Division of Neuroscience, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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ABSTRACT |
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Bennett, David J.,
Yunru Li,
Philip J. Harvey, and
Monica Gorassini.
Evidence for Plateau Potentials in Tail Motoneurons of Awake
Chronic Spinal Rats With Spasticity.
J. Neurophysiol. 86: 1972-1982, 2001.
Motor units of segmental tail
muscles were recorded in awake rats following acute (1-2 days) and
chronic (>30 days) sacral spinal cord transection to determine whether
plateau potentials contributed to sustained motor-unit discharges after
injury. This study was motivated by a companion in vitro study that
indicated that after chronic spinal cord injury, the tail motoneurons
of the sacrocaudal spinal cord exhibit persistent inward currents (IPIC) that cause intrinsically
sustained depolarizations (plateau potentials) and firing
(self-sustained firing). Importantly, in this companion
study, the plateaus were fully activated at recruitment and
subsequently helped sustain the firing without causing
abrupt nonlinearities in firing. That is, after recruitment and plateau activation, the firing rate was modulated relatively
linearly with injected current and therefore provided a good
approximation of the input to the motoneuron despite the plateau. Thus
in the present study, pairs of motor units were recorded
simultaneously from the same muscle, and the firing rate
(F) of the lowest-threshold unit (control unit) was used as
an estimate of the synaptic input to both units. We then examined
whether firing of the higher-threshold unit (test unit) was
intrinsically maintained by a plateau, by determining whether more
synaptic input was required to recruit the test unit than to maintain
its firing. The difference in the estimated synaptic input at
recruitment and de-recruitment of the test unit
(i.e., change in control
unit rate, F) was taken as an estimate of the plateau
current (IPIC) that intrinsically sustained the firing. Slowly graded manual skin stimulation was used to
recruit and then de-recruit the units. The test unit was recruited when
the control unit rate was on average 17.8 and 18.9 Hz in acute and
chronic spinal rats, respectively. In chronic spinal rats,
the test unit was de-recruited when the control unit rate (re:
estimated synaptic input) was significantly reduced, compared with at
recruitment (
F =
5.5 Hz), and thus a plateau participated in maintaining the firing. In the lowest-threshold motor
units, even a brief stimulation triggered very long-lasting firing
(seconds to hours; self-sustained firing). Higher-threshold units
required continuous stimulation (or a spontaneous spasm) to cause
firing, but again more synaptic input was needed to recruit the unit
than to maintain its firing (i.e., plateau present). In contrast, in
acute spinal rats, the stimulation did not usually trigger
sustained motor-unit firing that could be attributed to plateaus
because
F was not significantly different from zero. These results indicate that plateaus play an important role in sustaining motor-unit firing in awake chronic spinal rats and thus
contribute to the hyperreflexia and hypertonus associated with chronic injury.
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INTRODUCTION |
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Motoneurons in anesthetized animals respond relatively simply to
injected current or synaptic excitation with proportional depolarizations and firing, somewhat like a passive summation device
(Binder et al. 1996; Granit et al.
1966
). In contrast, in unanesthetized decerebrate cats,
motoneuron behavior is more complex because the motor output can be
substantially augmented and prolonged by voltage-dependent persistent
inward currents (IPIC)
(Hounsgaard and Kiehn 1989
; Hounsgaard et al.
1988
; Hultborn and Kiehn 1992
; Lee and
Heckman 1998b
). That is, a depolarizing input that
brings the potential above a critical threshold, even relatively
briefly, can cause the IPIC to
regeneratively activate and thus produce a sustained depolarization
(plateau) and discharge (self-sustained firing)
that outlasts the input (Bennett et al. 1998a
,b
;
Hounsgaard et al. 1988
). Studies of motor units in awake animals (Eken et al. 1989
; Gorassini et al.
1999a
) and humans (Gorassini et al. 1997
, 1998
,
2001a
,b
; Kiehn and Eken 1997
) have provided
evidence for self-sustained firing in normal motor function. Furthermore, indirect calculations suggest that the
IPIC (i.e., plateau) contributes a
surprisingly large portion (50%) of the estimated depolarizing current
to sustain moderate repetitive firing (Gorassini et al.
2001a
).
Plateaus are facilitated by neuromodulatory inputs from the brain stem
[e.g., serotonin (5-HT) and norepinephrine (NE)] (Hultborn and Kiehn 1992) and are thus eliminated by acute
spinalization (acute spinal rat, Bennett et al.
2001
; and cat, Conway et al. 1988
;
Hounsgaard et al. 1988
). However, over the months after injury, plateaus return in motoneurons of chronic spinal rats (Bennett et al. 2001
; see also Eken et al.
1989
), indicating that plateaus can also occur in motoneurons
by mechanisms intrinsic to the spinal cord. These results were obtained
by intracellular recordings in an in vitro chronic spinal preparation
(sacrocaudal spinal cord), where it was difficult to study the
motorneurons' natural firing behavior, cell type (flexor vs.
extensor), or role in spasticity following injury (Bennett et
al. 2001
). The purpose of the present study was to record from
the associated motor units in awake chronic spinal rats to
verify the existence of plateaus and further quantify their function.
Motor-unit recordings in the awake rat can be used to test for the
presence of self-sustained firing and associated plateaus by examining
whether a transient input (e.g., skin stimulation or muscle vibration)
can trigger a sustained motor-unit discharge that continues when the
estimated synaptic input to the motoneuron is held constant
or decreased (Gorassini et al. 1998, 2001a
,b
). That is,
if a plateau helps maintain the firing of the motoneuron intrinsically,
then more synaptic input should be required to initiate firing (and
activate the plateau) than to maintain the firing. Further, the amount
by which the synaptic input can be reduced before de-recruitment occurs
corresponds to the contribution of the plateau to sustaining firing.
The main difficulty with this is in estimating the synaptic
input to the motoneuron. However, we have recently developed a
paired motor-unit method in which the firing rate of a
second lower-threshold motor unit (control unit) recorded
simultaneously to the first (test unit; from same muscle) is used to
estimate the common synaptic input to both units (Gorassini et
al. 1998
, 2001a
). That is, the firing profile of the control
unit is taken as an estimate of the synaptic input to the test unit,
assuming that both units receive the same synaptic input or
at least linearly related synaptic input (common drive) (DeLuca
and Erim 1994
) and that the control unit responds relatively linearly to the common synaptic input (Bennett et al. 1998a
,
2001
).
Conceptually, it might be difficult to understand how the second
assumption could hold if the control unit has an active plateau. However, the term plateau does not indicate a fixed depolarization or
firing rate of the motoneuron but simply that there is an added depolarization produced by a persistent inward current,
IPIC (perhaps the term plateau
current might be less misleading than plateau potential) (see
Bennett et al. 1998b). Synaptic or injected current can
still produce changes in potential and firing rate modulation during a
plateau by summating with the IPIC
(i.e., membrane potential changes ride on top of the plateau)
(Bennett et al. 1998a
,b
; Hounsgaard et al.
1988
). Another potential difficulty is that nonlinear behavior (e.g., bistable firing) may occur during a plateau activation in the
control unit. For example, with intracellular current injection in cat
motoneurons, the IPIC (plateau) often
has a high threshold and is abruptly activated during firing, causing a
nonlinear jump in firing rate (bistable firing) (Bennett et al.
1998a
; Hounsgaard et al. 1988
). Thus the control
unit firing might not be linearly related to input if a plateau is
present. Fortunately, abrupt jumps in firing (bistable firing) are not
common during more physiological activation by synaptic inputs in cat
motoneurons (vs. intracellular) (see discussion in Gorassini et
al. 1998
) likely because the plateau threshold is much
lower in this situation (re dendritic origin) (Bennett et al. 1998a
). Instead the
IPIC is thought to be activated near
recruitment (Bennett et al. 1998a
), contributes to the
initial recruitment step (or initial steep increase in rate), and is
not further activated during increases in synaptic input; thus it does
not interfere with linear rate modulation. There are conditions where
bistable firing is seen with synaptic inputs in some motoneurons (Hounsgaard et al. 1988
), and in these cases, the
control unit might give an overestimation of the common synaptic input,
and thus an underestimation of the plateaus. In chronic spinal rat motoneurons, the plateau is often activated before
recruitment even with intracellular current injection
(Bennett et al. 2001
). Shortly after recruitment, during
a slowly graded intracellular current injection, the frequency-current
(F-I) relation is again relatively linear for most cells
(Bennett et al. 2001
). Thus the firing rate of a motor
unit (re control unit) should accurately reflect the input to the
motoneuron, as per the second assumption in the preceding text,
even if this motoneuron has a plateau.
In the present study, we have thus applied the paired-motor-unit method
to examine plateaus in awake chronic spinal rats. As anticipated, we
found that a brief synaptic excitation, such as skin stimulation,
caused sustained firing in a test unit that continued even when the
estimated synaptic input (control unit rate) was decreased
substantially compared with the input that was required to recruit the
test unit (self-sustained firing and plateau). Remarkably, our estimate
of the effect of the plateau on sustaining motor-unit firing (from
change in control unit rate) was quantitatively very similar to that
obtained with intracellular recording in vitro in the same population
of motoneurons (Bennett et al. 2001). The consistency of
the results from intracellular recording and motor-unit recordings
provides a validation of paired-motor-unit method. Indeed,
this validation was the second major purpose of the paper and is
important because the paired-motor-unit method has begun to be used to
study normal (Gorassini et al. 1998
, 2001a
,b
) and
spinal-cord-injured humans (Gorassini et al. 1999b
).
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METHODS |
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Single motor-unit action potentials were recorded in awake rats
following an S2 sacral spinal cord transection,
either within 1-2 days (acute spinal; n = 38 units
from 4 rats) or after >1 month (chronic spinal; n = 32 units from 5 rats). The details of the sacral spinal cord transection
surgery are provided elsewhere (Bennett et al. 1999).
The motor units were from the distal segmental tail muscles, which are
controlled by motoneurons in the sacrocaudal spinal cord below the
S2 sacral transection (Bennett et al.
1999
). The purpose was to examine plateau behavior from tail
motor-unit firing, employing the paired-motor-unit method of
Gorassini et al. (1998
, 2001a
). One month after sacral
transection, rats develop a syndrome of spasticity in the tail muscles
affected by the injury (hypertonus, coiling spasms, etc.)
(Bennett et al. 1999
), and all chronic spinal rats in
the present study had such spasticity. All procedures were approved by
a local animal-welfare committee.
Preparation for motor-unit recording
Just prior to motor-unit recording, acute and chronic spinal
rats were briefly anesthetized with isoflurane (1.5-3% in
O2), and the segmental tail muscles were exposed
by making a 5-cm longitudinal incision in the skin at the midpoint of
the tail, slightly ventral to the lateral dorsal vein in the left side.
The segmental muscles are three small intrinsic tail muscles (1 cm
each) that span between successive vertebra of the tail: the dorsal
muscle is involved in dorsolateral extension of the tail (lifting the
tail), while the ventral and ventrolateral muscles are involved in
ventrolateral flexion of the tail (see Fig.
1) (see also Steg 1964;
Thompson 1970
). These muscles are covered by tendons
projecting from larger muscles at the base of the tail (Thompson
1970
), and some of these tendons were cut and removed for
better access. Sterile gauze and oil were then placed over the exposed
muscles to prevent drying.
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The rat was transferred to a standard holding tube (Bennett et
al. 1999) with the tail sticking out a hole in the end of the tube. The tail was stabilized horizontally to minimize movements during
recordings. The anesthetic was then removed, and the rat remained in
the tube for the duration of the recording (~3 h) (as in
Bennett et al. 1999
). All rats were completely spinalized and
thus could not feel the tail or any associated manipulations. Further,
they tolerated the holding tube without distress (they usually rested
or slept), and indeed prior to surgery, they entered the tube on their own.
Motor-unit recording and activation
Fine tungsten microelectrodes (usually 2; 12 M; No. 5754, A-M
Systems) were manually inserted into one of the segmental tail muscles
and held in place by a coarse manipulator. Motor-unit activity was
recorded with a custom-built preamplifier (Dr. K. Yoshida) and an
Axoscope data-acquisition system (low-pass filter: 10,000 kHz,
high-pass filter: 100 Hz, gain: 2000; sampling rate: 20 kHz; Axon
Instruments). Activity was played through a speaker to aid in locating
motor units during electrode insertion. At least two similar threshold
motor units from the same muscle were recorded at a time
(often from the same electrode). The high impedance of the electrodes
assured pickup from only motor units in the same muscle, which was
verified by comparing how distant two electrodes could be when
recording from the same unit (0.2-0.5 mm).
The recorded data were analyzed off-line using custom software on a Linux system. Spikes were sorted manually by their shape, and the instantaneous frequency was computed. The frequency profile was at times averaged in 200-ms bins to facilitate analysis (see following text). For display purposes, the instantaneous firing rate of the motor unit was often plotted with the firing rate smoothed using a fifth-order polynomial (thick line on figures).
To follow the companion intracellular studies as closely as possible
(Bennett et al. 2001), we attempted to produce a slowly increasing, and then decreasing, synaptic input by applying a slowly
graded manual stimulation to the skin, hair or muscles of the tail. The
most common stimulation was to gently lift the tail hairs by entangling
them in the fibers of a cotton swab. In chronic spinal rats, touching a
single hair could often trigger a sudden sustained motor-unit discharge
or even a full spasm, and it was thus necessary to very carefully apply
the stimulus to get a slow, graded recruitment and discharge of the
motor units.
Paired motor-unit method
VALIDATION USING INTRACELLULAR RECORDINGS FROM TWO MOTONEURONS. As described in INTRODUCTION, we have used the firing rate of two simultaneously recorded motor units to study plateaus: one lower-threshold unit (control unit) to estimate the common synaptic input, and a second higher-threshold unit (test unit) to examine the involvement of the plateau in sustaining firing as the synaptic input was decreased. Specifically we measured the difference between the control unit's firing rate (estimated synaptic input) at recruitment and de-recruitment of the test unit to determine the amount by which the plateau helped maintain the firing of the test unit.
To validate this method, we have applied it to the firing of two tail motoneurons recorded intracellularly, both of which we know have plateaus a priori (Fig. 2). The intracellular recordings were obtained from a chronic spinal rat in the companion study and the methods are detailed there (Bennett et al. 2001
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SUMMARY AND REQUIRED CONDITIONS.
Returning to motor-unit recordings, if two motor units are recorded
simultaneously and we assume that they have the same synaptic input
(current or at least linearly related input), then the input to the
higher-threshold unit can be estimated from the firing profile of the
lower-threshold control unit. The degree of self-sustained firing and
plateau current (I) can be estimated, as in Fig.
2D. For demonstration purposes, suppose that the data in
Fig. 2D were from two such motor units. If the test unit was
recruited at a particular estimated input level (8-Hz control unit rate
in Fig. 2D; left dashed line), then the plateau and
IPIC contribute to maintaining firing
if the estimated input has to be reduced below this level before
de-recruitment occurs (to 6-Hz control unit rate; right dashed line;
F =
2 Hz). This analysis is possible even if the
control unit itself has a plateau. The only requirement is that the
plateau in the control unit is activated a few seconds before the test
unit is recruited.
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RESULTS |
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Coordination of tail muscles in sacral spinal rats
The chronic spinal rats included in this study had spasticity in
their tail muscles with associated hyperreflexia and hypertonus (Bennett et al. 1999). Single motor-unit recordings from
the different muscle types revealed that all three segmental tail
muscles (ventral, ventrolateral, and dorsal) had considerable
spontaneous or reflex-evoked activity involved in this spastic
behavior. This activity was often well coordinated, which was
convenient for the purposes of experimentally controlling the motor
units (see following text). For example, a light touch on the dorsal
surface of the tail caused activation of the ventral muscles and
deactivation of the dorsal muscles, which resulted in a contraction
that produced a ventral flexion of the tail, withdrawing it from the
skin contact point. Likewise, a light touch on the ventral surface
caused reciprocally controlled dorsal muscle activation and a
contraction that tended to extend the tail (dorsally) away from the
contact point. The ventral muscles were usually most active, and this
corresponds to the previous observation that the tail often flexed
ventrally in these spastic rats, especially during spasms (which coils
the tail under the body) (Bennett et al. 2001
). We have
thus mostly focused on the ventrolateral and ventral segmental muscles.
Acute spinal rats had much weaker muscle contractions (no spasms)
although similar patterns of muscle activity could be evoked with more forceful skin stimulation.
Plateaus and sustained firing of low-threshold motor units in chronic spinal rats
A striking feature of the chronic spinal rats was that
low-threshold motor units often produced very long-lasting discharges in response to a relatively brief skin stimulation. In the ventral muscles, this discharge could go on for hours (up to 3 h tested); in the dorsal muscles, it lasted for many seconds. Further, the discharge could only be stopped by a substantial inhibitory skin stimulation to the opposite skin surface. Figure
3 demonstrates the firing of a pair of
such low-threshold motor units recorded from the same muscle (ventral
muscle). Although both units had a very similar threshold, making them
difficult to recruit separately, we were able to recruit unit
1 without unit 2 by carefully touching the tail (not
shown). Interestingly, although it was difficult to recruit unit
1 alone (because of their close threshold), once unit 1 was activated, it produced sustained firing that was easy to modulate
by a further brief stimulation (Fig. 3A, asterisk), without recruiting the second unit. If the firing of
unit 1 depended on a sustained increase in synaptic input to
the motoneuron pool, then one would expect that unit 2 would
instead be recruited by even the slightest stimulation and associated
increase in the firing rate of unit 2, considering their
closeness in threshold. The fact that this did not occur suggests that
the firing of unit 1 was instead intrinsically maintained by
the activation of a plateau at recruitment, as previously described for
these motoneurons in Bennett et al. (2001).
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If we assume that the firing rate of unit 1 (control unit) provides an estimate of the synaptic input and this input was similar to the synaptic input received by unit 2 (test unit), we can compare the control rate (estimated synaptic input) before and after recruitment of the test unit and thus determine the effect of the plateau on sustaining the firing of this test unit (if present; see METHODS and INTRODUCTION). That is, before recruitment of the test unit (unit 2), the control unit reached 7-10 Hz (Fig. 3A, asterisk), following a brief dorsal stimulation (left arrow). A second dorsal stimulation caused a spasm that recruited the test unit. After recruitment of the test unit, the control unit rate could be reduced to as low as 3 Hz while the test unit continued to fire (Fig. 3A, double asterisk), suggesting that a plateau activated at recruitment provided a depolarizing current that allowed the input to be reduced without de-recruiting the test unit. In this case, the control unit firing rate was slowed by inhibitory stimuli to the ventral tail skin (Fig. 3A, at right-most arrows).
Even though the test unit (unit 2) required a moderately
high estimated synaptic input to be recruited, as judged by the control unit firing rate (unit 1), it continued to fire when the
estimated synaptic input was reduced so much that the control unit
reached near its minimum rate (Fig. 3A, right; see summary
in the following text). Occasionally, the control unit (unit
1) even stopped firing while the test unit (unit 2)
continued (3/16 units; Fig. 3B, left) (de-recruitment
reversal as in Gorassini et al. 2001b). If both units
stopped firing and were then recruited again, the original recruitment
order was restored (not shown; see DISCUSSION).
Plateaus in higher-threshold motor units in chronic spinal rats
Other motor units in chronic spinal rats required a continuous
tail skin/hair stimulation to cause them to fire (except during muscle
spasms). However, these higher-threshold motor units also exhibited
pronounced plateau behavior. For example, during a slowly graded skin
stimulation, the test unit (Fig.
4A) was recruited when the
estimated synaptic input was moderately high (i.e., control unit rate
of F = 18 Hz; left dashed line), whereas it was
de-recruited when the stimulation was reduced and the estimated
synaptic input was much lower (control unit rate F = 4 Hz; right dashed line). The drop in control unit rate, and thus
estimated synaptic input, at test de-recruitment was 14 Hz (=
F), which suggests that the plateau in the test unit
contributed a substantial depolarization that sustained its firing even
when the synaptic input was reduced. Given that the average
frequency-current (F-I) slope for chronic spinal motoneurons
was ~5 Hz/nA (Bennett et al. 2001
), this 14-Hz drop
corresponds approximately to a 2.8-nA persistent inward current (IPIC=
I =
F/slope) that helped sustain the test unit's firing (see Fig. 2 and METHODS for further explanation).
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As already discussed, the paired-motor-unit method that we have
employed assumes that the test and control unit received the same
input. This assumption can be verified by examining the correlation between the firing rates of both units, as shown in Fig. 4B.
Shortly after the onset of firing of the test unit, the firing rates of the test and control units were well correlated (;
r2 = 0.77), both tracking the
spontaneous fluctuations in synaptic input. Interestingly, for the
first couple seconds after recruitment, the test unit firing rate (Fig.
4A) lagged behind the control unit rate (Fig. 4B,
). This could be explained by a slow activation of the plateau in
the test unit, such that the plateau activation continued for a few
seconds after recruitment (see Bennett et al. 2001
). The
steeper slope of this portion of the plot in Fig. 4B
is consistent with the activation of a plateau at this time. Similar
delayed activation of plateaus has been seen during intracellular recordings from chronic spinal rat motoneurons, and indeed the plot in
Fig. 4B looks remarkably similar to counterclockwise
hysteresis of the F-I plot of Fig. 2D of
Bennett et al. (2001)
. Finally, this delayed plateau
activation was at times also manifested in low-threshold units that had
self-sustained firing triggered by a brief stimulation. That is, it was
at times necessary for these units to fire for a second before
self-sustained firing was obtained. With shorter activations, the unit
stopped firing immediately after stimulation (not shown).
The second assumption of the paired-unit analysis method was that the
firing rate of the control unit was linearly related to its input.
Although the linear correlation of the test and control units in Fig.
4B is consistent with a linear input-output relation, this
assumption cannot be directly tested. It should, however, be noted that
it is unlikely that nonlinearities associated with a plateau activation
occurred in the control unit during the period of analysis (i.e.,
during the firing of the test unit). That is, the control unit fired
for many seconds before the test unit was recruited, at which point the
control unit's plateau would be fully activated (if present)
(Bennett et al. 2001). Furthermore, there were no jumps
in control unit firing rate during this period (i.e., no delayed
plateau activation).
Lack of plateaus in acute spinal rats
Motoneurons of acute spinal rats usually required continuous
stimulation to maintain their firing and had no clear indications of a
plateau activation. For example, when the test motor unit (Fig.
5) was recruited during a slowly graded
skin stimulation, the estimated synaptic input corresponded to a
control unit firing rate of ~28 Hz (left dashed line). At
de-recruitment (at right dashed line), the estimated synaptic input was
similar, or even slightly higher (F = +3 Hz in this
case; i.e., no plateau, and firing stopped early). We found that the
acute spinal rat motor units were harder to study because the control
unit rate was more variable and difficult to control, perhaps because
recruitment of motor units in acute spinal rats often required more
vigorous skin stimulation than in chronic spinal rats. Accordingly, we found considerable variability in the estimated change in synaptic input at de-recruitment compared with recruitment (
F).
For a given motor unit, in some trials,
F was positive
and in others, negative (re plateau). On average, however, it was
positive, suggesting that plateaus were not present (as in Fig. 5; see
summary described in the following text).
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Summary of firing properties and estimation of plateau current
On average, the control unit firing rate (F, estimated
synaptic input) at test unit recruitment was 17.8 ± 5.3 Hz in
acute spinal rats and 18.9 ± 5.2 Hz in chronic spinal rats. For
acute spinal rats, the control units rates at de-recruitment simply covaried with the rates at recruitment (; i.e., fell on 45° line in Fig. 6A), with no
significant drop in rate at de-recruitment (
F = +0.25 ± 1.9 Hz; n = 19 motor-unit pairs; Fig.
6, B and C). In contrast, in chronic spinal rats,
the control unit rate at de-recruitment nearly always fell below the
recruitment rate (
below 45° line, Fig. 6A), with a
significant drop at de-recruitment (
F =
5.5 ± 4.0 Hz; n = 16 motor-units pair; Fig. 6,
B and C). This drop in estimated synaptic input
(
F) suggests that, after recruitment, a persistent inward
current (IPIC, plateau) was present that aided in maintaining firing. De-recruitment only occurred when the
current provided by the plateau was cancelled by the reduction in the
effective current provided by the synaptic input (
I =
IPIC). Because we know the average
slope of the F-I relation in chronic spinal rats (from
Bennett et al. 2001
), we can convert
F
into an equivalent change in current at de-recruitment
(
I =
F/slope; where slope = 5 Hz/nA; see METHODS and Fig. 2). Thus the persistent inward
current that aided in sustaining the motoneuron firing can be directly
computed (IPIC =
I =
F/slope). The average computed
IPIC is ~1.1 nA (Fig. 6C)
(compared with 0.8 nA in Bennett et al. 2001
).
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The firing rate of the control unit at the recruitment of the test unit
approximately reflected the test unit's recruitment threshold (and
corresponded to the ease with which the unit could be recruited with
cutaneous stimulation) since control unit was usually chosen as a
low-threshold, easily recruited unit. Thus Fig. 6B indicates
that plateaus (F < 0) occurred in motoneurons with
a moderate range of thresholds in chronic spinal rats (about half the
range, considering that the control units could reach ~50 Hz). There
was, however, no significant difference in
F in test
units recruited with control unit rates above, compared with below 18 Hz (mean rate; despite the slight slope in regression line in Fig.
6B). Motor-unit pairs from both flexor (n = 7/8 ventral and n = 5/5 ventrolateral) and extensor
(n = 2/3 dorsal) muscles exhibited plateau behavior
(assuming a plateau was significant if
F <
1.0
Hz). However, we have by no means made a complete survey of all
motor-unit types because the highest-threshold units were often hard to
activate and were thus not amenable to analysis (not shown), and we
have only studied a few extensor muscles.
Anomalies in repetitive firing after chronic spinal cord injury
In our companion in vitro studies (Bennett et al.
2001), we noticed that the control of repetitive firing is
significantly altered in motoneurons of chronic spinal rats with
plateaus, in at least two respects: the firing often starts at a higher
rate than it stops and the minimum repetitive firing rate is unusually low (1 Hz). Motor units in awake chronic spinal rats also exhibited both of these characteristics, as shown in Fig.
7. That is, the test unit in Fig. 7
started firing at ~15 Hz and stopped at <2 Hz (the control unit
de-recruitment is not shown). This test unit had a plateau, with a drop
in estimated synaptic input at de-recruitment of
F =
5.0 Hz (control unit rate), as described in the preceding text for
Fig. 4. Possibly the high initial rate was related to an abrupt
plateau activation that occurred at recruitment, as in Bennett
et al. (2001)
. For all units tested, on average the firing rate
at recruitment (12.7 ± 5.7 Hz) was significantly higher than at
de-recruitment (8.0 ± 3.6 Hz; n = 16; Fig.
8). In contrast, in acute spinal rats,
the firing rates at recruitment and de-recruitment were not
significantly different (9.1 ± 5.1 and 9.1 ± 3.7 Hz, respectively; n = 19).
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DISCUSSION |
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The results demonstrate that, after chronic sacral spinal cord
transection, motor units of segmental tail muscles often exhibit very
long-lasting discharges in awake rats that are not seen in acutely
transected rats. These motor units are controlled by the sacrocaudal
spinal cord below the injury site (Bennett et al. 1999)
and are therefore chronically denervated from descending inputs. Thus
the discharges are very much uncontrolled and contribute to the
spasticity seen in this preparation (as has been suggested in humans)
(Gorassini et al. 1999b
; Thomas and Ross
1997
). In the lowest-threshold units, this sustained firing
could be triggered by a brief skin stimulation and was often only
stopped by applying an inhibitory stimulation to the opposite skin
surface. Higher-threshold units required more stimulation to activate,
but again once firing had begun, it was maintained even when the
stimulation was reduced substantially. This sustained motor-unit firing
could not be attributed to sustained increases in estimated
synaptic input to the motoneuron pool (control unit rate; see following
text); instead, we suggest that it resulted from the activation of
IPIC and associated plateaus intrinsic
to the motoneurons. This conclusion is consistent with the plateaus
found in motoneurons of the sacrocaudal spinal cord in chronic spinal
rats in our companion in vitro study (Bennett et al.
2001
; also see Eken et al. 1989
;
Gorassini et al. 1999b
).
The present results contribute new concepts related to the functional
role of plateaus after chronic injury: plateaus can be triggered by
natural synaptic activation, such as nonnoxious skin stimulation;
plateaus contribute to sustained firing in both low- and
medium-threshold units, although the highest were not tested; and
plateaus occur in motor units of both flexor and extensor segmental
muscles. Interestingly, the tail flexor motor units were in general
most active; this might indicate that plateaus may be more prominent in
flexor motoneurons of chronic spinal rats (as in clonidine or
L-DOPA-treated acute spinal cats, compared with decerebrate or
5-HTP-treated cats) (Conway et al. 1988). However, the numbers of extensor units in the present study were not
sufficient to settle this issue. Considering the high incidence of
plateaus in motoneurons in vitro (Bennett et al. 2001
),
other muscles innervated by the sacrocaudal cord likely also have
plateaus in chronic spinal rats. However, we did not study these
muscles, because they are situated in the base of the tail (with long
tendons running down the length of the tail) and are thus less
accessible for study in the awake animal.
In using motor-unit firing to study plateaus, one approach is to look
for known anomalies in motoneuron firing that sometimes occur with
plateaus, including a high initial firing rate due to plateau
activation at recruitment (as in Fig. 7) (also see Bennett et
al. 1998a; Gorassini et al. 1998
, 1999
, 2001a
);
a lowering of the plateau threshold with repeated activation, and
associated lowering of the recruitment threshold (warm-up)
(Bennett et al. 1998b
; Gorassini et al. 1998
,
2001b
); bistable firing caused by plateau activation slightly
after recruitment (Eken et al. 1989
); and a very low
minimum firing rate, possibly related to repeated activation and
de-activation of the plateau currents
(IPIC) just subthreshold to the firing
level (Bennett et al. 2001
; Kernell 1999
;
see also similar low rate after injury in Carp et al.
1991
; Powers and Rymer 1988
; Thomas and
Ross 1997
). However, each of these anomalies in firing could
also be explained by variations in synaptic input rather than plateaus,
and thus while useful, they do not by themselves demonstrate the
presence of plateaus intrinsic to the motoneurons (see discussions in
Gorassini et al. 2001a
,b
).
It is critical that we have a reliable estimate of the synaptic input
to the motoneurons so as to be able to clearly distinguish pre- and
postsynaptic events and ultimately demonstrate the presence of plateaus
intrinsic to the motoneurons. We have thus estimated the synaptic input
to a particular motor unit (test unit) by examining the firing rate of
a similar, though slightly lower-threshold unit recorded from the same
muscle (control unit). This paired-motor-unit method has been used
before (Gorassini et al. 1998, 2001
a), although in the
present study, we have the unique advantage of knowing a priori that
plateaus are present and knowing how they affect the input-output
properties of the motoneurons (Bennett et al. 2001
). In
particular, for slowly graded low-amplitude current inputs (as in Fig.
2), the firing frequency usually tracks the injected current linearly
in sacrocaudal motoneurons (Bennett et al. 2001
) and
accordingly produces linear F-I plots as in Fig. 2C. Importantly, the F-I plot is linear
regardless of whether or not there is a plateau (in chronic
vs. acute spinal rats), because the plateau (when present) is usually
initiated at the time of recruitment and is fully activated immediately
or at least within a second or so of recruitment (depending on the
amplitude of the input) (Bennett et al. 2001
). Thus
presuming that the current provided by synaptic input acts like the
intracellularly injected current (Granit et al. 1966
;
though see Lee and Heckman 2000
), this synaptic input
should be proportional to the firing rate.
Finally, because the control and test motor units are from the same
muscle and have similar thresholds, they both likely receive the same
synaptic input, and thus the firing rate of the control unit can be
used to estimate the synaptic input to the test unit. This can be
confirmed by examining how well the test and control units covary with
each other (Fig. 4B) (Gorassini et al.
2001a,b
) or with the net muscle force (Gorassini et al.
1998
; common drive, DeLuca and Erim 1994
).
Interestingly, in cats the recruitment order of gastrocnemius motor
units is preserved for different types of synaptic input (stretch vs.
cutaneous) (Sokoloff et al. 1999
; though see
Garnett and Stephens 1981
), suggesting that variations in stimulation/synaptic input are seen equally by similar motor units.
Because we know the mean slope of the F-I relation
(Bennett et al. 2001), we can approximately convert the
control unit rate into estimated synaptic current (again assuming a
rough equivalence of synaptic and injected current, see above). In this
way, we have been able to show that after recruitment the estimated
synaptic current can be reduced substantially without de-recruiting a
unit. This reduction corresponds to an intrinsic current that assists in maintaining firing (presumably
IPIC), which is ~1 nA (see
RESULTS for details). This value corresponds reasonably
well with the estimates of IPIC
obtained directly during intracellular recording (0.8 nA)
(Bennett et al. 2001
). Further, the percent drop in
estimated synaptic input at de-recruitment, compared with recruitment,
is ~50% (Bennett et al. 2001
), indicating that the
plateaus provide about half the net depolarization to
maintain moderate contractions (at 10- to 30-Hz rates). Interestingly,
similar results were obtained from motor-unit recordings in awake
normal humans (Gorassini et al. 2001a
), indicating that
plateaus in chronic spinal animals are at least as large as normal
although without the usual descending inhibitory control to turn them off.
In some pairs of motor units, the test unit was recruited well after
the control unit (at a moderate control unit firing rate and thus
estimated synaptic input; Fig. 3A), but de-recruitment only
occurred when the synaptic input was reduced so much that the control
unit stopped firing before the test unit (Fig. 3). We have seen similar
de-recruitment reversals in human motor units before and have argued
that they result from a larger plateau in test unit compared with the
control unit (see alternative explanations in Gorassini et al.
2001b). That is, while both units likely had plateaus, the
current provided by the plateau (IPIC)
in the initially higher-threshold test unit may have been at times
slightly greater than in the control unit. Thus the test unit could
sustain its firing longer when the common synaptic input was brought
down even though it initially had a higher recruitment threshold.
Likely, de-recruitment reversals are anomalies because they require a very gradual de-recruitment, so only one unit stops without the other.
In practice, once both units stop firing for a few seconds, the
original recruitment order is restored (i.e., both have their presumed
plateaus deactivated).
In anesthetized animals, the firing rate of a motoneuron can decline
slowly over time during a steady intracellular activation. Such
late spike frequency adaptation has been attributed to
various mechanisms, including sodium inactivation and the accumulation of hyperpolarization from slow calcium-dependent potassium currents (Kernell 1999; Kernell and Monster 1982
).
In unanesthetized preparations, spike frequency adaptation also occurs,
although to some extent it may be countered by the activation of
persistent inward currents (IPIC)
(Bennett et al. 2001
; Lee and Heckman
1998a
,b
). Spike frequency adaptation might complicate the
interpretation of the control unit firing rate in terms of synaptic
input because over time the rate may drop even though the synaptic
input does not. However, several arguments suggest this was not a major
problem in the present study. 1) Most motoneurons in the
sacrocaudal cord did not exhibit marked spike frequency
adaptation for the low-amplitude slow current ramps employed
in our companion in vitro study (Bennett et al. 2001
).
This may be partly because spike frequency adaptation is greater for
higher firing rates and thus larger inputs (Kernell and Monster
1982
). Thus we used low-amplitude slow synaptic inputs in the
present motor-unit study to mimic the slow current inputs used in
vitro. 2) We preactivated the control unit for many seconds before recruiting the test unit so that if any spike frequency adaptation was present, it would have to some degree taken place before
the test unit was studied. 3) Most of the units studied were
low- or moderate-threshold units, and thus the highest-threshold units
that fire at the highest rates and are most likely to have spike
frequency adaptation (Bennett et al. 2001
;
Kernell and Monster 1982
) were not studied. And
4) if spike frequency adaptation did occur in the control
unit, it likely also occurred to a greater extent in the
higher-threshold test unit (with higher rates), and this would have
caused an underestimation of the IPIC
and plateau.
In conclusion, we have found that, after chronic spinal cord injury,
the discharge of motor units is sustained by plateaus intrinsic to the
motoneurons. Functionally, these plateaus and associated sustained
discharges contribute to hypertonus and long-lasting exaggerated
reflexes associated with spasticity following injury. We have used a
paired-motor-unit method to detect the plateaus and found that there
was a clear similarity between the plateaus estimated in this way and
those measured directly with intracellular recording in the same
population of motoneurons (Bennett et al. 2001). Also
the method was verified by applying it to pairs of motoneurons, rather
than motor units, where plateaus were independently tested with current
injection (Fig. 2). These results provide strong support for the
validity of the paired-motor-unit method and its previous use in human
motor-unit studies (Gorassini et al. 1998
, 1999b
,
2001a
,b
). Thus those human studies, taken together with the
present study, indicate that plateaus are important to normal behavior,
disappear with acute spinal transection, and re-appear after chronic
spinal cord injury and participate in generation of muscle spasms (see
also Eken et al. 1989
; Hounsgaard et al.
1988
).
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ACKNOWLEDGMENTS |
---|
We thank L. Sanelli and Y. Han for expert technical assistance, including surgeries and histology. We also thank F. Giddes for help in preparing the manuscript.
Funding was provided by the National Sciences and Engineering Research Council, Canadian Foundation for Innovation, and the Medical Research Council of Canada, and the Alberta Heritage Foundation for Medical Research.
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FOOTNOTES |
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Address for reprint requests: D. J. Bennett, Div. of Neuroscience, 513 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada (E-mail: david.bennett{at}ualberta.ca).
Received 25 September 2000; accepted in final form 3 May 2001.
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REFERENCES |
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