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Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada
Submitted 14 October 2008; accepted in final form 21 October 2008
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ABSTRACT |
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INTRODUCTION |
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), the Vth for action potentials in lumbar motoneurons is hyperpolarized during fictive locomotion induced by stimulation of the mesencephalic locomotor region (MLR) and returns to the control level shortly after the termination of stimulation of MLR. A persistent change in Vth is also observed in 16-wk endurance-trained rats, where the Vth is found to be hyperpolarized in hindlimb motoneurons compared with the untrained rats (Beaumont et al. 2003). In contrast, the Vth of lumbar motoneurons is depolarized in rats in which the hindlimbs are unweighted for 2 wk (Cormery et al. 2005) and in cats that are chronically spinalized for 6 wk (Hochman and McCrea 1994). These studies demonstrated that Vth can be modulated for different motor states or behavior and the modulation of Vth could be mediated by different pathways. We have previously shown that application of serotonin (5-hydroxytryptamine [5-HT]) or norepinephrine or activation of descending serotonergic fibers produced a Vth hyperpolarization of ventral horn neurons in the isolated spinal cord of the neonatal rats (Fedirchuk and Dai 2004b; Gilmore and Fedirchuk 2004). However, the mechanisms underlying the depolarization of Vth remain unknown. Our modeling studies suggest that sodium channels play a dominant role in regulation of Vth and can result in either hyperpolarization (Dai et al. 2002; Gardiner et al. 2006) or depolarization of Vth (Cormery et al. 2005). Therefore we expect sodium channel modulation to be a key mechanism regulating Vth.
It has become evident that sodium channels themselves are the target of modulation mediated by phosphorylation at specific sites (see Cantrell and Catterall 2001; Catterall 2000). In particular, phosphorylation of the 
subunit by protein kinase C (PKC) decreases the peak Na+ current in reconstituted brain sodium channels (Costa and Catterall 1984; Murphy and Catterall 1992; Numann et al. 1991). Na+ channels can be phosphorylated through activation of different neurotransmitter pathways. Phosphorylation of Na+ channels by cAMP-dependent protein kinase A (PKA) decreases peak INa in cultured brain neurons (Li et al. 1992), mammalian cells (Li et al. 1992, 1993), and Xenopus oocytes (Gershon et al. 1992; Smith and Goldin 1996). This phosphorylation has been shown to be voltage dependent and mediated by activation of D1-like dopamine receptors in acute isolated hippocampal neurons (Cantrell et al. 1997, 1999). Phosphorylation of Na+ channels by activation of PKC also reduces sodium currents and this PKC-mediated phosphorylation is shown to be activated by muscarinic acetylcholine receptors in rat hippocampal neurons (Cantrell et al. 1996). In mouse prefrontal cortex neurons, activation of PKC through 5-HT2a/c receptors decreases the rapid inactivating INa by reducing the maximal INa and shifting the fast inactivation voltage dependence (Carr et al. 2002). Many studies have explored the PKC pathway activated by 5-HT1A and 5-HT2 receptors (for review see Raymond et al. 2001). All these studies suggest that PKC-mediated modulation of INa via neurotransmitters is a fundamental way for the nervous system to regulate the neuronal excitability, although little information is available about this modulation in spinal ventral neurons.
Since PKC activation reduces peak sodium currents, it may be expected that PKC activation decreases neuronal excitability. This has been shown as the suppression of intrinsic bursting (Alroy et al. 1999) and the down-regulation of the persistent Na+ current in rat hippocampal pyramidal cells (Mittmann and Alzheimer 1998) and a reduction of dendritic excitability in mouse cortex pyramidal neurons (Carr et al. 2002) when PKC pathways are activated by PKC activators in these neurons. In contrast, however, activation of PKC has also been shown to increase neuronal excitability through a hyperpolarization of voltage threshold for action potentials in mouse neocortical neurons (Astman et al. 1998) or amplification of subthreshold depolarization in rat pyramidal neurons (Franceschetti et al. 2000). The increased neuronal excitability is attributed to the PKC-mediated hyperpolarization of onset voltages of persistent inward current (PIC). These contradictory findings suggest that 1) PKC is involved in regulating neuronal excitability; 2) the modulation of PIC by the PKC pathway could play an important role in this regulation; and 3) activation of PKC may exert a different influence on neuronal excitability in different systems.
Modulation of sodium currents via the PKC pathway has been studied intensively in many neuron types and systems (Cantrell and Catterall 2001; Catterall 2000) and the PKC-mediated modulation of PIC has also been reported in rodent brain neurons (Astman et al.,1998; Franceschetti et al. 2000). However, little is known about the modulation of INa and PIC in the spinal motor system, especially the interactions between the PKC and serotonergic pathways in modulating neuronal excitability. The goals of this study are to test the hypothesis that alterations in activity in the PKC pathway modulate the transient sodium currents and persistent inward currents of spinal ventral neurons of neonatal rats and alter the voltage threshold and neuronal excitability. This study also explores the interaction between the PKC- and 5-HT–mediated modulation of the Vth and demonstrates multiple pathways existing for modulation of Vth. Preliminary data have been reported in abstract form (Fedirchuk and Dai 2004a).
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METHODS |
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Preparation of slices
The slice experiments were carried out on neonatal (postnatal day 1 [P1] to P5) Sprague–Dawley rats. The rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg). After decapitation, the rats were eviscerated and pinned ventral side up in a Sylgard-lined dish filled with 4°C dissecting artificial cerebral spinal fluid (aCSF) bubbled with 95% O2-5% CO2. The spinal cords were then dissected. The lumbar segments (T13–L6) were isolated and introduced into a plastic dish filled with 1–5% agar solution (34–36°C). The dish was then placed in ice water for cooling down the temperature for about 1 min. The spinal cord was blocked and mounted into a Leica (VT 1000S) vibrating microtome filled with 4°C dissecting aCSF and bubbled with 95% O2-5% CO2. Transverse slices were cut at 200- to 250-µm thickness and transferred to recording aCSF for 
1 h before the patch-clamp recordings. The slices were then transferred to a recording chamber mounted in the stage of an upright Olympus BX50 microscope fitted with differential interference contrast optics. The chamber was perfused with recording aCSF at a rate of 0.5–1 ml/min, bubbled with 95% O2-5% CO2.
Neurons in ventral areas (lamina VII–X) were visualized using an infrared cube and recorded in whole cell patch clamp using glass pipette electrodes. The pipette electrodes were pulled from borosilicate glass (MTW 150F-4, WPI) using a P-87 puller (Sutter Instruments) and had resistances of 5–8 M
when filled with intracellular solution. A MultiClamp 700A patch-clamp amplifier, Digidata 1322A A/D converter, Minidigi 1A, and pClamp 9.0 software (all from Axon Instruments) were used for data acquisition. The whole cell patch recordings were made in both voltage- and current-clamp mode. Bridge balance and capacitance compensation were made before starting the recordings. Series resistance was monitored (usually <30 M) and compensated. Data were low-pass filtered at 3 kHz and sampled at 10 kHz. The data were analyzed using self-written codes with Igor Pro (4.0) and Axon Clampfit (9.0). Student's t-test and 
2 test were performed with statistical significance defined as P < 0.05. Results are shown as means ± SD. Activation and inactivation curves are presented as fitted means ± SE.
Measurement of cell membrane properties
The fast transient sodium current and persistent inward current (PIC) were generally recorded with potassium channel blockers (tetraethylammonium [TEA] and 4-aminopyridine [4-AP]) applied to the recording solution in this study. In some cells, however, these blockers were not used to study the membrane properties of the cells in both voltage- and current-clamp recordings. In this case the transient sodium current would not be isolated. Instead, it became a dominant component of the whole transient inward current (IT). Therefore we use IT to represent all the transient inward currents recorded in the present study, which could be either purely mediated or dominated by the fast transient sodium current. The IT and PIC were recorded in voltage-clamp protocol shown in Fig. 1. IT was recorded through a succession of 2-s voltage steps, with a step of either 2 or 5 mV, and a holding potential of –70 mV (Fig. 1A1). The maximal IT elicited by the voltage steps was used to calculate a set of IT parameters including the amplitude, half-width, maximal rise rate, and maximal decay rate. The step voltage at which the first IT was elicited was defined as the voltage threshold (Vth). In some cells the voltage dependence of IT was constructed by fitting the Boltzmann equation f(V) = 1/{1 + exp[(V1/2 – V)/Vc]} with the data recorded with protocols shown in Fig. 1A1 (for activation) and Fig. 1A2 (for inactivation), where the V1/2 is half-maximal activation (or inactivation) of IT and Vc is the slope of activation (or inactivation). To obtain the best-fitting values for the Boltzmann equation, interpolating points were used in some cells. The IT was recorded in the aCSF with normal sodium concentration (152 mM) in most of the experiments. In some cells, however, a lower sodium (50 mM) aCSF was used to measure IT to reduce the IT amplitudes and series-resistance voltage-clamp errors. In normal sodium concentration solution the IT is usually seen as all-or-none currents in most of the cells due to the limited space clamp of neurons recorded in fresh slice. Poor space clamp could cause a distortion of IT, which included the repetitive spikes (unclamped spikes) in one voltage step, delayed inward currents (longer time to reach peak), and bumps and notches in the inward currents. Recordings with any of these phenomena were excluded for calculation of IT parameters. Poor space clamp could also cause unchanged amplitude of IT in subsequent one or more depolarizing steps. Data characterized by this phenomenon were excluded for fitting the Boltzmann equation.
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Current-clamp recordings were also made to study the properties of the action potential (AP) in some cells. A 3-s step current with a step of 10–20 pA and a 10-s biramp current with peak of 300–500 pA were used to evoke repetitive firing. The rheobase, amplitude, half-width, maximal rise rate, and maximal decay rate of action potentials were measured from the spikes evoked with the minimum step current for repetitive firing. The voltage threshold for generation of action potentials in current-clamp recording was defined as the voltage at which the dV/dt 10 mV/ms in the rising phase of the APs and measured from the first spike in the spike train evoked by the ramp current protocol. The AP amplitude was measured from the Vth.
During the recording, the protocols of voltage or current clamp were repeated three to five times in each condition (control, drugs, washout, etc.), with about a 30-s interval between the recordings. Recordings were made 2–8 min after the drug application and repeated for 10–20 min before switching the conditions. The data were averaged from two or three trials for measurement of the parameters for IT, PIC, and AP described earlier. All recording were made at room temperature (20–22°C).
Difference in Vth measured in voltage- and current-clamp recordings
The term "Vth" is used in the present study as voltage threshold either for eliciting the fast transient inward current (IT) in voltage-clamp recording or for generating an action potential in current-clamp recording. Although the IT recorded in voltage clamp should be dominated by the same current underlying the APs recorded in current clamp, the Vth measured in voltage clamp is not the same as the Vth measured in current clamp. However, the validity of using both voltage- and current-clamp methods to assess Vth in this study was based on our experimental observations. Figure 1C shows Vth measured from 11 cells recorded in both voltage- and current-clamp protocols. Except for one cell, the Vth determined by voltage clamp is equal to or less than the Vth determined by current clamp. The difference between the mean Vth for the voltage-clamp protocol (–36.8 ± 7 mV) and Vth for the current-clamp protocol (–33.7 ± 6 mV) is only 3 ± 3 mV. The Vth determined by voltage-clamp protocol is also shown to be well correlated with the Vth determined by current-clamp protocol, suggesting that any change in either Vth would correspond to a change in the same direction for Vth measured with the other protocol, although the amount of change might be different (see Figs. 6 and 7 as examples). Based on these results we concluded that the Vth values obtained from either protocol describe the common property of the cells in initiating spikes. Thus for simplicity we use the Vth for values obtained from either protocol in the present study.
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EXTRACELLULAR SOLUTIONS. The dissecting aCSF (for slice preparation only) contained (in mM): NaCl (25), sucrose (188), KCl (1.9), NaH2PO4 (1.2), MgSO4 (10), NaHCO3 (26), kynurenic acid (1.5), glucose (25), and CaCl2 (1.0). The recording aCSF contained (in mM): NaCl (125), KCl (2.5), NaHCO3 (26), NaH2PO4 (1.25), D-glucose (25), MgCl2 (1), and CaCl2 (2.5). The pH of these solutions was adjusted to 7.3 with KOH. Osmolarity was adjusted to 305 mOsm by adding sucrose to the solution. In some experiments, the CaCl2 was replaced by BaCl2; CdCl2 (0.1–0.3) was added to the solutions to block the calcium currents. The low sodium concentration aCSF contained (in mM): HEPES (10), NaCl (50), MgCl2 (1), TEA-Cl (10), 4-AP (4), CsCl (50), CdCl2 (0.4), BaCl2 (2.5), and glucose (20) (pH = 7.3 with CsOH; osmolality 300–305 mOsm/L).
INTRACELLULAR SOLUTIONS. Solutions contained (in mM): CsCl (135), TEA-Cl (20), MgCl2 (5), BAPTA (2), HEPES (10), Na2ATP (5), and Na3GTP (0.5). In current-clamp recordings the intracellular solutions contained (in mM): K-gluconate (120), NaCl (5), HEPES (10), EGTA (5), MgCl2 (2), CaCl2 (1), Mg-ATP (5), and GTP (0.5) (pH was adjusted to 7.3 with KOH; osmolality was adjusted to 305 mOsm).
BLOCKERS. TEA (10 mM), 4-AP (4 mM), APV (20 µM), CNQX (10 µM), bicuculline (10 µM), and strychnine (10 µM) were used in most experiments. In some experiments tetrodotoxin (TTX, 2 µM) or nifedipine (20–30 µM) was used to differentiate the calcium or sodium component of PIC.
The PKC activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) was prepared as concentrated stock solutions and diluted to a final concentration of 10–30 µM with 0.02% DMSO immediately prior to recording in all slice experiments. Our experiments showed that DMSO in a concentration of 0.02% had no effect on IT and PIC.
The PKC inhibitor PKCI19–36 (PKCI, Calbiochem) was dissolved in intracellular solutions with a final concentration of 10–30 µM. 5-HT (10–30 µM) was added to the extracellular solution in some experiments. Drugs were applied using a gravity-based perfusion system. All chemicals were purchased from Sigma unless otherwise noted.
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RESULTS |
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; Franceschetti et al. 2000). In this study we first characterized the basic properties of these two currents and then investigated the modulation of these properties by PKC activation. Finally, we studied the interactions between the PKC and 5-HT pathways in the regulation of Vth. Characterization of basic properties of IT and PIC
The IT and PIC are characterized with the recordings from voltage-clamp protocols (see METHODS for details). Results from 95 cells show that the Vth for eliciting IT is at –42.7 mV, with IT amplitude of 422 pA, half-width of 10 ms, maximal rise rate of 533 pA/ms, and maximal decay rate of 284 pA/ms. Results from the same group of the cells indicate that the onset voltage for activation of PIC is –53.4 mV with PIC amplitude of 64 pA (see "Whole sample" in Table 1 for details). The cells recorded in this study were widely distributed from lamina VII to X. To explore differences in the properties of IT and PIC for neurons in different lamina, we classified these neurons into laminar groups ranging from VII to X. The results are shown in Table 1. These results indicate that cells in lamina IX, presumably including motoneurons, usually have a lower Vth for IT, larger IT amplitude, faster rise and decay rates of IT, higher onset voltage of PIC, and larger PIC amplitude, compared with the parameters of cells in other laminae. However, the statistically significant differences are observed only for the amplitudes of IT and PIC among the neurons from lamina VII to X. This might be due to fact that motoneurons are generally larger in size than interneurons and could possess a larger total conductance of IT and PIC than the interneurons in lamina VII, VIII, and X.
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Activation of PKC induced varied effects on Vth, although the OAG-induced reduction of IT was observed in all cells (35/35) and the reduction of PIC amplitude was seen in 94% of the cells (33/35). Figure 2 is an example in which OAG induced a reduction of IT and PIC and a depolarization of Vth. The IT of –450.3-pA amplitude was elicited by the voltage step to –35 mV in control (Fig. 2A, left). Bath application of 15 µM OAG produced a reduction of IT by 18% with 5-mV depolarization of Vth (Fig. 2A, right). The half-width of IT was increased by 15% and the maximal rise and maximal decay rates of IT were reduced by 19 and 28%, respectively (Fig. 2A, inset, dashed line for control). The current–voltage (I–V) curve (Fig. 2B, left) constructed by the peak of IT indicated that OAG reduced the IT and depolarized the activation of IT. This resulted in a 4.7-mV positive shift in half-maximal activation of IT, with an almost unchanged slope for the activation in Boltzmann equation (Fig. 2B, right panel and table). The OAG-induced reduction of PIC was also observed in the same cell. A 10-s biramp voltage was applied to the cell (Fig. 2C) and the PIC of 89.9 pA was evoked at an onset voltage of –51 mV (Fig. 2C, gray trace). A small reduction of PIC was observed in 5 min after bath perfusion of 15 µM OAG. By 15 min later, OAG reduced the PIC by 35% with a 3.2-mV depolarization of the onset voltage (Fig. 2C, black traces). Of 35 cells recorded with OAG, 7 of the cells showed a depolarization of Vth with reduction of IT in all these cells and reduction of PIC in 6 of the 7. One cell showed a depolarization of Vth with an increase in PIC. Although PKC activation reduced IT and PIC with depolarization of Vth, the alteration in Vth did not necessarily accompany the OAG-induced reduction of IT and PIC (see the following text).
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In more than half of the cells recorded with OAG, activation of PKC altered the properties of IT and PIC but did not change the Vth for IT. An example of OAG-induced reduction of IT and PIC with an unchanged Vth is shown in Fig. 3. The recordings were made in low sodium concentration (50 µM) aCSF in this cell. IT was measured as –607.6 pA with a Vth of –40 mV in control. OAG (10 µM) did not alter the Vth in this cell, but reduced the peak amplitude of IT by 60% to –240.8 pA, with a 46% increase in half-width and 60 and 77% decreases in maximal rise and decay rates, respectively. The reduction of peak IT in the whole range of step voltages is shown in the I–V curves in Fig. 3A (left). The changes in size of the IT at Vth = –40 mV is shown in the inset in Fig. 3A (dashed line for control; solid line for OAG). The voltage dependencies of IT described by using the Boltzmann equation (Fig. 3A, right) show that OAG induced a 3.6-mV right shift in the half-maximal activation of IT and a 2.3-mV left shift in half-maximal inactivation of IT, with small changes in the slopes (see table in Fig. 3A). PIC was reduced by OAG in the same cell. As shown in Fig. 3B, the PIC was measured as 146.9 pA with an onset voltage of –58.8 mV (Fig. 3B, gray trace). A small reduction of PIC was observed in only 3 min after OAG application. Eight minutes later, the PIC was reduced by about half. By 15 min after OAG administration, OAG reduced PIC amplitude by 83% to 25.3 pA and depolarized the PIC onset voltage by 7.3 mV (Fig. 3B, black traces). In a total of 35 cells recorded with OAG (2 of these cells were recorded in low sodium concentration aCSF), 20 of the cells showed no substantial changes in Vth, whereas the reduction of IT was observed in all of the cells and a reduction of PIC in 19 of the 20 cells. One of the cells showed an increase in PIC.
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It has been shown in previous studies that activation of PKC could induce variable changes in voltage dependence of fast inward sodium currents. Although no substantial change in voltage dependence of the sodium currents could be observed in many studies (Cantrell et al. 1996; Numann et al. 1991; Sigel and Baur 1988; West et al. 1991), both the depolarization of activation (Dascal and Lotan 1991) and hyperpolarization of activation and inactivation (Franceschetti et al. 2000) of the sodium currents have been reported. In the present study we show that activation of PKC could induce either a depolarization of Vth (Fig. 2) or leave Vth unchanged (Fig. 3). Activation of PKC could also result in a complete blockade of transient inward currents or hyperpolarization of Vth. Results from 35 cells recorded with OAG showed (Fig. 4A, white bars) that more than half of the cells (57%, 20/35) showed no change in Vth in the presence of OAG, whereas 20% of the cells (7/35) showed a depolarization of Vth and 9% (3/35) demonstrated a hyperpolarization of Vth. For another 14% (5/35) of the cells OAG blocked the IT.
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170 pA), in the maximal rise rate by 19% (153 pA), in the maximal decay rate by 14% (73 pA), and an increase in half-width by 25% (2 ms). The depolarization of Vth was small (4%, 2 mV) and not significant. OAG also produced a significant depolarization of PIC onset voltage by 8% (5 mV) and reduction of PIC by 49% (36 pA). The detailed results of OAG-induced changes in IT and PIC are summarized in Table 2. The data were classified into laminar groups ranging from VII to X to show the different responses of neurons in different lamina to OAG. In general, most of the effects of OAG were seen in all cell classes.
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Although PKC activation could produce variable effects on Vth, the reduction of IT and PIC were observed in almost all of the cells (100% for IT and 94% for PIC, n = 35). To confirm that the OAG effect on IT and PIC was mediated through activation of the PKC pathway we conducted additional experiments with PKCI in the recording pipette. A blockade of OAG effects on IT and PIC is shown in Fig. 5. The IT was measured as –467 pA with Vth of –35 mV in control with 20 µM PKCI in recording electrode (Fig. 5A). Ten minutes after bath application of 20 µM OAG, the IT was measured as –487 pA with an unchanged Vth. Although a small change was seen in spike shape and amplitude (Fig. 5A, inset), PKCI blocked the reduction of IT usually induced by OAG and Vth was unaltered. The PKCI also blocked the OAG-induced change in voltage dependence of IT in this cell (not shown). The blockade of OAG-induced changes in PIC by PKCI was also observed in the same cell. The PIC was evoked by a voltage ramp in both control (Fig. 5B, gray trace) and OAG (black trace) conditions. The PIC was measured as –182 pA with onset voltage of –60.3 mV in control. At 7 min after application of OAG, the PIC was measured as –180 pA, with an almost unchanged onset voltage (for detail see Fig. 5B, inset). Therefore PKCI blocked the OAG effect on PIC.
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To test the effect of PKC activation on Vth for generation of action potentials, we did some experiments with current-clamp recordings (see METHODS for details). In five cells, we made both voltage- and current-clamp recordings. Figure 6 shows an example of one of the cells recorded in both protocols. A 3-s current pulse (Fig. 6A1) and 10-s current ramp (Fig. 6A2) were delivered to the cell to evoke repetitive firing in both control and OAG conditions. Traces recorded from both conditions were overlapped (dashed for control and solid for OAG) and Vth was measured from each spike with circles marked on the traces (open for control and closed for OAG). OAG (20 µM) did not change the rheobase in this cell but depolarized the Vth of the first spike by 1.8 mV (3.1 mV on average for all spikes) with pulse current injection and by 3.9 mV (2.0 mV on average) with ramp current injection. The firing frequency was reduced by 1 Hz from 18 to 17 Hz in the pulse current injection. There was no change in rheobase (40 pA) but a small increase in AP width by 0.5 ms from 4.7 ms. Small reductions were observed in AP amplitude by –3.4 mV from 37.6 mV, maximal rise rate by –1 mV/ms from 15 mV/ms, and maximal decay rate by –0.2 mV/ms from 7 mV/ms. (Note: the AP amplitude was measured from Vth.) Voltage-clamp recordings made in the same cell showed that OAG depolarized the Vth for IT by 5 mV with a 17% (120 pA) reduction of the amplitude of IT (Fig. 6B). This cell demonstrated that activation of PKC could decrease the neuronal excitability by depolarization of Vth for action potentials and a reduction of repetitive firing frequency.
In 12 cells recorded with the current-clamp protocol, OAG depolarized the Vth for action potentials by 2.4 ± 0.8 mV in 8 of the cells and hyperpolarized the Vth by 1.8 ± 0.4 mV in 3 of them. One of the cells showed no change in Vth. Of the 12 cells, OAG produced either an increase (n = 4) or no change (n = 8) in rheobase. Results from the 12 cells are summarized in Fig. 7A. The averaged values measured in control include the rheobase (64 ± 43 pA), Vth (–39 ± 6 mV), AP amplitude (43 ± 13 mV measured from Vth), width (6 ± 2 ms), maximal rise rate (18 ± 7 mV/ms), and maximal decay rate (9 ± 4 mV/ms). Results in Fig. 7A indicate that activation of PKC could produce a small but significant depolarization of Vth (2 ± 1 mV, P < 0.05) with a significant increase in AP width (0.6 ± 0.2 ms, P < 0.05) and decrease in AP amplitude (8 ± 3 mV, P < 0.05), maximal rise rate (5 ± 2 mV/ms, P < 0.05), and maximal decay rate (2 ± 1 mV/ms, P < 0.05). The rheobase was increased by 14 ± 17 pA but not significantly (P > 0.08). Five of the 12 cells were recorded in both current and voltage protocols. The averaged activation of IT was fitted by Boltzmann equation and is shown in Fig. 7B. In current-clamp recording OAG depolarized the Vth for action potential by 1.8 ± 0.5 mV in these cells, whereas in voltage-clamp recording OAG induced a positive shift in the half-maximal activation of IT by 2.3 ± 1.7 mV, with little change in activation slope.
A different pathway is used by PKC and 5-HT for modulation of IT
Activation of PKC with OAG can induce a depolarization of Vth, which is opposite to the hyperpolarization of Vth induced by bath application of 5-HT or noradrenaline (Fedirchuk and Dai 2004b) or activation of descending serotonergic fibers (Gilmore and Fedirchuk 2004). In prefrontal cortex neurons the PKC pathway mediates 5-HT–induced inhibition of sodium currents (Carr et al. 2002). In spinal ventral neurons, however, it is unclear whether the PKC and serotonergic pathways could play a common role in modulation of Vth. In this study we tested the possible interaction between these two systems. We assessed the ability of 5-HT to induce Vth hyperpolarization with or without PKCI when OAG was applied. Figure 8 shows recordings from two cells recorded without PKCI. In control, the IT of –1,324 pA was elicited at Vth of –35 mV (single asterisks, black traces in Fig. 8A1). Bath application of 20 µM 5-HT hyperpolarized Vth by 5 mV to –40 mV (double asterisks, red traces in Fig. 8A1). Bath application of 20 µM OAG with 5-HT completely blocked the IT (Fig. 8A1, blue traces). Note that IT could not be elicited even when the voltage step was raised to –25 mV, 10 mV higher than that in control. However, the IT could partially recover after a 25-min washout with fresh aCSF (Fig. 8A1, green traces). This cell showed a 5-HT–induced hyperpolarization of Vth "antagonized" by OAG (see inset in Fig. 8A1 for the changes in shape and size of IT). The OAG- and 5-HT–induced changes in PIC were also recorded in the same cell. As shown in Fig. 8A2, the PIC of –60 pA was evoked by a voltage bi-ramp at an onset voltage of –69.4 mV (Fig. 8A2, black trace). 5-HT induced some EPSC in this cell but did not change the properties (amplitude and onset voltage) of the PIC (Fig. 8A2, red trace). However, OAG completely removed the PIC (Fig. 8A2, blue trace), which was then partially recovered from washout (Fig. 8A2, green traces). Note that the reduction of peak of the passive ramp current with OAG could be caused by an inhibition of potassium conductance by PKC activation (Murbartian et al. 2005), which could be partially washed out with fresh aCSF. To further test the interaction between the pathways of 5-HT and PKC, we switched the sequence of application of 5-HT and OAG in Fig. 8A. An example is shown in Fig. 8B. In this cell the IT was reduced (not blocked) by OAG. The IT was measured as –780 pA with Vth of –45 mV in control (single asterisks, black traces in Fig. 8B). Bath application of 20 µM OAG did not alter the Vth but reduced the IT by –270 pA to –510 pA (single asterisks, blue traces in Fig. 8B). Following bath application of 15 µM 5-HT with OAG, the Vth was hyperpolarized by 5 mV to –50 mV and the IT was further reduced by –115 pA to –395 pA (double asterisks, red traces). Both OAG and 5-HT induced a reduction of IT in this cell (Fig. 8B, inset). This cell demonstrated that OAG did not block the ability of 5-HT to induce a hyperpolarization of Vth.
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17%) reduction in IT amplitude (see inset in 5-HT condition, dashed trace for control). PKCI blocked the effect of OAG but not that of 5-HT on IT and Vth.
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Activation of PKC modulated both calcium and sodium components of PIC
The persistent inward current is composed of two currents: 1) the L-type calcium current (Ca–PIC), which is dihydropyridine sensitive, and 2) the persistent sodium current (Na–PIC), which is TTX sensitive. Since no blockers of these components were used in the preceding experiments, the PIC present in the above-cited study is presumably mediated by both calcium and sodium currents. To investigate the effect of PKC activation on either component of PIC, we did additional experiments with either TTX (2 µM) or nifedipine (20–30 µM) administrated in the recording solution. Examples are shown in Fig. 10. Our results showed that activation of PKC significantly reduced both calcium and sodium components of PIC. Of four cells recorded with TTX, bath application of OAG (20–30 µM) reduced Ca–PIC by about 25% with 1.3-mV depolarization of PIC onset (see Fig. 10A), whereas the similar amount of OAG reduced Na–PIC (n = 4) by about 70% with 8.7-mV depolarization of PIC onset (Fig. 10B). Although the Ca–and Na–PIC onset was not significantly depolarized by OAG due to the small number of samples, the significant reduction of the PIC amplitude clearly indicated that Ca–PIC and Na–PIC were modulated by activation of PKC pathway. Furthermore, the effect of PKC activation might be stronger on Na–PIC than that on Ca–PIC in terms of reduction of PIC and depolarization of PIC onset. See Table 4 for the details.
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To study the mechanisms underlying the changes in IT and PIC by PKC activation we did correlation analysis among some parameters measured from IT and PIC. It is known that the ascending phase of IT is dominated by activation of sodium currents, which could be described by the amplitude and rise rate of IT, whereas the descending phase of IT is determined by inactivation of IT and activation of some potassium conductances such as the delayed rectifier, which could be described by decay rate and width of the IT. To explore some putative mechanisms underlying the OAG-induced changes in IT and PIC, we analyzed the correlation between the maximal rise and decay rates (Fig. 11A) and the correlations between the amplitude of IT and the maximal rise (Fig. 11B) and decay rates (Fig. 11C). For example, alteration of both the activation and inactivation properties of sodium channels might be expected to alter both the rise rate and decay rate of IT. We also examined the correlations between the OAG effect on PIC and the effect on the amplitude of IT (Fig. 11D). Results from 15 cells are shown in Fig. 11, where the plots in the left column show the relations between two selected parameters measured from the recordings in control and OAG conditions (dashed line with open circles is for control and the solid line with closed circles is for OAG) and the plots in the right column show the relations of the changes induced by OAG between the two parameters. These results are discussed in the following section.
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Phosphorylation of sodium channels through the PKC pathway was previously reported to reduce the peak of the transient sodium currents and slow inactivation of sodium channels (Cantrell et al. 1996; Dascal and Lotan 1991; Lotan et al. 1990; Numann et al. 1991; Sigel and Baur 1988; West et al. 1991). Consistent with these reports (for review see Catterall 2000) we show in this study that activation of PKC reduces the amplitude of IT (or even blocks the IT in some cells) in neonatal rat spinal ventral neurons. In addition, our study also shows that PKC activation increases the half-width of IT and decreases the maximal rise and decay rates of IT. The mechanisms underlying these changes in IT have not been determined in this study. However, the correlation analysis suggests that it could be the same mechanisms that induce most of these changes. As shown in Fig. 11A1, there is a strong and almost unchanged correlation between the maximal rise and maximal decay rates before and after OAG application. Furthermore, the reduction of maximal rise rate is also highly correlated to the reduction of the maximal decay rates (Fig. 11A2), suggesting that mechanism(s) mediating the reductions of these two rates might be the same. Further analysis shows that the relations between the IT amplitude and the maximal rise (Fig. 11B1) and decay rates (Fig. 11C1) are fairly correlated in both control and OAG conditions. These correlations are maintained between the reduction of IT amplitude and the reductions of the maximal rise (Fig. 11B2) and maximal decay rates (Fig. 11C2), suggesting that it could be the same mechanism(s) mediating the reductions of the amplitude, the maximal rise rate, and the maximal decay rate of IT.
Relations between the half-width and other parameters of IT are more complicated than those discussed earlier. First, there is no significant correlation observed between the amplitude and half-width of IT (not shown), neither is there a significant correlation in the OAG-induced changes in these two parameters, suggesting that the mechanism mediating the decrease in IT amplitude may not be directly related to the increase in IT half-width. Second, the relations between the half-width and the maximal rise rate and the maximal decay rate are nonlinear and no significant correlation (linear or nonlinear) can be established between the OAG-induced increase in the half-width and the decrease in the maximal rise and decay rates (not shown). These results indicate that the mechanism(s) mediating the decrease in maximal rise and decay rates of IT may be only partially responsible for the increase in half-width of IT. This is contradictory to our original expectation. However, considering the fact that the width of transient inward currents could be determined by a balance of several conductances such as the sodium (inward), calcium (inward), and potassium (outward) currents, modulation of potassium conductances (e.g., Covarrubias et al. 1994; Jonas and Kaczmarek 1996; Murbartian et al. 2005), calcium conductances (e.g., Park et al. 2006) or both (e.g., Doerner et al. 1988) by activation of PKC might also contribute to the changes in the width of the inward currents observed in this study.
Mechanisms mediating the OAG-induced changes in PIC
In this study we show that activation of PKC generally results in a depolarization of PIC onset voltage and reduction of PIC amplitude (and conductance, as well) in neonatal rat spinal ventral neurons. The mechanisms underlying the OAG-induced decrease in PIC are unknown. However, from the correlation analysis we expect that the reduction of PIC could be related to the reduction of IT. As shown in Fig. 11D1, there is no correlation between the amplitudes of PIC and IT in both control and OAG conditions. However, the reduction of PIC amplitude is correlated to the reduction of IT amplitude after OAG administration (Fig. 11D2), indicating that the OAG-induced reduction of PICs might result in part from the reduction of IT. Supporting evidence can be seen in Figs. 3 and 8. In Fig. 3, the OAG-induced shifts in voltage dependence of IT resulted in a reduction of window current (Fig. 3A, right), which could partially account for the reduction of the PIC (Fig. 3B). In Fig. 8A, the PIC was completely removed with the blockade of IT by OAG (Fig. 8A, blue traces), which partially recovered with a partial recovery of IT after washout (Fig. 8A, green traces). Interestingly, the 5-HT–induced reduction of IT did not change the PIC in this cell (Fig. 8A, red traces). The hyperpolarization of Vth by 5-HT, which could enhance the window current, might play a role in maintaining the PIC in this cell. Ptak et al. (2005) showed that in the pre-Bötzinger complex region the transient sodium current can be reduced by a low concentration (3 µM) of riluzole via a higher affinity for the inactivation state. This could lead to a reduction of Na–PIC. In hippocampal neurons PKC-induced phosphorylation of transient sodium current via muscarinic acetylcholine receptors results in a reduction of the sodium current as well as Na–PIC (Cantrell et al. 1996). Based on these findings we expect that the PKC-dependent reduction of PIC observed in neonatal rat spinal ventral neurons could partially result from the modulation of IT inactivation (Cantrell et al. 1996; Chen et al. 2006) and partially from the modulation of Ca–PIC.
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DISCUSSION |
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Alteration of neuronal excitability by activation of PKC
Modulation of transient sodium current by activation of protein kinase alters neuronal excitability (for review see Catterall 2000). It has been shown in previous studies that the neuronal excitability could be reduced via the PKA pathway being activated by dopaminergic receptors (Calabresi et al. 1987; Cantrell et al. 1997, 1999). A similar conclusion about the effect of PKC activation on neuronal excitability might be drawn since the peak of transient sodium current is reduced by phosphorylation of sodium channels via PKC. However, previous studies have shown that the PKC activation increases the neuronal excitability by hyperpolarization of voltage threshold in mouse neocortical neurons (Astman et al. 1998) or by amplification of subthreshold depolarization in rat pyramidal neurons (Franceschetti et al. 2000). The increased excitability was attributed to the hyperpolarization of onset voltage of PIC. Contradictory observations have also been reported. For example, activation of PKC via muscarinic receptors suppresses the intrinsic bursting in hippocampal CA1 pyramidal cells (Alroy et al. 1999). This inhibitory effect is mediated by a down-regulation of the persistent Na+ current (Mittmann and Alzheimer 1998). Activation of PKC is also found to reduce dendritic excitability in prefrontal cortex pyramidal neurons (Carr et al. 2002). In the present study we show that the PKC activation reduces the excitability of neonatal rat spinal ventral neurons. This decreased neuronal excitability could result from a mixed effect of a depolarization of Vth for IT (or AP) and onset voltage for PIC and a reduction of amplitude of IT (or AP) and PIC. In some cells the Vth depolarization was also accompanied by an elevation of rheobase currents. On the other hand, PICs have been shown to play a role in initiation of spikes in repetitive firing not only in spinal interneurons (Theiss et al. 2007; Zhong et al. 2007) but also in cultured cells (Kuo et al. 2006). Therefore our findings suggest that neuronal excitability might be regulated by a balance of multiple effects of the PKC pathway and that the different effects of PKC activation could be manifest in different neuron populations.
The effect of PKC activation on the neuronal input–output relation was not tested in this study. Using a five-compartment model built with cat lumbar motoneuron properties (Dai et al. 2002) we showed that a 2- to 3-mV depolarization of INa activation and inactivation in the initial segment did not produce a substantial change in the primary range of the frequency–current (f–I) relation but could induce a fair amount of change in the secondary range of the f–I relation (shifting the f–I curve and altering the f–I slope; unpublished data). Therefore it is unlikely that OAG-induced changes in Vth alone could significantly alter the input–output relation of the cells in normal conditions (primary range). The effect of a small depolarization of Vth on motoneuron recruitment remains unknown. The recruitment of motoneurons was determined by several mechanisms including the current and voltage threshold of the motoneurons and strength of synaptic inputs to the motoneuron pools (Heckman and Binder 1990, 1993; Pinter 1990). In a previous study with a large-scale simulation we showed that alteration of Vth (7 mV) produced only a small change (<3 Hz) in firing frequency of the motoneurons, although it could alter the number (10–45%) of recruited motoneurons in the pools, depending on the cell type and excitatory synaptic strength (Dai et al. 1999). Therefore a 2-mV depolarization of Vth by activation of PKC might not be expected to alter the firing frequency of the neurons, but it might reduce the number of recruited neurons, depending on the functional role, synaptic connectivity, and membrane properties of the neurons. Future studies are required to test this prediction.
PKC activation modulated IT and PIC conductances
Although PKC activation induced large reductions of IT and PIC amplitudes, it did not induce a large depolarization of Vth. This apparent paradox might be explained in several ways. First, any change in Vth is mainly dominated by a modulation of IT activation property. Reduction of IT, however, could be induced by a reduction of IT conductance (availability of the channels) and/or alteration of voltage dependence of IT (a depolarization of activation and/or hyperpolarization of inactivation). Our data presented in this study suggest that modulation of IT conductance instead of its activation property might be the major effect of PKC activation on neonatal rat spinal ventral neurons. Second, our data also show that a large reduction of PIC (50%) by OAG is not accompanied by a large depolarization of PIC onset either (only 4.4 mV; see Table 2). This result suggests that OAG does not induce a substantial change in the subthreshold depolarizing current that might be mediated by PIC and play a role in regulating the Vth or PIC onset. Thus similar to the case of IT, modulation of PIC by PKC activation might mainly target the PIC conductance rather than its voltage dependence. Third, we have demonstrated that PIC was mediated by sodium and calcium currents (Fig. 10). Compared with IT the mixed currents of PIC have a larger time constant (over tens of milliseconds) for activation and might not make a significant contribution to the rising phase of the IT, which is activated within 1–2 ms and therefore determined the Vth.
Variable effects of PKC activation on Vth
As shown in Fig. 4, activation of PKC could induce various changes in Vth, whereas the reduction of the IT was consistent and observed in all cells recorded. Reduction of transient sodium current by PKC activation has been reported uniformly in many cell types (for review see Cantrell and Catterall 2001; Catterall 2000). However, the alterations of voltage dependence of the transient sodium current by PKC activation are different for different cell types, experimental conditions, and concentration of PKC agents. Activation of PKC could induce varied changes in voltage dependence of sodium channels, which are shown as the depolarization of activation (Dascal and Lotan 1991), hyperpolarization of activation and inactivation (Franceschetti et al. 2000), or an unchanged voltage dependence (Cantrell et al. 1996; Numann et al. 1991; Sigel and Baur 1988; West et al. 1991). It is unclear whether the variable effects of PKC activation on Vth observed in this study could be attributable to the different populations of cells in spinal cord. The cells recorded in our experiments were from a heterogeneous population distributed in lamina VII–X from T13–L6 (see Table 1). Significant differences were observed in amplitudes (conductances) of IT and PIC among the cells from lamina VII–X (Tables 1 and 2). Therefore the variable effects of PKC activation on Vth (and PIC) could be related to the different functions of the cells having different intrinsic membrane properties and laminar distributions. It may therefore reflect a complex modulation of the cell membrane properties through the PKC pathway. The multiple mechanisms also provide the nervous system with more flexibility to regulate the neuronal excitability to be appropriate for different motor tasks.
Modulation of IT and PIC versus animal ages
There is little information about PKC mediated modulation of IT and PIC with developmental age. Reduction of sodium currents through phosphorylation of sodium channels has been reported in different ages of animals and types of neurons. For example, PKC-mediated reduction of sodium currents was observed in brain neurons from P20 embryos of rats (Numann et al. 1991), in hippocampal neurons of adult (>P25) rats (Cantrell et al. 1996, 1997, 1999), in prefrontal cortex (PFC) neurons of 3- to 5-wk-old C57/BL6 mice (Carr et al. 2002), and in pyramidal neurons of Sprague–Dawley rats aged 10–25 days (Franceschetti et al. 2000). In this study we demonstrate that activation of PKC reduces the transient and persistent inward current in the spinal ventral neurons of P1–P5 neonatal rats. It appears to be a uniform phenomenon that phosphorylation of sodium channels via PKC decreases peak sodium current. It is unclear, however, if variation of voltage dependence of sodium channels with PKC activation is related to animal ages. More studies are required to address this issue in the future.
Multiple pathways for modulation of Vth by 5-HT and PKC
The present study demonstrates multiple pathways for modulation of Vth by 5-HT and PKC in neonatal rat spinal ventral neurons. Activation of 5-HT receptors hyperpolarizes the Vth in neonatal rat spinal neurons (Fedirchuk and Dai 2004b) and also facilitates the sodium (Harvey et al. 2006a,b) and calcium PIC (Li et al. 2006) in rat spinal motoneurons. These 5-HT–mediated effects on the Vth and PIC are opposite to the effects of PKC activation observed in this study. Furthermore, we have shown in this study that the ability of 5-HT to induce Vth hyperpolarization is not altered by PKC activation or inactivation, suggesting that these two pathways are independent of each other. The functional role of these two pathways in modulation of IT and PIC during locomotion remains unknown. However, activation of the serotonergic pathway generally enhances the neuronal excitability, whereas activation of the PKC pathway reduces the excitability. The balance of these two pathways may provide the motor system with more precise control of the configuration of the motor system for different motor tasks.
Space-clamp issues
Incomplete space clamp is a problem for almost all studies using whole cell patch-clamp techniques (Armstrong and Gilly 1992; Hodgkin and Huxley 1952; Rall and Segev 1985; Taylor et al.1961), especially to those studies using fresh slices or whole tissues in the recording. Any voltage-dependent current could be contaminated by the unclamped currents (Bar-Yehuda and Korngreen 2008). Incidents of distortion of the inward currents by poor space clamp were observed in the present study (see METHODS for details). The unclamped currents could cause an increase in the inward currents that could not be estimated in the present study. In theory, these distorted inward currents could lead to an overestimation of the inward current conductance. However, this error in our study is limited and small. First we excluded data with overt space-clamp issue from further analysis (see METHODS). For the remaining cells the portion of the unclamped currents in the recorded whole cell currents should be small. Second, our study focused on the OAG-induced changes in IT and PIC properties that were given in both absolute and relative values in our results (see Fig. 4 and Table 2). The absolute values of IT and PIC amplitude alone, which might theoretically be distorted by unclamped currents, are not essential to the overall findings of this study. The fact that activation of the PKC pathway could induce about 30% reduction of IT, which was comparable to the reduction of INa in other type of neurons (Cantrell and Catterall 2001), suggests that the unclamped inward currents did not confound our results.
Poor space clamp could also cause an error in calculation of the kinetics of IT. In this study, a steep slope (small Vc) in the Boltzmann function was observed. However, the half-maximal activation and inactivation of IT usually fell within the normal range in most of the cells. Only cells that showed a normal range of voltage dependence and flat slope in the Boltzmann function were selected for kinetics study. Further, the conclusions of our study again rely more on the relative changes in the kinetics rather than on the absolute values.
Poor space clamp had a relatively small effect on the parameters of IT (width, maximal rise and decay rates) since they are dependent more on the gating properties of the channels rather than the total conductance. Calculation of these parameters from the maximum IT (see METHODS) could also reduce the errors from the poor space clamp since these parameters are very stable as soon as a spike was completely elicited. On the other hand, however, we could not absolutely rule out possible error from poor space clamp in determination of Vth. This might account for some small variation in Vth. However, since the OAG-induced changes in Vth were small (2 mV on average) and the PKCI could significantly block these changes (Fig. 4), the variable Vth observed in this study should be due to the modulation of intrinsic membrane properties by PKC activation and not to error introduced by poor space clamp.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. Fedirchuk, Department of Physiology, University of Manitoba, 745 Bannatyne Ave., Winnipeg, MB, Canada R3E 0J9 (E-mail: brent{at}scrc.umanitoba.ca)
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REFERENCES |
|---|
|
Armstrong CM, Gilly WF. Access resistance and space clamp problems associated with whole-cell patch clamping. Methods Enzymol 207: 100–122, 1992.[Web of Science][Medline]
Astman N, Gutnick MJ, Fleidervish IA. Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices. J Neurophysiol 80: 1547–1551, 1998.
Bar-Yehuda D, Korngreen A. Space clamp problems when voltage clamping neurons expressing voltage-gated conductances. J Neurophysiol 99: 1127–1136, 2008.
Beaumont E, Gardiner PF. Endurance training alters the biophysical properties of hindlimb motoneurons in rats. Muscle Nerve 27: 228–236, 2003.[CrossRef][Web of Science][Medline]
Calabresi P, Mercuri N, Stanzione P, Stefani A, Bernardi G. Intracellular studies on the dopamine-induced firing inhibition of neostriatal neurons in vitro: evidence for D1 receptor involvement. Neuroscience 20: 757–771, 1987.[CrossRef][Web of Science][Medline]
Cantrell AR, Catterall WA. Neuromodulation of Na+ channels: an unexpected form of cellular plasticity. Nat Rev Neurosci 6: 397–407, 2001.
Cantrell AR, Ma JY, Scheuer T, Catterall WA. Muscarinic modulation of sodium current by activation of protein kinase C in rat hippocampal neurons. Neuron 16: 1019–1026, 1996.[CrossRef][Web of Science][Medline]
Cantrell AR, Scheuer T, Catterall WA. Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons. J Neurosci 19: 5301–5310, 1999.
Cantrell AR, Smith RD, Goldin AL, Scheuer T, Catterall W. Dopaminergic modulation of sodium current in hippocampal neurons via cAMP-dependent phosphorylation of specific sites in the sodium channel subunit. J Neurosci 17: 7330–7338, 1997.
Cantrell AR, Tibbs VC, Westenbroek RE, Scheuer T, Catterall WA. Dopaminergic modulation of voltage-gated Na+ current in rat hippocampal neurons requires anchoring of cAMP-dependent protein kinase. J Neurosci 19: RC21, 1999.
Carr DB, Cooper DC, Ulrich SL, Spruston N, Surmeier DJ. Serotonin receptor activation inhibits sodium current and dendritic excitability in prefrontal cortex via a protein kinase C-dependent mechanism. J Neurosci 22: 6846–6855, 2002.
Catterall WA. From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron 26: 13–25, 2000.[CrossRef][Web of Science][Medline]
Chen Y, Yu FH, Surmeier DJ, Scheuer T, Catterall WA. Neuromodulation of Na+ channel slow inactivation via cAMP-dependent protein kinase and protein kinase C. Neuron 49: 409–420, 2006.[CrossRef][Web of Science][Medline]
Cormery B, Beaumont E, Csukly K, Gardiner P. Hindlimb unweighting for 2 weeks alters physiological properties of rat hindlimb motoneurones. J Physiol 568: 841–850, 2005.
Costa MR, Catterall WA. Phosphorylation of the alpha subunit of the sodium channel by protein kinase C. Cell Mol Neurobiol 4: 291–297, 1984.[CrossRef][Web of Science][Medline]
Covarrubias M, Wei A, Salkoff L, Vyas TB. Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate. Neuron 13: 1403–1412, 1994.[CrossRef][Web of Science][Medline]
Dai Y, Bashor DP, Fedirchuk B, Jordan LM. Motoneuron threshold hyperpolarization: alteration of population output predicted by a large scale simulation. Soc Neurosci Abstr 25: 664.10, 1999.
Dai Y, Jones KE, Fedirchuk B, McCrea DM, Jordan LM. A modelling study of locomotion-induced hyperpolarization of voltage threshold in cat lumbar motoneurones. J Physiol 544: 521–536, 2002.
Dascal N, Lotan I. Activation of protein kinase C alters voltage dependence of a Na+ channel. Neuron 6: 165–176, 1991.[CrossRef][Web of Science][Medline]
Doerner D, Pitler TA, Alger BE. Protein kinase C activators block specific calcium and potassium current components in isolated hippocampal neurons. J Neurosci 8: 4069–4078, 1988.[Abstract]
Fedirchuk B, Dai Y. Activation of protein kinase C (PKC) depolarizes the voltage threshold for activation of spinal neurons in the neonatal. Soc Neurosci Abstr 171.11, 2004a.
Fedirchuk B, Dai Y. Monoamines increase the excitability of spinal neurones in the neonatal rat by hyperpolarizing the threshold for action potential production. J Physiol 557: 355–361, 2004b.
Franceschetti S, Taverna S, Sancini G, Panzica F, Lombardi R, Avanzini G. Protein kinase C-dependent modulation of Na+ currents increases the excitability of rat neocortical pyramidal neurones. J Physiol 528: 291–304, 2000.
Gardiner P, Dai Y, Heckman CJ. Effects of exercise training on alpha-motoneurons. J Appl Physiol 101: 1228–1236, 2006.
Gershon E, Weigl L, Lotan I, Schreibmayer W, Dascal N. Protein kinase A reduces voltage-dependent sodium current in Xenopus oocytes. J Neurosci 12: 3743–3752, 1992.[Abstract]
Gilmore J, Fedirchuk B. The excitability of lumbar motoneurones in the neonatal rat is increased by a hyperpolarization of their voltage threshold for activation by descending serotonergic fibres. J Physiol 558: 213–224, 2004.
Harvey PJ, Li X, Li Y, Bennett DJ. 5HT2 receptor activation facilitates a persistent sodium current and repetitive firing in spinal motoneurons of rats with and without chronic spinal cord injury. J Neurophysiol 96: 1158–1170, 2006a.
Harvey PJ, Li X, Li Y, Bennett DJ. Endogenous monoamine receptor activation is essential for enabling persistent sodium currents and repetitive firing in rat spinal motoneurons. J Neurophysiol 96: 1171–1186, 2006b.
Heckman CJ, Binder MD. Neural mechanisms underlying the orderly recruitment of motoneurones. In: The Segmental Motor System, edited by Binder MD, Mendell LM. New York: Oxford Univ. Press, 1990, p. 182–204.
Heckman CJ, Binder MD. Computer simulations of the effects of difference synaptic input systems on motor unit recruitment. J Neurophysiol 70: 1827–1840, 1993.
Hochman S, McCrea DA. Effects of chronic spinalization on ankle extensor motoneurons. II. Motoneuron electrical properties J Neurophysiol 71: 1468–1479, 1994.
Hodgkin AL, Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117: 500–544, 1952.
Jonas EA, Kaczmarek LK. Regulation of potassium channels by protein kinases. Curr Opin Neurobiol 6: 318–323, 1996.[CrossRef][Web of Science][Medline]
Krawitz S, Fedirchuk B, Dai Y, Jordan LM, McCrea DA. State-dependent hyperpolarization of voltage threshold enhances motoneurone excitability during fictive locomotion in the cat. J Physiol 522: 271–281, 2001.[Web of Science]
Kuo JJ, Lee RH, Zhang L, Heckman CJ. Essential role of the persistent sodium current in spike initiation during slowly rising inputs in mouse spinal neurones. J Physiol 574: 819–834, 2006.
Li M, West JW, Lai Y, Scheuer T, Catterall WA. Functional modulation of brain sodium channels by cAMP-dependent phosphorylation. Neuron 1151–1159, 1992.
Li M, West JW, Numann R, Murphy BJ, Scheuer T, Catterall WA. Convergent regulation of sodium channels by protein kinase C and cAMP-dependent protein kinase. Science 1439–1442, 1993.
Li X, Murray K, Harvey PJ, Ballou EW, Bennett DJ. Serotonin facilitates a persistent calcium current in motoneurons of rats with and without chronic spinal cord injury. J Neurophysiol 97: 1236–1246, 2006.[CrossRef][Web of Science][Medline]
Lotan I, Dascal N, Naor Z, Boton R. Modulation of vertebrate brain Na+ and K+ channels by subtypes of protein kinase C. FEBS Lett 267: 25–28, 1990.[CrossRef][Web of Science][Medline]
Mittmann T, Alzheimer C. Muscarinic inhibition of persistent Na+ current in rat neocortical pyramidal neurons. J Neurophysiol 79: 1579–1582, 1998.
Murbartian J, Lei Q, Sando JJ, Bayliss DA. Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels. J Biol Chem 280: 30175–30184, 2005.
Murphy BJ, Catterall WA. Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C. J Biol Chem 267: 16129–16134, 1992.
Numann R, Catterall WA, Scheuer T. Functional modulation of brain sodium channels by protein kinase C phosphorylation. Science 254: 115–118, 1991.
Park JY, Kang HW, Moon HJ, Huh SU, Jeong SW, Soldatov NM, Lee JH. Activation of protein kinase C augments T-type Ca2+ channel activity without changing channel surface density. J Physiol 577: 513–523, 2006.
Pinter MJ. The role of motoneuron membrane properties in the determination of recruitment order. In: The Segmental Motor System, edited by Binder MD, Mendell LM. New York: Oxford Univ. Press, 1990, p. 165–181.
Ptak K, Zummo GG, Alheid GF, Tkatch T, Surmeier DJ, McCrimmon DR. Sodium currents in medullary neurons isolated from the pre-Botzinger complex region. J Neurosci 25: 5159–5170, 2005.
Rall W, Segev I. Space clamp problems when voltage clamping branched neuron with intracellular microelectrodes. In: Voltage and Patch Clamping with Microelectrodes, edited by Smith TG Jr, Lecar H, Redman SJ, Gage PW. Bethesda, MD: Am. Physiol. Soc., 1985, p. 191–215.
Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, Grewal JS, Garnovskaya MN. Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther 92: 179–212, 2001.[CrossRef][Web of Science][Medline]
Sigel E, Baur R. Activation of protein kinase C differentially modulates neuronal Na+, Ca2+, and gamma-aminobutyrate type A channels. Proc Natl Acad Sci USA 85: 6192–6196, 1988.
Smith RD, Goldin AL. Phosphorylation of brain sodium channels in the I-II linker modulates channel function in Xenopus oocytes. J Neurosci 16: 1965–1974, 1996.
Taylor RE, Moore JW, Cole KS. Analysis of certain errors in squid axon voltage clamp measurements. Biophys J 1: 161–202, 1961.[Medline]
Theiss RD, Kuo JJ, Heckman CJ. Persistent inward currents in rat ventral horn neurones. J Physiol 580: 507–522, 2007.
West JW, Numann R, Murphy BJ, Scheuer T, Catterall WA. A phosphorylation site in the Na+ channel required for modulation by protein kinase C. Science 254: 866–868, 1991.
Zhong G, Masino MA, Harris-Warrick RM. Persistent sodium currents participate in fictive locomotion generation in neonatal mouse spinal cord. J Neurosci 27: 4507–4518, 2007.
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ABSTRACT
INTRODUCTION
Vth
: Vth is depolarized by OAG; 3) 
: Vth is hyperpolarized by OAG; and 4) 
