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The Journal of Neurophysiology Vol. 80 No. 2 August 1998, pp. 583-593
Copyright ©1998 by the American Physiological Society
Department of Physiology, Northwestern University Medical School, Evanston, Illinois 60201
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Lee, R. H. and C. J. Heckman. Bistability in spinal motoneurons in vivo: systematic variations in persistent inward currents. J. Neurophysiol. 80: 583-593, 1998. Bistable behavior in spinal motoneurons consists of self-sustained firing evoked by a brief period of input. However, not all motoneurons possess an equal capacity for bistable behavior. In the companion paper, we found that self-sustained firing was persistent for long periods only in motoneurons with low rheobases and slow axonal conduction velocities. High rheobase, fast conduction velocity motoneurons tend to be only partially bistable in that self-sustained firing lasts at most 1-2 s. The mechanisms underlying these differences between fully and partially bistable motoneurons were investigated by measuring their current voltage (I-V) relationships in the decerebrate cat preparation after administration of the noradrenergic 
1 agonist methoxamine. Slow (8 mV/s) triangular voltage commands were applied using the discontinuous single-electrode voltage-clamp technique. Both fully and partially bistable cells exhibited a region of negative I-V slope due to activation of a strong, persistent inward current. The peak amplitude of the total persistent inward current (IPIC) was equally large in fully and partially bistable cells, but there were substantial differences in how IPIC was activated and deactivated. In fully bistable cells, the offset of IPIC on the descending phase of the triangular voltage command occurred at a substantially more hyperpolarized voltage then its onset on the rising phase. Thus the I-V function of fully bistable cells exhibited marked hysteresis. Partially bistable cells had significantly less hysteresis. The lack of hysteresis in partially bistable cells was due to a greater decay of IPIC with time than that seen in fully bistable cells. Furthermore, the range over which activation and deactivation of IPIC occurred was more depolarized in partially than in fully bistable cells. The I-V functions were compared with frequency-current (F-I) functions from the same cells, the characteristics of which were presented in the companion paper. The strong onset-offset difference in IPIC in fully bistable cells corresponded to a similarly large hysteresis for the thresholds of their F-I functions. The reduced onset-offset difference for IPIC in partially bistable cells corresponded to a lack of hysteresis in F-I thresholds. Thus the properties of IPIC accounted for the main differences in the F-I behavior seen between fully and partially bistable cells.
The monoamines norepinephrine and serotonin can cause spinal motoneurons to generate long-lasting plateau potentials and, consequently, to exhibit self-sustained firing (Conway et al. 1988 Measurements of I-V relations
Single-electrode voltage-clamp data were obtained in 27 of the sample of 49 cells characterized during current clamp presented in the companion paper (Lee and Heckman 1998
Measurements of spike voltage thresholds
To provide a reference level for the voltage levels on the I-V functions, we also measured the voltage threshold for action potential generation. The voltage threshold for a spike was defined as the peak of the second time derivative of voltage in the depolarizing phase of the action potential. Spikes were digitized at 5-10 kHz, resulting in an accuracy of about ±2 mV for measurement of the spike threshold. We chose records in which tonic firing was generated by steady firing in muscle spindle Ia afferents, which was the same synaptic input used to assess the duration of self-sustained firing in the accompanying paper (Lee and Heckman 1998
Statistical analyses
Statistical procedures were identical to those in the accompanying paper. We used t-tests assuming unequal variances to compare the properties of fully and partially bistable cells and linear regression analyses to assess relations between variables. The significance level, alpha, was set at P = 0.05. Where results from multiple t-tests were compared (see Tables 1-3), we chose a conservative alpha level of 0.05/10 = 0.005.
Of the 27 cells in which I-V functions were measured, 10 were fully bistable and 17 were partially bistable. In 10 of these 27 cells, only data on the duration of self-sustained firing was obtained in addition to the I-V function data. In the other 17 cells (6 fully bistable, 11 partially bistable), we also obtained data for F-I functions (see Lee and Heckman 1998 Input conductance
Previous studies of motoneurons have shown that input conductance is correlated closely with rheobase, which is a good indicator of motor unit type (Zengel et al. 1985
Basic I-V relationships
Figure 2 illustrates the responses of a fully and a partially bistable cell to the standard triangular shaped voltage command. In the fully bistable cell, the current initially increases in proportion to voltage, but a departure from this linearity leads to a region of approximately zero slope (Fig. 2A). This is followed by a negative slope region, a second zero slope region, and then a return to a positive slope. This is the classic "N" shape produced by a strong inward current (e.g., Schwindt and Crill 1977 Activation/deactivation of IPIC in fully versus partially bistable cells
The examples shown in Fig. 2 suggest that the behavior of IPIC differs markedly between fully and partially bistable cells. Quantitative support for this conclusion is provided in Table 1, where the behaviors of IPIC in fully and partially bistable cells are compared. Table 1 also shows the results for regression analyses of how the properties of IPIC vary with input conductance. There was a significantly greater difference along both the voltage and current axes between the onset and offset of IPIC in fully bistable cells than in partially bistable cells (see Table 1). In addition to these differences in hysteresis, there was a clear trend for the onset and offset of IPIC to be more hyperpolarized in fully bistable cells than in partially bistable cells. Figure 3 illustrates the relationship between IPIC onset (A) and offset (B) voltages and input conductance. On average, the onset of IPIC was ~7 mV more hyperpolarized in fully than in partially bistable cells (Table 1). The analogous difference for offset voltages was even larger, about ~13 mV (Table 1).
Relationship between characteristics of IPIC and rhythmic firing behaviors
To investigate how the properties of IPIC influence rhythmic firing behaviors, data from cells in which both F-I and I-V characterizations had been made were analyzed. Figure 4 shows that the current levels for the onset and offset of IPIC exhibited a near one-to-one correspondence with the current levels for ascending (Fig. 4A) and descending thresholds (Fig. 4B) of the cells' F-I functions (determined from slow triangular current inputs) (see Lee and Heckman 1998 Amplitude and kinetics of IPIC
Although a natural assumption would be that IPIC is larger in fully bistable cells than in partially bistable cells, this was not the case. Instead, there was a slight, nonsignificant tendency for the initial peak of IPIC to be larger in partially bistable cells (Table 3), and the magnitude of the sustained peak did not differ in the two cell types. However, the reduction in the amplitude of IPIC (defined as the difference between the initial and sustained peaks) was positively correlated with input conductance (Table 3). This pointed to a time-dependent decay of IPIC as the main factor affecting hysteresis and so the kinetics of IPIC were examined further.
In the presence of the Effect of IPIC on rhythmic firing
The negative slope imparted to the I-V function by the activation of IPIC plays a major role in determining the form of the F-I function. Figure 7 illustrates the sudden shifts in voltage that tend to occur with negative I-V slope (Hille 1992
Origin of low rheobases
The range of rheobases and rhythmic firing thresholds in the present sample of motoneurons was shifted ~10 nA downward in comparison with the range seen in pentobarbitalanesthetized preparation (e.g., Gustafsson and Pinter 1984 Origin of IPIC
A persistent inward current was first documented in cat spinal motoneurons by Schwindt and Crill (1977) Mechanism of hysteresis in IPIC
What properties of the channels generating IPIC produce the hysteresis? One possibility is extremely slow kinetics for the channels involved, so that IPIC activation and deactivation fail to keep up with the rate of change of voltage. One source of slow kinetic behavior is that L-type Ca2+ channels exhibit voltage-dependent facilitation, which is a slow activation process that could contribute to hysteresis (Svirskis and Hounsgaard 1997 Origin of decay of IPIC
Two possible explanations for the decay of IPIC with time (Fig. 6) are inactivation of IPIC and activation of outward currents. Schwindt and Crill (1980)
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Hounsgaard and Kiehn 1989; Hounsgaard et al. 1988). Self-sustained firing can be eliminated by a brief hyperpolarizing pulse of sufficient magnitude to deactivate the underlying plateau potential. Thus motoneurons are said to be bistable in the absence of input, in that they can be either be quiescent or firing tonically. However, not all motoneurons have an equal capacity for bistable behavior (Hounsgaard et al. 1988; Lee and Heckman 1996b). In the companion paper, we found that fully bistable motoneurons, which can generate self-sustained firing for long periods, tend to have low current thresholds for firing action potentials and slow conduction velocities in their axons (Lee and Heckman 1998). This makes good functional sense in that these motoneurons are most likely to innervate fatigue resistant muscle fibers (Binder et al. 1996). In contrast, partially bistable motoneurons, which exhibit at most 1-2 s of self-sustained firing, have high thresholds and fast conduction velocities. Thus they likely innervate fatiguable muscle fibers.
). The primary difference is that, in fully bistable cells, the threshold current level for rhythmic firing decreases once firing has begun. In partially bistable cells, the current levels for initiation and cession of rhythmic firing are similar. In addition to these differences in F-I hysteresis in fully and partially bistable cells, differences in the firing acceleration due to the onset of plateau potential also were noted. The goal of this paper is to investigate the characteristics of the current-voltage (I-V) relations that generate these distinctive F-I behaviors.
, 1980). A persistent inward current is also evident in turtle motoneurons in spinal slice preparations. This current is mediated by L-type calcium (Ca2+) channels and, in the presence of serotonin, generates plateau potentials and bistable behavior (Hounsgaard and Kiehn 1989; Svirskis and Hounsgaard 1997). In neonatal mouse brain stem motoneurons (Rekling and Feldman 1997) and in crab stomatogastric motoneurons (Zhang and Harris-Warrick 1995), Ca2+-mediated nonselective cation channels play essential roles in sustaining the inward current during plateau potentials. Thus in motoneurons in the adult cat, the total persistent inward current (IPIC) generating the plateau potential and bistable behavior may involve sustained activation of more than one type of channel.
). A similar behavior for IPIC in cat motoneurons could account for the hysteresis in the threshold current level in the F-I function in fully bistable cells (Lee and Heckman 1998). The lack of F-I hysteresis in partially bistable cells then might reflect a lack of I-V hysteresis due to lack of a significant difference in the onset and offset IPIC.
for the F-I data). Our results showed that, as predicted, partially bistable cells have much less I-V hysteresis. This was found to be due to a tendency for a greater decay in IPIC with time. An unexpected finding was that IPIC was activated at a substantially higher voltage in partially bistable cells than in fully bistable cells. A portion of these data has been presented in abstract form (Lee and Heckman 1996a, 1997).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). All details of the decerebrate cat preparation and the current-clamp protocols are given there. Briefly, all studies were carried out after ventral topical application of the noradrenergic 1 agonist methoxamine. Thus the electrical properties of motoneurons were influenced by both this agent and the tonic activity in reticulospinal monoaminergic tracts, a characteristic of the decerebrate preparation (see Lee and Heckman 1998). The key current-clamp protocols were measurement of the duration of self-sustained firing, which we used to classify cells as either fully (duration >3 s) or partially (<3 s) bistable, and measurement of the F-I function, which was done by applying triangular-shaped injected currents in bridge balance mode. Once these data were obtained, we changed to discontinuous, single-electrode voltage-clamp mode (Axoclamp 2A amplifier, Axon Instruments). Switching rates varied between 6 and 15 kHz. Headstage output was monitored at all times to assess settling of electrode transients. Data with inadequate settling were rejected. The clamp feedback gains ranged from 10 to 40 nA/mV. In addition, an external low-frequency feedback loop (gain of 100, 
3 dB at 3 Hz) was used to virtually eliminate baseline offsets in voltage. Voltage-clamp data were filtered at 3 kHz, digitized at 5 kHz, and stored on removable hard disks for off-line analysis. The relatively high filter cutoff meant that the stored records were noisy, but this had the advantage of revealing any rapid spikes due to momentary loss of clamp control (see further text). Before any analyses were done, data records were further smoothed by digital filtering (typical 3 dB point of ~0.3 kHz).
) under relatively poor space clamp. Data with more than a few breakthrough spikes were rejected. As long as the number of these spikes was less than about five, they did not produce significant distortion of the measured current. We compared records with and without a few breakthrough spikes in four cells where slight increases in feedback gain eliminated the spikes in a repeat trial. Smoothed current in the two cases differed by <10%, which was about the same as the variance of associated with repeated trials lacking any spikes. Breakthrough spikes also occurred at the onset of the voltage steps, distorting the first 10-20 ms. This was not a problem because the peak current did not occur until 
200 ms, and our primary concern was the subsequent slow decay over the following 10 s.
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FIG. 2.
Behavior of IPIC during triangular voltage commands. A: record of current (bottom) vs. time for a fully bistable cell when subjected to a slow (10-s total duration) triangular voltage command (top, labeled line). Onset, first 0 slope point on ascending phase denoting the onset of IPIC. Initial peak, 2nd 0 slope point on ascending phase denoting peak of IPIC. Sustained peak, 1st 0 slope point on descending phase denoting peak IPIC after prolonged depolarization. Offset, 2nd 0 slope point on descending phase denoting offset of IPIC. B: same data as in A but plotted as current vs. voltage. Onset can be seen to occur at a substantially higher voltage and current level than offset, whereas the sustained peak occurs at a slightly lower voltage and is slightly smaller than the initial peak. C: record of current vs. time for a partially bistable cell. D: same data as in C but plotted as current vs. voltage. Onset and offset voltage and current levels are closer than for the fully bistable cells, and the sustained peak is substantially smaller than the initial peak.
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FIG. 1.
Rheobase vs. input conductance for fully and partially bistable cells. Fully bistable cells ( 
) have lower values for both parameters than do partially bistable cells (
). - - -, regression relationship (slope: 12.65; intercept: 5.22; r = 0.75; P < 1 × 10
9).
10 mV below the onset of IPIC. As pointed out in the DISCUSSION, the onset and offset of IPIC are important determinants of the shape of the F-I function. Because our primary goal was to understand why F-I functions differ between fully and partially bistable cells, the voltage and current levels for the onset and offset of IPIC were assessed from the I-V function without subtracting the leak conductance. However, leak conductances were subtracted from the measurements of the amplitude and time course of IPIC. For the amplitude measurements, this was done by fitting a regression line to the subthreshold region of the I-V function and then subtracting this line from the whole function (Fig. 5 shows I-V functions after this leak subtraction). Time course measurements were taken from 10-s voltage steps. The effect of the leak conductance was compensated by subtracting the measured clamp current from a waveform obtained by multiplying the voltage command step by the input conductance. Rate of decay of IPIC was defined as the current remaining at the end of a 10-s step expressed as a percentage of the peak current.
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FIG. 5.
Effect of voltage ramp rate of rise on IPIC. Leak current has been subtracted from all records. A portion of the gradual onset of IPIC may be due to closing of subthreshold H channels. 
, direction of the voltage ramp. A: ascending ramp at 3 different rates of rise in a fully bistable cell. Slowest ramp (- - -, 2 mV/s) gives behavior is similar to the standard rate ramp (
, 8 mV/s). Fastest ramp (···, 40 mV/s) gives a delayed onset and peak for IPIC, indicating that activation of IPIC was not keeping up with the depolarization. B: descending ramp at same rates and in the same fully bistable cell as in A. Although sustained peak currents are similar, offset is delayed as ramp speed increases. C: ascending ramp at same speeds as in A in a partially bistable cell. Slow ramp initial peak slightly precedes the standard ramp but is diminished, indicating that decay of the current is already underway. Peak of IPIC is delayed in the fast ramp is delayed substantially, much as in the fully bistable cell in A. D: descending ramp at same rates and in same cell as in C. Slow ramp sustained peak exhibits substantially more decay than the standard and fast ramps that have similar peak levels. However, as in the fully bistable cell in B, the offset of IPIC is more delayed during the fastest ramp than during the standard ramp.
). Because firing acceleration began immediately in many fully bistable cells, it was often not possible to get a clear assessment of the preacceleration spike threshold. In 11 cells with at least four to five spikes before onset of firing acceleration, comparison of pre- and postacceleration spike thresholds showed that the latter were 4.3 ± 1.0 mV (means ± SD) more depolarized than the former. This finding is consistent with the tendency of spike threshold to increase with increasing firing rate (Schwindt and Crill 1982). On the basis of these considerations, spike threshold for the initiation of steady firing was determined as the average spike threshold from 10 to 20 spikes immediately after the completion of acceleration minus 4.3 mV.
View this table:
TABLE 1.
Onset and offset levels for persistent inward current, IPIC
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FIG. 3.
Relationships between onset and offset voltages of IPIC and input conductance. A: IPIC onset voltage vs. input conductance. Regression line (- - -) shows a tendency for increasing onset voltage with increasing input conductance. Consistent with this, onset voltage is lower in fully bistable cells (see Table 1). Regression line slope: 13.21; intercept: 58.8; r = 0.71; P < 1 × 104. B: offset voltage vs. input conductance. Regression slope (- - -) is steeper than in A because fully bistable cells have a greater spread between onset and offset voltages than do partially bistable cells. Regression line slope: 21.75; intercept: 72.4; r = 0.76; P < 1 × 105.
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FIG. 4.
Relationships between the rhythmic firing thresholds of the F-I relationship and the current levels for the onset and offset of IPIC. A: onset of IPIC coincides with ascending firing threshold. , fully bistable cells; 
, partially bistable cells; - - -, regression line (slope: 0.76; intercept: 2.25; r = 0.95; P < 1 × 107). Note that there is a small bias toward lower threshold cells being slightly above the unity line () while higher threshold cells tend to be slightly below the unity line. B: offset of IPIC coincides with descending firing threshold. Lines and symbols as in A. Regression line slope: 0.919; intercept: 2.47; r = 0.92; P < 1 × 106.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
for the main results on F-I functions).
). However, the range of rheobases were found to be shifted downward in the present sample (Lee and Heckman 1998), presumably due to tonic activity in descending monoaminergic tracts in the decerebrate preparation and the ventral topical application of methoxamine. Despite this alteration in rheobase, there remained a good correlation between rheobase and conduction velocity (Lee and Heckman 1998). Figure 1 shows that the downward shift in rheobase range also left the well-known correlation between rheobase and input conductance (e.g., Gustafsson and Pinter 1984) relatively unaffected. Thus the low rheobase, low input conductance cells in this study are likely to innervate fatigue resistant muscle fibers. The overall range of input conductances in our sample (0.43-1.61 µS) is similar to that in previous studies (e.g., Gustafsson and Pinter 1984). This suggests that most of the decrease in rheobase occurred because of a depolarization without a large change in input conductance (cf. Larkman and Kelly 1992) (see DISCUSSION).
View this table:
TABLE 2.
Spike voltage threshold compared to IPIC onset and offset voltages
View this table:
TABLE 3.
Magnitude of IPIC
; Svirskis and Hounsgaard 1997). An N shape is also evident as the voltage command descends back to baseline. Figure 2B shows the same data plotted as an I-V function, with ascending and descending functions superimposed. Rates of rise and fall for the voltage command were slow (8 mV/s), so that these N shapes could not be due to a rapidly inactivating inward current such as the fast sodium current underlying the action potential. Thus the negative slopes must necessarily be due to the activation and deactivation of a highly persistent inward current, denoted here as IPIC. On the ascending I-V function derived from depolarizing phase of the voltage command, the two zero slope points are referred to hereafter as the onset (see labels on Fig. 2) and initial peak of IPIC. On the descending (repolarizing) phase the zero slope points are referred to hereafter as the sustained peak and offset of IPIC. In the voltage range traversed between the initial and sustained peaks of IPIC, the ascending and descending slopes of the I-V (Fig. 2B) function markedly increased, presumably due to the activation of outward currents. It is likely too that IPIC undergoes both a voltage- and time-dependent inactivation during this period because the sustained peak of IPIC is smaller than its initial peak. In spite of its reduced amplitude, IPIC has a substantially lower offset voltage than its onset voltage. These differences result in a strong hysteresis to the I-V function.
). This suggests that the hysteresis between IPIC onset and offset largely accounts for the analogous hysteresis in the F-I function (see DISCUSSION). However, the slope of the regression line in Fig. 4A was not exactly 1.0 because IPIC onset tended to be slightly below ascending F-I threshold in fully bistable cells, whereas the opposite tended to be true in partially bistable cells. These results suggested the existence of differences between fully and partially bistable cells for the position of IPIC onset and offset voltages with respect to spike voltage threshold.
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FIG. 6.
Decay of IPIC with time during voltage steps. A: 10-s, 10-mV step to the initial peak of IPIC in a fully bistable cell (leak subtracted) showing only a small degree of time-dependent decay. B: 10-s, 10-mV step to the peak of IPIC in a partially bistable cell. Slow decay of IPIC is substantial.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
1 agonist methoxamine and tonic activity in reticulospinal monoaminergic tracts (see the discussion of Lee and Heckman 1998), the I-V relationships of the motoneurons observed in this study revealed a negative conductance region due to activation of a persistent inward current, IPIC. An important finding was that IPIC exhibited systematic differences in fully bistable motoneurons compared with partially bistable motoneurons. In fully bistable cells, IPIC had a greater difference between onset and offset for both voltage and current, a lower range of voltages and currents over which it is activated, and less decay with time. The following discussion considers how these differences in IPIC relate to the differences in the F-I behaviors between fully and partially bistable cells. The origin of the differences in IPIC also are discussed.
). As the cell is depolarized past point a in Fig. 7, the negative slope causes voltage to rapidly jump to point b. The same process occurs as voltage then is hyperpolarized, but note that the jump occurs from point c to d. The key to understanding the relationship between the I-V functions presented here and the F-I functions presented in the companion paper (Lee and Heckman 1998) is the position of the negative I-V slope with respect to the spike voltage threshold. Figure 8 summarizes our I-V and spike threshold data and is used as the basis of the following discussion. For convenience, Fig. 8C provides a summary of the F-I relationship data from the companion paper. In the following, it is assumed that the dynamics of the afterhyperpolarization are too fast to significantly change the activation of IPIC and that the average membrane potential during firing is the key variable.
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FIG. 7.
Theoretical effect of negative I-V slope on changes in voltage. I-V function shown was obtained by heavily smoothing an I-V function for a partially bistable cell. Points a and c denote 0 slope points for increasing and decreasing inputs, respectively. Dashed arrows indicate the jumps in voltage that tend to occur due to negative I-V slope. Increasing input that exceeds a rapidly reaches a new stable point at b. Decreasing input that goes below c jumps to point d.
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FIG. 8.
Summary of I-V functions and how they relate to the F-I functions presented in the companion paper (Lee and Heckman 1998 ). All functions were constructed to represent the sample averages for the behaviors of either fully bistable cells (continuous lines in all parts) or partially bistable cells (interrupted lines). A: I-V functions for the ascending phase of the triangular voltage command. Points indicated by the triangles show the average values for the onset and initial peak of IPIC for the fully (filled triangle) and partially bistable (inverted triangle) cell samples. Vertical dashed line shows the average value for spike voltage threshold for the entire sample of cells, both fully and partially bistable (spike thresholds were not significantly different for the 2 cell types
see Table 2). I-V functions for each cell type were created by fitting 3 measured points (the average values for onset and peak of IPIC and the resting membrane potential) and 2 slope values using the technique of cubic splines. Fitted slope in the subthreshold range was specified by the average input conductance. Slope of the most depolarized portion of the function was not measured and was arbitrarily set to give a conductances of 3.0 and 4.0 µS in the fully and partially bistable cells, respectively. Labels with arrows and the 2 regions denoted a and b are important for the genesis of rhythmic firing and are explained in the text. B: I-V functions for the descending phase of the triangular voltage command. Inverted and filled triangles, average sample values for the offset and sustained peak of IPIC. Functions constructed as in A to fit these average values. C: representative F-I functions for fully bistable cells (solid line) and partially bistable cells (dashed line) based on the results of the companion paper (Lee and Heckman 1998). Symbols as in A. Functions were constructed by connecting the average values (denoted by triangles) for ascending threshold, acceleration onset, and peak acceleration firing by straight lines. Transition points not denoted by triangles are for portions of the F-I functions that were not quantified and thus should be considered qualitative estimates.
but, in contrast to fully bistable cells, this occurs slightly above spike voltage threshold. This provides a region where steady rhythmic firing can be maintained (b in Fig. 8A) before the onset of acceleration. Thus the ascending F-I threshold and acceleration onset are separate events (see 4 and 5 in Fig. 8C). The time-dependent decay in IPIC (see Fig. 6B) results in negative postacceleration gain (6 in Fig. 8C) and allows IPIC to largely subside before voltage again reaches spike threshold on the descending phase of the F-I function (7 in Fig. 8C). The correspondence between IPIC offset and descending F-I threshold in partially bistable cell occurs because IPIC offset is placed only slightly above the spike threshold. This means that the F-I functions in partially bistable cells lack hysteresis because IPIC onset is above spike threshold and because it lacks sufficient offset hysteresis to overcome this depolarized activation range.
; Kernell and Monster 1981), where neuronal activity is suppressed and there is no tonic activity in bulbospinal monoaminergic fibers. Previous in vitro studies of various types of motoneurons have shown that both serotonin and norepinephrine depolarize motoneurons and lower rheobase values, an effect in part mediated by reduction of a barium-sensitive resting potassium conductance (Larkman and Kelly 1992; Lindsay and Feldman 1993; Parkis et al. 1995). By itself, this would tend to decrease input conductance, but at least in the case of serotonin, this tendency can be offset by a depolarizing shift in the voltage sensitivity of the H channel (Larkman and Kelly 1992). This may well have occurred in our preparation, because the marked reduction in rheobase was not accompanied by an equally large reduction in input conductance (see Fig. 1). However, the preceding mechanism would not likely produce the negative rheobases seen in some cells. This appeared to occur because the onset voltage for IPIC was at a sufficiently hyperpolarized level in these cells to be activated even at the resting membrane potential.
. They suggested that it was primarily generated by a Ca2+ channel (Schwindt and Crill 1980). Consistent with this, Hounsgaard and colleagues have demonstrated that, in turtle motoneurons in a slice preparation, activation of a L-type Ca2+ channel was necessary for generation of a persistent inward current and the resulting plateau potentials (Hounsgaard and Kiehn 1989; Svirskis and Hounsgaard 1997). Although L-type Ca2+ channels usually are considered to be high-voltage activated, the data of Hounsgaard and colleagues clearly show it can be activated at low voltages. Furthermore, Magee and colleagues (1996) recently showed that L-type Ca2+ currents in CA1 pyramidal cells were activated at very low voltages as long as extracellular Ca2+ concentration was kept within its physiological range.
; Svirskis and Hounsgaard 1997), having similar voltage ranges for activation and deactivation and a similar degree of onset-offset hysteresis. Consequently it is very likely a L-type Ca2+ channel plays a fundamental role in generating IPIC in cat motoneurons. Consistent with this, plateau potentials can be generated in motoneurons in the decerebrate cat preparation when an intracellular sodium channel blocker is present (Brownstone et al. 1994). However, L-type Ca2+ channels may not be the sole source of IPIC. The studies done thus far have not ruled out a role for Ca2+-mediated currents. In fact, a Ca2+-mediated nonspecific cation current plays an important role in plateau potential generation in esophageal motoneurons in neonatal rats and in stomatogastric motoneurons in the crab (Rekling and Feldman 1997; Zhang and Harris-Warrick 1995). Future investigations with intracellular Ca2+ chelators and also intracellular K+ blockers are needed to help clarify the mechanisms responsible for generation of the persistent inward current in adult spinal motoneurons.
). Voltage-dependent facilitation was not directly investigated here, but it is clearly present in both turtle and cat motoneurons (Svirskis and Hounsgaard 1997, 1998) (D. J. Bennett, H. Hultborn, B. Fedirchut, and N. Gorassini, unpublished data). However, slow activation and deactivation of IPIC may not be sufficient to fully account for our results. Slowing the rate of change of our triangular voltage commands from 8 to 2 mV/s did not seriously alter the rate of onset of IPIC, suggesting that its activation kinetics were near steady state (see Fig. 5). Voltage steps with sufficient amplitude to reach the region of peak activation of IPIC were associated with a time to onset of peak current on the order of 1 s and a time to offset of 1-2 s (Fig. 6). These lags do not appear to be sufficient to fully account for the hysteresis for triangular inputs lasting a total of 10-40 s (corresponding to rates of change of 8-2 mV/s).
; Lee and Heckman 1996b). Furthermore, computer simulations with simple two compartment models of motoneurons show steady-state I-V hysteresis very similar to that observed here (Baginskas et al. 1993; Booth et al. 1997; Lee and Heckman 1996a). Finally, a steady-state hysteresis is likely to be necessary to achieve truly long-term self-sustained firing. This is because the voltage swings due to the AHP eventually would eliminate firing if the current were only slow to deactivate. However, these considerations do not rule out an important role for slow kinetics, and it seems likely that the hysteresis arises from a combination of kinetics and dendritic location.
found that the potassium AHP channel obscured the persistent inward current, and Hounsgaard (Hounsgaard and Mintz 1988) showed that apamin, a Ca2+-dependent potassium channel blocker, enhanced plateau potentials. Although serotonin reduces the amplitude of the AHP (Bayliss et al. 1995; Hounsgaard and Kiehn 1989), it is not entirely suppressed and may still play a role in the time-dependent decay of IPIC. However, it is not clear how this would account for the difference in decay magnitudes for partially versus fully bistable cells.
had an inactivating component with inactivation time constants of 60 and 200 ms. IPIC did sometimes show an initial decay with a similar time course in some cells (not shown), however, the partially bistable cell illustrated in Fig. 6B makes it clear that a much slower time constant also is present. Moreover, IPIC in fully bistable cells decayed very slowly, with a time constant of about 10 s. If the inactivation is at least partly voltage dependent, then a greater tendency toward inactivation might be expected in partially bistable cells. This is because the activation of IPIC is shifted to more depolarized levels in partially bistable cells as compared with fully bistable ones (see Fig. 4). The sources of these differences in voltage range for IPIC are unclear at present. However, computer simulations indicate the onset of IPIC can be shifted to more hyperpolarized current levels either by reducing the AHP conductance or by increasing the L-type Ca2+ conductance, or both (Booth et al. 1997).
; Kernell and Zwaagstra 1980). Thus the persistent inward current could be generated at a greater electrical distance from the soma in partially as compared with fully bistable cells. We are presently investigating these issues via computer simulation.
; Sawczuk et al. 1995). Furthermore, IPIC in our preparation appears to simply be an enhanced version of the persistent inward current seen in motoneurons in pentobarbital-anesthetized preparations (cf. Schwindt and Crill 1977, 1980). The amount of decay of firing in our results for partially bistable cells (see Fig. 5 in Lee and Heckman 1998) is greater than that seen in the pentobarbital-anesthetized preparation or in the slice preparation, but this difference may simply reflect a much larger amplitude of the persistent inward current in our preparation. Thus slow decay in the persistent inward current may be a major factor underlying long term rate adaptation in several different preparations. This leads to the prediction that agents that affect L-type Ca2+ channels will have a strong impact on long-term rate adaptation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jack F. Miller for assisting in some of these experiments and K. D. Paul for developing our data acquisition and analysis software. We also thank two anonymous referees for recommendations for improving this paper.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-34382. R. H. Lee was supported in part by a National Institute of Child Health and Human Development Training Grant (T32-HD-07418).
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FOOTNOTES |
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Address for reprint requests: C. J. Heckman, Dept. of Physiology M211, Northwestern University Medical School, Evanston, IL 60201.
Received 2 February 1998; accepted in final form 4 May 1998.
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Abstract
Introduction
Methods
Results
Discussion
References
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