Enhancement of Bistability in Spinal Motoneurons In Vivo by the Noradrenergic alpha 1 Agonist Methoxamine

R. H. Lee and C. J. Heckman

Department of Physiology and Department of Physical Medicine and Rehabilitation, Northwestern University Medical School, Chicago, Illinois 60611


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lee, R. H. and C. J. Heckman. Enhancement of bistability in spinal motoneurons in vivo by the noradrenergic alpha 1 agonist methoxamine. Like many types of motoneurons, spinal motoneurons in the adult mammal can exhibit bistable behavior. This means that short periods of excitatory input can initiate long periods of self-sustained firing and that equally short periods of inhibition can return the cell to the quiescent state. Usually, the presence of one of the monoamines (either serotonin or norepinephrine) is required for spinal motoneurons to express bistable behaviors. Because the decerebrate cat preparation has tonic activity in monoaminergic fibers that originate in the brain stem and project to spinal motoneurons, these cells sometimes exhibit bistable behavior. However, exogenous application of the noradrenergic alpha 1 agonist methoxamine greatly enhances bistable behavior in the decerebrate. The goal of this study was to identify the mechanisms of this action of methoxamine. The total persistent inward current (IPIC) in spinal motoneurons in the decerebrate cat was measured from I-V functions generated by triangular voltage commands applied using discontinuous single electrode voltage clamp. The effect of methoxamine on IPIC was assessed by comparing its properties in a control cell sample without methoxamine to its properties in a sample of cells obtained after application of methoxamine. In most experiments, at least one cell was obtained from each sample. Our results showed that methoxamine approximately doubled the amplitude of IPIC without changing its onset voltage, its offset voltage, or its persistence. The reduced amplitude was a consistent finding within experiments and so was unlikely to be caused by interanimal variability. In addition, methoxamine depolarized motoneurons without altering their input conductances, so that a smaller amount of current was required to reach the onset voltage of IPIC. These effects of methoxamine were approximately equal in all cells. As a result of these changes, methoxamine greatly enhanced the tendency for motoneurons to become bistable. It is proposed that the methoxamine-induced increase in the amplitude of IPIC is effective in enhancing the duration of bistable firing because this increase makes IPIC more resistant to the deactivating effects of the afterhyperpolarizations between spikes.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Spinal motoneurons in the adult mammal have the capacity for bistable behavior, in which brief periods of excitatory input can induce tonic self-sustained firing (Bennett et al. 1998; Hounsgaard et al. 1988; Lee and Heckman 1998b). The cell can be returned to the quiescent state by a brief inhibitory input. However, bistable behavior is a latent property in spinal motoneurons that is manifest in the presence of serotonin or norepinephrine (Conway et al. 1988; Hounsgaard et al. 1988). The decerebrate cat preparation has tonic activity in the reticulospinal fibers that release serotonin (Baldissera et al. 1981; Hounsgaard et al. 1988), and it also may exhibit tonic activity in reticulospinal noradrenergic fibers (Sastry and Sinclair 1976; see the discussion in Lee and Heckman 1998b). Consequently, motoneurons in the decerebrate cat sometimes exhibit bistable behavior (Bennett et al. 1998; Hounsgaard et al. 1988). We recently have shown that application of the noradrenergic alpha 1 agonist methoxamine in the decerebrate cat significantly enhances overall excitability of the motoneuron pool and causes all motoneurons to exhibit at least some degree of self-sustained firing (Lee and Heckman 1998b). However, there is a wide range in the duration of this self-sustained firing. Partially bistable cells only generate short periods (~1 s) of self-sustained firing. These cells tend to have high input conductances and fast axonal conduction velocities and thus likely innervate fatiguable muscle fibers (Binder et al. 1996). In contrast, fully bistable cells exhibit self-sustained firing that lasts for many seconds. Fully bistable cells have low input conductances and slow conduction velocities and likely innervate fatigue resistance slow twitch fibers.

Bistable behavior is generated in motoneurons by a persistent inward current, which may consist of more than one component (Hounsgaard and Kiehn 1989; Hsiao et al. 1998; Rekling and Feldman 1997; Schwindt and Crill 1980; Zhang et al. 1995). Single-electrode voltage-clamp analysis of the total persistent inward current (IPIC) in motoneurons in the decerebrate preparation with methoxamine showed that IPIC was very large in all cells (Lee and Heckman 1998a). Although IPIC was of approximately equal amplitude in the fully and partially bistable subgroups, it exhibited a much greater tendency for decay with time in the partially bistable cells.

The purpose of this study was to investigate the effect of the applied methoxamine on the properties of IPIC. This was done by obtaining data on IPIC in a sample of cells in the decerebrate preparation without application of methoxamine to provide a comparison with our previously published data in the decerebrate preparation with methoxamine (Lee and Heckman 1998a). Most experiments included at least one cell from each sample. We considered three possible ways that methoxamine might enhance IPIC and increase the tendency for self-sustained firing: methoxamine would increase the amplitude of IPIC, make it more persistent, or hyperpolarize its voltage threshold. Our results strongly support the first possibility and tend to exclude the second and third ones.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Data were obtained from 15 adult cats (average weight ~2.5 kg). In 14 of these experiments, one to four cells were recorded before we administered the noradrenergic agonist alpha 1 methoxamine to generate consistent bistable behavior in subsequently recorded cells. In addition, one experiment was devoted entirely to recording cells without methoxamine. The cells recorded without methoxamine constituted the control sample. The characteristics of the sample of cells recorded after methoxamine application have been presented in our previous work (Lee and Heckman 1996, 1998a,b).

Preparation

Surgical preparations followed standard procedures in our lab (see Heckman et al. 1994; Lee and Heckman 1998b for details). Briefly, all surgical preparations of the spinal cord and hindlimb were done under deep gaseous anesthesia (1.5-3.0% isoflurane in a 3:1 mixture of O2 and N2O). The deep anesthesia was maintained throughout a precollicular decerebration. The gaseous anesthesia then was discontinued, and the animal was allowed to breathe room air. The preparation was paralyzed with gallamine triethiodide (Flaxedil) and artificially respired. In addition, a bilateral pneumothorax was done to enhance intracellular recording stability. At the end of the experiment, the animals were killed with a lethal dose of pentobarbital. All procedures were approved by the animal care committee at Northwestern University.

Intracellular recording protocols

Protocols for the motoneurons in the control sample were very similar to those for the cell sample with methoxamine (see Lee and Heckman 1998a,b for details). Intracellular recordings were obtained with sharp microelectrodes. Microelectrode tips were broken back under microscopic observation and control. Because of the large currents required for successful single-electrode voltage-clamp techniques in spinal motoneurons, resistances of the electrodes were kept low---typically ~3-4 MOmega in saline before entering the cord.

In each cell, the standard protocol began with measurement of a spike antidromically evoked by single shocks to either the medial gastrocnemius (MG) or lateral gastrocnemius-soleus (LGS) nerve. The stimuli were repeated 20 times at 2 Hz to provide an average. The electrode resistance was compensated with a bridge circuit, and then rheobase, defined as the amplitude of a 50-ms current pulse required to elicit a single spike, was measured. These initial measurements were followed by one or more of the following three protocols: measurement of the cell's I-V function to characterize IPIC, measurement of the cell's relationship between firing frequency and injected current to define its F-I function, and measurement of the tendency for the cell to exhibit self-sustained firing. These protocols are described next.

To measure the characteristics of IPIC, we used the discontinuous, single-electrode voltage-clamp mode of our amplifier (Axoclamp 2A amplifier, Axon Instruments). Switching rates typically varied between 8 and 11 kHz. Headstage output was monitored at all times to assess settling of electrode transients. Data with inadequate settling were rejected. The clamp feedback gains ranged from 10 to 40 nA/mV. In addition, an external low-frequency feedback loop (gain of 10, -3 dB at 30 Hz) was used to virtually eliminate baseline offsets in voltage (cf. Misgeld et al. 1989; Richter et al. 1996). Details of the limitations for this techinique in motoneurons in vivo are given in Lee and Heckman (1998a). IPIC was characterized by applying a slow triangular voltage command with a rise time of 5 s, a total duration of 10 s, and amplitude of 30-40 mV.

The frequency-current (F-I) function of the cell was assessed via injection of a current waveform with a triangular shape, as in our previous study (Lee and Heckman 1998b; see also Bennett et al. 1998; Hounsgaard et al. 1988). The rates of rise and fall were equal at 5 s each. In most cells, the amplitude of the triangular-shaped current was 30 nA, resulting in a rate of rise of 6 nA/s. A steady DC bias current was used to control the maximum current reached during the input. The bias was adjusted so that maximum firing rates reached ~40-70 Hz.

The capacity for bistable behavior was assessed by application of a 1.5 s period of high-frequency, low-amplitude vibration (a 160-Hz sinusoid with a peak to peak amplitude of 80 µM) of the Achilles tendon (Lee and Heckman 1996, 1998b). This evokes steady high-frequency firing in muscle spindle Ia afferents, which form monosynaptic connections to MG and LGS motoneurons (Matthews 1972). The maximum duration of self-sustained firing evoked by the Ia input was assessed when the cell was depolarized to a steady level just below its firing threshold (see Lee and Heckman 1998b; also see Fig. 10). Hyperpolarizing baseline currents were used to eliminate the self-sustained firing and confirm that the Ia input had a crisp offset.

Data analysis

Conduction time was measured as the difference between time of stimulation in the muscle nerve and time of onset of the antidromic spike. Conduction times were normalized by the average for each experiment (between 4 and 12 cells were included in these averages). We then assumed the average conduction time corresponded to a conduction velocity of 97 m/s (Zengel et al. 1985). Thus multiplication of the normalized conduction time by 97 m/s gave an estimate of conduction velocity that was weighted for interexperiment differences in animal size. The use of averaging to account for interanimal variations in conduction distance appears to work well. The correlations between conduction velocity and other motoneuron properties in our data (see RESULTS) are as least as high as studies where conduction distance is measured in each animal (cf. Fleshman et al. 1981).

Figure 1 illustrates how the properties of IPIC were measured from the I-V functions generated by the triangular voltage commands. The same properties were assessed in our previous study of IPIC (Lee and Heckman 1998a). The onset of IPIC was specified as the point on the I-V function where the total inward current overcame the leak conductance sufficiently to give a net I-V slope of zero. The initial peak of IPIC then was defined to occur where the negative slope decreased to again give a net slope of zero. The offset and sustained peaks of IPIC were defined on the descending phase analogously to onset and initial peak on the ascending phase. In cells where IPIC on the descending phase was too small to impart a negative slope to the I-V function, the sustained peak was measured at the point where slope reached its minimum. Offset was measured where I-V slope underwent a downward inflection as the curve rejoined the subthreshold region. We did not subtract the leak from the I-V function before assessing the points for IPIC onsets, peak values, and offsets because we wished to define the contribution of IPIC to the functional behavior of the cell. We have shown previously, for example, that there is a good correlation between IPIC onset current and the threshold current for rhythmic firing (Lee and Heckman 1998a).



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Fig. 1. Motoneuron I-V function and measurement of the properties of persistent inward current (IPIC) in a motoneuron from the control cell sample. A: current and voltage as functions of time in a typical low-input conductance (0.55 µS) motoneuron. Ascending phase of a slow triangular voltage command evoked a net persistent inward current, IPIC. As voltage increased, IPIC reached its peak amplitude and then was overwhelmed by outward currents. On the descending phase, the offset of IPIC usually occurred at a lower voltage than its onset. Thick trace: current. Thin trace: voltage. B: data in A plotted as an I-V function. Note that the onset of IPIC defined the start of a negative slope region and that an additional negative slope region occurred at its offset. Labels indicate the properties of IPIC that were quantified in this study. Input conductance was calculated as the linear slope of the I-V function in its subthreshold range (see arrow). Amplitude of the initial peak (4.3 nA in this case) was calculated as the vertical difference between the peak and a line that originated at onset and had a slope equal to the input conductance (see the dashed line). In the bistable current range, each value of current is associated with two stable values of voltage. Ascending phase: thick line. Descending phase: thin line.

The initial peak amplitude of IPIC was defined as the difference in current between its onset and initial peak on the ascending phase, with the effect of the leak conductance across the two voltages compensated in the following way. The input conductance of the cell was measured from the slope of the I-V function in a 5-mV region centered ~10-15 mV below the onset of IPIC. A line with a slope equal to the input conductance then was extrapolated from IPIC onset voltage to the voltage corresponding to the initial peak of IPIC. The initial peak amplitude of IPIC was then the difference along the current axis between the top of this line and the initial peak of IPIC (see Fig. 1B). Leak was compensated by a similar method in the calculations of the sustained peak amplitude. In this case, the line with a slope equal to input conductance was extrapolated from the offset of IPIC. Note that these procedures yielded a slightly lower value for IPIC amplitude than would have been obtained by simply subtracting the leak conductance from the I-V relation. This was because the subthreshold I-V function in motoneurons usually has a significant curvature due to the interaction between the deactivation of the H current and the activation of IPIC (Binder et al. 1996). This curvature makes the amplitude of IPIC sensitive to the voltage range chosen for the definition of input conductance. The procedure for leak compensation we chose minimized the impact of estimation of input conductance on IPIC amplitude. It also is functionally relevant, because, as noted earlier, the onset of negative slope has a large impact on firing behavior.

Finally, we also measured two additional parameters of the I-V function that play an important role in the generation of bistability. First the I-V functions of spinal motoneurons exhibit hysteresis due to differing behavior between the ascending and descending portions of the triangular voltage command. Voltage hysteresis was defined as IPIC's onset voltage minus its offset voltage. Voltage hysteresis probably reflects the dendritic origin of much of IPIC (see DISCUSSION). Along the current axis, we measured the bistable current range, which was defined as the current at IPIC onset minus the current at its sustained peak (see Fig. 1B). This definition encompasses the I-V region in which the existence of a negative slope allows a single value of current to be associated with two stable values of voltage. This duality is what makes bistable behavior possible (Booth et al. 1997; Gutman 1991; Hsiao et al. 1998; Schwindt and Crill 1980) (see DISCUSSION). In some high-input conductance cells, decay of IPIC with time resulted in a sustained peak that was at or slightly above the onset, as shown by the example in Fig. 2. In these cases, the bistable current range was defined as the difference between the current levels at offset and sustained peak. The large region of negative slope on the ascending phase gives a false impression of the capacity for bistable behavior in high-input conductance cells because of the relatively rapid time-dependent decay of IPIC (see RESULTS).



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Fig. 2. I-V function of a high-input conductance (1.21 µS) motoneuron from the control cell sample. Note the strong decay of IPIC resulted in its sustained peak occurring at a higher current level than its initial peak, giving a small bistable current range.

For the F-I functions, we measured the currents at onset and offset of rhythmic firing. The gain of the F-I function was defined as the slope of the function during the ascending phase. Hysteresis in the F-I function was defined as the current at the offset of steady firing on the descending phase minus the current at firing onset on the ascending phase. We also measured the peak amplitude of the afterhyperpolarization (AHP) from the data used to generate the F-I function. To define a flat baseline, the digitized record of the cell's response to the triangular current was subjected to a digital low-frequency filter (-3 dB point of ~3-5 Hz). This had no effect on the shape of the AHP and eliminated polarization artifacts due to imperfect electrode behavior. The amplitude of the AHP was defined as the difference between the baseline and the point of maximum hyperpolarization between spikes. The AHP values for the first and second interspike intervals were averaged. Note that the durations of the first two interspike intervals were not significantly different (t-test, P > 0.60) in the present study as compared with our previous study with methoxamine (Lee and Heckman 1998b), so that the AHPs of the two data sets were compared at similar frequencies.

The Ia input generated by tendon vibration was used to test the strength of bistable behavior in each cell, which was defined as the duration of self-sustained firing after the cessation of the 1.5-s period of tendon vibration. In addition, the voltage threshold for the action potential was measured during the rhythmic firing evoked by the Ia input. The trial with the lowest firing rate was used to avoid the increase in spike threshold that accompanies increased rates (Schwindt and Crill 1982). The voltage threshold for the spike was defined as the voltage level at the first peak of the second differential of the spike on its ascending phase.

The main acceptance data criteria were that the amplitude of the antidromic spike exceeded 70 mV and the resting membrane potential did not vary by more than ±5 mV during the course of the data collection. The properties of the control data sample were compared with those of the sample with methoxamine in two ways. Average values were compared with t-tests, assuming unequal sample variances. Linear regression analyses were used to assess the relationships between variables, and the slopes for the regression relations in the two data samples sometimes were compared using the standard slope comparison test based on the t-distribution. The significance level, alpha, was set at P = 0.05. Where results from multiple t-tests were compared, we chose a conservative alpha level by dividing 0.05 by the number of t-tests (see Tables 1-4).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To identify the effects of the noradrenergic alpha 1 agonist methoxamine on the electrical properties of motoneurons, we measured the I-V functions, F-I functions, and tendencies for self-sustained firing in a sample of motoneurons in the decerebrate preparation without any methoxamine or other exogenous agent present. The characteristics of the cells in this control data sample then were compared with a sample of cells previously obtained in the decerebrate after application of methoxamine (Lee and Heckman 1998a,b). The control sample consisted of 31 cells. I-V functions were obtained in 20 of these 31 cells, F-I functions were obtained in 16 of 31 cells, and the duration of bistable firing was assessed in 26 of 31 cells. In all but one experiment, one or more control cells were recorded in the same experiment as one or more cells after methoxamine application. Thus the differences in motoneuron properties between the two cell samples were unlikely to be due to interanimal variations.

Basic electrical properties

Spinal motoneurons in anesthetized preparations are known to exhibit strong correlations among their basic properties, with low input conductance cells tending to have low rheobases and slow axonal conduction velocities (reviewed in Binder et al. 1996; Burke 1981). Conversely, high input conductance motoneurons tend to have high rheobases and fast axonal conduction velocities. These basic correlations are preserved in the decerebrate preparation (Burke 1968; Hultborn et al. 1988). For example, in the control cell sample, input conductance correlated with rheobase (r = 0.83; P < 0.01) and conduction velocity correlated with both input conductance (r = 0.69; P < 0.01) and rheobase (r = 0.76; P < 0.01). Similar correlations were observed in the cell sample with methoxamine (Lee and Heckman 1998a,b). This issue is important because motoneurons with low input conductances tend to innervate slow twitch, fatigue resistant muscle units, whereas high input conductance motoneurons tend to innervate fast twitch, fatiguable muscle units (Bakels and Kernell 1993; Burke et al. 1973; Gardiner 1993; Zengel et al. 1985). Thus these basic differences in motoneuron electrical properties play a major role in specifying the normal pattern of recruitment of motor units (Binder et al. 1996), which proceeds from low to high force units [i.e., Henneman's "size principle" (Henneman and Mendell 1981)].

Persistent inward currents

IPIC is the total persistent current in spinal motoneurons and likely consists of more than one component (Hounsgaard and Kiehn 1989; Hsiao et al. 1998; Rekling and Feldman 1997; Schwindt and Crill 1980; Zhang et al. 1995). Figure 1A illustrates the standard slow (8 mV/s) triangular voltage command used to assess each cell's I-V function. The cell shown is from the control sample. Figure 1B illustrates the resulting I-V function and defines the standard parameters of IPIC that were measured from this function. The cell in Fig. 1 had a relatively low input conductance (0.55 µS). Figure 2 illustrates the I-V function for a high-input conductance cell (1.21 µS) from the control sample. Note that the rapid onset of outward currents above the onset of IPIC sometimes evoked clamp currents >50 nA in the control cells. In 5 of 20 control cells in which I-V functions were obtained, these large currents caused a 1-3 s loss of adequate settling of electrode transients. The onset and initial peak of IPIC occurred well before loss of adequate settling, but data for the descending phase were rejected in these five control cells.

AMPLITUDE OF IPIC. Figure 3 illustrates the tendency for the initial peak amplitude of IPIC to be smaller in the control cell sample than in the sample with methoxamine. Although there is a considerable overlap in ranges, the average value for the control sample was about half that of the sample with methoxamine and this difference was statistically significant (see Table 1). Similarly, on the descending phase, the sustained peak of IPIC in the control sample was also about half that in the sample with methoxamine (Table 1). The reduced amplitude for IPIC in control cells was also clear in comparisons between control and methoxamine cells within individual experiments. In the cell sample with methoxamine (Lee and Heckman 1998a), there was a weak but significant trend for the initial peak of IPIC to be somewhat larger in high input conductance motoneurons (see Fig. 4; statistics are given in the figure legend). A similar trend was observed in the control sample (again, see Fig. 4). The slopes of the regression lines for the two data sets were not significantly different (P > 0.05). Therefore methoxamine had an approximately equal effect on both low- and high-input conductance cells.



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Fig. 3. Methoxamine increased the initial peak amplitude of IPIC. Top: frequency histogram of initial peak amplitudes of IPIC in the control sample (n = 20). Bottom: frequency histogram of initial peak amplitudes of IPIC in the sample (n = 27) with methoxamine. Although the ranges of the 2 data sets overlapped extensively, the average value for the control sample was much lower (see Table 1). Data from cells with methoxamine are from previously published work (Lee and Heckman 1998a).


                              
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Table 1. Characteristics of IPIC obtained from I-V functions



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Fig. 4. Methoxamine generated an approximately equal increase in the amplitude of IPIC in all motoneurons. Initial peak amplitude of IPIC is plotted as a function of input conductance for the control data set (black-lozenge ) and the methoxamine data set (triangle  and box-dot , with triangle  indicating partially bistable cells and · indicating fully bistable cells). ---, regression relation for control sample: slope: 4.7, intercept: 6,8; r = 0.29, P < 0.05. - - -, regression relation for sample with methoxamine: slope: 8.6; intercept: 12.0; r = 0.42; P < 0.01. Data from cells with methoxamine are from previously published work (Lee and Heckman 1998a).

VOLTAGE THRESHOLDS. Despite its clear effect on the amplitude of IPIC, methoxamine did not significantly influence IPIC's voltage thresholds. Figure 5A shows IPIC onset voltage plotted as a function of input conductance for cells with (Lee and Heckman 1998a) and without methoxamine. In both data sets, there is a strong tendency for onset voltage to be more depolarized in high input conductance cells. There were no significant differences in slopes of the functions for the two data sets (P > 0.05). The range of values for input conductance in the control data sample reached a higher level than in the sample with methoxamine, but this extension of the range was due to only two cells. The average values of input conductance were not significantly different in the two data sets (see Table 1). Figure 5B shows offset voltage as a function of input conductances. Again, the two data sets are virtually superimposable, with high input conductance cells tending to have a much more depolarized offset. Overall, the average values of onset and offset voltages for the two data sets were not significantly different (Table 1). Thus methoxamine had little or no impact on the voltage ranges for activation and deactivation IPIC.



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Fig. 5. Methoxamine did not alter the onset and offset voltages for IPIC. A: onset voltage for IPIC as a function of input conductance for both control and methoxamine samples (symbols as in Fig. 4). ---, regression relation for control sample: slope: 9.5; intercept: -55.9; r = 0.43; n = 20; P < 0.05. - - -, regression relation for sample with methoxamine: slope: 13.1; intercept: -58.7; r = 0.71; n = 27; P < 0.01. B: offset voltage vs. input conductance. Symbols and lines as in Fig. 4. Control regression relation: slope: 20.3; intercept: -70.3; r = 0.69; n = 15; P < 0.001. Methoxamine regression relation: slope: 21.6; intercept: -72.3; r = 0.76; n = 27; P < 0.01. Data from cells with methoxamine are from previously published work (Lee and Heckman 1998a).

HYSTERESIS AND PERSISTENCE. Figure 6 shows the relation between the input conductance and voltage hysteresis in IPIC. Voltage hysteresis is the tendency for the offset of IPIC to occur at a more hyperpolarized level than its onset (see Fig. 1B for an example). It is likely that this hysteresis in IPIC occurs because much of this current originates in dendritic regions under poor space clamp (see DISCUSSION). There was a moderate tendency for greater voltage hysteresis in low input conductance motoneurons in both cell samples (Fig. 6). There is also a weak tendency for the average value of voltage hysteresis to be larger in the cell sample with methoxamine, but this tendency was not statistically significant (see Table 1). The slopes of the regression relations for the two data sets were not significantly different (P > 0.05). In the cell sample with methoxamine (Lee and Heckman 1998a), the differences between voltage hysteresis in low and high input conductance cells were probably due to differences in the persistence of IPIC. Hysteresis was small in low input conductance, partially bistable cells because IPIC underwent considerable decay in the time that elapsed between the initial and sustained peaks. Because the relationship between voltage hysteresis and input conductance in the control sample was similar to that in the sample with methoxamine, it seems likely that methoxamine had little or no influence on the persistence of IPIC.



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Fig. 6. Methoxamine did not significantly influence the hysteresis along the voltage axis. Slope of the relationship between voltage hysteresis and input conductance was not significantly different for the 2 sets of data nor were the average values (see Table 1). Symbols as in Fig. 4. Regression relations: control sample (solid line): slope: -9.0; intercept: 11.6; r = -0.63; n = 15; P < 0.01. Methoxamine sample (- - -): slope: -8.5; intercept: 13.6; r = -0.62; n = 27; P < 0.01. Data from cells with methoxamine are from previously published work (Lee and Heckman 1998a).

To confirm that IPIC was highly persistent in low input conductance cells in the control sample, we applied long-duration (10 s) voltage steps in five cells with input conductances <1.0 µS (range: 0.42-0.85 µS). These steps were 10-20 mV in amplitude, and baseline levels were adjusted so that the step just reached the voltage for the initial peak of IPIC. In all five cells, IPIC decayed by <20% during the 10-s step. An example is shown in Fig. 7. These results are very similar to those obtained with 10-s duration steps in low input conductance cells in the presence of methoxamine (see Fig. 6 in Lee and Heckman 1998a). Thus it seems unlikely that methoxamine acts to increase the persistence of IPIC.



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Fig. 7. Methoxamine did not alter the persistence of IPIC in low input conductance cells. Top: current generated in a control cell in response to the long-duration (10 s) voltage step (bottom). Step just reached the voltage level at the initial peak of IPIC for this cell. Leak conductance has been compensated. Note that, just as in low input conductance cells in the presence of methoxamine, there is little or no decay with time. Cell input conductance: 0.42 µS.

BISTABLE CURRENT RANGE. Although voltage hysteresis was not altered by methoxamine, it had a clear impact on the characteristics of IPIC along the current axis. In fact, the bistable current range in the control sample was <30% of the bistable current range in the sample with methoxamine (see Table 1). In both the control sample and the sample with methoxamine, there was a modest trend for low input conductance cells to have a larger bistable current range, as shown in Fig. 8. As for the relation between voltage hysteresis and input conductance in Fig. 6, the reduced bistable current range in high input conductance cells occurred because these cells exhibit a stronger tendency for IPIC to decay with time. The I-V function shown in Fig. 2 provides an example of a high-input conductance cell in which IPIC underwent considerable decay by the time of its sustained peak, resulting in a small bistable current range. The greater bistable current range seen in low-input conductance cells is illustrated in Fig. 1B. The overall increase in bistable current range induced by methoxamine is a particularly important result because this parameter plays a key role in determining the duration of self-sustained firing (see DISCUSSION). Note that, in Fig. 8, all of the fully bistable cells in the sample with methoxamine have large bistable current ranges.



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Fig. 8. Methoxamine markedly increased the bistable current range. Although the slopes of the relationships between bistable current range and input conductance were not significantly different, on average, the bistable current range was much less in the control sample as compared with the sample with methoxamine (see Table 1). Symbols as in Fig. 4. Regression relations: Control sample (---): slope: -4.4; intercept: 7.3; r = -0.49; n = 15; P < 0.05. Methoxamine sample (- - -): slope: -4.3; intercept: 11.9; r = -0.38; n = 27; P < 0.05. Data from cells with methoxamine are from previously published work (Lee and Heckman 1998a).

BASELINE DEPOLARIZATION. Although methoxamine did not have a significant impact on the voltage for the onset of IPIC, it did influence the amount of current required to reach this level. Table 2 shows that the average current for IPIC onset in the control cell sample was ~7 nA higher than the average value for the sample with methoxamine. This difference was statistically significant (see Table 2). A similar but smaller difference existed for the offset current of IPIC that did not reach statistical significance (Table 2). Similarly, the resting membrane potential was somewhat more depolarized in the sample with methoxamine than in the control sample, but this difference also failed to reach statistical significance (Table 2). Taken together, however, these results suggest that methoxamine acted to depolarize motoneurons. This conclusion is in accord with previous studies of brain stem motoneurons in slice preparations (e.g., Hsiao et al. 1998; Larkman and Kelly 1992; Lindsay and Feldman 1993; Parkis et al. 1996). Note, however, that depolarization occurred without a significant change in input conductance (see Table 1). This may have occurred because noradrenergic agents can simultaneously decrease a leak current while increasing the H current (Larkman and Kelly 1992). Both of these effects depolarize motoneurons but they have opposite actions on input conductance.


                              
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Table 2. Changes in baseline excitability

Frequency-current functions

The activation of IPIC also has a potent impact on the motoneuron F-I function generated by a slow (typically 6 nA/s) triangular pattern of injected current. In the fully bistable cells in the sample with methoxamine, the onset of IPIC causes a strong acceleration in firing rate (Lee and Heckman 1998b). Furthermore the offset of the F-I function occurs at lower current than its onset. We define F-I hysteresis as offset current minus onset current, so that fully bistable cells have positive values for onset-offset hysteresis in both their F-I and I-V functions. In partially bistable cells in the sample with methoxamine, firing acceleration is also very strong but it starts well above firing threshold. IPIC undergoes considerable decay in these partially bistable cells during the course of the slow triangular injected current protocol, so that offset of firing occurs at a similar current level to onset and hysteresis is small.

In the control cell sample, positive F-I hysteresis was scarcely evident. At best, onset and offset occurred at similar current levels, as shown by the F-I function for the cell on the left in Fig. 9. In the other cells, offset occurred at a substantially higher current than offset, giving a negative hysteresis to these motoneurons (see the F-I function for the cell on the right in Fig. 9). This negative hysteresis is typical of cells that lack bistable behavior (Hounsgaard et al. 1988). Overall, the hysteresis of the F-I function was significantly correlated with rheobase (r = -0.63, n = 16, P < 0.01), being near zero in low rheobase cells and moderately negative in high rheobase cells (as illustrated by the examples in Fig. 9). The F-I hysteresis also was correlated strongly with the bistable current range of the I-V function in both the control (r = 0.72; n = 10; P < 0.01) and methoxamine (r = 0.74; n = 16; P < 0.01) cell samples. Thus the larger the bistable current range, the greater the tendency for F-I hysteresis.



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Fig. 9. F-I relations in the absence of methoxamine. Instantaneous frequency is plotted as a function of injected current for both the ascending and descending phases of the triangular shaped input. Arrows indicate ascending and descending phases. Low-input conductance cells tended to have F-I functions like the one on the left with the lower current threshold, whereas high-input conductance cells tended to have F-I functions like the one with the higher current threshold.

Clear acceleration in firing rate due to onset of IPIC is a standard feature of cells with methoxamine (Lee and Heckman 1998b) but was evident in only four of the control cells. The relatively small amplitude of IPIC in the control cells may have prevented a clear acceleration in firing rate being detected, especially because firing at high rates tend to be noisy (see Fig. 9). As a result, the gains of the F-I functions in the control sample [1.61 ± 0.38 (SD) Hz/nA] were similar to those seen in pentobarbital anesthetized preparations (reviewed in Binder et al. 1996). In contrast, the acceleration in firing rate in the cells with methoxamine produced very high F-I gains (usually > 10 Hz/nA) (Lee and Heckman 1998b).

The differences in the F-I function between the two data sets were not due to differences in the voltage threshold for spike initiation. In the control sample, the spike voltage threshold was -47.9 mV ± 5.7 (SD) mV. In the sample with methoxamine, the spike voltage threshold was -48.9 ± 5.4 mV (Lee and Heckman 1998a). These two sample averages were not significantly different (t-test; P > 0.40). Thus in both samples, spike threshold tended to be slightly more depolarized than IPIC onset in low input conductance cells and slightly more hyperpolarized than IPIC onset in high input conductance cells (see Fig. 5A). However, the AHP in the sample with methoxamine was somewhat smaller than in the control sample (control: 7.0 ± 1.6 mV; methoxamine: 5.9 ± 2.0 mV) (cf. Parkis et al. 1996). This reduction fell just short of statistical significance (t-test; P < 0.06). A methoxamine-induced reduction in the AHP would be expected to promote bistable behavior (see DISCUSSION).

Self-sustained firing

One of the main effects of IPIC is to generate bistable behavior in motoneurons. Our standard test to evoke self-sustained firing consisted of a 1.5-s period of monosynaptic excitatory input applied during a steady baseline injected current that brought the cell near firing threshold (see METHODS). Figure 10 shows examples of cells with and without self-sustained firing. Only 8 of the 26 control in which the standard test was applied exhibited self-sustained firing. None of these eight cells generated self-sustained firing that lasted >3 s. In contrast, in the presence of methoxamine, all cells generated at least some self-sustained firing. Further, ~30% of the cells with methoxamine were fully bistable and generated self-sustained firing for many seconds (Lee and Heckman 1998b). Thus the methoxamine-induced increase in the amplitude of IPIC was associated with a large increase in the tendency of motoneurons to generate self-sustained firing.



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Fig. 10. Self-sustained firing in the absence of methoxamine. Synaptic input was generated by Ia afferents activated by vibration of the Achilles tendon. Top: example of 1 of the cells with the longest duration bistable firing seen in the control data set. This cell exhibited tonic firing at rest and so a baseline holding current of -3 nA was applied. Bottom: more typical result, a lack of self-sustained firing. Spikes are truncated for clarity in both records. A 10-nA bias current was applied.

I-V functions were obtained in 15 of the 26 control cells in which self-sustained firing was evaluated. Of these 15 cells, 5 were in the group with self-sustained firing. These five control cells with self-sustained firing tended to have low input conductances as well as slow conduction velocities and low rheobases (Table 3). In the sample with methoxamine, these are the cells that tend to be fully bistable (Lee and Heckman 1998b). Within the control sample, Table 4 shows that the amplitude of IPIC was about the same cells with and without self-sustained firing. However, as expected for low input conductance cells from Fig. 5, IPIC onset and offset voltages tended to be hyperpolarized in the control cells with self-sustained firing (Table 4). Voltage hysteresis was somewhat larger as well but this difference did not quite attain statistical significance (cf. Fig. 6 and see Table 4). The most important parameter for bistable behavior, the bistable current range, was significantly larger in the cells with self-sustained firing (cf. Fig. 8 and see Table 4). Overall, these results suggest that methoxamine acted to convert cells with a weak tendency for self-sustained firing to the fully bistable state while allowing cells with no previous bistable behavior to become partially bistable.


                              
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Table 3. Comparison of electrical properties in control cells with and without self-sustained firing


                              
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Table 4. Comparison of IPIC in control cells with and without self-sustained firing


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

The results of this study show that spinal motoneurons in the decerebrate cat preparation possess a substantial persistent inward current, IPIC. The properties of IPIC in the motoneurons in the present study were compared with the properties of IPIC obtained in our previous work (Lee and Heckman 1998a,b), where the noradrenergic alpha 1 agonist methoxamine was applied to the decerebrate preparation to enhance bistable behavior. Although IPIC was rather large in the motoneurons of this study, application of methoxamine approximately doubled its amplitude without altering its onset and offset voltages or its persistence. Thus the primary differences in IPIC between fully and partially bistable cells in the sample with methoxamine (Lee and Heckman 1998a,b) are already present in the control cell sample. The dose of methoxamine chosen for enhancement of bistable behavior in our studies appeared to give the maximum increase in reflex excitability for this agent (see the METHODS section of Lee and Heckman 1998b). We have not investigated the effects of lower doses, but we suspect that lower doses would reduce the amplitude of IPIC to a value closer to that of the control data. The amplification of IPIC by methoxamine is consistent with recent data from turtle motoneurons showing that the L-type Ca2+ current that produces bistable behavior in these cells can be modulated by several different agents (Svirskis and Hounsgaard 1998).

Because bistable behavior was weak in the absence of methoxamine (see for example Fig. 10), it is likely that maintaining self-sustained firing for long periods of time requires more than just a highly persistent inward current. The persistent current must also attain a sufficiently large amplitude (see following text). Another effect of methoxamine was to provide a significant baseline depolarization. This steady depolarization would make it easier for synaptic inputs to reach threshold for activation of IPIC and, consequently, self-sustained firing.

Self-sustained firing in the decerebrate without exogenous agents

In the presence of methoxamine, all motoneurons exhibit at least some degree of bistable firing (Lee and Heckman 1998b). The percentage of cells exhibiting self-sustained firing in the present study without methoxamine was similar to but somewhat less than that seen in a previous study in the decerebrate without any exogenous agents (Hounsgaard et al. 1988). The minor discrepancy is likely accounted for by differences in the method of decerebration (anoxic destruction of the forebrain in Hounsgaard et al. 1988 vs. the precollicular transection employed here), which could easily impact the tonic activity in the reticulospinal monoaminergic fibers. In addition, we used relatively large electrodes in both this and our previous work (Lee and Heckman 1996, 1998a,b). It is likely that fully bistable cells tend to be small. This is because fully bistable cells tend to have low input conductances (Lee and Heckman 1998b), and low input conductance cells tend to be the smallest in terms of soma diameter (Binder et al. 1996). Thus we may have undersampled the population of cells that exhibit self-sustained firing.

Lack of hysteresis in the F-I function

In the presence of methoxamine, low input conductance cells exhibited a strong tendency for positive hysteresis in their F-I functions because offset of firing occurred at a lower current level than onset (Lee and Heckman 1998b). This F-I hysteresis correlates well with the hysteresis in the I-V function, which is probably due to the dendritic origin of a substantial portion of IPIC (Lee and Heckman 1998a). In this study, as in previous work (Hounsgaard et al. 1988), cells lacking in bistable behavior exhibit negative F-I hysteresis: offset occurs at a substantially higher current than onset (see the F-I function in Fig. 9, right). Thus the greater the amplitude and persistence of IPIC, the greater the tendency for F-I offset to be shifted to a lower current. In low input conductance cells in the present study, IPIC is sufficiently large and persistent to shift firing offset down to a current near firing onset, resulting in near zero hysteresis (as illustrated by the F-I function on the left in Fig. 9). The methoxamine-induced increase in IPIC may push firing offset to a still lower current value, converting this zero F-I hysteresis to a strong positive value.

Role of voltage hysteresis in the I-V function

Bistability becomes possible in neurons when a persistent inward current is large enough to induce a negative slope in the I-V function (Booth et al. 1997; Gutman 1991; Hsiao et al. 1998; Schwindt and Crill 1980). This allows a single value of current to be associated with two stable values of voltage. When the high-voltage value is above spike threshold, as is the case in spinal motoneurons in the decerebrate, the cell can toggle between a quiescent state and self-sustained rhythmic firing. The range of currents that allow for bistability is defined by the onset and the peak of the persistent inward current---i.e., by the amplitude of the "N" shape in the I-V function (see points 1 and 2 in Fig. 11). In spinal motoneurons, an additional factor comes into play. IPIC displays a clear hysteresis in that its offset tends to occur at a lower voltage and current than its onset (Lee and Heckman 1998a; Svirskis and Hounsgaard 1997) (see Fig. 1B). This hysteresis expands the range of currents that generate bistable behavior by pushing the sustained peak of IPIC to a more hyperpolarized current level than the initial peak (see point 3 on Fig. 11). This is especially true in low input conductance cells where IPIC is the most persistent and consequently its voltage hysteresis is the largest. The primary source of the voltage hysteresis is probably the dendritic origin of much of IPIC (Hounsgaard and Kiehn 1993; Lee and Heckman 1996). We previously have shown that voltage hysteresis in low-input conductance cells is not a time dependent phenomenon but persists even when the ascending and descending phases of the triangular voltage command each last 20 s (Lee and Heckman 1998a). The most likely explanation is that dendrites are under relatively poor space clamp from a voltage clamp applied by an electrode at or near the soma (Gutman 1991; Lee and Heckman 1998a). Consequently on the descending phase of the I-V function, the dendrites remain depolarized by the dendritic portion of IPIC even when the somatic voltage becomes more hyperpolarized than the level at which IPIC was activated on the ascending phase. This interpretation is supported strongly by computational models with persistent currents in dendritic compartments (Booth et al. 1997; Gutman 1991). Thus the dendritic origin of IPIC enhances bistable behavior.



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Fig. 11. This is the average I-V function for control cells with low input conductances, which are the ones most likely to exhibit some self sustained firing. - - - and numbers on the figure are used in the text to explain why the relatively small amplitude of IPIC in control cells limits the duration of self-sustained firing. I-V function was generated by averaging together the I-V functions for each control cell with an input conductance <= 0.8 µS.

Key role for the amplitude of IPIC

Why does increasing the amplitude of IPIC have such a strong impact on the duration of self-sustained firing? In high input conductance cells, bistable behavior is weak even in the presence of methoxamine (Lee and Heckman 1998b). It is not surprising that reducing the amplitude of IPIC eliminates the tendency for self-sustained firing in cells in which IPIC tends to decay. However, in low input conductance cells, IPIC is highly persistent even in the control cell sample. Furthermore, the average bistable current range in these cells seems reasonably large at ~5 nA (see Table 4). The key point is probably that, during rhythmic firing, the interaction between the spike and the AHP provides a perturbation that easily can push the cell below the bistable range of currents. In the following discussion of this interaction, we treat the AHP as a fluctuation in current around the steady-state I-V function. This oversimplification ignores dynamic changes in the I-V function due to the spike and AHP but appears to adequately capture the basic effects of the AHP on IPIC.

In low input conductance cells, the spike voltage threshold lies somewhat above the onset of IPIC and, consequently, activation of IPIC initiates rhythmic firing. In our standard test for self-sustained firing, a baseline current is injected to bring the cell to as close to firing threshold as possible without synaptic noise evoking spikes (see Fig. 10). Thus the net baseline current is probably sitting within the bistable current range (see the lower dashed line in Fig. 11). The addition of the synaptic current is then sufficient to exceed the threshold of IPIC and initiate rhythmic firing (upper dashed line). However, after synaptic input ceases, average firing rate drops considerably (see Fig. 10, top) and the average current level returns to a level close to the baseline current (lower dashed line in Fig, 11). The AHP between spikes tends to push the current below the average level. Although the offset of IPIC is slow (time constant of ~0.5 s) (Lee and Heckman 1998a) compared with the time course of the AHP (50-100 ms at slow firing rates), nonetheless each AHP may provide some deactivation of IPIC. This could result in a progressive decline in average level that causes the cell to reach the bottom of the bistable current range (pt. 3 in Fig. 11). Just such a progressive decline in average membrane potential is evident in Fig. 10 (top). As a consequence, self-sustained firing declines in frequency and ceases. Note that spike voltage threshold (~48 mV) tends to be somewhat more depolarized than the voltage at the sustained peak in Fig. 11, suggesting that the cell should stop firing before IPIC fully deactivates. However, our measurement of spike threshold is set where voltage begins its steep rate of change. This steep change is likely to occur well into the range of activation of the fast, inactivating Na+ current. The functional voltage threshold probably is set by the persistent component of the Na+ current, which has a threshold a few millivolts more hyperpolarized than the threshold for the fast Na+ current (e.g., Brown et al. 1994). Consequently, the cell likely will continue to fire until IPIC deactivates.

When methoxamine increases the amplitude of IPIC, especially its sustained peak, then the cell's bistable current range is larger. Consequently, the lowered average current level due to the AHPs during self-sustained firing remains well above the bottom of the bistable current range. Thus it would be expected that there is some minimum amplitude for IPIC needed to generate long-duration self-sustained firing. If, as proposed above, the fluctuations in voltage and current due to the AHP play a crucial role in terminating self-sustained firing in the control cells, then elimination of spikes by blocking the fast sodium current may allow these cells to generate long-lasting plateau potentials.

If baseline current is increased so that the cell fires tonically before the synaptic input is applied, then would it be more difficult for the AHP to deactivate the IPIC. However, the cell could not exhibit bistable behavior in that case because the voltage threshold for spike generation is slightly above the voltage threshold for onset of IPIC. Consequently currents large enough to generate firing would fully activate IPIC before the application of Ia synaptic input.

The above arguments also suggest that amplitude of the AHP is very important in determining the duration of self-sustained firing. In slice preparations, noradrenergic agonists do not always affect the AHP (e.g., Larkman and Kelly 1992), but the small reduction in AHP amplitude due to methoxamine in our results is consistent with the modest effect seen in hypoglossal motoneurons (Parkis et al. 1996). As AHP amplitude declines, there should be a lessor tendency for deactivation of IPIC. It is likely that firing F-I gain also will be increased (cf. Parkis et al. 1996), so that self-sustained firing may reach a higher frequency. At higher frequencies, the average membrane potential tends to be more depolarized; this would help maintain the cell in the bistable range. Thus methoxamine acts to potentiate self-sustained firing in at least three ways: by depolarizing the cell, by reducing the AHP amplitude, and, most importantly, by increasing the amplitude of IPIC. Because IPIC may consist of more than one component (Hounsgaard and Kiehn 1989; Hsiao et al. 1998; Schwindt and Crill 1980) and because it could be strongly affected by outward currents, further work is required to identify the mechanism of how methoxamine increases IPIC.


    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, Chicago, IL 60611.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 October 1998; accepted in final form 19 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society