1Department of Physiology, Emory University, Atlanta, Georgia 30322; and 2Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195
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ABSTRACT |
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Prather, Jonathan F.,
Randall K. Powers, and
Timothy C. Cope.
Amplification and Linear Summation of Synaptic Effects on
Motoneuron Firing Rate.
J. Neurophysiol. 85: 43-53, 2001.
The aim of this study was to measure
the effects of synaptic input on motoneuron firing rate in an
unanesthetized cat preparation, where activation of voltage-sensitive
dendritic conductances may influence synaptic integration and
repetitive firing. In anesthetized cats, the change in firing rate
produced by a steady synaptic input is approximately equal to the
product of the effective synaptic current measured at the resting
potential (IN) and the slope of the
linear relation between somatically injected current and motoneuron discharge rate (f-I slope). However, previous
studies in the unanesthetized decerebrate cat indicate that firing rate
modulation may be strongly influenced by voltage-dependent dendritic
conductances. To quantify the effects of these conductances on
motoneuron firing behavior, we injected suprathreshold current steps
into medial gastrocnemius motoneurons of decerebrate cats and measured
the changes in firing rate produced by superimposed excitatory synaptic
input. In the same cells, we measured
IN and the f-I slope to
determine the predicted change in firing rate (F = IN * f-I slope).
In contrast to previous results in anesthetized cats, synaptically
induced changes in motoneuron firing rate were greater-than-predicted. This enhanced effect indicates that additional inward current was
present during repetitive firing. This additional inward current amplified the effective synaptic currents produced by two different excitatory sources, group Ia muscle spindle afferents and caudal cutaneous sural nerve afferents. There was a trend toward more prevalent amplification of the Ia input (14/16 cells) than the sural
input (11/16 cells). However, in those cells where both inputs were
amplified (10/16 cells), amplification was similar in magnitude for
each source. When these two synaptic inputs were simultaneously
activated, their combined effect was generally very close to the linear
sum of their amplified individual effects. Linear summation is also
observed in medial gastrocnemius motoneurons of anesthetized cats,
where amplification is not present. This similarity suggests that
amplification does not disturb the processes of synaptic integration.
Linear summation of amplified input was evident for the two segmental
inputs studied here. If these phenomena also hold for other synaptic
sources, then the presence of active dendritic conductances underlying
amplification might enable motoneurons to integrate multiple synaptic
inputs and drive motoneuron firing rates throughout the entire
physiological range in a relatively simple fashion.
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INTRODUCTION |
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Motoneurons transform synaptic
inputs into frequency-coded spike train outputs. Increments of current
cause firing rate increases that can be predicted from the linear slope
of the cell's intrinsic frequency-current (f-I)
relation (Granit et al. 1966; Kernell 1970
; Schwindt and Calvin 1973
). In the
decerebrate cat preparation studied here, excitatory synaptic currents
have been shown to activate dendritic voltage-sensitive conductances
that can contribute substantial amounts of additional depolarizing
current (Bennett et al. 1998a
; Lee and Heckman
1998b
). The effects of those conductances are evident in
motoneuron firing rate data obtained in humans (Gorassini et al.
1998
; Kiehn and Eken 1997
) and decerebrate
animals (Bennett et al. 1998a
; Lee and Heckman
1998a
). However, the influence of those conductances on
synaptic integration remains poorly understood. Therefore direct
examination of motoneuron repetitive firing is necessary to more fully
describe the relation between synaptic input and firing rate output in
the presence of voltage-sensitive dendritic conductances.
In interpreting the impact of a synaptic input on firing rate, the most
functionally relevant parameter is the current reaching the site of
spike initiation, thought to be an axonal segment in close proximity to
the soma (Colbert and Johnston 1996; Coombs et
al. 1957
; Stuart et al. 1997
). Here we
use intrasomatic microelectrodes to record the total synaptic current
reaching the soma (Bernarder et al. 1994
; Heckman
and Binder 1988
; Redman 1976
), which we will refer to as the "effective synaptic current" [or
IN, following the terminology of
Heckman and Binder (1988)
]. If effective synaptic current and microelectrode current injected into the soma have equivalent effects, this can lead to a simple prediction of
synaptically evoked changes in firing rate. The steady-state relation
between injected current (I) and motoneuron firing rate
(f) is linear over most of the
physiological range of firing rates (Binder et al.
1996
). As a result, the steady-state change in firing rate produced by a steady synaptic input (
F) can be predicted
from the product of the effective synaptic current
(IN) and the slope of the
f-I relation:
F = IN * f-I slope
(Powers and Binder 1995
).
It is not known whether injected and effective synaptic currents always
modulate motoneuron firing in an equivalent fashion. A given amount of
effective synaptic current can indeed have the same effect on firing
rate as the same amount of injected current (e.g., Granit et al.
1966; Kernell 1970
; Schwindt and Calvin
1973
). In those cases, amplification is absent, and the
addition of excitatory synaptic current to a background of injected
current causes a uniform firing rate increment across all levels of
injected current. Alternatively stated, the f-I relation in
the presence of such a synaptic input is parallel and shifted to the
left along the current axis compared with the relation in the absence
of synaptic input. However, much of the previous quantitative analysis
of firing rate modulation in motoneurons is based on data obtained in
anesthetized preparations (Granit et al. 1966
;
Powers and Binder 1995
; Schwindt and Calvin
1973
; reviewed in Binder et al. 1996
; Crill 1983
), in which there is likely to be little or no
tonic activity in descending monoaminergic fibers (cf.
Hounsgaard et al. 1988
). In contrast, in the
unanesthetized decerebrate preparation where descending monoaminergic
fibers are thought to be tonically active (Hounsgaard et al.
1988
), or in the presence of exogenously applied monoamines,
the behavior of motoneuron dendrites can be dominated by the activation
of a voltage-sensitive persistent inward current (Bennett et al.
1998b
; Hounsgaard and Kiehn 1993
; Lee and
Heckman 1996
). In addition, voltage-dependent mechanisms not
associated with monoaminergic facilitation in this preparation, such as
persistent sub-threshold sodium current (Hsiao et al. 1998
; Nishimura et al. 1989
),
N-methyl-D-aspartate (NMDA) receptor currents
(Brownstone et al. 1994
), or calcium-dependent mixed cation (CAN) current (Perrier and Hounsgaard 1999
;
Rekling and Feldman 1997
), may also be activated during
repetitive firing. It has been proposed that induction of any or all of
these persistent inward currents would amplify the synaptic current
reaching the soma (Hounsgaard and Kiehn 1993
;
Kiehn 1991
; Lee and Heckman 1996
; Schwindt and Crill 1982
).
Activation of voltage-dependent dendritic conductances makes it
difficult to predict the effects of synaptic input on motoneuron firing
rate. These effects are likely to depend on the relation between the
voltage dependence of the persistent inward current and the range of
membrane voltages present in the dendrites during repetitive discharge
(cf. Schwindt and Crill 1982). For example, if the
persistent inward current were fully activated within the depolarized
voltage range traversed before the cell fires repetitively, then the
IN present during firing would be
amplified compared with that measured at the resting membrane
potential. If that amplification remained approximately constant across
injected current settings, then the resulting synaptically induced
shift in the f-I relation would be parallel to control
conditions and greater-than-predicted (Schwindt and Crill
1995
). Alternatively, the dendritic current could be maximal at
relatively low levels of depolarization and become systematically
smaller with increasing injected current due to decreased driving force
(Burke 1967
; Cope et al. 1987
;
Lev-Tov et al. 1983
; Rall 1977
;
Rose and Cushing 1999
; Segev et al.
1990
). In that case, the f-I slope would be less in
the presence of synaptic input than in its absence. In contrast,
progressive increases in the activation of a dendritic current with
increasing depolarization would cause
IN to be systematically greater across
increasing injected current magnitudes. The f-I slope in the
presence of such a synaptic input would therefore be greater than in
the absence of synaptic input. In fact, both synaptically induced
increases and decreases in f-I slope have been reported
(Bennett et al. 1998a
; Brownstone et al.
1992
). Either type of change in f-I slope during
synaptic activation (Kernell 1965
;
Shapovalov 1972
) or a greater-than-expected shift in the
f-I relation will lead to a difference between predicted and
observed synaptic effects on firing rate. Given this variety of
mechanisms that contribute to uncertainty in predicting the effect of
synaptic current on firing rate, direct examination is essential for a
full understanding of this process.
The present study was designed to determine whether the simple model of
synaptically evoked firing rate modulation based on data obtained in
anesthetized cats (Powers and Binder 1995) also applies
to motoneurons studied in unanesthetized, decerebrate cats. The effects
of activating two different populations of primary afferents, one
carrying muscle length information (group Ia muscle spindle afferents)
and the other carrying cutaneous information (caudal cutaneous sural
nerve afferents), were directly compared to examine possible
input-specific differences. In addition, effects of concurrent
activation of both inputs were investigated to compare summation of
synaptic effects to that recently reported in anesthetized cats
(Powers and Binder 2000
). We measured the effective
synaptic currents near the resting potential produced by the two
different excitatory inputs, using the modified "voltage-clamp"
technique of Heckman and Binder (1988)
. In the same
motoneurons, we measured the steady-state relation between injected
current and motoneuron firing rate. We found that the shifted
f-I relations evoked by a steady synaptic input were nearly
always parallel to the control f-I slopes over the range of
injected current and synaptic input magnitudes studied here. In
addition, the magnitude of the f-I shift was nearly always
greater than the firing rate change predicted by the product of
IN and the f-I slope,
indicating amplification of the effective synaptic current during
repetitive firing. The amount of amplification did not appear to be
related to those intrinsic properties recorded in the present study for
each motoneuron and was similar for the two different excitatory
inputs. When the two inputs were applied concurrently, the observed
change in firing rate was approximately equal to the linear sum of
their individual effects. Portions of these results have been
previously presented in abstract form (Prather et al.
1998
).
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METHODS |
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Surgical and experimental procedures
Data were collected from 16 medial gastrocnemius (MG) motoneurons recorded in nine adult cats (2.5-3.5 kg) with the approval of the Emory University Institutional Animal Care and Use Committee. Anesthesia was induced in a closed chamber and maintained via a tracheal cannula throughout the initial dissection with a gaseous mixture of halothane (1.5-2.5%) in a 1:1 mixture of NO2:O2. Artificial respiration was adjusted to hold end-tidal CO2 between 3 and 4%. The right carotid artery and jugular vein were cannulated for monitoring blood pressure and administering fluids, respectively. Atropine sulfate (0.54 mg/ml, 1 ml/20 lbs. body wt) was given intramuscularly to reduce bronchial secretion, and dexamethasone phosphate (1.0 mg/kg) was delivered intravenously to minimize edema. The lumbosacral enlargement was exposed by a laminectomy from L4 to S1 to provide access to MG motoneurons. The left hindlimb was dissected to expose the MG muscle nerve and caudal cutaneous sural nerve, and the triceps surae muscles were separated from their surrounding tissues. After separating the plantaris tendon, the remainder of the Achilles tendon was cut and attached to a servomotor that provided the muscle stretch stimulus. The animal was then mounted in a recording frame, and following ligation of the left carotid artery an intercollicular decerebration was performed. Anesthesia was discontinued after the decerebration. At the end of the recording session, animals were killed using a lethal dose of intravenous pentobarbital sodium.
Intracellular recordings were made from MG motoneurons using glass
micropipettes filled with 2 M K-acetate (resistances of 5-10 M)
connected to an Axoclamp-2A amplifier operated in bridge mode. When
resting membrane potential was steady and action potential amplitude
exceeded 70 mV, records were collected (DC to 10-kHz band-pass) and
stored on computer (22-kHz digitization). Rheobase current
(Irh), input resistance
(Rin), action-potential
afterhyperpolarization half-decay (AHP), and axonal conduction velocity
(CV) were measured using the protocols of Zengel et al.
(1985)
.
A steady excitatory synaptic input was introduced from two different
sources: repetitive electrical stimulation of afferents in the intact
caudal cutaneous sural nerve (40-µs pulses, 100 Hz, <5 T stimulus
intensity) and activation of primary muscle spindle (group Ia)
afferents by mechanical vibration of the triceps surae muscles (167-Hz
sinusoid, 80-µm amplitude). The intensity of sural nerve stimulation
and the background level of muscle stretch were adjusted for each cell
so that each source produced about the same mean level of
depolarization. The effective synaptic current
(IN) produced by each source alone and
in combination was then measured using an intrasomatically placed
microelectrode and the modified "voltage-clamp" technique of
Heckman and Binder (1988) (Fig.
1A). In this stimulus
procedure, microelectrode injected current is combined with
high-frequency repetitive activation of a synaptic input and consists
of three consecutive 500-ms epochs: 1) injected current
alone, 2) simultaneous injected current and synaptic input,
and 3) synaptic current alone (see Fig. 1A). In practice, it is not necessary to precisely clamp the membrane potential
at the resting value on a given trial, since
IN can be estimated from the responses
to synaptic input in combination with several different levels of
injected current (cf. Powers and Binder 1995
,
2000
). The value of IN
is determined by interpolating a line to the relation between injected
current and membrane potential (relative to rest) during epoch 2 and
determining the current value at which that relation crosses the zero
voltage axis. In the example illustrated in Fig. 1A, when 10 nA of hyperpolarizing current was combined with repetitive activation
of the sural nerve, the mean membrane potential was 3.1 mV below the
resting potential (horizontal dashed line), whereas the membrane
potential was 3.5 mV above rest when sural was activated in combination
with 5 nA of hyperpolarizing current and 0.9 mV above rest with 7 nA of hyperpolarizing current (not shown). The effective synaptic current produced by the sural input was estimated by linear interpolation to be
7.7 nA in this cell.
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Stimulus and recording procedures
Motoneurons were stimulated to fire repetitively using the
following stimulus protocol. A range of suprathreshold 1-s current steps were injected to determine the slope of the f-I
relation. Motoneurons were then stimulated to fire by various
combinations of injected current and synaptic input. Midway through a
1-s period of injected depolarizing current, the synaptic input was
initiated and maintained for 1 s. Each trial of injected current
plus synaptic input was preceded and followed by a control trial of
injected current alone (cf. Powers and Binder 1995). The
interval between synaptic stimuli was
30 s to avoid changes in
interneuronal excitability and possible wind-up of plateau mechanisms
(Bennett et al. 1998a
; Svirskis and Hounsgaard
1997
).
Data analysis
In the decerebrate cat preparation, motoneuron excitability can
fluctuate over time. To ensure that changes in excitability were not
responsible for the observed rate changes, the firing rate induced by
injected current alone during the initial 500 ms of the stimulus
protocol was compared with the control trials of the same injected
current that preceded and followed each stimulus. Similarity in firing
rates during these periods ensured that motoneuron excitability was
similar between stimulus and control conditions. The effect of synaptic
input on firing rate was then assessed by calculating the difference
between the mean firing rate during the 500-ms epoch of injected
current plus synaptic input and the mean firing rate of the bracketing
control trials. This observed change in motoneuron firing rate
(Fobs) was compared with the predicted change (
Fpr) estimated
from the product of the f-I slope and
IN measured at the resting potential.
Figure 1C provides a graphic illustration of the calculation
of
Fpr. If effective synaptic
current (IN) has the same effect on
firing rate as an equivalent amount of injected current (see
INTRODUCTION), the predicted change in firing rate is
calculated by moving along the control f-I relation (solid
line) by the amount IN. The effects of
synaptic input on firing rate were examined using at least two
different levels of injected current, and in many cases at several
different levels (Fig. 3). The best fit linear regression was
determined for the relation between injected current and firing rate
for trials in which synaptic input was present (dotted line in Fig. 3)
as well for the bracketing control trials (solid line). The
f-I relation in the presence of synaptic input was then
compared with that predicted based on shifting the control relation by an amount equal to IN (dashed line).
Changes in motoneuron firing rate that were significantly larger than
the predicted values (see Statistical analysis) were
taken as evidence of an amplified effect of synaptic current.
Statistical analysis
Synaptically induced changes in firing rate were measured at a number of different levels of injected current. Those data were used to generate f-I relations for electrode current alone and electrode current plus synaptic stimulus conditions. For each cell, the regression line of f-I data in the presence of synaptic input was calculated (dotted line in Fig. 3) and compared with a regression line for control (injected current alone) data that had been shifted along the x-axis (current) by an amount equivalent to the effective synaptic current measured at rest (dashed line in Fig. 3). If the activation of synaptic input did not change the slope of the f-I relation, then amplification could be inferred as a difference in the y-intercepts (firing rate axis) of the observed and expected regression lines. An analysis of covariance (ANCOVA) was used to test whether the f-I slope with combined injected current and synaptic activation was not significantly different from the expected regression. In those cells where the two slopes were not different, ANCOVA was further used to test the hypothesis that the y-intercepts of the observed and expected relations were significantly different, indicating the presence of amplification.
Potential sources of error
Both the estimates of IN and
f-I slope are subject to error, due to variability in the
synaptic responses, slow drifts in resting potential, errors in
electrode bridge balance and changes in cell excitability. Previous
calculations of the errors associated with estimation of
IN suggest a 15% uncertainty in these
estimates (Powers and Binder 1995). Measurements of
synaptically evoked changes in firing rate will be affected by slow
changes in the motoneuron's repetitive discharge properties. We
attempted to minimize the effect of these changes by bracketing the
responses to injected current in the presence of synaptic input with
trials in which injected current was presented alone. Nonetheless,
collection of firing rate data at several different levels of injected
current with and without different synaptic inputs typically took
10-30 min, and changes in cell excitability over this time period were likely to contribute to the scatter in the f-I relations
(see Fig. 3). Two different variations of the stimulus protocol were used in a subset of the cells to minimize this source of error (described in RESULTS). Although these various sources of
error could contribute to differences between the predicted and
observed changes in firing rate produced by a given synaptic input in
individual cases, they should not have caused the systematic
amplification observed across the entire sample.
There is another potential source of error that might cause systematic
differences between predicted and observed changes in firing rate;
however, this type of error should produce an underestimate of
amplification magnitude. Predictions of the change in firing rate
produced by a synaptic input were based on the effective synaptic
current measured at the resting potential. For excitatory synaptic
inputs, the membrane depolarization during repetitive discharge will
reduce the driving force for synaptic current (Burke
1967; Cope et al. 1987
; Lev-Tov et al.
1983
; Rall 1977
; Rose and Cushing
1999
; Segev et al. 1990
). As a result, in the
absence of amplification by active conductances, the effective synaptic
current present during repetitive discharge will be less than that
observed at rest (cf. Powers and Binder 1995
,
2000
). Consequently, our predicted firing rate change
represents an overestimate of that expected to occur in a neuron that
does not exhibit amplification. Comparison of this overestimated
predicted firing rate against observed rates will cause an
underestimation of synaptic effects on firing rates.
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RESULTS |
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Amplified effect of synaptic current on motoneuron firing
A greater-than-expected increase in motoneuron firing rate caused
by sural synaptic excitation is illustrated in Fig.
2. The top traces represent
the instantaneous firing rate produced by 1-s steps of injected current
(bottom traces) in the presence (right) or
absence (left) of superimposed 100-Hz stimulation of the
sural nerve (indicated by the solid bar). The firing rate elicited by
15 nA of injected current was 20 pps. That rate was increased to 41 pps
when another 10 nA of injected current were added (superimposed firing
rate traces on the left). It was expected that the cell
would display the same firing rate increase if an equivalent amount of
effective synaptic current were added instead of injected current.
However, the addition of sural input (estimated to produce an effective
synaptic current of 7.7 nA using the modified voltage-clamp technique
in Fig. 1A) to the 15 nA of injected current increased
firing rate to 67 pps. This enhanced effect of synaptic input can also
be seen in Fig. 1A, since sural synaptic current alone
caused the motoneuron to fire repetitively, whereas 10 nA of injected
current did not (Fig. 1B). These data demonstrate that
activation of the synaptic input under repetitive firing conditions
caused a change in firing rate that was greater than predicted from
measurements of IN at resting
potential. This result is consistent with the activation of a
voltage-dependent persistent inward current (Lee and Heckman
1996; Schwindt and Crill 1995
).
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Amplification was present at several different background firing rates.
Figure 3 illustrates the firing rates
elicited in another motoneuron by various amplitudes of injected
current alone (, solid regression line) and in combination with
excitatory Ia input (
, dotted regression line). The dashed line
illustrates the predicted f-I relation in the presence of
the Ia input, produced by shifting the control f-I relation
along the current axis by an amount equal to the effective synaptic
current measured at rest (4 nA). Over the whole range of currents
tested in this cell, the increments in firing rate generated by the
addition of synaptic current exceeded expected values. While the slopes
of the dashed and dotted lines are not identical (1.00 and 1.21, respectively), their differences are not significant (ANCOVA test for
parallelism, P > 0.05). Furthermore, the magnitude of
observed amplification is greater than could be explained by such a
difference. The possibility of changing f-I slope during
activation of synaptic input is further investigated in subsequent
sections.
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The greater-than-expected increment in firing might reflect activation
of a mechanism that relies on sustained somatic depolarization to
develop, rather than one activated by synaptic input. This possibility
was assessed by implementing the first of two modifications to our
primary stimulus protocol. Firing rate increments were measured in
response to the addition of either synaptic input or additional
injected current introduced with a 1-s delay on a background of
suprathreshold injected current. Figure
4A illustrates sequential
stimulus trials in which either a synaptic input (current and firing
rate records at left) or injected current (records at
right) was added after a 1-s delay to a background of 23 nA of injected current. The added injected current (6.5 nA) was selected to match the effective synaptic current estimated at the resting potential in response to the combined stimulation of sural and Ia
inputs. Despite the equivalence of conditions in these two trials, the
synaptic input produced substantially larger increases in motoneuron
firing rate than the injected current, 26 pps versus 11 pps,
respectively. These findings demonstrate that the enhanced effectiveness of synaptic inputs could not be reproduced by injected current alone, perhaps because the synaptic currents have access to
amplification mechanisms in the dendrites that are not accessible to
current injected in the soma. These data are consistent with the notion
that voltage-sensitive plateau conductances, which are present along
motoneuron dendrites (Bennett et al. 1998b; Carlin et al. 2000
; Lee and Heckman
1996
), participate in amplification of the synaptic current
delivered to the soma.
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The observed amplification could also have been caused by a
long-lasting synaptically induced increase in the slope of the f-I relation (see INTRODUCTION). This was
unlikely to have affected the results since the control f-I
relation was calculated from responses to injected current alone, which
were interspersed with responses to combined injected current and
synaptic input. However, to demonstrate the stability of the control
f-I relation more convincingly, a second modification was
made to the stimulus protocol. Each cell was driven to fire using four
levels of injected current, ranging from twice the rheobase current to
the maximum positive current that did not polarize the electrode. The
two intermediate current settings were evenly spaced between those
limits. In Fig. 4B, the motoneuron was driven using current
settings of 8, 15.3, 22.6, and 30 nA. The responses to a given amount
of injected current alone were quite similar even though they occurred
at different times following the application of the synaptic input
(gray shaded regions in Fig. 4B). The control f-I
relation is illustrated by the solid line in Fig. 4C, and it
can be seen that there is relatively little scatter in the individual
responses at a given level of injected current (). Further, firing
rates evoked by simultaneous application of injected current and
activation of Ia afferents (
---
) were consistently and
appreciably greater than predicted values (- - -).
Analyses of variance and covariance were used to compare f-I
relations in the presence and absence of synaptic input, based on data
obtained using either the modified technique described in Fig.
4B (4 of 16 cells) or the standard protocol (12 of 16). The
distributions of f-I slopes were similar in the presence and absence of synaptic input (control: 1.72 ± 0.48 pps/nA, mean ± SD, range = 0.84-2.63 pps/nA; Ia: 1.63 ± 0.52 pps/nA,
0.79-2.42 pps/nA; sural: 1.43 ± 0.56 pps/nA, 0.41-2.52 pps/nA;
Ia + sural: 1.77 ± 0.55 pps/nA, 1.06-2.30 pps/nA;
ANOVA: F = 1.01, P = 0.40). The
f-I slope in the presence of synaptic activation was
indistinguishable from the slope of the expected regression in 16 of 16 cells for the Ia input and 15 of 16 for the sural input (ANCOVA test of parallelism, P > 0.05). Synaptic current was amplified
during repetitive firing, as indicated by a significant increase in
y-intercept between observed and expected regression lines,
in 14 of 16 cells (88%) for Ia input and 11 of 16 cells (69%) for
sural input. Therefore in almost all cases, the difference between
observed and expected synaptically induced rate changes was not due to
a change in f-I slope, but rather a change in the amount of
current reaching the soma during synaptic stimulation. The parallelism
of f-I relations in the presence and absence of synaptic
input indicates that for the injected current and synaptic input
magnitudes studied here, the increment in effective synaptic current
was not voltage dependent beyond its activation. These data are
consistent with the notion that most of the amplification demonstrated
by each cell occurs in the voltage range between resting potential and
the threshold for repetitive discharge (cf. Lee and Heckman
1996; Schwindt and Crill 1995
).
Figure 5, A and B,
illustrates the relation between observed and predicted firing rate
changes for Ia (A) and sural (B) inputs. Those
cases in which the observed firing rate changes were significantly different from predicted values are indicated by open symbols and the
nonsignificant cases by filled symbols. In both panels, nearly all of
the significant cases are above the line of identity (diagonal line).
In the one case of a cell in which the sural input caused a firing rate
decrease, the sural input was transiently inhibitory and produced a net
decrease in firing rate. Only one cell failed to amplify either input.
That cell was not different from other motoneurons in the synaptic
current it received or its intrinsic properties, except that it had the
largest observed Irh (22 nA). However,
amplification of one or both inputs was evident in other cells that
also had high rheobase (e.g., 16, 17, and 21 nA). There was no tendency
for the magnitude of amplification to be less in cells with larger
rheobase (Pearson's correlation, Ia: R = 0.14,
P = 0.60; sural: R =
0.06,
P = 0.82).
|
Direct comparison of Ia and sural amplification
Ia input was amplified in 14 of 16 motoneurons (88%). Cells that did or did not amplify Ia input were indistinguishable in their cellular properties (Irh, Rin, AHP, CV, f-I slope; t-test for independent samples, P > 0.31 all cases). The two cells that failed to amplify Ia input had Irh of 6 and 22 nA, demonstrating that failure to amplify was not limited to only the least excitable motoneurons. Those two cells both received small synaptic currents (1.0 and 1.5 nA); however, amplification of Ia input was evident in six other cells where Ia IN was between 0.5 and 1.5 nA. Therefore failure to amplify was not simply due to insufficient excitatory synaptic drive onto the amplification mechanisms. In addition, there was no correlation between the magnitude of Ia IN and the amount of amplification observed in each cell (Pearson's correlation, R = 0.33, P = 0.21). Therefore with respect to both cellular properties and IN magnitudes, the two cells that failed to amplify Ia input were indistinguishable from those cells that did display amplification.
Sural input was amplified in 11 of 16 motoneurons (69%). Cells that did or did not amplify sural input were indistinguishable in their cellular properties and IN magnitudes (t-test for independent samples, P > 0.19 all cases). In 10 of those 11 cells that amplified sural input, Ia input was also amplified. The cell that amplified sural but not Ia input was not anomalous in its intrinsic properties or in the amount of effective synaptic current it received. As was the case for the Ia input, there was no correlation between sural IN magnitude and the amount of amplification observed in each cell (Pearson's correlation, R = 0.13, P = 0.63). Therefore neither Ia nor sural synaptic input was uniformly amplified across the population of MG motoneurons. However, it was not apparent from these data which motoneuron characteristics regulated whether or not amplification was expressed in a given cell.
Ia and sural inputs were both amplified in 10 of 16 motoneurons (63%).
Those 10 cells were indistinguishable from the remaining motoneurons
that amplified only one or neither input (t-test for independent samples, P > 0.24 all cases). Over the
observed range of injected currents and firing rates in those 10 cells,
ANCOVA revealed that the shift in the f-I relation was
parallel and significant for both inputs in the same motoneuron.
Observed firing rates exceeded expected values for both inputs in 9 of
10 cells. The lone exception was sural input in the cell for which
sural was transiently inhibitory. On average, the observed change in
firing rate was about three times larger than the predicted change in rate (3.3 ± 3.9). The average difference between observed and expected rate changes was slightly but not significantly larger for Ia
input (14.2 ± 9.7 pps) than for sural input (9.6 ± 12.7 pps, paired t-test, P = 0.13). However, this
difference was primarily due to the influence of the motoneuron for
which sural was transiently inhibitory and another cell in which the
change in rate for Ia exceeded that of sural by 20 pps. Overall, Ia and
sural inputs were indistinguishable in their amplified effects on
firing rate (paired t-test, P = 0.13). The
solid line in Fig. 5C is the line of unity, and the dotted
lines indicate a range of ±6 pps around this line. Data from 7 of 10 motoneurons fall within this range. The motoneuron in which the firing
rate change due to Ia input exceeded that of sural by 20 pps lies well
away from unity. That motoneuron was not exceptional in its intrinsic
properties (Irh 11 nA,
Rin 1.8 M, AHP 14.3 ms), nor did it
receive especially large subthreshold synaptic currents (Ia: 2.6 nA;
sural: 3.2 nA). It is not apparent why the response of this cell was so
different from the general trend for Ia and sural to be amplified similarly.
Amplified synaptic effects exhibit linear summation
The potential for nonlinear interactions [e.g., reduction in
driving force, shunting of current by adjacent conductances
(Oakley et al. 1999)] leaves uncertainty about the
influence of simultaneously active synaptic inputs on motoneuron
firing. Direct examination revealed that the amplified Ia and sural
inputs exhibited approximately linear summation (Fig.
6) over the amplitudes of
IN studied here. The observed
increases in firing rate during combined Ia and sural activation
correlated very well with the linear sum of rate increases due to Ia
and sural separately (R = 0.94, slope = 0.77, y-int = 4.2 pps, P < 0.001). The slope
of this relation is slightly less than unity, suggesting that summation
may have been slightly less than linear. However, the nine data points
are scattered about the line of identity, and the average increase in
firing rate due to activation by combined input (26.0 ± 16.7) was
statistically indistinguishable from the linear sum of the average
effects of each input individually (Ia: 14.2; sural: 9.6 pps; paired
t-test, P = 0.42).
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Amplification is dissociated from expression of plateau properties
Plateau properties were investigated in each motoneuron. A cell
was classified as possessing plateau properties if it displayed one or
more of the following traits: firing rate hysteresis during linearly
increased and decreased injected current amplitude (Bennett et
al. 1998a), firing rate acceleration during constant-amplitude injected current pulses, or sustained firing following termination of
excitatory stimulus (Eken et al. 1989
). Eight of the 16 cells exhibited clear evidence of these properties. Consistent with earlier reports (Lee and Heckman 1998a
), the population
expressing plateau properties had lower rheobase currents
(t-test for independent samples, P < 0.01)
and a nonsignificant trend toward higher input resistances
(P = 0.07) than those cells that did not express
plateaus. Motoneurons that did express plateaus were indistinguishable
from cells that did not, with respect to AHP (P = 0.28)
and axonal CV (P = 0.38). Interestingly, cells
expressing plateau properties had steeper f-I slopes
(2.3 ± 0.8) than cells without plateau properties (1.5 ± 0.5, t-test for independent samples, P = 0.02). Both Ia and sural inputs were amplified in seven of the eight cells with plateau properties. In the remaining cell, Ia input was
amplified, but sural input was not. Cells that did or did not express
plateaus were indistinguishable in their amplification magnitude of Ia
input (t-test for independent samples, P = 0.26) and sural input (P = 0.47). Therefore the amount
of amplification expressed by each motoneuron, while varying across the
population, did not vary systematically across any of the measured
intrinsic properties or as a function of the expression of plateau properties.
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DISCUSSION |
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The change in motoneuron firing rate produced by activation of
synaptic inputs was significantly greater-than-expected in 14 of 16 cells in which the Ia muscle spindle afferent input was tested and in
11 of 16 where the sural nerve input was tested. Our results show that
in the decerebrate cat, synaptic currents are amplified during
repetitive firing, and this voltage-dependent amplification of synaptic
current is an integral element of the motoneuron input/output relation.
In contrast, in intact, pentobarbital-anesthetized cats, changes in
firing rate produced by a variety of synaptic inputs are quite close to
those predicted on the basis of the effective synaptic current measured
at rest (Powers and Binder 1995). The differences
between these two preparations probably reflect the relative
predominance of a persistent inward current (Schwindt and Crill
1982
) in the decerebrate preparation (Kiehn and Eken
1998
; Lee and Heckman 1999
), likely as a result
of tonic activity in neurons providing monoaminergic inputs to
motoneurons (cf. Hounsgaard et al. 1988
).
Although the ionic basis of this persistent inward current in cat
motoneurons is not known, in other motoneurons it has been shown to be
mediated primarily by either an L-type calcium current (Hounsgaard and Mintz 1988), a persistent sodium current
(Nishimura et al. 1989
), or a mixture of these two
currents (Hsiao et al. 1998
). In some motoneurons,
activation of a calcium-dependent mixed cation (CAN) conductance may
lead to a persistent inward current, although multiple calcium spikes
are generally required for significant activation (Perrier and
Hounsgaard 1999
; Rekling and Feldman 1997
).
Alternatively, a voltage-dependent persistent inward current could
result from a receptor-mediated effect, such as a voltage-dependent
increase in synaptic current through NMDA-activated receptors, which
has been suggested as a potential contributor to voltage-dependent
amplification of synaptic potentials associated with fictive locomotion
(Brownstone et al. 1994
). However, NMDA receptors are
unlikely to contribute significantly to the amplification process
reported here, as sural and Ia inputs are similarly amplified but the
contribution of NMDA receptors to the Ia excitatory postsynaptic potential (EPSP) is only minimal or entirely absent in adult cat spinal
motoneurons (Engberg et al. 1993
; Jahr and
Yoshioka 1986
; Kalb et al. 1992
; Miller
et al. 1997
; Walmsley and Bolton 1994
; cf.
Flatman et al. 1987
). Further study involving
pharmacological manipulation of the conductances described above is
needed to elucidate the mechanisms of amplification. Regardless of the
exact mechanisms underlying persistent inward currents in cat
motoneurons, the functionally relevant features of this class of
current are its persistence, its relatively low threshold for
activation, and the fact that a significant proportion of the
responsible channels appear to have a dendritic location (cf.
Bennett et al. 1998a
; Carlin et al. 2000
;
Hounsgaard and Kiehn 1993
; Lee and Heckman
1996
).
Previous analysis of the effects of these persistent inward currents on
motoneuron repetitive firing have focused on bistable discharge
behavior, i.e., a specific type of plateau property characterized by
self-sustained tonic discharge following brief presentation of an
excitatory stimulus (e.g., Kiehn and Eken 1998; Lee and Heckman 1998a
,b
). The strength of this
bistability varies systematically across the motoneuron pool and is
strongest in motoneurons with the lowest thresholds for excitation
(Lee and Heckman 1998a
,b
). If the mechanisms underlying
bistability and amplification are similar, one might expect that cells
that demonstrated bistability, or any other type of plateau property,
might also amplify synaptic current more than cells that did not
express plateaus. However, we found that almost all cells exhibited
amplification, whereas only 8 of 16 cells exhibited plateau properties.
This discrepancy might suggest that amplification and the induction of
plateau potentials are executed by different mechanisms. Alternatively, the two behaviors may share a common mechanism, but dendritically located synaptic currents have better access to the underlying conductances than somatically injected currents. In addition, plateaus
may already have been active at the time of recruitment in some cells,
not permitting us to observe any of the criteria used to characterize
the expression of plateaus (Bennett et al. 1998a
;
Lee and Heckman 1996
). Several previous studies have
suggested that amplification may be an important correlate of plateau
expression (e.g., Dickenson and Nagy 1983
;
Hartline et al. 1988
; Kiehn 1991
; Kiehn et al. 1996
; Rekling and Feldman
1997
; Stafstrom et al. 1985
); however, the
requirements for amplification are less restrictive than those needed
for activation of plateau potentials. For example, the sudden
depolarization or acceleration in firing rate that is characteristic of
plateau induction may require two stable equilibrium points on the
steady-state current-voltage (I-V) relation of the cell
(i.e., an N-shaped I-V relation that crosses the zero current axis) (Gutman 1991
; Lee and Heckman
1998b
; Schwindt and Crill 1980
), whereas
amplification will occur whenever the persistent inward current leads
to a decrease in the slope of the I-V relation.
Figure 7 provides a graphical illustration of this point, and shows that over a given range of membrane voltages, amplification could be similar in a cell that expresses plateau properties and another neuron that does not. Figure 7A shows hypothetical steady-state I-V relations for a portion of dendritic membrane in a neuron expressing a plateau (bold line) and another neuron without a plateau (thin line). For the neuron indicated by the bold line, the dendritic I-V curve exhibits a region of negative slope conductance, whereas in the neuron indicated by the thin line, the slope of the I-V curve is always positive. The point is illustrated more clearly by Fig. 7B, which provides an expanded view of the portion of the I-V curves indicated by the dotted rectangle in A. Over the voltage region bounded by the vertical dotted lines, both curves show a continuous decrease in slope conductance compared with that due to the slope of the leak conductance (dashed diagonal line). As a result, in both neurons there will be voltage-dependent amplification of synaptic inputs, since a given increment in synaptic current will lead to an increasingly larger local dendritic depolarization. If sufficient steady depolarizing current is applied to bring the membrane to the voltage represented by the rightmost vertical line, the dendritic membrane represented by the bold I-V curve will jump to a depolarized voltage, i.e., it will exhibit the abrupt firing rate increase that characterizes the induction of a plateau. Nonetheless, for voltages below this point the membranes represented by the two I-V curves will exhibit identical amplification of synaptic inputs.
|
When Ia and sural inputs were presented simultaneously, the consequent
firing rate changes were approximately equal to the linear sum of the
amplified rate changes elicited by each input individually. Linear
summation might be due to spatial segregation of different inputs onto
different dendrites of motoneurons, but this seems unlikely because Ia
monosynaptic inputs are widely distributed across the dendritic tree of
MG motoneurons (Burke and Glenn 1996) and would
therefore overlap with sural inputs. Linearity may be achieved instead
by a balanced activation of different types of active dendritic
conductances that boost or shunt the inputs (e.g., Bernarder et
al. 1994
; Cash and Yuste 1999
; Lee and
Heckman 1996
; Margulis and Tang 1998
;
Nettleton and Spain 2000
; Schwindt and Crill
1995
; for review, see Yuste and Tank 1996
). It
is likely that the combined action of many types of active conductance
are responsible for the observed linear summation; however, the current
data do not allow their individual contributions to be discerned.
Even under the influence of current modulation by active conductances,
the slope of the f-I relation during synaptically induced amplification was nearly always indistinguishable from that observed during activation by injected current alone. This similarity suggests that activation of amplification mechanisms does not disturb the processes of synaptic integration (cf. Binder et al.
1993). Our results thus illustrate a phenomenon by which
excitatory inputs like those used here can smoothly grade motoneuron
firing rate over the entire physiological range.
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ACKNOWLEDGMENTS |
---|
The authors thank M. Binder, V. Haftel, and F. Frost for providing comments on the manuscript.
This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-21023, NS-31925, and NS-26480.
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FOOTNOTES |
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Address for reprint requests: J. F. Prather, Dept. of Physiology, 1648 Pierce Dr., Emory University, Atlanta, GA 30322 (E-mail: jprathe{at}emory.edu).
Received 29 December 1999; accepted in final form 14 September 2000.
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REFERENCES |
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