Departments of Physiology and Physical Medicine and Rehabilitation, Northwestern University Medical School, Chicago, Illinois 60611
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lee, R. H. and
C. J. Heckman.
Paradoxical Effect of QX-314 on Persistent Inward Currents and
Bistable Behavior in Spinal Motoneurons In Vivo.
J. Neurophysiol. 82: 2518-2527, 1999.
Spinal motoneurons can
exhibit bistable behavior, which consists of stable self-sustained
firing that is initiated by a brief excitatory input and terminated by
brief inhibitory input. This bistable behavior is generated by a
persistent inward current (IPIC). In cat
motoneurons with low input conductances and slow axonal conduction
velocities, IPIC exhibits little decay with time and thus self-sustained firing is long-lasting. In contrast, in
cells that have high input conductances and fast conduction velocities,
IPIC decays with time, and these cells
cannot maintain long duration self-sustained firing. An alternative way
to measure bistable behavior is to assess plateau potentials after the
action potential has been blocked by intracellular injection of QX-314 to block sodium (Na+) currents. However, QX-314 also blocks
calcium (Ca2+) currents and, because
IPIC may be generated by a mixture of Ca2+ and Na+ currents, a reduction in amplitude
of IPIC was expected. We therefore systematically compared the properties of
IPIC in a sample of cells recorded with
QX-314 to a control sample of cells without QX-314, which was obtained
in a previous study. Single-electrode voltage-clamp techniques were
applied in spinal motoneurons in the decerebrate cat preparation
following administration of a standardized dose of the noradrenergic
1 agonist methoxamine. In the sample with QX-314, the average value
of IPIC was only about half that in the
control sample. However, the reduction of
IPIC was much greater in cells with slow as
compared with fast conduction velocities. Because a substantial portion
of IPIC originates in dendritic regions and
because conduction velocity covaries with the extent of the dendritic
tree, this result suggests that QX-314 may fail to diffuse very far
into the dendrites of the largest motoneurons. The analysis of the
decay of IPIC and plateau potentials in
cells with QX-314 also produced an unexpected result: QX-314 virtually
eliminated time-dependent decay in both IPIC and plateau potentials. Consequently, IPIC
became equally persistent in high and low input conductance cells.
Therefore the decay in IPIC in high input
conductance cells in the absence of QX-314 is not due to an intrinsic
tendency of the underlying inward current to decay. Instead it is
possible that the decay may result from activation of a slow outward
current. Overall, these results show that QX-314 has a profound effect
on IPIC and thus plateau potentials obtained
using QX-314 do not accurately reflect the properties of
IPIC in normal cells without
QX-314.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many types of motoneurons have been shown to
exhibit bistable behavior, in which tonic self-sustained firing can be
toggled on and off by brief excitatory and inhibitory inputs (e.g.,
Bennett et al. 1998; Hounsgaard and Kiehn
1989
; Hounsgaard et al. 1988
; Hsiao
et al. 1998
; Lee and Heckman 1998b
;
Rekling and Feldman 1997
; Svirskis and Hounsgaard
1998
; Zhang and Harris-Warrick 1995
; Zhang et al. 1995
). In most motoneurons,
neuromodulators, such as the monoamines serotonin and norepinephrine,
are required for expression of bistable behavior (Hounsgaard and
Kiehn 1989
; Hounsgaard et al. 1988
; Hsiao
et al. 1998
; Lee and Heckman 1999b
). It has also
been shown that, at least in spinal motoneurons in the cat and the
turtle, much of the persistent inward current that generates bistable
behavior originates in the dendrites (Bennett et al. 1998
; Hounsgaard and Kiehn 1993
; Lee and
Heckman 1996
).
One striking characteristic of spinal motoneurons in the cat is the
existence of systematic differences in their bistable behaviors.
Motoneurons with low input conductances and slow axonal conduction
velocities have the capacity to be fully bistable, in that
they can generate long periods of self-sustained firing (Lee and
Heckman 1998a,b
). This strong bistability is possible because
the total persistent inward current
(IPIC) in low input conductance cells
has a large amplitude and exhibits little or no decay with time.
Motoneurons with high input conductances and fast conduction velocities
possess an equally large amplitude IPIC, but it tends to slowly decay
over the course of a few seconds. Consequently, high input conductance
motoneurons are only partially bistable, often generating
<1 s of self-sustained firing.
However, the duration of IPIC does not
precisely match the duration of self-sustained firing (Lee and
Heckman 1998a). In the high input conductance cells,
self-sustained firing decays more rapidly than does
IPIC. Further, we have recently shown
that the amplitude of IPIC is a key
factor affecting the duration of self-sustained firing in low input
conductance cells (Lee and Heckman 1999b
). When the
amplitude of IPIC is decreased,
self-sustained firing fails within 1-2 s even though
IPIC remains highly persistent. These
differences between the persistence of
IPIC and the duration of
self-sustained firing may be partly due to the deactivating effects of
the afterhyperpolarization (AHP) following each action potential. This
suggests that if action potentials were eliminated by intracellular
injection of the lidocaine derivative QX-314 to block sodium
(Na+) currents (Connors and Prince
1982
; Narahashi et al. 1972
), then the resulting
plateau potentials should have properties very similar to those of
IPIC.
However, QX-314 is now known to not only block
Na+ currents but to also reduce calcium
(Ca2+) currents (Talbot and Sayer
1996). Because IPIC may be
generated by a combination of persistent Ca2+ and
Na+ currents (Hounsgaard and Kiehn
1989
; Hsiao et al. 1998
; Lee and Heckman
1998a
), we anticipated that QX-314 might reduce the amplitude of IPIC in addition to eliminating
spiking. Therefore the primary goal of this paper was to assess the
effects of QX-314 on IPIC. This
assessment was done by comparing the properties of
IPIC in the sample of cells recorded
with QX-314 to a sample of cells recorded without QX-314 in a previous
study (Lee and Heckman 1998a
). We encountered two
unanticipated results. IPIC amplitude
was reduced by QX-314, but the reduction was far greater in low input
conductance cells than in high input conductance cells (see
DISCUSSION). Moreover, QX-314 entirely eliminated the decay
in IPIC in high input conductance cells (see DISCUSSION). These large effects of QX-314
preclude quantitative comparisons between plateau potentials and
bistable behavior in cells without QX-314. A preliminary version of
these results has been published in abstract form (Lee and
Heckman 1998c
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Data were obtained in 16 adult cats (average weight ~2.5 kg).
Data from eight of these experiments were used to evaluate a new method
for calculating axonal conduction velocity (see Data analysis). The other eight experiments provided the primary data for this paper on the effects of QX-314. In all of these eight primary
experiments, we used the decerebrate cat preparation and administered
the noradrenergic 1 agonist methoxamine via a
modified intrathecal method before recording from any cells. The
standard dose, given in all experiments, was 5 µmol of methoxamine
dissolved in 100 µl of distilled water. We have shown that
methoxamine substantially increases the amplitude of
IPIC in motoneurons in our decerebrate preparation (Lee and Heckman 1999b
). The increased
amplitude of IPIC greatly enhanced the
probability that motoneurons would exhibit bistable behavior. All
details of the surgical preparation, decerebration, and administration
of methoxamine are given in our previously published studies
(Lee and Heckman 1998a
,b
, 1999b
). All procedures were
approved by the local animal care committee at Northwestern University.
To assess the effects of QX-314 on
IPIC, we compared the properties of
IPIC in the sample of cells recorded
in this study using electrodes containing QX-314 (referred to as the
"QX-314 sample") to the properties obtained in a sample of cells
without QX-314 (the control sample). The control sample consisted
primarily of data from 27 cells, which were obtained in a previous
study in the decerebrate preparation with the same dose of methoxamine as in this study (Lee and Heckman 1998a). However, this
previous data set only had seven cells in which the rate of decay of
IPIC was measured (see Data
analysis). Because measurements of decay of
IPIC were a particularly important component
of this study, data on decay for 4 more cells without QX-314 from 2 additional experiments were added to the control sample, to give a
total of 31 cells. For the QX-314 sample, electrodes were filled with a
solution consisting of 50-100 mM of the bromide salt of QX-314 and 3 M
KCl. This sample consisted of 18 cells from 6 experiments. We have
found the effects of methoxamine to be remarkably consistent from
experiment to experiment (Lee and Heckman 1998a
,b
), so
the results of this study are unlikely to be due to interanimal variations.
Experimental protocols
Protocols were similar for both the QX-314 sample and the control sample. Data collection began with identification of the cell as a motoneuron by antidromic activation from either the medial gastrocnemius (MG) or lateral gastrocnemius/soleus (LGS) nerves. A series of 20 antidromic spikes were averaged for conduction time measurements. We then injected a series of steady currents, 5-10 s in duration, to induce rhythmic firing and allow QX-314 to block the Na+ spikes. Typically, spike generation for rhythmic firing failed after ~1-2 min. In some cells, a single small (~10 mV) spike continued to occur at the onset of each current step, and additional applications of current did not further reduce its amplitude. Thus our criterion for sufficient block of action potentials was the total failure of rhythmic firing.
The single-electrode voltage-clamp technique was then used to measure
IPIC (see Lee and Heckman
1998a for details and limitations). As in our previous work
(Lee and Heckman 1998a
), the primary characteristics of
IPIC were assessed from the pattern of
currents evoked by a slow triangular voltage command (usually 40 mV in
amplitude applied at a rate of 8 mV/s for both ascending and descending
phases). This triangular voltage command was applied ~1-3 min after
rhythmic firing was eliminated. The persistence of
IPIC was then measured from
voltage-clamp steps with a long duration (10 s). In some cells, we also
measured the plateau potentials generated by
IPIC. The plateau potentials were
evoked either by a triangular shaped injected current (typically 30 nA
in amplitude with rates of rise and fall of 6 nA/s) or by a brief
period of synaptic input. For this, a 1.5-s period of monosynaptic
input was generated by low-amplitude, high-frequency vibration of the
Achilles tendon, which activates muscle spindle Ia afferents
(Matthews and Stein 1969
).
Data analysis
PROPERTIES OF IPIC.
Data analysis techniques for IPIC in
the QX-314 sample were identical to those for the control sample (see
Lee and Heckman 1998a). Figure
1 illustrates the standard parameters we
assess for IPIC, which are taken from
the I-V function generated by the triangular voltage
command. These parameters are the onset and the initial peak on the
ascending phase and the sustained peak and offset on the descending
phase. Leak conductance was subtracted to define the amplitudes of the
initial and sustained peaks (see Lee and Heckman 1998a
for leak subtraction details). However, onsets and offsets were
measured without leak subtraction because we wished these parameters to
be functionally relevant in terms of the firing behavior of the cell
(Lee and Heckman 1998a
,b
). Input conductance was
measured from the slope of the subthreshold region of the
I-V function in a 5- to 10-mV range ending at least 5 mV
below IPIC onset.
|
PERSISTENCE OF IPIC AND PLATEAU POTENTIALS. The persistence of IPIC was assessed during a long-duration (10 s) voltage step. The baseline for the step was set ~10 mV below the onset of IPIC, and the step amplitude was adjusted so that it reached the voltage level at the initial peak of IPIC on the cell's I-V function. Persistence of IPIC was calculated by expressing the amplitude of IPIC in the final second of the 10-s step as a percentage of the maximum amplitude in the first second (an example is shown in Fig. 9A in RESULTS). In the QX-314 sample, we measured the persistence of plateau potentials evoked by our standard 1.5-s period of monosynaptic Ia input (an example is shown in Fig. 9B in RESULTS). If necessary, a baseline current was applied to allow the depolarization produced by this monosynaptic input to exceed the voltage for plateau activation. The persistence of the plateau potential was calculated similarly to that of IPIC. The average plateau potential amplitude in a 1-s time window starting 9 s after the end of the Ia input was expressed as a percentage of its average amplitude in the first second after the Ia input.
CONDUCTION TIME MEASUREMENTS.
Conduction time was measured as the difference between time of
stimulation in the muscle nerve and time of onset of the antidromic spike, measured as the average of 20 trials. In some cells, spike amplitude began to decline during the series of 20 spikes that went
into the average, even though resting potential remained stable.
Presumably this was due to QX-314. However, QX-314 did not influence
conduction time. In six cells, we compared the conduction time measured
from the initial antidromic spike, which was >70 mV in amplitude, to
that measured after QX-314 had eliminated both the somatic-dendritic
and initial segment components of the spike, leaving only the M-spike
[the M-spike typically has amplitude of ~5-7 mV and is assumed to
result from passive propagation of the axonal spike through the initial
segment and soma (Eccles et al. 1957)]. In each cell,
the times of onset of the full spike and the M-spike were the same, and
hence conduction time was unchanged.
CALCULATION OF CONDUCTION VELOCITY.
Interanimal variations in size have a significant effect on conduction
times (Emonet-Denand et al. 1988). Measurements of conduction distances and calculation of the resulting conduction velocities are presumed to account for interanimal variations. Despite
this normalization, pooling conduction velocity from more than one
experiment can increase the variance in the sample of conduction
velocities, resulting in a degradation in the relations between
conduction velocity and other motor-unit properties
(Emonet-Denand et al. 1988
). In the present study, we
have used an alternative normalization method for the effect of
interanimal variations in size on conduction velocity and performed a
series of control experiments to demonstrate its validity (see the
following subsection). The alternative method was done as follows.
After all intracellular data were collected, the electrode was
positioned just outside the cell (the standard procedure to assess the
resting membrane potential). The extracellular field due to antidromic
stimulation was averaged (32 trial per average) and superimposed on the
intracellular record of the first part of the spike (an average of 20 trials). Figure 2 shows three examples of
this superimposition. The time of spike initiation was taken as the
first digitized point in which the spike diverged from the waveform
defined by the extracellular field (arrow labeled "S" in each
record; digitization rate: 100 kHz). The onset time of the
extracellular field (arrow labeled "F" in each case) was assumed to
be generated by the initiation of the action potentials for nearby
motoneurons with the shortest conduction times. Thus we normalized each
cell's conduction time by the onset time for the extracellular field.
The actual conduction velocity was estimated by assuming the
motoneurons producing the earliest onset of the field had conduction
velocities at the high end of the range for motoneurons. We chose a
value of 110 m/s (cf. Zengel et al. 1985
).
|
CONTROL EXPERIMENTS FOR CONDUCTION VELOCITY CALCULATIONS.
To assess how well normalization by the field onset worked in reducing
interanimal variations in conduction velocity estimates, we measured
conduction time, field onset time, and conduction distance in a sample
of 147 cells in 8 experiments (no QX-314 was used). Two of these
experiments primarily consisted of posterior biceps-semitendinosus
(PBSt) motoneurons, which have a substantially shorter conduction
distance than MG or LGS motoneurons. All cells with antidromic action
potentials >20 mV were accepted for this sample because, as noted
above, we have found that conduction time is not affected by spike
height (cells with good spike heights provided data for experimental
protocols that were not part of the work present here). The average
conduction velocity based on the assumption that field onset
corresponded to 110 m/s was 93.5 ± 9.6 (SD) m/s. This was not
significantly different from the average conduction velocity calculated
from estimated conduction distance, which was 94.2 ±15.4 m/s
(t-test, P > 0.6). Note, however, the
standard deviation for the field onset normalization was less than that
for conduction distance normalization. This reduced standard deviation
suggested that the field onset time might have more accurately
reflected interanimal variations in size. To test this possibility
further, the relationship between conduction velocity and rheobase was
compared for the two normalization methods. For this analysis, we
included only cells where spike height was >70 mV because rheobase is
sensitive to impalement injury (Binder et al. 1996).
Further, we only included data from the 3 experiments with at least 10 cells with rheobase measurements to allow comparison of variance both
within and between experiments. In the resulting sample of 33 cells,
the correlation between rheobase and conduction velocity estimated by
conduction distance was r = 0.32. The correlation based
on field onset was higher, at r = 0.51. The latter
correlation fell within the range of the three within-experiment
correlation coefficients, which were 0.44, 0.50, and 0.70. Thus use of
the onset of the field to estimate conduction velocity is at least as
good if not better than use of conduction distance when attempting to
reduce the effects of interanimal variations in size. The field onset
method was therefore used in the present study. However, it should be
noted that we restricted our sampling of motoneurons to areas where
large extracellular fields were evoked from the appropriate muscle
nerves to assure that the field was generated by a reasonably large
sample of cells. Also, it is essential to generate the field solely
from the nerve that antidromically activates the muscle, as the field
onsets for different nerves at a given recording locus can vary substantially.
DATA ACCEPTANCE CRITERIA.
The main data acceptance criteria were that, throughout the time period
for data collection, the resting membrane potential remained more
hyperpolarized than 55 mV and did not vary by more than ±5 mV. The
properties of the control data sample were compared with those of the
QX-314 sample in two ways. Average values were compared using
t-tests, assuming unequal sample variances. Linear regression analyses were used to assess the relationships between variables. 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 Table 1).
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The primary goal of this study was to assess the effects of QX-314
on IPIC in spinal motoneurons. This
goal was accomplished by comparing the characteristics of
IPIC in the sample of 18 cells in this
study, which were obtained after intracellular injection of QX-314, to
the characteristics of IPIC in a
control sample of cells [n = 31; most of control data
were obtained in our previously published study (Lee and Heckman
1998a)]. As noted in METHODS, all studies, both
with and without QX-314, were carried out after administration of a
standardized dose of the noradrenergic
1 agonist methoxamine. This was done to enhance the probability of
encountering bistable behavior in our decerebrate preparation (Lee and Heckman 1999b
).
Intracellular QX-314 reduces the amplitude of IPIC
Because intracellular injection of QX-314 reduces both
Na+ and Ca2+ currents
(Talbot and Sayer 1996), we expected that intracellular injection QX-314 would tend to reduce the amplitude of
IPIC in spinal motoneurons. The
histograms in Fig. 3 summarize the
distribution of values for the initial peak of
IPIC in the two cell samples. On
average, QX-314 reduced the initial peak amplitude of
IPIC to ~42% of its value in the
control sample, and this reduction was statistically significant (see
Table 1). The sustained peak of IPIC
in the sample of cells with QX-314 was similarly reduced, averaging
~62% of its value in the control sample (also significant; see Table
1). The average values for initial and sustained peaks in the QX-314
sample were not significantly different from each other
(t-test; P > 0.4). These results show that
QX-314 markedly reduces IPIC. However,
it is clear from Fig. 3 that QX-314 had a wide range of effects on the
amplitude of IPIC and that, at least
in some cells, its amplitude fell well within the control range. Figure
4 shows an I-V function for a
cell in which IPIC retained a
reasonably large amplitude (~16 nA) despite injection of QX-314. For
comparison, the I-V function for the cell illustrated in
Fig. 1 is also included in Fig. 4. This comparison suggests that QX-314
tends to reduce IPIC to a greater
extent in low than high input conductance cells. In fact there was a
correlation between IPIC amplitude and
input conductance among the cells with QX-314 (for the initial peak,
r = 0.70, P < 0.001; for the sustained peak, r = 0.62, P < 0.01). However,
input conductance in part depends on cell size (Binder et al.
1996
), so the possibility that the effects of QX-314 vary with
cell size is considered in the next section.
|
|
Intracellular QX-314 effects and motoneuron size
A significant portion of IPIC
originates in dendritic regions (Bennett et al. 1998;
Hounsgaard and Kiehn 1993
; Lee and Heckman 1996
), so one factor that might influence the effectiveness of QX-314 is how well it diffuses into the dendritic tree. Because high
input conductance motoneurons have more extensive dendritic trees than
low input conductance motoneurons (Binder et al. 1996
), the lesser effect of QX-314 on IPIC in
high input conductance cells may result from a failure of QX-314 to
diffuse very far into the dendrites of these larger cells. To evaluate
this possibility, we focused on the electrical property of motoneurons
that is most closely related to their size, the conduction velocity of
the axon. Conduction velocity is directly correlated with dendritic surface area (Burke et al. 1982
; Kernell and
Zwaagstra 1981
) and is unaffected by QX-314 (see
METHODS). If QX-314 is less effective in reducing
IPIC in larger cells, then there
should be a strong correlation between
IPIC amplitude and conduction velocity
in the QX-314 sample. Furthermore, the same correlation in the control sample should be weaker.
Figure 5 shows that, in the control cell
sample, there was a relation between estimated conduction velocity (see
METHODS) and the initial peak of
IPIC, but it was indeed very weak and was not significant (intercept: 1.51; slope: 0.22; r = 0.25; P > 0.05). In contrast, in the QX-314 sample,
there is a very strong but highly nonlinear relationship between the
initial peak of IPIC and estimated
conduction velocity (Fig. 5). IPIC is
markedly reduced in cells with conduction velocities below ~100 m/s.
Above this point, IPIC amplitude
increases very steeply with conduction velocity. The relation between
the sustained peak of IPIC and conduction velocity was similarly nonlinear (not shown). Log
transformations of the initial and sustained peaks of
IPIC resulted in correlation coefficients of r = 0.78 (P < 0.001)
and r = 0.68 (P < 0.01), respectively.
These results suggest that QX-314 may fail to diffuse far into the
dendrites of the higher conduction velocity cells. An alternative
explanation, which is considered in the DISCUSSION, is that
the ionic currents that generated IPIC
differ in cells with slow and fast conduction velocities.
|
QX-314 alters several other aspects of the motoneuron I-V function
In addition to its strong impact on IPIC amplitude, QX-314 had several other subtle but interesting effects. The following sections consider alterations in the onset and offset of IPIC and also changes in the subthreshold region of the I-V function.
QX-314 LINEARIZES THE SUBTHRESHOLD REGION OF THE I-V
FUNCTION.
Figure 6 compares the average ascending
I-V functions for the QX-314 sample to the average for the
control sample. The reduction in the initial peak of
IPIC due to QX-314 is clear, but this
figure also reveals a difference in the subthreshold region of the
I-V function. The average subthreshold I-V
function in the QX-314 sample lacks the curvature, or rectification, of
the average subthreshold function in the control sample. To quantify
these differences in curvature, quadratic functions were fit to the
region of each cell's I-V function below
IPIC activation. The term describing the curvature of the fitted function was significantly smaller for the
QX-314 sample than for the control sample (QX-314: 0.001 nA/mV2; control:
0.018
nA/mV2; t-test, P < 0.003). This difference in curvature did not have major impact on
average input conductance, which was calculated over a 5- to 10-mV
range in the subthreshold region and was not significantly different in
the two data sets (QX-314 data sample: 0.82 ± 0.43 µS; control
data sample: 0.94 ± 0.28 µS; t-test,
P > 0.3). The linearity of the subthreshold
I-V function in the presence of QX-314 was seen in both low
and high input conductance cells, as is apparent from the examples in
Fig. 4. At depolarized levels, the linearity is probably due to the
suppression in amplitude of the Ca2+ and
Na+ currents that generate
IPIC (cf. Schwindt and Crill
1980
). At hyperpolarized levels, the linearity may reflect
suppression of an H-current, because previous studies have shown that
QX-314 is an effective H-current blocker (Perkins and Wong
1995
).
|
QX-314 DEPOLARIZES THE ONSET VOLTAGE FOR IPIC IN SOME CELLS. In the control population, the voltages for the onset of IPIC tended to occur at more depolarized levels in partially bistable cells than in fully bistable cells. As a consequence, onset correlated with input conductance in the control sample, as shown in Fig. 7A. In the QX-314 sample, the relation between onset voltage and input conductance was seriously degraded (Fig. 7A). However, the average voltage level for onset in the QX-314 sample was not significantly different from in the control sample (see Table 1). Most of the reduced slope for the onset-input conductance relationship with QX-314 appears to be due to a depolarizing shift in onset in many of the low input conductance cells. One possible reason for poor onset-input conductance relation may be that QX-314 has its greatest effect in the soma and proximal dendrites. Thus much of IPIC remaining after QX-314 injection would originate in the distal dendrites, where it would be electrically distant from a voltage clamp applied at the soma (see DISCUSSION).
|
QX-314 HYPERPOLARIZES THE OFFSET VOLTAGES FOR IPIC IN SOME CELLS. Like onset, the offset of IPIC tends to be more hyperpolarized in low input conductance cells. Figure 7B shows that the strong relation between offset voltage and input conductance seen in the control cell sample was still very evident in the QX-314 sample. Note however that, in the high input conductance cells, offset voltage appears to be shifted to a more hyperpolarized level in the QX-314 sample than in the control sample. As a result, average voltage level in the QX-314 sample was ~6 mV lower than in the control sample (this difference did not quite reach our conservative level of statistical significance during multiple comparisons; see Table 1). This modest shift in offset voltage for high input conductance cells may reflect a change in the persistence of IPIC (see the final section of RESULTS).
QX-314 INCREASES THE HYSTERESIS IN IPIC
ONSETS AND OFFSETS.
Comparison of Fig. 7, A and B, reveals an
important characteristic of the activation and deactivation of
IPIC: offset occurs at a substantially
more hyperpolarized voltage level than onset (see also Figs. 1 and 4).
The primary source of this hysteresis is probably that a major portion
of IPIC originates in dendritic regions, which are likely under poor space clamp (Lee and
Heckman 1998a). In addition, in the control cell sample,
voltage hysteresis in IPIC was
greatest in fully bistable cells. As a result, there was a strong
inverse relation between hysteresis and input conductance. Figure
7C shows that QX-314 actually increased hysteresis by
shifting this relationship upward without much altering its slope. This suggests that, even in the presence of QX-314, a substantial portion of
IPIC still originates in dendritic
regions. The tendency for increased hysteresis in the cells with QX-314
in low input conductance cells may occur because this agent is most
effective in reducing IPIC near the
soma, leaving the dendritic portion relatively unaffected (see
DISCUSSION).
Plateau potentials in cells with QX-314
The application of QX-314 to bistable motoneurons reveals the
existence of plateau potentials during current clamp. A triangular shaped injected current was applied in six cells to reveal the plateau
potential generated by IPIC. The time
course of the injected current was slow (5 nA/s), giving overall rates
of change of voltage that roughly matched those during the
voltage-clamp protocols used to measure
IPIC. Figure
8A shows that the plateau
potential evoked by the triangular current input had a sharp onset and
also that its offset current was considerably below its onset current. This plateau behavior was seen in all six cells and is very
similar to that seen in previous studies of motoneurons with QX-314
(Bennett et al. 1998; Brownstone et al.
1994
). In Fig. 8B, the relations between current
and voltage obtained in both current-clamp and voltage-clamp conditions
for one cell (same as in 8A) are superimposed. The
resulting functions are very similar, with the exception that, during
current clamp, the onset of the plateau produces a sharp jump in
voltage. This is expected from the instability generated by the
negative slope conductance evident in the same region during voltage
clamp. The offset of the plateau was then more gradual than during
voltage clamp, which presumably reflects the lesser degree of control
over dendritic regions during current clamp as compared with voltage
clamp. Similar results were obtained for all six cells. It was
noteworthy that even though QX-314 dramatically reduces
IPIC, it still provided sufficient current
to generate a substantial plateau potential (~10 mV in the case of
Fig. 8A). Overall, the plateau potential behaviors
observed in this study were very similar to the behaviors seen in
previous studies (Bennett et al. 1998
; Brownstone
et al. 1994
).
|
Intracellular QX-314 eliminated the slow decay of IPIC in high input conductance cells
In the control cell sample, IPIC
exhibited a much greater tendency for decay with time in high input
conductance cells than in low input conductance cells (Lee and
Heckman 1998a). However, Fig.
9A shows a high input
conductance cell with QX-314 in which there is no detectable decay in
IPIC. Figure 9B shows a
nondecaying plateau potential in another high-input conductance cell
with QX-314. Figure 10 compares the
persistence of IPIC for 11 cells with
QX-314 compared with that seen in the 11 cells from the control population, with each data set plotted as a function of input conductance. In the QX-314 sample, persistence in 3 of the 11 cells was
assessed from the plateau potential, whereas, in the other 8 cells,
persistence was measured from IPIC (in
3 of these 8 cells, plateau potential persistence was found to be
nearly identical to that of IPIC). In
the control data set, persistence declines precipitously with input
conductance. In contrast, IPIC was
highly resistant to decay in all of the cells with QX-314, regardless
of input conductance.
|
|
An additional way of estimating the persistence of IPIC, using the I-V function, strongly supported the conclusion that QX-314 made IPIC highly resistant to decay. The triangular voltage command used to generate the I-V function was slow, so that ~3-4 s usually elapsed between the initial and sustained peaks of IPIC (see Fig. 1). Thus the closer the amplitude of the sustained peak to the initial peak, the greater the persistence of IPIC. Table 1 shows that the sustained peak was reduced by ~9 nA compared with the initial peak in the control data set, whereas this reduction was much smaller in the cells with QX-314, ~1.5 nA. As noted above, this meant that the initial and sustained peaks in the QX-314 data set were not significantly different from each other. In five of the cells in the QX-314 sample, the sustained peak was actually slightly larger than the initial peak (as illustrated by the I-V function for the cell in Fig. 1). The lack of decay of IPIC in the cells with QX-314 indicates that a major portion of the inward current that generates IPIC is highly persistent even in high input conductance motoneurons. Thus the tendency for decay of IPIC in high input conductance motoneurons without QX-314 may be due to activation of slow outward currents (see DISCUSSION).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The effect of QX-314 on IPIC in spinal motoneurons in the cat was paradoxical in the sense that QX-314 greatly reduced the amplitude of IPIC but nonetheless increased its persistence. However, these effects of QX-314 varied in a systematic fashion between high and low input conductance motoneurons. The largest reduction in amplitude of IPIC with QX-314 was seen in low input conductance, slow conduction velocity motoneurons, which probably correspond to the fully bistable cells in the control sample. QX-314 did not noticeably increase persistence of IPIC in these low input conductance cells because fully bistable cells already exhibit little or no time-dependent decay in IPIC. In high input conductance, fast conduction velocity motoneurons, which probably correspond to the partially bistable cells in the control cell sample, QX-314 had a lesser impact on the amplitude of IPIC but entirely eliminated its tendency to decay.
These results show that, as in other cells (e.g., Perkins and
Wong 1995; Talbot and Sayer 1996
), QX-314 does
much more than block action potentials in motoneurons. Thus
measurements of plateau potentials in motoneurons with QX-314 do not
provide an accurate reflection of the characteristics of bistable
behavior or the properties of IPIC in
cells without QX-314. In some types of studies, the suppression of
IPIC along with the spike may in fact
be desirable. IPIC provides very
potent amplification of synaptic inputs (Lee and Heckman
1996
), which might tend to obscure the precise pattern of
synaptic inputs generated by different sources onto motoneurons. Thus
intracellular QX-314 might aid in investigating the functional anatomy
of synaptic inputs onto motoneurons. On the other hand, it is likely
that suppression of IPIC and other
currents by QX-314 would dramatically alter synaptic integration within
the motoneuron. This alteration precludes conclusions about normal
synaptic integration based on cells with QX-314, but comparisons of
synaptic integration between cells with and without QX-314 might
provide useful insights about this fundamental issue.
Relation between the amplitude of IPIC and conduction velocity
One possible explanation for the strong relationship between
IPIC amplitude and conduction velocity
(Fig. 5) is that the ionic composition of
IPIC may differ in fully and partially
bistable cells. Because QX-314 is more effective in blocking
Na+ than Ca2+ currents, the
reason that IPIC was smallest in low
input conductance cells may be that a larger proportion of the total
current in fully bistable cells is due to Na+
rather than Ca2+ channels. Our results do not
exclude this possibility, but one key aspect of the data suggests that
a different ionic composition is not the whole explanation. In fast
conduction velocity cells, the amplitude of
IPIC was only slightly reduced (see
the high conduction velocity region of Fig. 5). Based on measurements
in hippocampal cells in a slice preparation (Talbot and Sayer
1996), the concentration of QX-314 that blocks
Na+ spikes should reduce
Ca2+ currents to ~20% of their control
amplitude. If a Ca-mediated current contributes to
IPIC, it too would be drastically
reduced by such a sharp drop in Ca2+ entry. Thus
QX-314 did not reduce the amplitude of
IPIC in high input conductance
motoneurons to the extent expected from its actions on both
Na+ and Ca2+ currents in
other types of neurons.
For this reason, we suggested an alternative explanation in
RESULTS, namely the failure of QX-314 to diffuse far enough
into the dendrites of the fast conduction velocity cells to suppress the dendritic component of IPIC.
Although the rate of diffusion of QX-314 into the narrow branches of
dendritic trees is not known, it is clear that motoneurons with fast
conduction velocities tend to have more extensive dendritic trees than
motoneurons with slow conduction velocities (Burke et al.
1982; Kernell and Zwaagstra 1981
). Furthermore,
it is clear that a substantial portion of IPIC originates in dendritic regions
(Bennett et al. 1998
; Hounsgaard and Kiehn
1993
; Lee and Heckman 1996
). In motoneurons with
conduction velocities of ~100 m/s or slower, QX-314 did in fact
reduce the initial peak of IPIC to the
level expected on the basis of its effect on Ca2+
currents (Talbot and Sayer 1996
), i.e., ~20% of the
control amplitude (see Fig. 5). In contrast, in some cells with
conduction velocities above 100 m/s, the amplitude of
IPIC was well within the range of
control values. This lack of effect is consistent with a failure of
diffusion of QX-314 into dendritic regions of the large, fast conduction velocity cells. Note that QX-314 was effective in blocking rhythmic firing in all cells, including the fastest conduction velocity
neurons. Thus it is unlikely that the lesser reduction of
IPIC in the fastest conduction
velocity cells was due to a failure to attain sufficient entry of
QX-314 from the electrode into the soma and initial segment.
One prediction of slow diffusion of QX-314 is that the amplitude of IPIC should decline with time in the fast conduction velocity cells. This prediction was difficult to test in our in vivo preparation because IPIC seems to be very sensitive to recording quality and often tends to decline with time even in the control cells without QX-314. Therefore all comparisons were based on data in a similar time window, 1-3 min post spike failure. Higher concentrations of QX-314 in the electrodes may have increased the rate of diffusion into the dendrites, but we found that concentrations above 100 mM tended to degrade transient behavior of our electrodes and prevent good discontinuous voltage clamp.
A differential impact of QX-314 on the somatic versus dendritic
components of IPIC may also account
for the increased onset-offset hysteresis seen in low input conductance
cells with QX-314 (Fig. 7C). Computer simulations with a
simple motoneuron model consisting of a somatic compartment coupled to
a single dendritic compartment showed that increasing the electrical
isolation between the two compartments increased onset-offset
hysteresis (Lee and Heckman 1999a). Increasing the
proportion of IPIC in the dendrites
versus the soma has a similar effect (unpublished observations). Thus a
differential effect of QX-314 on the soma versus dendritic components of IPIC should also increase
hysteresis. However, this explanation may not apply to high input
conductance cells. It was argued above that the nonlinear relationship
between conduction velocity and IPIC
meant that QX-314 did not much affect the dendritic component of
IPIC in high input conductance cells.
In these cells, the increased hysteresis with QX-314 may largely be due
to the hyperpolarization of the offset of
IPIC. This hyperpolarization is
probably due to the increased persistence of
IPIC in high input conductance cells
(see the following section).
Persistence
The most surprising result in this study was the marked enhancement of the persistence of IPIC in the high input conductance cells with QX-314. This enhanced persistence provides an important insight into why IPIC tends to decay in these cells in the absence of QX-314. IPIC decay could be due to slow inactivation of one or more channels producing IPIC or to slow development of an outward current to counterbalance IPIC. The elimination of decay by QX-314 shows that at least part of IPIC in high input conductance cells does not undergo inactivation over the course of 10 s. Furthermore, because QX-314 was less effective in suppressing the amplitude of IPIC in these high input conductance motoneurons, the proportion of IPIC that does not inactivate forms the majority of the total current. Therefore QX-314 probably does not alter decay by simply removing an inactivating component of IPIC. It seems reasonable to suppose that the decay of IPIC in partially bistable cells is due to slow activation of an outward current instead of inactivation of IPIC.
How is it that QX-314 can have such a large impact on persistence in the cell in which it had the least effect on amplitude? As for many of the actions of QX-314, this paradox probably arises from a differential impact on somatic versus dendritic regions of the cell. The strong effect on persistence suggests that QX-314 is somehow reducing an outward current that is in or near the soma. The lack of effect on IPIC amplitude, as noted above, likely occurs for the converse reason: most of IPIC is generated in dendritic regions where the impact of QX-314 is limited by slow diffusion.
The present results do not reveal which outward current might produce
the slow decay in high input conductance cells, but one factor that is
likely to be important is that IPIC
onset and offset occur in a more depolarized range in these cells (see
Fig. 7, A and B). In fact, in high input
conductance, partially bistable cells,
IPIC is not activated until several
millivolts above spike threshold (Lee and Heckman
1998b). Thus one possibility is that onset of
IPIC is accompanied by some degree of
activation of the delayed rectifier K+ current.
However, it is not clear how activation of the delayed rectifier could
account for the slow time course of decay. Typically, the time constant
for decay of IPIC in high input
conductance cells is on the order of several seconds, whereas the
slowest time constants for activation of the delayed rectifier are on the order of milliseconds.
An alternative possibility is that the more depolarized level results
in a greater influx of Ca2+ in the soma, which
then slowly increases intracellular Ca2+
concentration. The increased Ca2+ concentration
could then slowly activate a Ca-mediated K+
current, such as that which generates the spike AHP. By limiting Ca2+ entry, QX-314 could prevent excessive
buildup of intracellular Ca2+ and reduce
activation of the Ca-mediated K+ current. Because
QX-314 may be ineffective in reaching dendritic regions, the crucial
area for reducing Ca2+ entry and buildup of
Ca2+ concentration may be at or near the soma.
The possibility that QX-314 acts directly on the
K+ channels cannot be excluded. QX-314 has been
shown to suppress the large-conductance, Ca-mediated
K+ channel in patch-clamp recordings in excised
patches from hippocampal neurons (Oda et al. 1992).
However, this suppression may only occur at very high concentrations of
QX-314 because this K+ conductance was not
affected by intracellular injection in hippocampal cells
(Connors and Prince 1982
).
Finally, it should be noted that reduction in net amplitude of
IPIC is not necessarily enough to
prevent its decay. We have recently completed a study of
IPIC in the decerebrate preparation without application of methoxamine (Lee and Heckman
1999b). In that study, IPIC
average amplitude was ~11 nA, which is only slightly larger than the
8 nA seen in the QX-314 sample in the present study in the decerebrate
with methoxamine. Despite the reduction in amplitude of
IPIC in the decerebrate without
methoxamine, the tendency for IPIC to
slowly decay in high input conductance cells was still clearly
apparent. We suspect this is because the main outward current that
provides this decay in high input conductance cells is located at or
near the soma, where it is strongly affected by QX-314 in the present study.
Overall, the enhanced persistence of IPIC in partially bistable cells in the presence of QX-314 is an important result because it suggests that IPIC in partially bistable cells is essentially similar to IPIC in fully bistable cells. This suggests that the ionic composition of IPIC is similar in both types of motoneurons. There remains, however, the important issue of understanding why IPIC is activated at a more depolarized level in high input conductance motoneurons than low input conductance ones. One possibility is that differences in electrical structures of the dendrites in low and high input conductance cells affect the apparent voltage threshold as seen from the soma. However, considerable further work examining the types and distributions of voltage-sensitive channels on motoneuron dendrites is required before this possibility can be evaluated.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-34382 and NS-28076.
![]() |
FOOTNOTES |
---|
Address for reprint requests: C. J. Heckman, Dept. of Physiology M211, Northwestern University Medical School, 303 E. Chicago Ave., 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 21 June 1999; accepted in final form 30 July 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|