al. 1988
; Larsson et
al. 1991
; Martin et al. 1988
;
Tötösy de Zepetnek et al. 1992
), cat
diaphragm (Sieck et al. 1996
), cat peroneus longus
(Kernell et al. 1983
), rat soleus (Chamberlain and Lewis 1989
; Kugelberg 1973
), and rabbit
masseter (Kwa et al. 1995
) but not in cat extraocular
muscles (Nelson et al. 1986
). In addition, it is
independent of differences in absolute contraction time among species
and muscles. For example, sag distinguishes fast- and slow-contracting
units in cat and rat MG motor units even though the CTtw of
cat F units overlaps that of rat S units (Burke et al.
1973
; Einsiedel and Luff 1993
).
The similarity between motor units with sag and with a rapidly decrementing pattern of force accumulation is not surprising because the methods are based on detecting different aspects of the same underlying deviations from linearity in the shape of the force envelope during unfused tetani. The absence of detectable sag in a given tetanus does not imply that the phenomenon does not exist under those conditions but rather that whatever is responsible for the diminished force production during the tetanus is present but of insufficient magnitude to cause an overt reduction in force. This is supported by the observation that the SFI and TTP of tetani without sag from units with sag more closely matched the behavior of sagS and sagC tetani than those from motor units that never sagged.
Although most studies in which sag was assessed have supported the
relationship between sag and motor unit type, Tötösy de Zepetnek et al. (1992) reported that only 31% of F motor
units (as identified by CT) displayed sag. In a subset of 23 histochemically identified F motor units, 5 displayed discrepancies
between histochemical and physiological classification: one represented
a failure to detect sag, whereas the other four reflected discrepancies
between oxidative status and fatiguability. The present data suggest
that the specific stimulation conditions used explained the failure to
detect sag in many motor units with short CTs. First, sag was tested
most thoroughly at two different ISIs. The limitations on correctly
detecting sag with small numbers of trains indicated by the present
study (Fig. 9) is consistent with the poor correlation between sag and
time course of contraction and relaxation in studies using one-or
two-train sag tests (Bakels and Kernell 1993b
;
Gardiner 1993
; Grottel and Celichowski
1990
; Reinking et al. 1975
) and the good
correlation observed in studies in which sag tests were performed at
three or more different ISIs (Chamberlain and Lewis 1989
; Einsiedel and Luff 1993
; Kanda and
Hashizume 1992
). Second, one of the ISIs used to test for sag
was at 1.25 × CTtw, an ISI multiple at which there is
a relatively low probability of detecting sag (Fig. 8). Third, tetani
for assessing sag were repeated at short intervals; this would have
reduced the likelihood of detecting sag in motor units capable of
expressing it (Fig. 2) (also see Celichowski 1992b
).
It is also possible that varying degrees of correlation between sag and
histochemical and/or other contractile properties arose at least in
part from differences in the criteria used to define sag. The
contribution of this factor is difficult to evaluate because criteria
for defining sag rarely have been discussed explicitly (but see
Kwa et al. 1995). In the present study, some tetani
exhibiting sagC2 did not have overt local minima in their
force envelopes (e.g., Fig. 3, middle). Although these were
unlikely to have been identified as having sag in other studies, these
tetani represented only 3% of the total number of tetani evaluated,
and their inclusion did not alter the sag status of any motor unit
(i.e., not considering these tetani as having sagC did not
cause any unit with sag to become a nonsagging unit nor did it
substantially alter the values of %sagS and
%sagC or their relationships with other contractile properties). Exclusion of these data from the post hoc analysis illustrated in Fig. 9 resulted in a decrease of <5% in the
probability of correctly detecting sag in any individual tetanus or
combination of tetani. Thus after taking into account the factors that
confound the ability to detect sag and the differences in methodology
among studies, it seems likely that the presence or absence of sag is a
reasonable physiological correlate of F and S motor unit type, respectively.
The present study has sought to identify factors that contribute to variability in sag conformation that could affect the classification of F and S motor units according to their sag status. The data demonstrate substantial variability in the tetanic frequencies at which sag can be elicited, suggesting that a large part of the variability in sag testing among studies has arisen from use of inappropriate tetanic ISIs and/or an insufficient number of tetani. The present analysis indicates that several tetani at ISIs between 1.5 and 2.5 × CTtw (or 1.3-2.0 × CTpot) should be tested to ensure detection of sag in those units capable of expressing it. Under most circumstances, the improvement in reliability of detecting sag would be worth the modest cost in time for performing the additional tests.
Mechanism of sag
The limited information available on the physiological expression
of sag has provided some insight into its mechanism. Burke et
al. (1976) observed that the TTP of individual contractions decreased during the course of an unfused tetanus. The present results
confirmed their observation and also showed: that the decrease in TTP
was delayed in tetani with sagS relative to those with
sagC (Fig. 5); this is consistent with the difference in location of maximum tetanic force in tetani with these sag subtypes (Fig. 7); that TTP did not decrease from its value during the first
contraction in motor units that never exhibited sag (Fig. 5); and that
the increment in force added by a given contraction during the tetanus
varied with its TTP (Fig. 6). These observations are consistent with
the hypothesis of Burke et al. (1976)
that the
diminished capacity for force production that characterizes sag
reflects a reduced duration of the contractile state. Their suggestion
that such an effect could be due to differences in the sarcoplasmic
reticulum between F and S muscle fibers is consistent with more recent
information on the many type-dependent differences in the calcium
release and sequestration properties of this structure (reviewed in
Stephenson et al. 1998
).
The present data also indicate that sag is not a single phenomenon. The initial maxima in the force envelopes of tetani with sagS and sagC have different ordinal and temporal locations, suggesting that these phenomena represent two discrete processes. The observations that the TTP decreases during tetani with either sagS and sagC and that the frequency dependence of the location of the initial maximum in the force envelope of the two sag subtypes is similar (Fig. 7) suggest that the mechanism underlying the initial decrement in force in sagS and sagC may be qualitatively similar. The subsequent increase in force seen only in tetani with sagC appears to be the result of a different mechanism. The relationship between force increment and TTP is strong only until the initial tetanic maximum is reached, becoming substantially weaker at later peaks (Fig. 6). The lack of a sustained relationship between force increment and TTP beyond the tetanic maximum suggests that additional factors play a role in the later components of sag.
The secondary rise at the end of the force envelope during
sagC could result from transient changes in the
phosphorylation state of the myosin light chain subunits responsible
for twitch potentiation (Sweeney et al. 1993). The
increase in twitch amplitude associated with repeated activation is
more pronounced in motor units with sag than in those without it (see
Ptw:Ppot of F and S units in Table 1). The most
fatiguable motor units with the shortest HRTtw (i.e., those
with the highest probability of exhibiting sagC; see Fig.
12, A and C) exhibit the greatest degree of
increase in HRT during potentiation (compare HRTtw and
HRTtw:HRTpot of FF with those of other types in
Table 1). With a time constant for phosphorylation on the order of
seconds (Moore and Stull 1984
), potentiation would not
have a substantial effect on force production until late in the tetanus
(median and range of mid-95% of time to last peak for all tetani with
sagC = 729 ms and 387-1,122 ms, respectively). The
prolonged course of recovery from potentiation (Brown et al.
1998
; Moore and Stull 1984
) could contribute to the changes in force envelope that occur with repeated tetanic activation at 2-s intervals but not at 10-s intervals (Fig. 2).
The presence of sag and the magnitude of CTtw are
distributed similarly among motor units. However, this relationship is
not invariant, in that sag and CTtw can be dissociated
after reinnervation after peripheral nerve injury to the extent that
some motor units defy classification as described in the preceding text
(Rafuse and Gordon 1998). The incomplete control by the
reinnervating motoneurons over the expression of muscle fiber protein
isoforms indicates that at least some of the determinants of sag and
CTtw are distinct but are co-expressed under normal
conditions. Twitch contraction time is determined by several factors
including calcium release and reuptake mechanisms of the sarcoplasmic
reticulum and by myosin heavy and light chain isoform content
(Pette and Staron 1990
; Stephenson et al.
1998
). In addition to the potential relationship between sag
and sarcoplasmic reticulum function discussed in the preceding text, an
intriguing relationship emerged between expression of sag and the
distribution of contractile properties of fibers containing different
myosin heavy chain (MHC) isoforms. MHC I and II are distributed
differentially among S and F motor units, respectively. The
distributions of CT among fibers expressing MHC IIA, IIX, or IIB
overlap, but fatiguability, Ptw, and Ptet increase with isoform expression in the following order: MHC IIA < MHC IIX < MHC IIB (Larsson et al. 1991
). In the
present study, these three contractile properties also varied with
%sagC. This raised the possibility that the presence of
any form of MHC II is related to the ability to express sag and that
differential expression of MHC II isoforms is associated with the
probability of exhibiting sagC. However, the present data
are not entirely consistent with this hypothesis in that the
differential distribution of HRTtw in motor units of
different %sagC and fatigue sensitivities was not
manifested among motor units containing different MHC II subtypes.
Differences in contractile properties among motor units could reflect
in part differences in the arrangement of their individual muscle
fibers such that F motor units tend to exert their force on adjacent
fibers or connective tissue more than S motor units (Ounjian et
al. 1991). However, cat caudofemoralis motor units display sag
despite the minimal contribution of series elastic elements
(Brown et al. 1998
). Thus systematic variation among motor unit types in the series elastic component is not necessary for
(but could contribute to) the differential expression of sag.
The deviations in linearity of force production that characterize sag appear to be related most directly to changes in the duration or efficacy of the contractile state. Such changes could result from transient alterations in the release of calcium from the sarcoplasmic reticulum and/or its subsequent sequestration, the sensitivity of the contractile machinery to calcium, or the efficacy of crossbridge formation. The covariation of sag expression with several different contractile properties indicates that sag reflects the interaction of multiple processes within the muscle fibers.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Dennis McFarland for thoughtful comments on this manuscript and Drs. Barry Botterman and Timothy Cope for helpful discussions of axonal recording methodology. We also thank H. Sheikh for help in preparing this manuscript.
This work was supported by National Institutes of Health Grants NS-22189 and HD-36020.
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FOOTNOTES |
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Address for reprint requests: J. Carp, Wadsworth Center, New York State Department of Health, Albany, NY 12201.
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 29 November 1998; accepted in final form 24 February 1999.
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REFERENCES |
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