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Centre for Neuroscience, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
Submitted 13 August 2003; accepted in final form 18 October 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Although studies have shown that exaggerated cutaneous/flexor reflexes (Bennett et al. 1999; Remy-Neris et al. 1999) and increased tonic stretch reflexes are involved in the production of this syndrome (Burke et al. 1970; Powers et al. 1989; Thilmann et al. 1991), the exact mechanisms underlying spasms and spasticity are still not fully understood. Increased excitatory spinal reflex pathways (Barbeau and Norman 2003; Schmit et al. 2002), decreased descending and segmental inhibition (Cavallari and Pettersson 1989; Crone et al. 2003; Mailis and Ashby 1990; Thompson et al. 1998), newly formed neuronal circuitry due to the sprouting after injury (Krenz and Weaver 1998), and increased neuronal excitability, including interneurons and motoneurons (Bennett et al. 2001c; Eken et al. 1989) may all contribute.
Our recent studies from spinal-cord-injured adult rats have shown that the development of muscle spasms is caused by large voltage-dependent persistent inward currents (PICs; e.g., calcium currents) that develop in motoneurons weeks to months after injury (chronic spinal) (Li and Bennett 2003). These PICs produce sustained depolarizations of the motoneuron (plateau potentials; lasting seconds) in response to brief depolarizing stimulations, resulting in sustained firing that outlasts the stimulation (self-sustained firing). Thus the PICs amplify and prolong the response of motoneurons to transient sensory inputs and ultimately generate exaggerated and sustained reflex responses characteristic of the spastic syndrome. Further study indicates that the PICs in these motoneurons are mediated by a subthreshold TTX-sensitive persistent sodium current (sodium PIC) and a low-threshold nimodipine-sensitive persistent calcium current (calcium PIC; Cav1.3 L-type calcium channel) (Li and Bennett 2003). One goal of the present study was to examine how these two currents contribute to the long-lasting reflexes associated with muscle spasms.
Plateaus and PICs are also activated in motoneurons of animals with intact spinal cord and brain stem (Bennett et al. 1998; Gorassini et al. 1999a; Lee and Heckman 1998a) and uninjured humans (Gorassini et al. 1998, 2002; Kiehn and Eken 1997) and thus amplify and prolong synaptic inputs in normal behavior. Normally PICs depend critically on descending facilitation from brain-stem-derived serotonin (5-HT) or noradrenaline (Hounsgaard et al. 1988a; Hsiao et al. 1998; Lee and Heckman 1998a,b), and thus plateaus are eliminated with acute spinal cord injury. Surprisingly, the redevelopment of PICs in motoneurons after chronic spinal cord injury occurs even though the monoamines that normally facilitate PICs, such as 5-HT, are greatly diminished below the injury site (Newton and Hamill 1988). The recovered PICs after chronic injury have amplitudes comparable to those recorded in spinal cord/brain stem intact animals (Bennett et al. 1998; Lee and Heckman 1998a,b) and normal awake humans (Gorassini et al. 2002). However, due to the loss of proper descending inhibitory control after spinal cord injury, even a brief stimulation is sufficient to trigger plateaus and produce very long-lasting reflexes and spasms (Bennett et al. 2001a).
The activation of the PICs may also contribute to the abnormally slow firing of motor units observed in humans with spasticity after chronic spinal cord injury. For example, the activated PICs may increase the conductance of the motoneurons (Bennett et al. 2001c), thus making it more difficult to increase the firing rate, which could contribute to the lower maximum firing rates reached during volitional contractions in humans after spinal cord injury (Zijdewind and Thomas 2003). Another interesting phenomenon seen in these injured humans is very slow clockwork-like motor unit firing, either occurring spontaneously or triggered after an innocuous stimulation (Zijdewind and Thomas 2001; M. A. Gorassini, unpublished data). Interspike intervals during this firing are up to half a second and are much longer than expected from normal motoneurons (Matthews 1996). A similar phenomenon has also been recorded directly in motoneurons of rats after chronic injury (Bennett et al. 2001c). It can be triggered by intracellular stimulation and thus is an intrinsic property of the motoneurons. Likely, it is mediated by repetitive re-activation of the voltage-dependent PICs with slow kinetics near firing threshold (as suggested by Bennett et al. 2001c; Carp et al. 1991; Hodgkin 1948; Kernell 1999). Thus the second goal of the present study was to specifically examine the role of PICs in this and other abnormal slow firing behaviors seen after injury.
Aside from abnormally slow firing, the firing behavior of motoneurons of chronic spinal rats is very much like that in normal motoneurons in the spinal-cord-intact state (i.e., in unanesthetized decerebrate cats, Bennett et al. 1998; Hounsgaard et al. 1988a; Lee and Heckman 1998b). Both exhibit self-sustained firing produced by large PICs (see preceding text), and both exhibit classic input-output properties with primary and secondary range firing responses to injected current (piecewise linear frequency-current, F-I, relations). However, with the chronic spinal rat preparation, recordings are made in vitro (Bennett et al. 2001c), making it possible to directly block the PICs pharmacologically and examine their role in the classic input-output firing properties of motoneurons. This was our third goal. We found that the activation voltage of the calcium PIC critically determines the firing behavior. When the calcium PIC is activated subthreshold to the initial firing level at recruitment (in about half the cells), then it assists in producing self-sustained firing, but this firing is linearly modulated with current, with a single F-I slope. However, when the calcium PIC is activated above the initial firing level, then it does not assist in low-frequency firing (in primary range) but causes a steep acceleration in firing when it is being activated (secondary range F-I slope). In all cells, after the calcium PIC is steadily activated, then it produces a paradoxically lower F-I slope (denoted as tertiary range), which we argue is due to the increased conductance provided by this calcium PIC. These results lead to the suggestion that steady-state firing in the primary, secondary and tertiary ranges can be directly defined in terms of the state of the calcium PIC (see DISCUSSION).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, 2001a,c). Usually, within 30 days dramatic spasticity developed in the tail muscles, which are innervated by sacrocaudal motoneurons below the level of the injury. Only rats >50 days (also usually <120 days) post injury with clear spasticity were included in the present study. See Bennett et al. (1999) for details of the chronic spinal transection and spasticity assessment. All experimental procedures were approved by the University of Alberta animal welfare committee. In vitro preparation
The experimental procedure has been previously described in detail (Li and Bennett 2003) and is only briefly summarized here. Normal and chronic spinal rats were deeply anesthetized with urethane (0.18 g/100 g; with a maximum of 
0.45 g for rats >250 g), and the whole sacrocaudal spinal cord was removed and placed in a dissection chamber filled with modified artificial cerebrospinal fluid (mACSF) maintained at 20°C. After an hour's rest in the dissection chamber, the cord was transferred to the recording chamber where it was immersed in continuously flowing (5 ml/min) normal ACSF (nACSF), maintained at 25°C. The long ventral roots (usually sacral S4 and caudal Ca1) and caudal equina (which had attached caudal dorsal roots) were mounted on silver-chloride wires above the nACSF and covered with high vacuum grease (Dow Corning). Sharp intracellular recording electrodes were made from thick-wall glass capillaries (Warner GC 150F-10, 1.5 mm OD) with a micropipette puller (Sutter P-87 puller), filled with a 1:1 mixture of 2 M KAcetate and 2 M KCl and beveled down to 20–30 M
on a rotary grinder (Sutter, BV-10, fine 006 beveling stone). Electrodes were advanced perpendicularly into the ventral surface of the cord with a stepper-motor micromanipulator (660, Kopf) to penetrate motoneurons. Motoneurons were identified by antidromic ventral root stimulation. Only motoneurons with a stable penetration, resting potential < –60 mV, spike amplitude >60 mV, and reliable repetitive firing were included in the study. An Axoclamp2b intracellular amplifier (Axon Instruments) running in either discontinuous current-clamp modes (DCC, switching rate: 7–10 kHz, output bandwidth: 3.0 kHz) or discontinuous voltage-clamp modes (gain: 1–2.5 nA/mV) were used to collect the data. The basic properties of the motoneurons, such as cell resistance, firing threshold, and firing level, were measured during current ramps in DCC mode as described in Li and Bennett (2003).
Drugs and solution
Two kinds of ACSF were used in the experiments: nACSF in the recording chamber and mACSF in the dissection chamber prior to recording. The composition of nACSF was (in mM) 122 NaCl, 24 NaHCO3, 2.5 CaCl2, 3 KCl, 1 MgSO4, and 12 D-glucose. The composition of mACSF was (in mM) 118 NaCl, 24 NaHCO3, 1.5 CaCl2, 3 KCl, 5 MgCl2, 1.4 NaH2PO4, 1.3 MgSO4, 25 D-glucose, and 1 kynurenic acid. Both kinds of ACSF were saturated with 95% O2-5% CO2 and maintained at pH 7.4. Drugs added to the nACSF in the experiments included: 0.5–2 µM TTX (RBI), 3–20 µM nimodipine (Sigma), and 400 µM Cd2+ (Sigma) as detailed in Li and Bennett (2003).
Plateau and PIC activation in current- and voltage-clamp recording
Slow triangular current ramps (0.4 nA/s) and voltage ramps (standard speed: 3.5 mV/s, varied from 2 to 5 mV/s) were applied to the motoneurons to evoke the plateaus and the associated PIC. During the current ramps (in current-clamp), the PIC that contributed to a subthreshold plateau and sustained firing was estimated from the difference in injected current required to terminate a plateau (Iend), compared with the current required to start the plateau (
I = Iend – Istart, see Fig. 1A) (see also Bennett et al. 2001c). Also, the subthreshold plateau was quantified by extrapolating the linear subthreshold voltage-current relation to just prior to the first spike (thin line in Fig. 1A) and subtracting this linear response from the actual depolarization (measured 5 ms prior to the 1st spike to avoid rapid upswing of the spike). The term plateau is used to denote any relatively sustained depolarization produced by a PIC. As discussed in Bennett et al. (1998), it does not imply a fixed depolarization. Instead, the depolarization produced by the PIC (plateau) can summate with other depolarizations, such as the passive depolarization during a current ramp, and thus a plateau can essentially ride on top of a passive current ramp response (Figs. 1A and Fig. 4B).
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). To obtain an estimation of the passive leak current that summed with the PIC to give the total recorded current, a linear relation was fit to the subthreshold current response in the linear region 10 mV below the negative slope region onset and extrapolated to more positive voltages (leak current, thin triangular line overlaying current; Fig. 1B). The PIC amplitude was then estimated by subtracting this leak current from the recorded current, and this difference is indicated by the arrow in Fig. 1B (see details in Fig. 1 of Li and Bennett 2003). In a previous paper (Li and Bennett 2003), we defined the first zero slope point on the up ramp in the recorded current as the onset current (Ion) of the PIC and the corresponding voltage as the onset voltage (Von); the second zero slope point of the recorded current in the up ramp is defined as the initial peak current (Ii) of the PIC. In the present paper, we measured the half-activation voltage (V1/2) of the PIC because this point corresponded well to when the plateau was activated in current-clamp (see Fig. 9 in Li and Bennett 2003) [V1/2 is voltage at which half PIC activated, at Ion – (Ion – Ii)/2]. Also, the PIC was activated over a broad range, and the onset voltage Von underestimates the voltage at which the PIC and plateau were functional, compared with V1/2. The slope of the subthreshold leak current was denoted S1. The slope of the I-V relation was also computed at suprathreshold voltages, above the negative slope region, and denoted S2. The S2 slope was measured over a small 1-mV range and at a common voltage when the PIC was fully activated (at Vj in Fig. 1 of Li and Bennett 2003) to minimize time dependent activation effects. Thus the S1 and S2 approximate the membrane conductance.
Instantaneous firing frequency as a function of injected current (F-I) was computed using a custom Linux-based program (G. R. Detillieux, Winnipeg). The F-I slope was computed for piecewise linear regions of the F-I relation with a regression. To compare the difference in firing frequency before and after drug applications (nimodipine), only ramps with matched amplitude and speed were employed, and thus some cells were discarded because we did not have matched ramps. This was particularly important for cells with significant rate adaptation (Bennett et al. 2001c) that were very sensitive to ramp speed amplitude and speed. In some cells, there were significant changes in subthreshold input resistance after drug application, presumably due to leakage or electrode blockage, and such cells were also discarded from the F-I analysis. The initial firing level (spike threshold) for each cell was measured from the first spike elicited by the current ramp, at the potential where there first began a rapid acceleration in the rate of depolarization to >10 V/s (Li and Bennett 2003).
Dorsal root reflexes
A single electrical stimulation pulse was applied to the dorsal caudal root (Ca1; 0.02–0.1 nA, 0.1 ms, 10-s minimum interval between stimuli) while the motoneuron membrane was held at different potentials with a bias current, and reflex responses were recorded in the motoneurons. This single shock was usually enough to elicit a long-lasting reflex.
Data analysis
Data were analyzed in Clampfit 8.0 (Axon Instruments). Data are shown as means ± SD. A Student's t-test was used to test for statistical differences with a significance level of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), and the purpose of the present paper was to determine how these PICs affected firing behavior. The PICs were measured with slow triangular voltage ramps, in voltage-clamp mode, as shown in Fig. 1B. During an upward voltage ramp, the current initially increased linearly due to the passive leak current (shown extrapolated with thin triangular line, Fig. 1B, middle), but after the onset of the PIC, the current deviated negatively and caused a negative-slope region in the I-V relation. The difference between the measured current and the extrapolated leak current was used as an estimate of the net PIC (vertical arrow in Fig. 1B; see METHODS). The half activation voltage of the PIC (V1/2; vertical thin line in Fig. 1B) was used to quantify the location of the negative-slope region, which we have previously shown corresponds to the point at which a plateau is activated during a current ramp (see Fig. 9 in Li and Bennett 2003). In all motoneurons of chronic spinal rats (n = 27), the V1/2 was on average –50.1 ± 4.72 mV, which was 3.79 ± 1.72 mV below the firing level (i.e., spike threshold, dashed line in Fig. 1B; significant difference), where the subthreshold plateau occurred (left arrow in Fig. 1A).
There are only two major currents that make up the PIC: a sodium PIC and a calcium PIC (Li and Bennett 2003). With the application of nimodipine, only the sodium PIC remains, and this current is shown in Fig. 1F (top), after leak current subtraction (see METHODS). The reduction in current with nimodipine represents the calcium PIC, and this is also shown in Fig. 1F (bottom). Nimodipine blocks the calcium PIC without affecting the fast sodium spike (Figs. 2 and 9, described later) and was thus particularly useful in studying the effects of PICs on firing as shown in the following text.
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Previously, we have shown that motoneurons of chronic spinal rats can be divided into two distinct types based on their unique firing patterns (Bennett et al. 2001c), and these were again found in the present population of motoneurons, as shown in Figs. 1 and 2. In the first type of motoneuron (Fig. 1; n = 16/27 cells), there was initially a linear increase in potential during a current ramp, but 5 mV below the firing level, the potential accelerated relatively steeply. This acceleration marked the onset of a subthreshold plateau from a PIC activation (left arrow in Fig. 1A) (Bennett et al. 2001c; Li and Bennett 2003) because if the current was reduced shortly afterward, there was a maintained depolarization and associated firing that continued even when the current was reduced far below the current that initiated firing (self-sustained firing). This self-sustained firing was quantified by I = Istart – Iend, where Istart and Iend are the currents at the start and end of plateau and firing. The I was on average 1.13 ± 0.19 nA for this cell type. The plateau stopped just after firing ceased as seen by the after-potential at the end of the descending current ramp (right arrow in Fig. 1A). In this type of cell, the PIC was as fully activated as possible shortly after recruitment because larger ramps did not produce more self-sustained firing (not shown, though see Fig. 5 in Bennett et al. 2001c). Furthermore, shortly after recruitment there was no further evidence of PIC activation (no firing rate jumps), and the firing changed linearly with the current, even during repeated current ramps and during self-sustained firing (at currents below the plateau activation threshold; Fig. 1C). Thus we refer to this type of cell as low-threshold linear self-sustained firing cells (LLS type).
In the second type of motoneuron, shown in Fig. 2 (n = 11/27 cells), during a slow current ramp, there was also a subthreshold plateau and associated PIC activation like in LLS cells (left arrow in Fig. 2, A and B). However, this PIC was not fully activated at recruitment because only a small degree of self-sustained firing was produced when the ramp was turned around shortly after recruitment (I = 0.6 in Fig. 2A; mean I = 0.34 ± 0.15 nA for all cells). In contrast, larger ramps evoked a late acceleration in firing (labeled "s" in Fig. 2B) that marked the further onset of a PIC because this extra firing was sustained despite reduced current (F-I hysteresis), and there was significantly more self-sustained firing (I = 1.2 in Fig. 2B; mean I = 0.98 ± 0.15 nA). These cells we refer to a late-accelerating self-sustained firing cells (LAS type). LAS type cells were on average lower rheobase cells (1.29 ± 1.53 nA) than LSS cells (2.53 ± 0.69 nA) (see Bennett et al. 2001c).
The linear increase in firing with current prior to the late acceleration we refer to as primary range firing (p in Fig. 2B), and the firing during the late acceleration we refer to as ramp-evoked secondary range firing (s in Fig. 2B), similar to the classic steady-state firing responses (Bennett et al. 1998; Kernell 1965a; Schwindt and Crill 1982). The firing after the late acceleration we refer to as tertiary range firing (t in Fig. 2B). Classically, the steady-state primary and secondary range firing is obtained from an increasing sequence of 1-s current steps and measured in the last half second of each step (Kernell 1965a). We instead used very slowly increasing current ramps (lasting 10–20 s), but for the primary and tertiary ranges, the firing obtained at a particular current was similar to the steady-state firing during a classic current step (data not shown). In the secondary range during a current ramp, the firing was not quite in steady state, and thus we denote this the ramp-evoked secondary range. While the F-I slope in this range is steep compared with the primary slope, the firing still increases over a 2-s period (Fig. 2) and thus is approximately comparable to the classic secondary range response to two 1-s current steps.
Subthreshold sodium and calcium PIC contributions to LLS-type cells
In LLS type cells, the subthreshold plateau activation was caused by both sodium and calcium PICs because nimodipine significantly reduced it (from 9.49 ± 3.53 to 3.17 ± 2.09 mV), but blocking it required both nimodipine and TTX (see details in Li and Bennett 2003). Furthermore, direct measurements of these two PICs in voltage clamp (described in the preceding text and in Fig. 3 described in the following text) indicate that they were both activated subthreshold and thus should indeed produce a subthreshold plateau. That is, in LLS cells the half activation voltage V1/2 of the calcium PIC (–44.2 mV in Fig. 1F; mean of 47.4 ± 5.52 mV) and sodium PIC (–43.7 mV in Fig. 1F; mean of 46.8 ± 4.01 mV) were always less than the firing level (–41.4 mV in Fig. 1F; mean of 44.6 ± 4.18 mV; significant differences; n = 7/7 cells with nimodipine) and not significantly different from each other. Nimodipine significantly increased the current required to initiate firing (from 2.53 ± 0.69 to 3.26 ± 0.55 nA) and in most cells, lowered the initial firing rate (in 4/7 cells; lowered from 7.73 ± 1.34 to 6.38 ± 1.53 Hz, although not quite significantly; P = 0.13), so the calcium PIC was involved in initiating firing. Both the sodium and calcium PICs were nearly fully activated by the time the firing level at recruitment was reached (Fig. 1F), and this is consistent with the preceding conclusion that the PICs and plateau were as fully activated as possible at recruitment and subsequently did not contribute to accelerations in firing rate during the ascending current ramp (unlike LAS cells). Nimodipine also significantly reduced the afterpotential after de-recruitment (from 6.39 ± 1.33 to 3.01 ± 2.14 mV; right arrows in Fig. 1, A and D) and eliminated the brief accelerations in firing that are sometimes seen just prior to de-recruitment (* in Fig. 1, A and C, but not D) (also see Fig. 5 of Bennett et al. 2001c), and thus these were calcium PIC mediated.
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I significantly lowered from 1.15 ± 0.22 to 0.26 ± 0.26 nA). A subthreshold calcium PIC occurred in all LLS cells and thus is a primary requirement for LLS type firing behavior. However, there did remain significant self-sustained firing in nimodipine, and this was due to slow firing caused by the sodium PIC, that under some conditions could last for very long periods (see Figs. 6, 7, 8 described in the following text).
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In contrast, when nimodipine was applied to LAS-type cells (n = 5), there was no significant effect on the subthreshold plateau activation, the recruitment current (1.29 ± 1.53 nA before and 1.40 ± 2.25 nA after), or low-frequency firing (see details in Fig. 5B, described later), and thus only the sodium PIC was involved in subthreshold plateau behavior and low-frequency firing. However, when these cells were brought to fire at higher frequencies with a larger current ramp, there was a characteristic late acceleration in firing that was always blocked by nimodipine (5/5 cells), and thus this nonlinearity in the F-I relation (s range in Fig. 2B) was caused by the calcium PIC. In LAS-type cells, the calcium PIC (seen in Fig. 2) was activated at a potential (V1/2 = –44.8 ± 5.30 mV; right arrow in Fig. 2F) significantly higher (by 3.50 ± 1.67 mV) than the firing level at recruitment (–48.3 ± 4.48 mV; firing level at dashed line in Fig. 2F), and much higher than the subthreshold sodium PIC (V1/2 = –51.3 ± 4.56 mV; left arrow in Fig. 2F). Thus this higher-threshold calcium PIC in LAS-type cells was only activated after recruitment when the membrane potential was sufficiently depolarized during higher-frequency firing. At this time the calcium PIC activation produced the late acceleration in firing rate (firing level at this time indicated by * in Fig. 2F).
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I = 0.97 ± 0.15 to 0.24 ± 0.31 nA), indicating that the calcium PIC played a major role in sustaining the firing, as for LSS cells. In summary, in LAS cells, the sodium and calcium PICs were distinctly separated in their activation voltages and, respectively, produced two distinct effects: an early subthreshold plateau and a late acceleration in firing respectively, followed by self-sustained firing.
Interestingly, the sodium PIC activation level (V1/2) was 4 mV higher in the LSS cells (–46.8 ± 4.01 mV) compared with LAS cells (–51.3 ± 4.56 mV, although not quite significant difference, P = 0.07, because between cell comparisons of absolute potentials are variable). Also, the firing level was 4 mV higher in LSS cells (–44.6 ± 4.18 mV) compared with LAS cells (–48.3 ± 4.48 mV, P = 0.09). In contrast, the calcium PIC activation was only 2 mV lower in LSS cells (–47.4 ± 5.52 mV) compared with LAS cells (–44.8 ± 5.30 mV, P = 0.65). These differences between LSS and LAS cells in the sodium PIC and sodium spike activation were largest and closest to significance, suggesting that the differences in the firing behavior in LLS cells compared with LAS could be mostly attributed to higher sodium PIC and spike activation levels and to a lesser extent to a lower calcium PIC activation level.
Variations in calcium PIC determines firing behavior
In many cells (n = 13), we added TTX first, rather than nimodipine, and in these cells, we could not study the firing because the spikes were blocked together with the sodium PIC. However, in these cells, the calcium PIC could be observed directly as the PIC remaining in TTX (Fig. 3, C and D) and compared with the firing level at recruitment prior to TTX. The half activation potential of the calcium PIC (V1/2 calcium) clearly separated all these cells into two groups corresponding to the LLS and LAS classification of their firing behavior (prior to TTX application) as expected from the nimodipine experiments described in the preceding text. This is shown in Fig. 3E in which the degree of self-sustained firing (I) is plotted as a function of the V1/2 calcium relative to the firing level. When the V1/2 calcium was below firing level (n = 7; Fig. 3C), pronounced sustained firing occurred (I = 1.12 ± 0.16 nA) even for small ramps reaching just above the recruitment threshold (as in Fig. 3A; 
at left of vertical line in Fig. 3E), and these cells all corresponded to LLS type cells (n = 7/7) as described in the preceding text. When the V1/2 calcium was above the firing level (n = 6; Fig. 3D), significantly less self-sustained firing occurred (I = 0.35 ± 0.16 nA) for the same small current ramps (Fig. 3B and at right of vertical line in Fig. 3E), even though these cells had as large PICs (Fig. 3D) as the LLS cells (Fig. 3C). However, with larger ramps (like in Fig. 2B; 
in Fig. 3E) pronounced self-sustained firing (I = 1.02 ± 0.11 nA) could usually be evoked after a late acceleration in firing (n = 4/6), and these cells corresponded to LAS type cells (upper at right of Fig. 3E).
There were, however, a few cells with the V1/2 calcium above the firing level that always had weak self-sustained firing and did not have a firing rate acceleration regardless of the ramp amplitude (lower at right of Fig. 3E; n = 2/6 cells tested with TTX). These cells had large calcium PICs, but the calcium PIC threshold was too high to produce a late acceleration during firing evoked with a current ramp [due to accumulated afterhyperpolarization (AHP) effects] (see Li and Bennett 2003). Interestingly, in these cells the calcium PIC and firing rate acceleration could be activated by synaptic input (described in a later section), suggesting a difference due to dendritic location of the synaptic inputs and calcium PIC (Bennett et al. 1998).
Low F-I slope in chronic spinal rats results from Ca2+-mediated conductance
As we have shown previously, motoneurons from chronic spinal rats fired with a significantly shallower F-I slope (mean: 4.56 Hz/nA, see following text) than the motoneurons of acute spinal rats (6.7 Hz/nA) (acute data from Bennett et al. 2001c) when tested with current ramps as in Fig. 2. We proposed that the main reason for this lower slope was the activation of the large PICs in chronic spinal rats that should have increased the conductance of the cells and thus increased the difficulty in depolarizing the cells with injected current and ultimately decreased the F-I slope (Bennett et al. 2001c).
To examine this issue, we first verified that the membrane conductance increased after the PIC activation by estimating the conductance from the slope of the I-V relation during the ramp voltage-clamp experiments (see METHODS). Indeed as shown in Fig. 4A (bottom trace), at voltages below the negative-slope region, prior to PIC activation, the conductance (I-V slope S1) was much shallower than above the negative-slope region with the PIC activated (S2, measured at common potential at vertical line). In motoneurons of chronic spinal rats, the ratio of these two slopes (S2/S1) was significantly >1 (S2/S1 = 5.28 ± 3.42, n = 13), whereas in motoneurons of acute spinal rats, the ratio was not significantly different from 1 (because there was usually a linear I-V relationship, with no PIC activation; S2 slopes measured at same voltage as in chronic spinal rats) (see Li and Bennett 2003). This increased conductance in chronic spinal rats was mediated by both sodium and calcium PICs because the ratio of S2/S1was significantly decreased when TTX or nimodipine were added to the bath (TTX shown in Fig. 4A, middle trace), and it was decreased to 1 after both drugs were applied (Fig. 4A, top linear trace). Importantly, the increased conductance after PIC activation was not caused by a voltage-dependent potassium conductance in the voltage range studied (< –30 mV) because TTX and nimodipine eliminated the increased conductance when measured at a common voltage (see linear responses in top trace of Fig. 4A). Thus persistent sodium and calcium PICs mediated the increased conductance. The calcium-PIC-mediated increase in conductance (S2) likely includes a Ca2+-activated K+ current (unpublished data).
The increased conductance after calcium PIC activation was also seen during current-clamp experiments where TTX was present (Fig. 4B), and a calcium-mediated plateau occurred without spiking. That is, on the plateau with the calcium PIC activated, the slope of the voltage curve (resistance, R2 = 1/conductance) was only 48% of the slope before the plateau started (R1; significantly lower; R1/R2 = 2.10 ± 0.83; n = 12), suggesting that the conductance of the membrane was doubled by the activated calcium PIC.
Finally, to prove that the presence of the calcium PIC contributed to the shallow F-I slope seen in motoneurons of chronic spinal rats, we blocked this current with nimodipine (Fig. 5), which again, had no direct affect on the sodium spikes or AHPs. As expected, the F-I slope was found to be significantly steeper after nimodipine compared with before (7.37 ± 3.32 Hz/nA compared with 4.56 ± 1.19 Hz/nA, n = 9, Fig. 5A), provided that the slope before nimodipine application was measured in a region where the calcium PIC was known to be active (above calcium PIC threshold). The cell in Fig. 5A is an LLS cell with the calcium-PIC-activated subthreshold to firing, and the F-I slope was clearly affected throughout by nimodipine. Whereas, the cell in Fig. 5B is an LAS cell with the calcium PIC only activated after the late acceleration in firing, and the slope was only increased by nimodipine after this late acceleration, in the tertiary range, as described next.
Secondary and tertiary range firing caused by calcium PIC
In LAS-type cells (Fig. 5B), nimodipine had no significant effect on the F-I slope when the cell fired in the primary range subthreshold for late acceleration in firing (Fig. 5, B and C; mean slope: 3.04 ± 1.02 Hz/nA in the primary range before nimodipine, p, and 3.70 ± 1.02 Hz/nA after nimodipine, p'), as would be expected of the lack of calcium PIC in this range. In contrast, after the late acceleration in firing, and thus calcium PIC activation, the F-I slope (in tertiary range, t in Fig. 5B; mean: 2.10 ± 1.31 Hz/nA) was significantly less than the slope in nimodipine, even at matched frequency ranges (Fig. 5C; mean: 3.70 ± 1.02 Hz/nA), consistent with the activated calcium PIC reducing the F-I slope. Also consistent with this interpretation, the slope with the calcium PIC fully activated, in tertiary range (Fig. 5B; t; mean: 2.10 Hz/nA, as in the preceding text), was significantly less than the slope measured prior the calcium PIC activation, in the primary range (p in Fig. 5B; mean: 3.04 Hz/nA). Of course, during the late acceleration in firing (ramp-evoked secondary range), the F-I slope was very steep (s in Fig. 5B; mean: 6.32 ± 3.63 Hz/nA), and this steep secondary range firing was eliminated with nimodipine (as discussed in the preceding text), leaving only simple linear primary range firing for all currents (Fig. 5C; mean: 3.70 ± 1.02 Hz/nA). This is consistent with the idea that the secondary range firing is caused by the calcium PIC onset (Schwindt and Crill 1982).
Very slow firing caused by persistent sodium currents
When a current ramp was applied to a motoneuron of a chronic spinal rat, the last few spikes of firing were usually very slow (mean: 2.28 ± 0.67 Hz; Figs. 1A, 2A, 3A, and 5B) and less than half the minimum repetitive firing rate that occurs in motoneurons of acute spinal rats (7.51 ± 3.53 Hz; significant difference) (Bennett et al. 2001c). We demonstrate in this section that this unusually slow firing is mediated by a subthreshold oscillation of the large sodium PIC seen in motoneurons of chronic spinal rats. Very slow firing was most clearly studied when evoked by a brief current pulse in a cell held close to its firing threshold with a bias current (Fig. 6A). After the pulse, there was usually a pause in firing during which a plateau was slowly activated (at arrow in Fig. 6A, top), and then very slow self-sustained firing began. This self-sustained firing continued for many seconds, or even minutes, and then either stopped spontaneously or was terminated by a hyperpolarizing pulse (Fig. 6A). Typically, the firing rate was very low (mean: 2.82 ± 1.21 Hz) and extremely regular (SD in firing 0.26 ± 0.13 Hz), unlike the variable slow firing that can be driven by synaptic noise (Matthews 1996; Powers and Binder 2000). In some cells, we did see transient rate changes during the long periods of slow self-sustained firing (Fig. 7, asterisks, described later), but these were due to spontaneous synaptic events because excitatory postsynaptic potentials (EPSPs) of a similar duration could be seen prior to firing (not shown). The minimum firing rates corresponded to inter-spike intervals of 300–800 ms, which were much longer than the duration of the usual AHP (50–150 ms), measured in response to antidromic stimulation at rest (AHP indicated by length of box in the expanded section of data in Fig. 6A). Thus these cells fired like clockwork at much lower rates than predicted by the AHP duration.
In contrast, in motoneurons of acute spinal rats the minimum repetitive firing rate during a depolarizing pulse was 7.51 ± 3.53 Hz (Bennett et al. 2001c) (7.68 Hz in Fig. 6B), which corresponds to an inter-spike interval of 133 ms, and this is close to the AHP duration (125 ms in Fig. 6B) as would be expected for motoneurons with small PICs that fire in the absence of synaptic noise (Kernell 1965b). Also, self-sustained firing could not be evoked in acute spinal rats (Fig. 6B) (Bennett et al. 2001c), consistent with the small PICs and lack of negative-slope region in the I-V relation in these cells (Li and Bennett 2003).
During slow firing in chronic spinal rats, the trajectory of the membrane potential between spikes was very much like the subthreshold onset of the plateau prior to the first spike after a brief current pulse (Fig. 6A, top, left arrow) or during a current ramp (left arrows in Figs. 1A, 2A, 3A). That is, on the expanded time scale in Fig. 6A, after the current pulse, a plateau was activated (in the region of dotted line) that depolarized the cell above baseline (thin line). This plateau activation involved a slow ramp up and then a faster acceleration that ultimately triggered a spike (ramp&accelerate trajectory). After the 100-ms AHP from this first spike (region of box), the membrane potential rose again with the same ramp&accelerate trajectory (see ramp and acceleration labels in Fig. 6A) as though a plateau was again being activated (in region of dotted line in Fig. 6A), and this repeated with each spike. Thus a plateau was being activated prior to each spike and then deactivated by the AHP that followed that spike, and this process was repeated to cause slow firing.
Because the sodium PIC is rapidly deactivated by a hyperpolarization (in <10–50 ms) (Lee and Heckman 2001; Li and Bennett 2003), whereas the calcium PIC is not (remains on for >500 ms), it stands to reason that only the sodium portion of the plateau could be deactivated during each 100-ms AHP, and thus only the sodium PIC could participate in this slow firing. Indeed, in cells where the calcium PIC was activated far above the firing threshold and was only involved in high-frequency firing (LAS type; Fig. 6 shows such a cell), slow self-sustained firing was just as easily evoked as in other cells (LLS type).
Also, slow firing persisted when the calcium PIC was blocked with nimodipine (n = 4), with the same slow steady rate (3.37 ± 0.49 Hz before and 2.60 ± 0.67 Hz after nimodipine; Fig. 7) and the same characteristic subthreshold plateau activation and associated ramp&accelerate interspike trajectory (in region of dotted line in Fig. 7B). Thus this very slow firing did not involve the calcium PIC. In contrast, in cells that had a poor persistent sodium PIC, which did not by itself produce a negative slope region (Fig. 10D; described later), very slow firing could not be evoked (Fig. 10A). Instead the minimum firing rate corresponded to an interspike interval similar in duration to the AHP, as for motoneurons of acute spinal rats described in the preceding text. Thus a negative-slope region produced by the sodium PIC is the primary requirement for slow firing.
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), and this as usual did not require the calcium PIC because it was not blocked by nimodipine (Fig. 8B; LAS cell described in the preceding text). Likewise, just prior to de-recruitment on the downward current ramp, there was the usual very slow firing (mean rate: 2.31 ± 1.04 Hz), with one very long interval, with a characteristic ramp&accelerate trajectory similar to the plateau onset (Fig. 8A, right ). This slow firing again did not depend on the calcium PIC because it was not significantly altered by nimodipine (Fig. 8B; mean rate: 2.28 ± 0.67 Hz).
When low-dose TTX was added, with nimodipine present, the characteristic subthreshold plateau onset was eliminated (no acceleration at left of Fig. 8, C and D), and likewise the slow firing on the downward ramp and the characteristic ramp&accelerate interspike trajectory were eliminated. Thus the subthreshold plateau onset and slow firing are both mediated by the TTX-sensitive sodium PIC. The firing became very erratic and failed on the downward current ramp in low-dose TTX, even when the spike was unaffected. Long irregular firing intervals were seen on the upward ramp, but with a very different interspike trajectory, without the ramp&accelerate shape (Fig. 8, C and D). Instead, long intervals occurred on the upward ramp simply because a spike failed to be produced by the end of the AHP and then was only produced as the cell was further depolarized by the increasing current. Furthermore, it appears that this sodium PIC is critical in enabling the cell to generate rhythmic firing at all because when this current was disrupted, firing became irregular (Fig. 8, C and D) as has been previously reported (Lee and Heckman 2001).
Long-lasting spastic reflexes evoked by brief low-threshold afferent stimulation
In chronic spinal rats, a single low-threshold dorsal root stimulation shock produced a very long-lasting reflex response that we observed in most motoneurons during intracellular recording (stimulation 2x afferent threshold, T in Fig. 9B and typically 2–10 x T; n = 11/13), as described previously (Bennett et al. 2001c). This is consistent with the exaggerated reflexes recorded extracellularly from ventral roots of these chronic spinal rats with the same dorsal root stimulation (Li et al. 2004), and the associated tail spasms recorded with electromyography in awake chronic spinal rats (Bennett et al. 1999, 2001a).
To examine the role of plateaus/PICs in long-lasting reflexes, we have blocked the PICs with two separate methods: hyperpolarization and application of nimodipine. The first method relies on the voltage dependence of the PIC that underlies the plateaus (Bennett et al. 2001c) as demonstrated in a motoneuron of a chronic spinal rat in Fig. 9A. That is, although a brief depolarizing current pulse activated a large plateau and long-lasting self-sustained firing when the cell was held at rest, the same pulse could not activate a plateau when the cell was hyperpolarized (i.e., hyperpolarization abolished the plateau). Similarly, while a brief dorsal root stimulation triggered a long-lasting reflex in a motoneuron (Fig. 9B, top), no long-lasting reflex could be evoked when the motoneuron was held hyperpolarized (Fig. 9B). Therefore the long-lasting reflex was mediated by PICs/plateaus intrinsic to the motoneurons that were blocked by hyperpolarization (just as in Fig. 9A).
Hyperpolarization of the motoneuron did not block the synaptic input caused by the dorsal root stimulation, and this input produced a 0.5- to 1-s-long EPSP (Fig. 9B, bottom). It is this unusually long EPSP that triggered the slowly activating PICs and reflex when the cell was not hyperpolarized. This can be clearly seen in Fig. 9B in which the onset of spiking was delayed because the cell was very near threshold, and the EPSP could be observed first, followed by a PIC/plateau onset (at arrow) and then self-sustained firing. Also, cells without a clear long polysynaptic EPSP (n = 2/13) did not have long-lasting reflexes to a single low-threshold stimulation even though they had large PICs. They could, however, produce a long-lasting reflex (seconds) in response to repeated high-frequency stimulation (100 Hz, for 0.5 s) by the summated monosynaptic reflex (see following text) triggering a plateau/PIC.
Calcium PIC alone produces the long-lasting reflex in some motoneurons
In addition to the sodium and calcium PICs, any other voltage-dependent current facilitated by the dorsal root stimulation [i.e., N-methyl-D-aspartate (NMDA) receptors] could also be involved in the plateau and associated long-lasting reflex. In general, proving that only sodium and calcium PICs are involved is difficult because the sodium PIC cannot be easily blocked without blocking the synaptic input involved in triggering the long-lasting reflex (EPSP; TTX blocks the dorsal roots rapidly). Fortunately, there were some motoneurons (n = 2/9) that had clear long-lasting reflexes (Fig. 10A) and only weak sodium PICs (Fig. 10D). In these motoneurons, 20 µM nimodipine blocked the long-lasting reflex regardless of the holding potential (Fig. 10C), and thus the calcium PIC mediated these long-lasting reflexes. Furthermore, nimodipine did not block the EPSP evoked by the dorsal root stimulation (Fig. 10C), and thus nimodipine's primary effect was to block the L-type Ca2+ channels and associated self-sustained firing postsynaptically. In these cells, nimodipine also blocked the plateaus evoked with intracellular current injection (not shown), and this is consistent with the elimination of the calcium-mediated negative-slope regions in the I-V plot during voltage-clamp (compare Fig. 10, C and D). There was a small TTX-sensitive sodium PIC that remained in nimodipine (Fig. 10D) although not large enough to produce a plateau, as mentioned in the preceding text (no negative slope region). In summary, in these cells with weak sodium PICs, the long-lasting portion of the spastic reflexes were entirely due to calcium PICs on the motoneurons mediated by L-type calcium currents, and sustained postsynaptic NMDA-like currents were thus not involved in the long-lasting reflexes.
Sodium and calcium PICs produce long-lasting reflexes in other motoneurons
In other motoneurons (n = 7/9), persistent sodium currents were also involved in the long-lasting reflex. Again these cells had a long-lasting reflex mediated by a large PIC and self-sustained firing that was triggered by dorsal root stimulation, and this was blocked by hyperpolarization (Fig. 11A). However, this dorsal-root-evoked PIC and long-lasting reflex was not blocked by nimodipine. It was, however, reduced in amplitude, and there was slower firing and no acceleration in firing, presumably due to the block of the calcium PIC (compare Fig. 11, A and D). This PIC and long-lasting reflex that remained in nimodipine was due to the sodium PIC currents because there remained a large PIC in voltage-clamp ramps (Fig. 11, F compared with C), there was characteristic very slow firing (Fig. 11D, top), and this sodium PIC was blocked by TTX (not shown). Thus in this cell both sodium and calcium PICs produced the long-lasting reflexes.
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Interestingly, reflex evoked calcium PICs and plateaus were more easily triggered (at a lower potentials) than PICs evoked by intracellular current injection, perhaps related to the dendritic location of the Ca2+ channels and synaptic inputs (Bennett et al. 1998). That is, with intracellular current injection the calcium PIC was only activated in LAS-type cells with a substantial depolarization well above the initial firing level at recruitment (after frequency acceleration at left arrow in Fig. 9A, top; LAS-type cell), whereas with dorsal root stimulation, a subthreshold plateau and subsequent long-lasting reflex could be activated (e.g., left arrow in Fig. 9B, top). In the extreme case, there were cells that had high-threshold calcium PICs that could not be activated during firing evoked by intracellular current injection (described in the preceding text), but subthreshold calcium PICs and long-lasting firing could be evoked by dorsal root stimulation (n = 3/3).
Unusually long polysynaptic EPSPs in acute and chronic spinal rats
As mentioned in the preceding text, in chronic spinal rats, the low-threshold dorsal root stimulation evoked unusually long EPSPs that were clearly seen when the motoneurons were hyperpolarized to block the plateau and associated PICs (Figs. 11, A and B, 10, A and B, and 9B). These hyperpolarized EPSPs were on average 9.8 ± 4.8 mV in amplitude and lasted 960 ± 270 ms. Typically, the reflex response also had a monosynaptic component, and this was on average14.4 ± 8.3 mV (Fig. 11B). The long EPSP followed in the tail of the monosynaptic EPSP and thus was of short latency. However, the long EPSP was definitely of polysynaptic origin because when the monosynaptic reflex was not present, then the long EPSP had a latency of 3.3 ms beyond the monosynaptic latency. Both the mono- and polysynaptic EPSPs were not blocked by nimodipine (Figs. 10 and 11) and thus did not depend on pre- or postsynaptic L-type calcium channels (calcium PICs).
Interestingly, in acute spinal rats, there were also very similar long polysynaptic EPSPs (Fig. 9C), and thus these must have emerged acutely with injury because indirect evidence from EMG recordings suggest that this EPSP is not present prior to injury in the awake rat (D. J. Bennett and C. L. Cooke, unpublished results). When these EPSPs were measured with the cell hyperpolarized (Fig. 9C, bottom), as in the
), the initial linear F-I slope (primary range, p), then late acceleration to a steeper slope (ramp-evoked secondary range, s), followed by firing with a very shallow slope (tertiary range, t). On the downward ramp (
