Department of Physiology and Department of Physical Medicine and Rehabilitation, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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Lee, R. H. and
C. J. Heckman.
Enhancement of bistability in spinal motoneurons in vivo by the
noradrenergic 1 agonist methoxamine. Like many
types of motoneurons, spinal motoneurons in the adult mammal can
exhibit bistable behavior. This means that short periods of excitatory
input can initiate long periods of self-sustained firing and that
equally short periods of inhibition can return the cell to the
quiescent state. Usually, the presence of one of the monoamines (either
serotonin or norepinephrine) is required for spinal motoneurons to
express bistable behaviors. Because the decerebrate cat preparation has
tonic activity in monoaminergic fibers that originate in the brain stem
and project to spinal motoneurons, these cells sometimes exhibit
bistable behavior. However, exogenous application of the noradrenergic
1 agonist methoxamine greatly enhances bistable behavior
in the decerebrate. The goal of this study was to identify the
mechanisms of this action of methoxamine. The total persistent inward
current (IPIC) in spinal motoneurons in the
decerebrate cat was measured from I-V functions generated by
triangular voltage commands applied using discontinuous single
electrode voltage clamp. The effect of methoxamine on
IPIC was assessed by comparing its properties in
a control cell sample without methoxamine to its properties in a sample
of cells obtained after application of methoxamine. In most
experiments, at least one cell was obtained from each sample. Our
results showed that methoxamine approximately doubled the amplitude of
IPIC without changing its onset voltage, its offset voltage, or its persistence. The reduced amplitude was a
consistent finding within experiments and so was unlikely to be caused
by interanimal variability. In addition, methoxamine depolarized
motoneurons without altering their input conductances, so that a
smaller amount of current was required to reach the onset voltage of
IPIC. These effects of methoxamine were
approximately equal in all cells. As a result of these changes,
methoxamine greatly enhanced the tendency for motoneurons to become
bistable. It is proposed that the methoxamine-induced increase in the
amplitude of IPIC is effective in enhancing the
duration of bistable firing because this increase makes
IPIC more resistant to the deactivating effects
of the afterhyperpolarizations between spikes.
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INTRODUCTION |
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Spinal motoneurons in the adult mammal have the
capacity for bistable behavior, in which brief periods of excitatory
input can induce tonic self-sustained firing (Bennett et al.
1998; Hounsgaard et al. 1988
; Lee and
Heckman 1998b
). The cell can be returned to the quiescent state
by a brief inhibitory input. However, bistable behavior is a latent
property in spinal motoneurons that is manifest in the presence of
serotonin or norepinephrine (Conway et al. 1988
;
Hounsgaard et al. 1988
). The decerebrate cat preparation has tonic activity in the reticulospinal fibers that release serotonin (Baldissera et al. 1981
; Hounsgaard et al.
1988
), and it also may exhibit tonic activity in reticulospinal
noradrenergic fibers (Sastry and Sinclair 1976
; see the
discussion in Lee and Heckman 1998b
). Consequently,
motoneurons in the decerebrate cat sometimes exhibit bistable behavior
(Bennett et al. 1998
; Hounsgaard et al.
1988
). We recently have shown that application of the
noradrenergic
1 agonist methoxamine in the decerebrate
cat significantly enhances overall excitability of the motoneuron pool
and causes all motoneurons to exhibit at least some degree of
self-sustained firing (Lee and Heckman 1998b
). However,
there is a wide range in the duration of this self-sustained firing.
Partially bistable cells only generate short periods (~1
s) of self-sustained firing. These cells tend to have high input
conductances and fast axonal conduction velocities and thus likely
innervate fatiguable muscle fibers (Binder et al. 1996
).
In contrast, fully bistable cells exhibit self-sustained firing that lasts for many seconds. Fully bistable cells have low input
conductances and slow conduction velocities and likely innervate
fatigue resistance slow twitch fibers.
Bistable behavior is generated in motoneurons by a persistent inward
current, which may consist of more than one component (Hounsgaard and Kiehn 1989; Hsiao et al.
1998
; Rekling and Feldman 1997
; Schwindt
and Crill 1980
; Zhang et al. 1995
).
Single-electrode voltage-clamp analysis of the total persistent inward
current (IPIC) in motoneurons in the decerebrate
preparation with methoxamine showed that IPIC
was very large in all cells (Lee and Heckman 1998a
).
Although IPIC was of approximately equal
amplitude in the fully and partially bistable subgroups, it exhibited a
much greater tendency for decay with time in the partially bistable cells.
The purpose of this study was to investigate the effect of the applied
methoxamine on the properties of IPIC. This was
done by obtaining data on IPIC in a sample of
cells in the decerebrate preparation without application of methoxamine
to provide a comparison with our previously published data in the
decerebrate preparation with methoxamine (Lee and Heckman
1998a). Most experiments included at least one cell from each
sample. We considered three possible ways that methoxamine might
enhance IPIC and increase the tendency for
self-sustained firing: methoxamine would increase the amplitude of
IPIC, make it more persistent, or hyperpolarize
its voltage threshold. Our results strongly support the first
possibility and tend to exclude the second and third ones.
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METHODS |
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Data were obtained from 15 adult cats (average weight ~2.5
kg). In 14 of these experiments, one to four cells were recorded before
we administered the noradrenergic agonist 1 methoxamine to generate consistent bistable behavior in subsequently recorded cells. In addition, one experiment was devoted entirely to recording cells without methoxamine. The cells recorded without methoxamine constituted the control sample. The characteristics of the sample of
cells recorded after methoxamine application have been presented in our
previous work (Lee and Heckman 1996
, 1998a
,b
).
Preparation
Surgical preparations followed standard procedures in our lab
(see Heckman et al. 1994; Lee and Heckman
1998b
for details). Briefly, all surgical preparations of the
spinal cord and hindlimb were done under deep gaseous anesthesia
(1.5-3.0% isoflurane in a 3:1 mixture of O2 and
N2O). The deep anesthesia was maintained throughout a
precollicular decerebration. The gaseous anesthesia then was
discontinued, and the animal was allowed to breathe room air. The
preparation was paralyzed with gallamine triethiodide (Flaxedil) and
artificially respired. In addition, a bilateral pneumothorax was done
to enhance intracellular recording stability. At the end of the
experiment, the animals were killed with a lethal dose of
pentobarbital. All procedures were approved by the animal care
committee at Northwestern University.
Intracellular recording protocols
Protocols for the motoneurons in the control sample were very
similar to those for the cell sample with methoxamine (see Lee and Heckman 1998a,b for details). Intracellular recordings were obtained with sharp microelectrodes. Microelectrode tips were broken
back under microscopic observation and control. Because of the large
currents required for successful single-electrode voltage-clamp
techniques in spinal motoneurons, resistances of the electrodes were
kept low
typically ~3-4 M
in saline before entering the cord.
In each cell, the standard protocol began with measurement of a spike antidromically evoked by single shocks to either the medial gastrocnemius (MG) or lateral gastrocnemius-soleus (LGS) nerve. The stimuli were repeated 20 times at 2 Hz to provide an average. The electrode resistance was compensated with a bridge circuit, and then rheobase, defined as the amplitude of a 50-ms current pulse required to elicit a single spike, was measured. These initial measurements were followed by one or more of the following three protocols: measurement of the cell's I-V function to characterize IPIC, measurement of the cell's relationship between firing frequency and injected current to define its F-I function, and measurement of the tendency for the cell to exhibit self-sustained firing. These protocols are described next.
To measure the characteristics of IPIC, we used
the discontinuous, single-electrode voltage-clamp mode of our amplifier
(Axoclamp 2A amplifier, Axon Instruments). Switching rates typically
varied between 8 and 11 kHz. Headstage output was monitored at all
times to assess settling of electrode transients. Data with inadequate settling were rejected. The clamp feedback gains ranged from 10 to 40 nA/mV. In addition, an external low-frequency feedback loop (gain of
10, 3 dB at 30 Hz) was used to virtually eliminate baseline offsets
in voltage (cf. Misgeld et al. 1989
; Richter et
al. 1996
). Details of the limitations for this techinique in
motoneurons in vivo are given in Lee and Heckman
(1998a)
. IPIC was characterized by
applying a slow triangular voltage command with a rise time of 5 s, a total duration of 10 s, and amplitude of 30-40 mV.
The frequency-current (F-I) function of the cell was
assessed via injection of a current waveform with a triangular shape, as in our previous study (Lee and Heckman 1998b; see
also Bennett et al. 1998
; Hounsgaard et al.
1988
). The rates of rise and fall were equal at 5 s each.
In most cells, the amplitude of the triangular-shaped current was 30 nA, resulting in a rate of rise of 6 nA/s. A steady DC bias current was
used to control the maximum current reached during the input. The bias
was adjusted so that maximum firing rates reached ~40-70 Hz.
The capacity for bistable behavior was assessed by application of a
1.5 s period of high-frequency, low-amplitude vibration (a 160-Hz
sinusoid with a peak to peak amplitude of 80 µM) of the Achilles
tendon (Lee and Heckman 1996, 1998b
). This evokes steady
high-frequency firing in muscle spindle Ia afferents, which form
monosynaptic connections to MG and LGS motoneurons (Matthews 1972
). The maximum duration of self-sustained firing evoked by the Ia input was assessed when the cell was depolarized to a steady level just below its firing threshold (see Lee and Heckman
1998b
; also see Fig. 10). Hyperpolarizing baseline currents
were used to eliminate the self-sustained firing and confirm that the
Ia input had a crisp offset.
Data analysis
Conduction time was measured as the difference between time of
stimulation in the muscle nerve and time of onset of the antidromic spike. Conduction times were normalized by the average for each experiment (between 4 and 12 cells were included in these averages). We
then assumed the average conduction time corresponded to a conduction
velocity of 97 m/s (Zengel et al. 1985). Thus
multiplication of the normalized conduction time by 97 m/s gave an
estimate of conduction velocity that was weighted for interexperiment
differences in animal size. The use of averaging to account for
interanimal variations in conduction distance appears to work well. The
correlations between conduction velocity and other motoneuron
properties in our data (see RESULTS) are as least as high
as studies where conduction distance is measured in each animal (cf.
Fleshman et al. 1981
).
Figure 1 illustrates how the properties
of IPIC were measured from the I-V
functions generated by the triangular voltage commands. The same
properties were assessed in our previous study of
IPIC (Lee and Heckman 1998a). The
onset of IPIC was specified as the point on the I-V function where the total inward current
overcame the leak conductance sufficiently to give a net I-V
slope of zero. The initial peak of
IPIC then was defined to occur where the
negative slope decreased to again give a net slope of zero. The
offset and sustained peaks of
IPIC were defined on the descending phase analogously to onset and initial peak on the ascending phase. In cells
where IPIC on the descending phase was too small
to impart a negative slope to the I-V function, the
sustained peak was measured at the point where slope reached its
minimum. Offset was measured where I-V slope underwent a
downward inflection as the curve rejoined the subthreshold region. We
did not subtract the leak from the I-V function before
assessing the points for IPIC onsets, peak values, and offsets because we wished to define the contribution of
IPIC to the functional behavior of the cell. We
have shown previously, for example, that there is a good correlation
between IPIC onset current and the threshold
current for rhythmic firing (Lee and Heckman 1998a
).
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The initial peak amplitude of IPIC
was defined as the difference in current between its onset and initial
peak on the ascending phase, with the effect of the leak conductance
across the two voltages compensated in the following way. The input
conductance of the cell was measured from the slope of the
I-V function in a 5-mV region centered ~10-15 mV below
the onset of IPIC. A line with a slope equal to
the input conductance then was extrapolated from
IPIC onset voltage to the voltage corresponding
to the initial peak of IPIC. The initial peak
amplitude of IPIC was then the difference along
the current axis between the top of this line and the initial peak of
IPIC (see Fig. 1B). Leak was
compensated by a similar method in the calculations of the
sustained peak amplitude. In this case, the line with a
slope equal to input conductance was extrapolated from the offset of
IPIC. Note that these procedures yielded a
slightly lower value for IPIC amplitude than
would have been obtained by simply subtracting the leak conductance from the I-V relation. This was because the subthreshold
I-V function in motoneurons usually has a significant
curvature due to the interaction between the deactivation of the
H current and the activation of IPIC
(Binder et al. 1996). This curvature makes the amplitude
of IPIC sensitive to the voltage range chosen
for the definition of input conductance. The procedure for leak
compensation we chose minimized the impact of estimation of input
conductance on IPIC amplitude. It also is
functionally relevant, because, as noted earlier, the onset of negative
slope has a large impact on firing behavior.
Finally, we also measured two additional parameters of the
I-V function that play an important role in the generation
of bistability. First the I-V functions of spinal
motoneurons exhibit hysteresis due to differing behavior between the
ascending and descending portions of the triangular voltage command.
Voltage hysteresis was defined as
IPIC's onset voltage minus its offset voltage. Voltage hysteresis probably reflects the dendritic origin of much of
IPIC (see DISCUSSION). Along the
current axis, we measured the bistable current range, which
was defined as the current at IPIC onset minus
the current at its sustained peak (see Fig. 1B). This
definition encompasses the I-V region in which the existence of a negative slope allows a single value of current to be associated with two stable values of voltage. This duality is what makes bistable
behavior possible (Booth et al. 1997; Gutman
1991
; Hsiao et al. 1998
; Schwindt and
Crill 1980
) (see DISCUSSION). In some high-input
conductance cells, decay of IPIC with time
resulted in a sustained peak that was at or slightly above the onset,
as shown by the example in Fig. 2. In
these cases, the bistable current range was defined as the difference
between the current levels at offset and sustained peak. The large
region of negative slope on the ascending phase gives a false
impression of the capacity for bistable behavior in high-input
conductance cells because of the relatively rapid time-dependent decay
of IPIC (see RESULTS).
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For the F-I functions, we measured the currents at onset and
offset of rhythmic firing. The gain of the F-I function was
defined as the slope of the function during the ascending phase.
Hysteresis in the F-I function was defined as the current at
the offset of steady firing on the descending phase minus the current
at firing onset on the ascending phase. We also measured the peak
amplitude of the afterhyperpolarization (AHP) from the data used to
generate the F-I function. To define a flat baseline, the
digitized record of the cell's response to the triangular current was
subjected to a digital low-frequency filter (3 dB point of ~3-5
Hz). This had no effect on the shape of the AHP and eliminated
polarization artifacts due to imperfect electrode behavior. The
amplitude of the AHP was defined as the difference between the baseline
and the point of maximum hyperpolarization between spikes. The AHP values for the first and second interspike intervals were averaged. Note that the durations of the first two interspike intervals were not
significantly different (t-test, P > 0.60)
in the present study as compared with our previous study with
methoxamine (Lee and Heckman 1998b
), so that the AHPs of
the two data sets were compared at similar frequencies.
The Ia input generated by tendon vibration was used to test the
strength of bistable behavior in each cell, which was defined as the
duration of self-sustained firing after the cessation of the 1.5-s
period of tendon vibration. In addition, the voltage threshold for the
action potential was measured during the rhythmic firing evoked by the
Ia input. The trial with the lowest firing rate was used to avoid the
increase in spike threshold that accompanies increased rates
(Schwindt and Crill 1982). The voltage threshold for the
spike was defined as the voltage level at the first peak of the second
differential of the spike on its ascending phase.
The main acceptance data criteria were that the amplitude of the antidromic spike exceeded 70 mV and the resting membrane potential did not vary by more than ±5 mV during the course of the data collection. The properties of the control data sample were compared with those of the sample with methoxamine in two ways. Average values were compared with t-tests, assuming unequal sample variances. Linear regression analyses were used to assess the relationships between variables, and the slopes for the regression relations in the two data samples sometimes were compared using the standard slope comparison test based on the t-distribution. The significance level, alpha, was set at P = 0.05. Where results from multiple t-tests were compared, we chose a conservative alpha level by dividing 0.05 by the number of t-tests (see Tables 1-4).
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RESULTS |
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To identify the effects of the noradrenergic 1
agonist methoxamine on the electrical properties of motoneurons, we
measured the I-V functions, F-I functions, and
tendencies for self-sustained firing in a sample of motoneurons in the
decerebrate preparation without any methoxamine or other exogenous
agent present. The characteristics of the cells in this control data
sample then were compared with a sample of cells previously obtained in
the decerebrate after application of methoxamine (Lee and
Heckman 1998a
,b
). The control sample consisted of 31 cells.
I-V functions were obtained in 20 of these 31 cells,
F-I functions were obtained in 16 of 31 cells, and the
duration of bistable firing was assessed in 26 of 31 cells. In all but
one experiment, one or more control cells were recorded in the same
experiment as one or more cells after methoxamine application. Thus the
differences in motoneuron properties between the two cell samples were
unlikely to be due to interanimal variations.
Basic electrical properties
Spinal motoneurons in anesthetized preparations are known to
exhibit strong correlations among their basic properties, with low
input conductance cells tending to have low rheobases and slow axonal
conduction velocities (reviewed in Binder et al. 1996; Burke 1981
). Conversely, high input conductance
motoneurons tend to have high rheobases and fast axonal conduction
velocities. These basic correlations are preserved in the decerebrate
preparation (Burke 1968
; Hultborn et al.
1988
). For example, in the control cell sample, input
conductance correlated with rheobase (r = 0.83; P < 0.01) and conduction velocity correlated with both
input conductance (r = 0.69; P < 0.01)
and rheobase (r = 0.76; P < 0.01).
Similar correlations were observed in the cell sample with methoxamine (Lee and Heckman 1998a
,b
). This issue is important
because motoneurons with low input conductances tend to innervate slow
twitch, fatigue resistant muscle units, whereas high input conductance
motoneurons tend to innervate fast twitch, fatiguable muscle units
(Bakels and Kernell 1993
; Burke et al.
1973
; Gardiner 1993
; Zengel et al.
1985
). Thus these basic differences in motoneuron electrical properties play a major role in specifying the normal pattern of
recruitment of motor units (Binder et al. 1996
), which
proceeds from low to high force units [i.e., Henneman's "size
principle" (Henneman and Mendell 1981
)].
Persistent inward currents
IPIC is the total persistent current in
spinal motoneurons and likely consists of more than one component
(Hounsgaard and Kiehn 1989; Hsiao et al.
1998
; Rekling and Feldman 1997
; Schwindt and Crill 1980
; Zhang et al. 1995
). Figure
1A illustrates the standard slow (8 mV/s) triangular voltage
command used to assess each cell's I-V function. The cell
shown is from the control sample. Figure 1B illustrates the
resulting I-V function and defines the standard parameters
of IPIC that were measured from this function. The cell in Fig. 1 had a relatively low input conductance (0.55 µS).
Figure 2 illustrates the I-V function for a high-input
conductance cell (1.21 µS) from the control sample. Note that the
rapid onset of outward currents above the onset of
IPIC sometimes evoked clamp currents >50 nA in
the control cells. In 5 of 20 control cells in which I-V
functions were obtained, these large currents caused a 1-3 s loss of
adequate settling of electrode transients. The onset and initial peak
of IPIC occurred well before loss of adequate settling, but data for the descending phase were rejected in these five
control cells.
AMPLITUDE OF IPIC.
Figure 3 illustrates the tendency for the
initial peak amplitude of IPIC to be smaller in
the control cell sample than in the sample with methoxamine. Although
there is a considerable overlap in ranges, the average value for the
control sample was about half that of the sample with methoxamine and
this difference was statistically significant (see Table
1). Similarly, on the descending phase,
the sustained peak of IPIC in the control sample was also about half that in the sample with methoxamine (Table 1). The
reduced amplitude for IPIC in control cells was
also clear in comparisons between control and methoxamine cells within individual experiments. In the cell sample with methoxamine (Lee and Heckman 1998a), there was a weak but significant trend for the initial peak of IPIC to be somewhat larger
in high input conductance motoneurons (see Fig.
4; statistics are given in the figure
legend). A similar trend was observed in the control sample (again, see Fig. 4). The slopes of the regression lines for the two data sets were
not significantly different (P > 0.05). Therefore
methoxamine had an approximately equal effect on both low- and
high-input conductance cells.
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VOLTAGE THRESHOLDS.
Despite its clear effect on the amplitude of
IPIC, methoxamine did not significantly
influence IPIC's voltage thresholds. Figure
5A shows
IPIC onset voltage plotted as a function of
input conductance for cells with (Lee and Heckman 1998a)
and without methoxamine. In both data sets, there is a strong tendency
for onset voltage to be more depolarized in high input conductance cells. There were no significant differences in slopes of the functions
for the two data sets (P > 0.05). The range of values for input conductance in the control data sample reached a higher level
than in the sample with methoxamine, but this extension of the range
was due to only two cells. The average values of input conductance were
not significantly different in the two data sets (see Table 1). Figure
5B shows offset voltage as a function of input conductances.
Again, the two data sets are virtually superimposable, with high input
conductance cells tending to have a much more depolarized offset.
Overall, the average values of onset and offset voltages for the two
data sets were not significantly different (Table 1). Thus methoxamine
had little or no impact on the voltage ranges for activation and
deactivation IPIC.
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HYSTERESIS AND PERSISTENCE.
Figure 6 shows the relation between the
input conductance and voltage hysteresis in
IPIC. Voltage hysteresis is the tendency for the
offset of IPIC to occur at a more hyperpolarized
level than its onset (see Fig. 1B for an example). It is
likely that this hysteresis in IPIC occurs
because much of this current originates in dendritic regions under poor
space clamp (see DISCUSSION). There was a moderate tendency
for greater voltage hysteresis in low input conductance motoneurons in
both cell samples (Fig. 6). There is also a weak tendency for the
average value of voltage hysteresis to be larger in the cell sample
with methoxamine, but this tendency was not statistically significant
(see Table 1). The slopes of the regression relations for the two data
sets were not significantly different (P > 0.05). In
the cell sample with methoxamine (Lee and Heckman
1998a), the differences between voltage hysteresis in low and
high input conductance cells were probably due to differences in the
persistence of IPIC. Hysteresis was small in low
input conductance, partially bistable cells because IPIC underwent considerable decay in the time
that elapsed between the initial and sustained peaks. Because the
relationship between voltage hysteresis and input conductance in the
control sample was similar to that in the sample with methoxamine, it
seems likely that methoxamine had little or no influence on the
persistence of IPIC.
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BISTABLE CURRENT RANGE. Although voltage hysteresis was not altered by methoxamine, it had a clear impact on the characteristics of IPIC along the current axis. In fact, the bistable current range in the control sample was <30% of the bistable current range in the sample with methoxamine (see Table 1). In both the control sample and the sample with methoxamine, there was a modest trend for low input conductance cells to have a larger bistable current range, as shown in Fig. 8. As for the relation between voltage hysteresis and input conductance in Fig. 6, the reduced bistable current range in high input conductance cells occurred because these cells exhibit a stronger tendency for IPIC to decay with time. The I-V function shown in Fig. 2 provides an example of a high-input conductance cell in which IPIC underwent considerable decay by the time of its sustained peak, resulting in a small bistable current range. The greater bistable current range seen in low-input conductance cells is illustrated in Fig. 1B. The overall increase in bistable current range induced by methoxamine is a particularly important result because this parameter plays a key role in determining the duration of self-sustained firing (see DISCUSSION). Note that, in Fig. 8, all of the fully bistable cells in the sample with methoxamine have large bistable current ranges.
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BASELINE DEPOLARIZATION.
Although methoxamine did not have a significant impact on the voltage
for the onset of IPIC, it did influence the
amount of current required to reach this level. Table
2 shows that the average current for
IPIC onset in the control cell sample was ~7
nA higher than the average value for the sample with methoxamine. This
difference was statistically significant (see Table 2). A similar but
smaller difference existed for the offset current of
IPIC that did not reach statistical significance
(Table 2). Similarly, the resting membrane potential was somewhat more
depolarized in the sample with methoxamine than in the control sample,
but this difference also failed to reach statistical significance (Table 2). Taken together, however, these results suggest that methoxamine acted to depolarize motoneurons. This conclusion is in
accord with previous studies of brain stem motoneurons in slice preparations (e.g., Hsiao et al. 1998; Larkman
and Kelly 1992
; Lindsay and Feldman 1993
;
Parkis et al. 1996
). Note, however, that depolarization
occurred without a significant change in input conductance (see Table
1). This may have occurred because noradrenergic agents can
simultaneously decrease a leak current while increasing the
H current (Larkman and Kelly 1992
). Both of
these effects depolarize motoneurons but they have opposite actions on
input conductance.
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Frequency-current functions
The activation of IPIC also has a potent
impact on the motoneuron F-I function generated by a slow
(typically 6 nA/s) triangular pattern of injected current. In the fully
bistable cells in the sample with methoxamine, the onset of
IPIC causes a strong acceleration in firing rate
(Lee and Heckman 1998b). Furthermore the offset of the
F-I function occurs at lower current than its onset. We define F-I hysteresis as offset current minus onset current,
so that fully bistable cells have positive values for onset-offset hysteresis in both their F-I and I-V functions.
In partially bistable cells in the sample with methoxamine, firing
acceleration is also very strong but it starts well above firing
threshold. IPIC undergoes considerable decay in
these partially bistable cells during the course of the slow triangular
injected current protocol, so that offset of firing occurs at a similar
current level to onset and hysteresis is small.
In the control cell sample, positive F-I hysteresis was
scarcely evident. At best, onset and offset occurred at similar current levels, as shown by the F-I function for the cell on the
left in Fig. 9. In the other
cells, offset occurred at a substantially higher current than offset,
giving a negative hysteresis to these motoneurons (see the
F-I function for the cell on the right in Fig.
9). This negative hysteresis is typical of cells that lack bistable
behavior (Hounsgaard et al. 1988). Overall, the
hysteresis of the F-I function was significantly correlated
with rheobase (r =
0.63, n = 16, P < 0.01), being near zero in low rheobase cells and
moderately negative in high rheobase cells (as illustrated by the
examples in Fig. 9). The F-I hysteresis also was correlated strongly with the bistable current range of the I-V function
in both the control (r = 0.72; n = 10;
P < 0.01) and methoxamine (r = 0.74;
n = 16; P < 0.01) cell samples. Thus
the larger the bistable current range, the greater the tendency for
F-I hysteresis.
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Clear acceleration in firing rate due to onset of
IPIC is a standard feature of cells with
methoxamine (Lee and Heckman 1998b) but was evident in
only four of the control cells. The relatively small amplitude of
IPIC in the control cells may have prevented a
clear acceleration in firing rate being detected, especially because
firing at high rates tend to be noisy (see Fig. 9). As a result, the
gains of the F-I functions in the control sample [1.61 ± 0.38 (SD) Hz/nA] were similar to those seen in pentobarbital anesthetized preparations (reviewed in Binder et al.
1996
). In contrast, the acceleration in firing rate in the
cells with methoxamine produced very high F-I gains
(usually > 10 Hz/nA) (Lee and Heckman 1998b
).
The differences in the F-I function between the two data
sets were not due to differences in the voltage threshold for spike initiation. In the control sample, the spike voltage threshold was
47.9 mV ± 5.7 (SD) mV. In the sample with methoxamine, the spike voltage threshold was
48.9 ± 5.4 mV (Lee and
Heckman 1998a
). These two sample averages were not
significantly different (t-test; P > 0.40).
Thus in both samples, spike threshold tended to be slightly more
depolarized than IPIC onset in low input
conductance cells and slightly more hyperpolarized than
IPIC onset in high input conductance cells (see
Fig. 5A). However, the AHP in the sample with methoxamine
was somewhat smaller than in the control sample (control: 7.0 ± 1.6 mV; methoxamine: 5.9 ± 2.0 mV) (cf. Parkis et al.
1996
). This reduction fell just short of statistical significance (t-test; P < 0.06). A
methoxamine-induced reduction in the AHP would be expected to promote
bistable behavior (see DISCUSSION).
Self-sustained firing
One of the main effects of IPIC is to
generate bistable behavior in motoneurons. Our standard test to evoke
self-sustained firing consisted of a 1.5-s period of monosynaptic
excitatory input applied during a steady baseline injected current that
brought the cell near firing threshold (see METHODS).
Figure 10 shows examples of cells with
and without self-sustained firing. Only 8 of the 26 control in which
the standard test was applied exhibited self-sustained firing. None of
these eight cells generated self-sustained firing that lasted >3 s. In
contrast, in the presence of methoxamine, all cells generated at least
some self-sustained firing. Further, ~30% of the cells with
methoxamine were fully bistable and generated self-sustained firing for
many seconds (Lee and Heckman 1998b). Thus the
methoxamine-induced increase in the amplitude of
IPIC was associated with a large increase in the
tendency of motoneurons to generate self-sustained firing.
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I-V functions were obtained in 15 of the 26 control cells in
which self-sustained firing was evaluated. Of these 15 cells, 5 were in
the group with self-sustained firing. These five control cells with
self-sustained firing tended to have low input conductances as well as
slow conduction velocities and low rheobases (Table 3). In the sample with methoxamine, these
are the cells that tend to be fully bistable (Lee and Heckman
1998b). Within the control sample, Table
4 shows that the amplitude of
IPIC was about the same cells with and without
self-sustained firing. However, as expected for low input conductance
cells from Fig. 5, IPIC onset and offset
voltages tended to be hyperpolarized in the control cells with
self-sustained firing (Table 4). Voltage hysteresis was somewhat larger
as well but this difference did not quite attain statistical
significance (cf. Fig. 6 and see Table 4). The most important parameter
for bistable behavior, the bistable current range, was significantly
larger in the cells with self-sustained firing (cf. Fig. 8 and see
Table 4). Overall, these results suggest that methoxamine acted to
convert cells with a weak tendency for self-sustained firing to the
fully bistable state while allowing cells with no previous bistable
behavior to become partially bistable.
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DISCUSSION |
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The results of this study show that spinal motoneurons in the
decerebrate cat preparation possess a substantial persistent inward
current, IPIC. The properties of
IPIC in the motoneurons in the present study
were compared with the properties of IPIC obtained in our previous work (Lee and Heckman 1998a,b
),
where the noradrenergic
1 agonist methoxamine was
applied to the decerebrate preparation to enhance bistable behavior.
Although IPIC was rather large in the
motoneurons of this study, application of methoxamine approximately
doubled its amplitude without altering its onset and offset voltages or
its persistence. Thus the primary differences in
IPIC between fully and partially bistable cells
in the sample with methoxamine (Lee and Heckman 1998a
,b
)
are already present in the control cell sample. The dose of methoxamine
chosen for enhancement of bistable behavior in our studies appeared to
give the maximum increase in reflex excitability for this agent (see the METHODS section of Lee and Heckman
1998b
). We have not investigated the effects of lower doses,
but we suspect that lower doses would reduce the amplitude of
IPIC to a value closer to that of the control
data. The amplification of IPIC by methoxamine
is consistent with recent data from turtle motoneurons showing that the
L-type Ca2+ current that produces bistable behavior in
these cells can be modulated by several different agents
(Svirskis and Hounsgaard 1998
).
Because bistable behavior was weak in the absence of methoxamine (see for example Fig. 10), it is likely that maintaining self-sustained firing for long periods of time requires more than just a highly persistent inward current. The persistent current must also attain a sufficiently large amplitude (see following text). Another effect of methoxamine was to provide a significant baseline depolarization. This steady depolarization would make it easier for synaptic inputs to reach threshold for activation of IPIC and, consequently, self-sustained firing.
Self-sustained firing in the decerebrate without exogenous agents
In the presence of methoxamine, all motoneurons exhibit at least
some degree of bistable firing (Lee and Heckman 1998b).
The percentage of cells exhibiting self-sustained firing in the present study without methoxamine was similar to but somewhat less than that
seen in a previous study in the decerebrate without any exogenous agents (Hounsgaard et al. 1988
). The minor discrepancy
is likely accounted for by differences in the method of decerebration
(anoxic destruction of the forebrain in Hounsgaard et al.
1988
vs. the precollicular transection employed here), which
could easily impact the tonic activity in the reticulospinal
monoaminergic fibers. In addition, we used relatively large electrodes
in both this and our previous work (Lee and Heckman 1996
,
1998a
,b
). It is likely that fully bistable cells tend to be
small. This is because fully bistable cells tend to have low input
conductances (Lee and Heckman 1998b
), and low input
conductance cells tend to be the smallest in terms of soma diameter
(Binder et al. 1996
). Thus we may have undersampled the
population of cells that exhibit self-sustained firing.
Lack of hysteresis in the F-I function
In the presence of methoxamine, low input conductance cells
exhibited a strong tendency for positive hysteresis in their
F-I functions because offset of firing occurred at a lower
current level than onset (Lee and Heckman 1998b). This
F-I hysteresis correlates well with the hysteresis in the
I-V function, which is probably due to the dendritic origin
of a substantial portion of IPIC (Lee and
Heckman 1998a
). In this study, as in previous work
(Hounsgaard et al. 1988
), cells lacking in bistable
behavior exhibit negative F-I hysteresis: offset occurs at a
substantially higher current than onset (see the F-I
function in Fig. 9, right). Thus the greater the amplitude
and persistence of IPIC, the greater the
tendency for F-I offset to be shifted to a lower current. In
low input conductance cells in the present study,
IPIC is sufficiently large and persistent to
shift firing offset down to a current near firing onset, resulting in
near zero hysteresis (as illustrated by the F-I function on
the left in Fig. 9). The methoxamine-induced increase in
IPIC may push firing offset to a still lower
current value, converting this zero F-I hysteresis to a
strong positive value.
Role of voltage hysteresis in the I-V function
Bistability becomes possible in neurons when a persistent inward
current is large enough to induce a negative slope in the I-V function (Booth et al. 1997;
Gutman 1991
; Hsiao et al. 1998
; Schwindt and Crill 1980
). This allows a single value of
current to be associated with two stable values of voltage. When the
high-voltage value is above spike threshold, as is the case in spinal
motoneurons in the decerebrate, the cell can toggle between a quiescent
state and self-sustained rhythmic firing. The range of currents that allow for bistability is defined by the onset and the peak of the
persistent inward current
i.e., by the amplitude of the "N" shape in the I-V function (see points 1 and 2 in Fig.
11). In spinal motoneurons, an
additional factor comes into play. IPIC displays a clear hysteresis in that its offset tends to occur at a lower voltage
and current than its onset (Lee and Heckman 1998a
;
Svirskis and Hounsgaard 1997
) (see Fig. 1B).
This hysteresis expands the range of currents that generate bistable
behavior by pushing the sustained peak of IPIC
to a more hyperpolarized current level than the initial peak (see point
3 on Fig. 11). This is especially true in low input conductance cells
where IPIC is the most persistent and
consequently its voltage hysteresis is the largest. The primary source
of the voltage hysteresis is probably the dendritic origin of much of
IPIC (Hounsgaard and Kiehn 1993
;
Lee and Heckman 1996
). We previously have shown that
voltage hysteresis in low-input conductance cells is not a time
dependent phenomenon but persists even when the ascending and
descending phases of the triangular voltage command each last 20 s
(Lee and Heckman 1998a
). The most likely explanation is
that dendrites are under relatively poor space clamp from a voltage
clamp applied by an electrode at or near the soma (Gutman
1991
; Lee and Heckman 1998a
). Consequently on
the descending phase of the I-V function, the dendrites
remain depolarized by the dendritic portion of
IPIC even when the somatic voltage becomes more
hyperpolarized than the level at which IPIC was
activated on the ascending phase. This interpretation is supported strongly by computational models with persistent currents in dendritic compartments (Booth et al. 1997
; Gutman
1991
). Thus the dendritic origin of IPIC
enhances bistable behavior.
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Key role for the amplitude of IPIC
Why does increasing the amplitude of IPIC
have such a strong impact on the duration of self-sustained firing? In
high input conductance cells, bistable behavior is weak even in the
presence of methoxamine (Lee and Heckman 1998b). It is
not surprising that reducing the amplitude of
IPIC eliminates the tendency for self-sustained firing in cells in which IPIC tends to decay.
However, in low input conductance cells, IPIC is
highly persistent even in the control cell sample. Furthermore, the
average bistable current range in these cells seems reasonably large at
~5 nA (see Table 4). The key point is probably that, during rhythmic
firing, the interaction between the spike and the AHP provides a
perturbation that easily can push the cell below the bistable range of
currents. In the following discussion of this interaction, we treat the AHP as a fluctuation in current around the steady-state I-V
function. This oversimplification ignores dynamic changes in the
I-V function due to the spike and AHP but appears to
adequately capture the basic effects of the AHP on
IPIC.
In low input conductance cells, the spike voltage threshold lies
somewhat above the onset of IPIC and,
consequently, activation of IPIC initiates
rhythmic firing. In our standard test for self-sustained firing, a
baseline current is injected to bring the cell to as close to firing
threshold as possible without synaptic noise evoking spikes (see Fig.
10). Thus the net baseline current is probably sitting within the
bistable current range (see the lower dashed line in Fig. 11). The
addition of the synaptic current is then sufficient to exceed the
threshold of IPIC and initiate rhythmic firing
(upper dashed line). However, after synaptic input ceases, average
firing rate drops considerably (see Fig. 10, top) and the average current level returns to a level close to the baseline current
(lower dashed line in Fig, 11). The AHP between spikes tends to push
the current below the average level. Although the offset of
IPIC is slow (time constant of ~0.5 s)
(Lee and Heckman 1998a) compared with the time course of
the AHP (50-100 ms at slow firing rates), nonetheless each AHP may
provide some deactivation of IPIC. This could
result in a progressive decline in average level that causes the cell
to reach the bottom of the bistable current range (pt. 3 in Fig. 11).
Just such a progressive decline in average membrane potential is
evident in Fig. 10 (top). As a consequence, self-sustained
firing declines in frequency and ceases. Note that spike voltage
threshold (~48 mV) tends to be somewhat more depolarized than the
voltage at the sustained peak in Fig. 11, suggesting that the cell
should stop firing before IPIC fully deactivates. However, our measurement of spike threshold is set where
voltage begins its steep rate of change. This steep change is likely to
occur well into the range of activation of the fast, inactivating
Na+ current. The functional voltage threshold probably is
set by the persistent component of the Na+ current, which
has a threshold a few millivolts more hyperpolarized than the threshold
for the fast Na+ current (e.g., Brown et al.
1994
). Consequently, the cell likely will continue to fire
until IPIC deactivates.
When methoxamine increases the amplitude of IPIC, especially its sustained peak, then the cell's bistable current range is larger. Consequently, the lowered average current level due to the AHPs during self-sustained firing remains well above the bottom of the bistable current range. Thus it would be expected that there is some minimum amplitude for IPIC needed to generate long-duration self-sustained firing. If, as proposed above, the fluctuations in voltage and current due to the AHP play a crucial role in terminating self-sustained firing in the control cells, then elimination of spikes by blocking the fast sodium current may allow these cells to generate long-lasting plateau potentials.
If baseline current is increased so that the cell fires tonically before the synaptic input is applied, then would it be more difficult for the AHP to deactivate the IPIC. However, the cell could not exhibit bistable behavior in that case because the voltage threshold for spike generation is slightly above the voltage threshold for onset of IPIC. Consequently currents large enough to generate firing would fully activate IPIC before the application of Ia synaptic input.
The above arguments also suggest that amplitude of the AHP is very
important in determining the duration of self-sustained firing. In
slice preparations, noradrenergic agonists do not always affect the AHP
(e.g., Larkman and Kelly 1992), but the small reduction in AHP amplitude due to methoxamine in our results is consistent with
the modest effect seen in hypoglossal motoneurons (Parkis et al.
1996
). As AHP amplitude declines, there should be a lessor tendency for deactivation of IPIC. It is likely
that firing F-I gain also will be increased (cf.
Parkis et al. 1996
), so that self-sustained firing may
reach a higher frequency. At higher frequencies, the average membrane
potential tends to be more depolarized; this would help maintain the
cell in the bistable range. Thus methoxamine acts to potentiate
self-sustained firing in at least three ways: by depolarizing the cell,
by reducing the AHP amplitude, and, most importantly, by increasing the
amplitude of IPIC. Because IPIC may consist of more than one component
(Hounsgaard and Kiehn 1989
; Hsiao et al.
1998
; Schwindt and Crill 1980
) and because it
could be strongly affected by outward currents, further work is
required to identify the mechanism of how methoxamine increases IPIC.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-34382 and NS-28076.
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FOOTNOTES |
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Address for reprint requests: C. J. Heckman, Dept. of Physiology M211, Northwestern University Medical School, Chicago, IL 60611.
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 2 October 1998; accepted in final form 19 January 1999.
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REFERENCES |
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