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Centre for Neuroscience, University of Alberta, Edmonton, Alberta, Canada
Submitted 6 October 2006; accepted in final form 25 February 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-conotoxin MVIIC and was resistant to the L-type calcium channel blocker nimodipine, whereas the L-Ca and SKL currents were blocked by nimodipine. Furthermore, the SKAHP current activated within 10 ms of the spike, whereas the SKL current was delayed 
100 ms after the onset of the L-Ca current, suggesting that the SKL currents were not as spatially close to the L-Ca currents. Finally, the SKL and the L-Ca currents were poorly space clamped, with oscillations at their onset and hysteresis in their activation and deactivation voltages, consistent with currents of dendritic origin. The impact of these dendritic currents was especially pronounced in 15% of motoneurons, where apamin led to uncontrollable L-Ca currents that could not be deactivated, even with large hyperpolarizations of the soma. Thus, although the SKL currents are fairly small, they play a critical role in terminating the dendritic L-Ca currents. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Li and Bennett 2003; Powers and Binder 2003; Svirskis and Hounsgaard 1997). These PICs are facilitated by monoamines, such as serotonin (Hounsgaard and Kiehn 1989; Perrier and Hounsgaard 2003) and norepinephrine (NE) (Lee and Heckman 1999a), which arise mostly, although not exclusively, from the brain stem (Maxwell et al. 1996; Patel et al. 1997). Also, PICs are facilitated by other transmitters, such as glutamate and acetlycholine, acting on metabotropic receptors (Svirskis and Hounsgaard 1998). Functionally, PICs are important for bistable behavior, including production of plateau potentials and self-sustained firing, seen in the normal spinal cord intact animals (Lee and Heckman 1998a,b; Schwindt and Crill 1982) and chronic spinal animals (Bennett et al. 2001). Furthermore, bistable behaviors have been shown to play a major role in motor unit firing in normal and spinal cord–injured humans (Gorassini et al. 1998, 2004; Kiehn and Eken 1997). Because of their dependence on facilitation from monoamines, PICs are dramatically reduced, although not eliminated, by acute spinal transection, which eliminates brain stem monoamine innervation. However, these PICs return with chronic injury, possibly because of a developed supersensitivity to residual monoamines below the transection (Harvey et al. 2005a–c).
The persistent sodium currents (Na PIC) in motoneurons are TTX sensitive, activate subthreshold to firing, and partly inactivate over a few seconds after activation. The persistent calcium currents (Ca PIC) are nimodipine-sensitive, activate near the firing threshold, but persist for longer than the Na PIC (seconds to minutes); thus they play the major role in sustained depolarizations (plateaus) and firing (self-sustained firing) (Bennett et al. 2001; Li and Bennett 2003). The Ca PICs responsible for plateaus in motoneurons have been shown to be mediated by an L-type calcium current (termed L-Ca current, Perrier et al. 2002; Simon et al. 2003), likely acting through the newly identified low-threshold Cav1.3 calcium channels (Xu and Lipscombe 2001).
Besides acting as a charge carrier, the calcium ion is also capable of activating other channels, such as the calcium-activated small conductance potassium channel (SK channel), the calcium-activated big conductance potassium channel (BK channel), and the calcium-activated nonselective cationic (ICAN) channel. Therefore the Ca PIC recorded in motoneurons is likely the direct L-Ca current plus calcium-activated currents. The ICAN current has been shown to not play an important role in the plateau potentials in motoneurons (Perrier and Hounsgaard 1999). Also, BK currents have been found to be responsible for the fast afterhyperpolarization (fAHP) and are activated primarily by the N-type and P/Q-type calcium channels (Umemiya and Berger 1994) but are unlikely to play a major role in plateaus mediated by L-type calcium channels because of their relatively transient activation (McLarnon 1995).
SK channels have been found to be present in most types of neurons, including motoneurons, and are responsible for the medium-duration postspike afterhyperpolarization (mAHP) (McLarnon 1995). Thus the SK currents activated by spikes must hyperpolarize motoneurons by producing mAHPs and must counteract the net Ca PIC that helps sustain firing. However, it is unclear whether the L-Ca current that mediates the Ca PIC (through Cav1.3) directly activates SK currents. SK channels have little voltage dependence, but great Ca2+-dependent sensitivity. The conductance of SK channels increases with accumulation of intracellular Ca2+, and the channels are persistent, inactivating only slowly (McLarnon 1995). Thus in the presence of persistent L-Ca currents, the SK currents could very well produce a persistent outward current (that we term SKL) that opposes this persistent calcium current. Testing this possibility is the purpose of this paper.
Our study showed that the L-type calcium current (L-Ca) does indeed activate an SKL current, which subsequently diminishes the recorded Ca PIC. Using apamin to block the SK channel, the recorded Ca PIC is increased, and the calcium plateau is enlarged and prolonged. For comparison, we also quantified the SK currents that mediated the AHPs during firing (SKAHP) and examined the role of both SKAHP and SKL currents in firing. We recorded PICs and firing in motoneurons in an in vitro preparation (Bennett et al. 1999b) where the sacrocaudal spinal cord was removed either from normal adult rats (acute spinal) or from adult rats after 2 mo of spinal cord injury (chronic spinal). These two preparations allowed us to also examine whether the SK currents were smaller in chronic than acute spinal rats, which would contribute to the large net PICs seen in chronic spinal rats.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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, 2004). All recordings were made from the whole sacrocaudal spinal cord that was removed from the rat with an acute S2 sacral transection and maintained in vitro (as detailed below). This transection was made in chronic spinal rats just rostral to the original chronic spinal injury to not further damage the sacrocaudal cord. All experimental procedures were approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee. In vitro preparation
Details of the experimental procedures have been described in previous publications (Bennett et al. 2001; Li and Bennett 2003; Li et al. 2004b). Briefly, all rats were anesthetized with urethane (0.18 g/100 g; with a maximum dose of 0.45 g), and part of the caudal cord (between the T13 and L6 vertebrae) was exposed and kept wet with modified artificial cerebrospinal fluid (mACSF). The rat was given pure oxygen for 5 min before being transferred to a dissection chamber containing mACSF. All spinal roots were removed, except the S4 and all caudal ventral roots (the latter where taken as a single bundle and collectively referred to as Ca1, because Ca1 is the most prominent caudal root). The cord was secured by gluing its dorsal surface to a small piece of nappy paper. After 1.5 h of incubation in the dissection chamber at room temperature (20–21°C), the cord was transferred to a recording chamber containing normal ACSF (nACSF) maintained at 23–25°C with a flow rate >5 ml/min. The cord was fixed to the bottom of the recording chamber with the ventral side up by pinning the nappy paper to the Sylgard base of the chamber. After a 1-h wash period in the nACSF, to allow residual anesthetic and kynurenic acid to wash out, the nACSF was recycled as follows: it was oxygenated in a 200-ml source bottle, run through the recording chamber, collected, filtered, and finally returned to the source bottle with a pump (Harvey et al. 2005b). Because of the large volume (200 ml) of the source bottle and the small volume of the spinal cord (<0.05 ml), accumulation of metabolic byproducts from the spinal cord was likely negligible.
Intracellular recording
Intracellular recording methods were as described in Li and Bennett (2003) and are briefly summarized here. Sharp intracellular electrodes were filled with 1 M K-acetate and 1 M KCl and beveled to a resistance of 25–30 M
using a rotary beveller (Sutter BV-10). A stepper-motor (660, Kopf) was used to advance the electrodes into the ventral horn, and intracellular recordings from motoneurons were made with an Axoclamp 2b intracellular amplifier (Axon Instruments, Union City, CA) running in discontinuous current clamp (DCC; switching rate 5–8 kHz, output bandwidth 3.0 kHz) or discontinuous single-electrode voltage clamp (SEVC; gain 0.8–2.5 nA/mV) modes and sampled at 6.7 kHz with a Clampex system (Axon Instruments). The S4 and Ca1 ventral roots were wrapped around Ag/AgCl electrodes above the recording chamber and sealed with grease (Chemplex 825 silicon compound grease and Dow Corning High Vacuum grease), which allowed for antidromic stimulation identification of motoneurons. Motoneurons with a resting potential below –60 mV and antidromic spike overshoot >0 mV were considered healthy and used for recording.
Drugs and solutions
Two kinds of ACSF were used in these experiments: mACSF in the dissection chamber before recording and nACSF in the recording chamber. The mACSF consisted of (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. Normal ACSF consisted of (in mM) 122 NaCl, 24 NaHCO3, 2.5 CaCl2, 3 KCl, 1 MgCl2, and 12 D-glucose. Both types of ACSF were saturated with 95% O2-5% CO2 and maintained at pH 7.4. Additional drugs were added as required, including 2 µM TTX (Alamone Labs), 0.15 µM apamin (Alamone Labs), 15 µM nimodipine (Sigma), and 5 µM conotoxin G-VII-C (Sigma). The spinal cords were briefly exposed to nACSF solution containing 0.04% pronase E (Helixx Technologies) for 10 s before recording to weaken the pia of the spinal cords and to allow for easier penetration (Buschges 1994).
Persistent inward currents in current- and voltage-clamp recording
Slow triangular current ramps (0.4 nA/s) were applied to the motoneurons to induce firing and associated AHPs, measure basic cell properties, and evoke plateau potentials. The input resistance (R) was measured during the ramp over a 5-mV range near rest and subthreshold to PIC onset. Instantaneous firing frequency (F) was computed from the current ramp recordings using Clampfit 9.0 software. Without TTX present, the plateau potential was seen as a subthreshold rapid change in membrane potential before the firing threshold and a long afterdepolarization after cessation of firing. With TTX present, a full plateau was evoked during a current ramp that produced a sustained depolarization riding on top of the passive triangular ramp response (Li et al. 2004a). Input capacitance (C) was measured from the response to a 0.4-nA hyperpolarizing current step by estimating the time constant, 
, of an exponential fit to the response, using the relation: = R x C.
Slow triangular voltage ramps (3.5 mV/s) were applied to measure the PICs in voltage clamp. During the upward portion of the ramp, the current initially increased linearly with voltage in response to the passive leak conductance. A linear relation was fit in this region (5–10 mV below the PIC onset) and extrapolated to the whole range of the ramp. At more depolarized potentials, as the PIC threshold was reached, there was a downward deviation from the extrapolated leak current. The amplitude of the PIC was measured as the peak amplitude of this downward deviation. Large PICs usually caused a negative slope region (NSR) in the current response. The onset voltage for the PIC (Von) was defined as the voltage at which the I-V slope first reached zero (Li and Bennett 2003). The current value corresponding to Von was defined as Ion. The width of the PIC (Vw) was defined as the width of the valley formed by the NSR measured at the current Ion in the I-V plot. That is, Vw = Vjump – Von, where Von and Vjump were the first and second potentials, respectively, at which Ion occurred during the upward current ramp. Previously, it has been shown that the width of the PIC (Vw) corresponds to the amplitude of the plateau potential that is produced by the PIC (Li and Bennett 2003). The changes in conductance caused by the PIC were estimated by the measuring the slope of the I-V relation at rest (resting slope-conductance, S1; same as leak conductance) and comparing this to the slope-conductance well after the PIC was fully activated (after NSR ended), when the I-V slope had again reached a fairly steady state (linear region; termed steady-state S2 slope conductance; see details in RESULTS). Steady state was found to occur in the voltages above Vjump (typically –50 to –45 mV), and the S2 slope was thus measured by fitting a line to the segment of data 5 mV above Vjump. The spike voltage threshold (Vth), was averaged from five consecutive spikes, starting with the second spike on the up ramp, and was taken as the voltage at which the rate of change of potential was >10 V/s (Li et al. 2004a).
Analysis of AHP and its conductance and voltage dependence
The postspike AHP was quantified when the membrane potential was held subthreshold to repetitive firing by evoking spikes with antidromic stimulation of the ventral roots. The maximum AHP amplitude was taken as the amplitude of the medium-duration afterhyperpolarization (mAHP), and the duration was quantified as the duration of the mAHP at half its amplitude (half duration). The dependence of the AHP amplitude on potential was measured by systematically varying the potential before the antidromic stimulation with a bias current. As described in RESULTS, the AHPs measured in this way had a linear voltage dependence, and we thus fit a linear regression of the AHP versus potential relation and made the following calculations to quantify this voltage dependence. The conductance of the SK channels is known not to have a voltage dependence itself (Lancaster et al. 1991), and so it can be thought of a simple fixed conductance (GAHP). Therefore the SK current (ISK) underlying the AHP can be approximately modeled as the product of this SK conductance and the difference between the potential (V) and the reversal potential for potassium (EK). That is, ISK = GAHP(V – EK). This SK current (ISK) ultimately acts through the leak conductances GL and the SK conductance itself to produce the mAHP
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Data analysis
Data were analyzed in Clampfit 8.0 (Axon Instruments), and figures were made in Sigmaplot (Jandel Scientific). Data are shown as mean ± SD. Unless otherwise stated, a paired Student's t-test was used to test for statistical differences before and after drug applications, with a significance level of P < 0.05. Unpaired t-test were used to compare acute and chronic spinal rats. One motoneuron was recorded per rat for each experimental condition tested.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Intracellular recordings were made from motoneurons of chronic and acute spinal rats, whereas the whole sacrocaudal spinal cord was maintained in vitro. In the main group of cells, we first examined firing and AHP properties in nACSF (n = 18 chronic and n = 8 acute). Then, in about one half these motoneurons, we applied apamin alone to examine SK current contributions to firing (n = 9 chronic spinal, n = 5 acute spinal). In the remaining motoneurons and some additional motoneurons, we applied TTX followed by apamin to examine SK current involvement in the Ca PIC (n = 22 chronic, n = 8 acute spinal rats). The basic membrane properties (R, C, and Vm) of these motoneurons are summarized in Table 1. For most of the results, we focus on chronic spinal rats because they have larger, more easily quantifiable PICs, although both acute and chronic spinal rats were found to have similar SK currents.
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In control nACSF, the sodium spike that was evoked by either antidromic stimulation of the ventral roots (Fig. 1A) or current injection (Fig. 2, A and C) was always followed by a postspike AHP in both chronic (n = 18) and acute (n = 8) spinal rats. This AHP had a classic combination of a small fAHP followed by a pronounced mAHP (Viana et al. 1993). At the end of a series of spikes, there was a final AHP (Fig. 2, A and D), but this was not longer than a typical mAHP, and thus there was not the very slow AHP seen in other neurons (no sAHP) (Lasser-Ross et al. 1997).
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mAHP limits the firing rate and PIC activation
When activated by a current injection, motoneurons of chronic (n = 18) or acute (n = 8) spinal rats typically fired at <20 Hz (Fig. 2A), corresponding to intervals greater than the time to peak of the mAHP (36.5 ± 6.5 ms) (Kernell 1965). The firing rate was restricted to this low range in large part because of the mAHP, because when the mAHP was blocked with apamin (0.15 µM), much higher firing rats were achieved, reaching 100 Hz for the same standard current ramp (Fig. 2B). In chronic spinal rats in apamin, the firing rate jumped extremely rapidly to this high 100-Hz rate (in an all-or-nothing manner; in 8/9 chronic spinal rats tested) because of the activation of a large PIC and associated plateau potential (afterpotential) that could be seen after the termination of firing on the downward current ramp (Fig. 2B, arrow). Before apamin, there was a smaller plateau (Fig. 2A, afterpotential at arrow), because the mAHPs limited the PIC activation (Li et al. 2004a). Both before and after apamin, some firing persisted on the downward current ramps at currents well below the current to recruit the motoneuron (self-sustained firing, 8/9), although this firing was always much faster after apamin (8/8). This self-sustained firing has previously been shown to be largely caused by Ca PICs (nimodipine-sensitive; Li et al. 2004a) and confirms the presence of a Ca PIC.
Motoneurons without a large-enough Ca PIC to produce a plateau (i.e., in acute spinal rats, 5/5; and in 1/9 chronic spinal rats) fired roughly in proportion to the injected current before apamin, with no self-sustained firing (data not shown, but see Bennett et al. 2001 and Li et al. 2004a). However, with the application of apamin, even these cells reached very high rates (near 100 Hz) with standard-size current ramps that normally only produced 10- to 15-Hz firing without apamin, but this firing only reached a peak (100 Hz) at the peak of the current injection rather than at the onset of firing, because there was not a large Ca PIC (data not shown).
Slow firing does not require the mAHP
In nACSF, very slow firing often occurred near the threshold for derecruitment, as previously described for cells with large Na PIC, especially in chronic spinal rats (Harvey et al. 2005b; Li et al. 2004a). This very slow firing was at intervals that well exceeded the mAHP duration (see last few spike intervals in Fig. 2D; <5 Hz; n = 9/9 chronic spinal rats tested), and has previously been shown to result from a near-threshold slow oscillation of the Na PIC, where the Na PIC causes a characteristic slow ramp (at double arrows) and an acceleration in the membrane potential that triggers a spike (*; referred to as ramp and acceleration profile; this profile is blocked by a low dose of TTX that blocks the Na PIC but not the spike; see details in Li et al. 2004a). After each spike, the AHP deactivates the Na PIC, and the Na PIC-dependent ramp and acceleration profile occurs again and triggers another spike (see 2 interspike intervals shown in Fig. 2D). The Na PIC continues to oscillate in this slow manner, producing the slow firing, with one spike per oscillation cycle.
After the block of the mAHP with apamin, this slow firing persisted, again with a characteristic Na PIC–dependent ramp and acceleration profile (at double arrows and * in Fig. 2F), producing long interspike intervals (n = 9/9 chronic spinal rats). This Na PIC–dependent ramp and acceleration profile followed immediately after the fAHP (Fig. 2F), whereas, without apamin, it occurred later, after the mAHP (above dashed line in Fig. 2D). Thus the hyperpolarization from the fAHP alone is apparently sufficient to deactivate the Na PIC, after which, the Na PIC activates again in the characteristic ramp and acceleration profile (see 2 interspike interval at left of Fig. 2F).
Importantly, in apamin, this Na PIC–dependent ramp and acceleration profile also occurred during more rapid firing, with interspike intervals where it would normally be obscured by the mAHP. Thus the Na PIC plays a role at all firing rates, although its actions overlap with that of the AHP at higher rates.
Subthreshold to the Na PIC activation (near –60 mV, which is also subthreshold to repetitive firing; Li et al. 2004a) this ramp and acceleration profile never occurred after a spike evoked by an antidromic ventral root stimulation in apamin (Fig. 1B), consistent with it being mediated by the Na PIC. Instead, the membrane potential came back to rest within 20 ms after the spike (mAHP completely blocked by apamin).
AHP depends on N-, P/Q-type and not L-type calcium currents
When all the calcium currents were blocked with cadmium (400 µM), the mAHP was completely blocked (n = 4/4 cells tested), consistent with the above conclusion that the mAHP is mediated by a calcium-activated potassium current (SK current). A selective block of the high voltage–activated N- and P/Q-type calcium channels with -conotoxin MVIIC (5 µM, n = 5) mimicked the effects of cadmium, again completely eliminating the mAHP (fAHP also eliminated, n = 5/5; Fig. 3B). This conotoxin had no effect on the low voltage–activated Ca PIC (see Li and Bennett 2003). In contrast, a block of the L-type calcium channels with nimodipine had no effect on the AHP (mAHP or fAHP, n = 11/11; Fig. 3, C and D), whereas nimodipine completely blocked the Ca PIC (n = 12/12; which is thought to be Cav1.3 L-type calcium channel mediated; see Fig. 5 described below and Li and Bennett 2003). Thus the mAHP in motoneurons is produced by an apamin-sensitive SK current that is activated by calcium that flows through N- and P/Q-type high voltage–activated channels during the spike. Furthermore, the L-type calcium channels do not seem to play a major role in the AHP, in contrast to their major role in producing the low voltage–activated Ca PIC (see details below).
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To compare the AHP across cells and between acute and chronic spinal rats, we found it necessary to first quantify the strong voltage dependence of the mAHP, which occurs because the mAHP current is a potassium current close to its reversal potential (EK; see METHODS; the SK channel conductance GSK itself is voltage-independent; see Introduction). That is, when the mAHP was measured from antidromically evoked spikes while the membrane potential was systematically varied with a bias current (e.g., –70 mV shown in inset of Fig. 4A), the mAHP amplitude varied linearly with the membrane potential [subthreshold to repetitive firing, Fig. 4A; because SK current = GSK(V – EK)]. As expected, apamin blocked the mAHP at all potentials (Fig. 4A, triangles), and thus, regardless of the potential, the mAHP resulted from just an SK potassium current (n = 5 acute and 9 chronic spinal rats tested). At the fixed potential of –70 mV (near rest), the mAHP was not significantly different in acute and chronic spinal rats (n = 7 and n = 15, respectively, tested in nACSF, although not all subsequently tested with apamin). The reversal potential for the mAHP, and thus also EK, was on average –80.1 ± 4.6 and –82.0 ± 2.9 mV in acute and chronic spinal rats (computed where regression lines crossed 0 mV), respectively, and there was no significant difference between these two groups. Because the potassium concentration gradient was not likely to change from cell to cell, the variability in EK was probably just a result of error in recording absolute potential (e.g., tip potential drift). Thus to compensate for this error in Fig. 4C, we corrected the potentials in all cells so that they had a common EK, equal to the overall mean EK (–81 mV). After this correction, there was a very close overlap of the AHP-versus-potential relations, suggesting that, at a given potential, the AHP was very similar in all cells. In particular, the mAHP at the corrected –70 mV potential was again not significantly different in acute and chronic spinal rats (n = 7 and 15, respectively). Of course, the reversal potential for mAHP only represents the reversal potential as seen at the soma, and if the mAHP currents are distal to the soma, it might be expected to be more hyperpolarized. However, because mean reversal potential is only –81 mV and the mAHP varies remarkably linearly with potential, it is likely that the mAHP currents are from channels near the soma.
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L-type calcium current of the Ca PIC activates an SK current in chronic spinal rats
In chronic spinal rats, when the membrane potential was slowly increased under voltage-clamp conditions, a large PIC was activated and seen as a downward deviation from the extrapolated subthreshold linear leak current (thin line); this PIC usually produced an outright negative slope region in the I-V relation (NSR; Fig. 5C). Part of this PIC (about one half) was blocked rapidly by TTX, which has previously been attributed to a persistent sodium current in these motoneurons (Na PIC; n = 22/22 chronic spinal; Fig. 5A; Li and Bennett 2003). The remaining current in TTX was blocked by nimodipine (15 µM; leaving only a linear leak current-voltage relation below –40 mV; n = 12/12 tested) and thus was mediated by an L-type Ca PIC (Fig. 5C) (see details in Li and Bennett 2003). This Ca PIC is the focus of the remainder of the Results section. In chronic spinal rats, the Ca PIC was activated with an average onset threshold of –58.6 ± 4.1 mV and reached a peak of 2.2 ± 1.2 nA at –54 ± 3.7 mV (n = 22). With decreasing current ramps (Fig. 7, described later) the Ca PIC was deactivated at a significantly lower voltage than its onset voltage (Voff; 12.5 ± 3.3 mV lower). Acute spinal rats had similar, but much smaller, Ca PICs, which are described later (Fig. 9).
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Neither the resting membrane potential nor the leak conductance (1/R) was significantly affected by apamin (n = 22 chronic and 8 acute spinal rats tested); thus there was not a resting calcium current (different from L-Ca) that activated an SK current.
SK current produces a steep increase in conductance (the wall)
In nACSF, during the increasing voltage ramp, the slope of the I-V response relation was always much steeper after the full activation of the Ca PIC (termed slope-conductance S2 and measured after peak of Ca PIC at Vjump; near –45 mV; Fig. 5D, dashed vertical line) compared with before the Ca PIC activation (slope conductance S1; same as leak conductance; Fig. 5, A–D; n = 22/22 chronic spinal rats). The greater slope conductance after Ca PIC activation (S2/S1 = 2.5 ± 1.2) resulted from 1) the increased conductance arising from the L-Ca current (Li et al. 2004a) and 2) the activation of SKL currents. That is, application of apamin significantly lowered the S2 slope relative to the S1 slope (S2/S1 ratio = 1.6 ± 0.5; n = 22). Characteristically, in apamin, the current during the voltage ramp increased linearly after the initial onset of the L-Ca (after NSR; n = 22/22 chronic spinal), consistent with the onset of a steady L-Ca current that simply increased the overall conductance (by 20% of leak conductance in Fig. 6B). In contrast, before apamin (Fig. 6A; n = 22), the current during the voltage ramp increased much more steeply after the initial onset of the Ca PIC (after NSR), with a nonlinear parabolic shape induced by the SK current, as though intracellular calcium was accumulating. Thus the steep apamin-sensitive S2 slope provides a distinctive feature of the I-V relation that indicates the presence and size of the SKL current. By subtracting the S2/S1 conductance ratios before and after apamin, we get the ratio of the SKL conductance to the leak conductance (S1), which was 0.85 ± 0.8, indicting that the SKL current produced a conductance similar in size to the leak conductance (and AHP conductance; Table 2). This steep apamin-sensitive S2 slope conductance was eliminated by either nimodipine (n = 12/12; Fig. 5C) or apamin (n = 22/22; Fig. 5D), further supporting the idea that the L-Ca current activates an SK current (SKL).
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Apamin-sensitive oscillations at the onset of the Ca PIC
When the Ca PIC was large enough to produce an NSR in the I-V relation (in voltage clamp), a plateau potential was always seen in current clamp during a ramp current injection, induced by the instability of this NSR (Li and Bennett 2003; Schwindt and Crill 1982), which occurred in all chronic spinal rats (n = 22/22). This plateau had a characteristic sharp overshoot at its onset (Fig. 7, D and G), like a spike, although we specifically refer to it as an overshoot spike, to distinguish it from the much larger calcium spikes induced by TEA in motoneurons (Hounsgaard and Mintz 1988). After this calcium overshoot-spike, an afterhyperpolarization (CaAHP) always occurred (n = 22/22), which tended to reduce the plateau. The CaAHP was blocked by apamin (n = 22/22) and thus was mediated by an SKL current. In some cells, this CaAHP was sustained during the plateau and thus served to simply reduce the plateau amplitude (Fig. 7, G–I; n = 10/22 cells). However, in other cells (12/22), the CaAHP reached a peak hyperpolarization in 200–300 ms and was then turned off, presumably because the CaAHP reduced the L-Ca current underlying the plateau and overshoot-spike. A second calcium overshoot-spike then followed as the L-Ca current reactivated, followed by a second CaAHP. This oscillation of the L-Ca and the SK current continued for a few cycles until a steady-state activation of the L-Ca and the SK currents was reached. This oscillation was always blocked by apamin (n = 12/12), confirming the role of the SKL currents (Fig. 7, D–F). Furthermore, in apamin, the steady-state depolarization was greater than without apamin, showing that the SKL current also produced a steady hyperpolarization throughout the plateau, reducing its size.
Interestingly, during voltage ramps, a similar oscillation was also seen at the onset of the Ca PIC (Fig. 7A), even though this current was measured under voltage clamp, which should at least clamp the potential near the soma, avoiding local oscillations of channels. These oscillations were again blocked by apamin (n = 12/12; Fig. 7B), and thus again were mediated by SKL currents. Likely, these oscillations resulted from calcium overshoot-spikes interacting with CaAHPs, as described above under current clamp, but occurring in the distal unclamped dendrites. Indeed, all cells that had these Ca PIC oscillations under voltage clamp conditions (n = 12) also had calcium overhoot-spikes and CaAHP oscillations under current-clamp conditions (e.g., Fig. 7, A and D, is from same cell). This provides indirect evidence that the SKL currents are of dendritic origin, like the L-Ca currents, and perhaps unlike the SKAHP currents.
SKL current is slow to activate
To examine the time-course of the SKL currents, we used a series of long voltage steps in chronic spinal rats (n = 11 tested). Subthreshold to the Ca PIC, these steps produced a current response proportional to the leak current (trace 1 and 2 in Fig. 8, B and E and left of Fig. 8F). For larger steps above threshold, the Ca PIC was activated and reduced the current below the expected leak current (Fig. 8, B and E, traces 3 and 4, and right of F). As previously reported, with steps just above threshold for the Ca PIC (trace 3), the Ca PIC activated with a substantial delay of 1 s or more (Li and Bennett 2003). Usually, a few of the characteristic SKL-mediated oscillations (apamin-sensitive) occurred during this slow Ca PIC activation (like on current ramps; Fig. 7B). With larger voltage steps, Ca PIC activated more rapidly, starting at the onset of the step and reaching 10% of its maximum within 45 ± 7 ms (n = 11), although steady-state activation was not reached for 1 s. In contrast, the first sign of the SKL current onset, seen as small SK-mediated oscillation (1st inflection), did not occur until 170 ± 19 ms (n = 11). Thus the SK current onset was delayed by 100 ms, relative to the underlying L-Ca current that activated it, and the L-Ca activation of the SK currents was slower than the sodium spike-mediated activation of the SK currents (AHP onset in <10 ms), suggesting differences in spatial location.
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After termination of the voltage steps, the Ca PIC always took 0.5 s to turn off, producing a characteristic tail current, with a mean duration of 323 ± 372 ms before apamin (time to 50% decay in tail current; n = 11). Associated with this tail current, there was a residual SK current that also decayed off slowly (Fig. 8C). Blocking the SK current with apamin ultimately led to a significantly longer tail current (783 ± 965 ms; n = 11). Thus the outward SK current acted to speed the deactivation of the inward L-Ca current.
SKL currents are relatively larger in acute than chronic spinal rats
Motoneurons of acute spinal rats had small but significant Ca PICs (n = 8 tested), as previously described (Harvey et al. 2005b). These Ca PICs were not large enough to produce an NSR in the current response to a voltage ramp and instead only induced a negative deflection in current relative to the extrapolated leak current (n = 8/8; PIC indicated by arrow in Fig. 9A). Because of the lack of NSR, there were not plateaus seen with current injection in these motoneurons (data not shown). Apamin significantly increased these PICs (arrow larger in Fig. 9B; see also Fig. 10; n = 8), indicating that there was an apamin-sensitive SKL current (Fig. 9D), as in chronic spinal rats. Also, apamin significantly decreased the S2 slope (Fig. 9B), as in chronic spinal rats, indicating a substantial SKL conductance in the region of the wall near –40 mV.
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40%, compared with in chronic spinal rats (Fig. 10B; n = 22). Barium also eliminates apamin-sensitive SK currents
To further examine the calcium-activated currents, we substituted barium (Ba) for calcium (Ca) as the charge carrier through the calcium channels (n = 5 chronic spinal rats). This effectively eliminates all calcium-activated currents (including the SK current) because barium is much less effective than calcium in activating these currents (Enomoto et al. 1991). As shown in Fig. 11B, this barium substitution always led to an increase in the PIC (labeled L-Ba) compared with in normal calcium-containing ASCF (Ca PIC), very much as we saw with apamin. Unfortunately, because the barium current through calcium channels is generally known to be larger than the calcium current through the same channels (Hille 2001), part of this increase in the PIC may have been caused by the differing current-carrying capability of barium. However, the shape of the I-V relation in barium was remarkably like that in apamin (Fig. 6B), suggesting that the barium substitution caused an elimination of SK currents that produced the characteristic very steep S2 slope in nACSF. Indeed, this was the case because applying apamin while in barium had no significant effect on the PIC (L-Ba is apamin resistant; n = 5). Also, in barium alone, like in apamin, the S2 slope after the PIC activation was very shallow and close to the leak conductance (S1; Figs. 11A and 6A). On average, the ratio S2/S1 was significantly reduced by barium (n = 5; from 2.5 ± 0.86 to 1.5 ± 0.17; Fig. 11E), like with apamin. Furthermore, the S2/S1 ratio, at 1.5, was not significantly different in barium and apamin, indicating that both apamin and barium eliminated a similar conductance (barium and apamin sensitive) that was about equal to the leak conductance (2.5 – 1.5 = 1).
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). Thus it is difficult to determine whether barium blocks more calcium-activated currents than does apamin. Barium reveals an unusual high voltage–activated PIC and plateau
Surprisingly, in barium there was always a very large high voltage–activated PIC (high voltage–activated Ba PIC) that was activated above –30 mV (2 in Fig. 11C; n = 5) and not seen in apamin (n = 22). This was activated at a distinctly higher potential than the normal low voltage–activated PIC (1 in Fig. 11C; activated at about –50 mV, as in nACSF or apamin), and thus remarkably discrete low and high voltage–activated PICs were seen in the same cell (Fig. 11C, 1 and 2, respectively). This high voltage–activated Ba PIC produced a large sustained high voltage–activated Ba plateau potential in current clamp that was very difficult to turn off (Fig. 11D, 2), as described in detail below. This large high voltage–activated Ba PIC may have been in part an artifact of barium application because it was not seen without barium even with the large depolarizations possible in apamin (Fig. 6C). Probably, this high voltage–activated Ba PIC was a result of a loss of inactivation of high voltage–activated calcium channels (N, P/Q-type) resulting from removal of calcium from the bath because calcium channel inactivation is known to have a strong dependence on calcium itself (calcium-dependent calcium inactivation) (Cens et al. 2005; Zong et al. 1994).
During the onset of the high voltage–activated Ba plateau, there was at times a burst of very fast Ba spikes (at 40 Hz), as shown in the inset in Fig. 12C (n = 3/5). These spikes were much narrower, faster, and larger than the overshoot-spikes seen at the onset of the low voltage–activated Ca PIC, described in Fig. 7. Thus it seems that the channels that mediate the high voltage–activated PIC (likely N,P-type) are more rapid than the L-type calcium channels that mediate the low voltage–activated Ca PIC.
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The Ca PICs had a voltage threshold for activation (measured at electrode, in soma) that was significantly higher than the threshold for deactivation (by 10 mV, hysteresis described above). This has previously been attributed to the dendritic nature of these currents and the inevitable poor space-clamp over these distal dendrites in very large dendritic trees of motoneurons (Hounsgaard and Kiehn 1993; Hultborn 2002). However, in nACSF, this poor space clamp was not a problem because the Ca PICs or Ca plateaus could still be fully deactivated after they were activated (Fig. 12, A and D), and thus quantified, albeit at a distance from their dendritic location. However, in a few cells in apamin (n = 4/26; chronic) and most cells in barium (n = 4/5; chronic), the PIC (L-Ca current or Ba PIC) was uncontrollable, in the sense that, once activated, it could not be completely deactivated, regardless of the hyperpolarization applied. This loss of control over the L-Ca current (or Ba PIC) occurred in voltage clamp, as in Fig. 12E, or in current clamp, where associated plateaus could not be turned off, as in Fig. 12B.
Once we lost control over the L-Ca currents (or Ba PIC) on a voltage ramp (Fig. 13A and C), subsequent voltage ramps evoked smaller L-Ca currents (or Ba PICs) than before (Fig. 13, B and C) because a portion of the PIC was tonically activated (or inactivated). The situation was extreme in barium, where at times the Ba PIC remained fully activated, despite very hyperpolarized holding potentials, and a subsequent voltage ramp evoked no further PIC (Fig. 13C; n = 4/5). When held for many minutes at a hyperpolarized level, some Ba PIC could be evoked (data not shown), presumably because a portion of the Ba PIC eventually inactivated (allowing the dendrites to hyperpolarize and enabling some deactivation of the Ba PIC). Likewise, with a current ramp after the first uncontrolled plateau activation, a second current ramp produced a smaller plateau (data not shown). In these kinds of cells (15% of cells in apamin), we had to be cautious in comparing the pre- and postapamin results because, if the loss of control was not noticed, the L-Ca in apamin appeared smaller than the Ca PIC before apamin, leading to the erroneous conclusion that apamin blocked the calcium current itself. Thus in the results described above, only cell measured without loss of control of the PIC were used (22/26 cells). We interpret the loss of control over the calcium currents after a block of SK currents as indicating that a portion of the dendritic tree distal to the electrode had gone onto an uncontrolled L-Ca-mediated plateau. Why this only occurred in some cells in apamin (15%) is uncertain, although it may relate to how close the electrodes were to the center of L-Ca channel activation. In any event, these results show that the SK currents play a major physiological role of assuring that the L-Ca currents can be turned off after they are activated because such uncontrolled PICs were not seen without apamin or barium present.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Our results show that the low voltage–activated persistent calcium current in spinal motoneurons activates a calcium-activated potassium current that is directly blocked by apamin and indirectly blocked by nimodipine (SKL current). This outward SK current opposes the inward persistent calcium current, resulting in a smaller net current (Ca PIC, 20% smaller). Blocking this SK current leads to larger PICs and ultimately larger plateaus, thus explaining the earlier apamin studies of Hounsgaard and Mintz (1988). Previously, it has been suggested that SK currents oppose persistent calcium currents in this manner (Hultborn 1999), but this had not been directly confirmed in motoneurons. Because of its low activation voltage, its block with a relatively high dose of nimodipine (>10 µM; see RESULTS and Li and Bennett 2003), and its complete resistance to conotoxins (see RESULTS and Li and Bennett 2003), the persistent calcium current that activates these SKL currents is likely mediated by the Cav1.3 channels recently characterized by Xu and Lipscombe (2001). We refer to this calcium current as the Cav1.3 current.
High voltage–activated persistent calcium currents that are, in contrast, sensitive to conotoxins have been reported in motoneurons (N-, P-, Q-type; Carlin et al. 2000a; McCarthy and TanPiengco 1992; Powers and Binder 2003), and they may also activate SK currents. However, the role of these persistent currents is not obvious because, during normal firing, the memb
) and 7 cells from acute spinal rats (
). Thick straight lines show the average potassium reversal potential for motoneurons from chronic spinal rats and acute spinal rats, respectively, as labeled; there was no significant difference between these 2 groups. C: data from B corrected and replotted so that all cells have a common potassium reversal potential, Ek, corresponding to average Ek. D: mAHP current measured in voltage-clamp mode after a simulated spike (data not shown, 2-ms step of +70 mV). Gray trace shows corresponding mAHP evoked from a spike in current-clamp mode.
