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1Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry; 2Division for Interdisciplinary Dentistry, Osaka University Dental Hospital, Osaka; 3The Research Institute of Personalized Health Science, Health Sciences University of Hokkaido, Hokkaido, Japan; and 4Department of Oral Anatomy, School of Dentistry, Kyungpook National University, Daegu, South Korea
Submitted 9 November 2006; accepted in final form 16 January 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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). Given the expression of INaP in the dendrites or soma, the INaP is considered to play roles in the forward communication between the dendrites and soma (Chance et al. 2002) and considered to control the membrane excitability in the soma-dendrite in the voltage region just subthreshold for spike generation (Crill 1996). However, the upregulation of INaP by protein kinase C (PKC) caused a decrease in the threshold for spike activation in neocortical pyramidal neurons (Astman et al. 1998), in which spikes are initiated in the proximal axon (Stuart and Sakmann 1994). More recently, the local application of tetrodotoxin (TTX) to the proximal axon of neocortical layer V pyramidal neurons was shown to block the INaP recorded in the soma in response to soma depolarization, suggesting that INaP is primarily generated in the proximal axon (Astman et al. 2006). Nevertheless, there is still no explicit evidence that spikes are initiated by the activity of INaP in the proximal axon. On the other hand, a transient or inactivating and low-voltage–activated Na+ current was found to be expressed in the axon far beyond the initial segment (IS) of neocortical layer V pyramidal neurons (Colbert and Pan 2002) and blockade of those inactivating Na+ channels by locally applied TTX caused an increase in the threshold for spike activation (Colbert and Johnston 1996). Thus it is still unclear whether INaP is directly involved in the initiation of spikes in the proximal axon.
The primary sensory neurons supplying periodontal mechanoreceptors or jaw-closer muscle spindles are unique in generating spikes in response to synaptic inputs onto the somata (Verdier et al. 2004), thereby displaying two distinct types of spikes, one arising from sensory organs and the other arising from somatic inputs (Saito et al. 2006). This is because their somata are exceptionally located in the mesencephalic trigeminal nucleus (MTN) within the brain stem subsequently receiving various synaptic inputs (Hinrichsen and Larramendi 1970; Liem et al. 1992). We also previously demonstrated that MTN neurons display spike backpropagation from the spike-initiation site somewhere in the stem axon to the soma in response to injection of current pulses into the soma, and suggested that somatic inputs or impulses arising from sensory organs, whichever trigger spikes in the stem axon first, can be forwarded to their target synapses (Saito et al. 2006). Therefore we aimed to elucidate the role of INaP in spike initiation in the stem axon of MTN neurons by using a dual patch-clamp recording method. In the present study, we found that the process of spike initiation in the stem axon is highly sensitive to riluzole, 10 nM TTX, and QX-314, strongly suggesting an involvement of INaP in spike initiation in MTN neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The whole cell patch-clamp recording method was essentially similar to that used in our previous studies (Kang et al. 2004; Saito et al. 2006). Briefly, coronal slices of 200- to 250-µm thickness were made from the brain stem of Wistar rats (6–18 days postnatal). The standard extracellular solution had the following composition (in mM): 124 NaCl, 26 NaHCO3, 1.8 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.3 MgCl2, and 10 glucose. The internal solution of the patch pipettes had the following ionic composition (in mM): 123 K-gluconate, 18 KCl, 10 NaCl, 3 MgCl2, 2 ATP-Na2, 0.3 GTP-Na3, and 10 HEPES; pH 7.4 adjusted with KOH for whole cell recordings. The membrane potential values given in the text were corrected for the junction potential (10 mV) between the internal solution for the whole cell recording (negative) and the standard extracellular solution. The recording chamber with a volume of 1.0 ml was continuously perfused with the extracellular solution at a flow rate of 1.0–1.5 ml/min.
Using Axopatch 1D, 200A, 200B, and MultiClamp 700A (Molecular Devices, Foster City, CA), single or dual whole cell current-clamp recordings were made on MTN neurons viewed under Nomarski optics (BX-50WI, Olympus, Tokyo, Japan). Two patch pipettes used for simultaneous whole cell recordings were pulled using the same parameter configuration (P-97, Sutter Instrument, Novato, CA) and their pipette resistances were 4–6 M
. Because all experiments were carried out on the recordings in which the series resistance was <10 M, Axopatch 200A/B was used in the normal current-clamp mode, consequently causing no practical difference in the current-clamp performance between Axopatch 1D and 200A/B (Saito et al. 2006). Because the series resistance compensation was disabled in the current-clamp mode of Axopatch 1D and 200A or not used in Axopatch 200B, only the recordings that showed no sign of apparent bridge imbalance were included in the analysis. Moreover, because spikes were always triggered after the offset of current pulses, an inappropriate bridge balance, if any, would not affect the spike height. All recordings were made at room temperature (21–24°C). Records of currents and voltages were low-pass filtered at 5–10 kHz (three-pole Bessel filter), digitized at a sampling rate of 40 kHz (Digidata 1322A, Molecular Devices), and stored on a computer hard disk.
Stimulation and drug application
With a tungsten microelectrode (impedance: 1 M at 5 kHz), microstimulation (intensity: 0.5–5.0 µA; duration: 60 µs) was applied to the stem axon at a site 40–60 µm apart from the soma. Riluzole (Sigma–Aldrich, St. Louis, MO), tetrodotoxin (TTX, Wako Pure Chemical, Osaka, Japan), and 4-aminopyridine (4-AP, Sigma–Aldrich) were bath-applied at concentrations of 10–20 µM, 10–50 nM, and 0.2–0.5 mM, respectively. The membrane-impermeable lidocaine analogue QX-314 (Sigma–Aldrich) was included in the internal solution at a concentration of 0.5–5 mM.
Subcellular application of QX-314 by a dual whole cell recording method
To apply QX-314 locally to a subcellular region, we performed a dual whole cell recording using QX-314–containing and QX-314–free patch pipettes. The QX-314–containing patch pipette was placed on the soma of an MTN neuron and the QX-314–free patch pipette on the axon hillock (AH) whose patch membrane was ruptured first. This would create a decreasing concentration gradient of QX-314 from the soma toward the AH. This was simulated with Lucifer yellow (LY, Sigma–Aldrich). One patch pipette filled with the internal solution containing 0.1% LY was placed on the soma of an MTN neuron and the other patch pipette filled with the LY-free internal solution was placed, not right on the AH site, but on a site near the AH (Fig. 1). This is because the possible mechanical distortion of the AH membrane by the patch pipette may prevent LY from diffusing into the axon if the patch pipette is placed right on the AH site. A steady-state concentration gradient decreasing from the soma to the AH was achieved 5–7 min after the establishment of the dual whole cell (DWC) recording (Fig. 1A). The fluorescent intensity was higher around the LY-containing soma pipette (a), whereas it was much lower around the LY-free pipette placed near the AH (b). However, 2 min after the removal of the AH pipette from the MTN neuron, the LY fluorescence spread homogeneously all over the soma and subsequently revealed the stem axon (*, Fig. 1B). Thus it was assumed that, similar to LY, a steady-state concentration gradient of QX-314 would also be achieved by establishing a DWC recording. This would be useful to examine the subcellular localization of QX-314–sensitive channels.
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RESULTS |
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In our previous study (Saito et al. 2006), simultaneous patch-clamp recordings from the soma and axon hillock (AH) revealed a spike backpropagation from the spike-initiation site in the stem axon to the soma through the AH in response to injection of a short current pulse into the soma. Soma spikes (S-spikes) emerged with a delay after the offset of the short current pulse (Fig. 2), partly as a result of the backpropagation and partly as a result of the electrotonic separation from the current-pulse injection site in the soma to the spike-initiation site in the stem axon. As shown in Fig. 2Aa, with an increase in the peak voltage level (v, inset) of depolarizing responses to current pulses, the S-spike was triggered with less delay. If INaP is involved in the spike initiation in MTN neurons, the activation of S-spikes would be further delayed by inhibiting INaP with selective blockers, riluzole or a low concentration of TTX (Schwindt and Crill 1996; Stafstrom et al. 1985). Therefore we first examined whether riluzole or a low concentration of TTX causes a further delay in spike initiation in response to injection of current pulses into the soma, without affecting the spike itself.
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). Bath application of 10 µM riluzole increased the threshold for evoking S-spikes with the same delay (TMRR) (inset, superimposed traces *1 in Fig. 2A, a and b) and caused a greater delay in response to the same depolarization (v) (inset, superimposed traces *2 in Fig. 2A, a and b). Thus riluzole shifted the v–TMRR relationship positively (compare black and red circles, Fig. 2A, c and d), without appreciably attenuating the spike amplitude (compare interrupted lines in Fig. 2A, a and b). These observations suggest that INaP is directly involved in spike initiation. This could be more clearly seen in the presence of 4-AP, which revealed a ramplike depolarization preceding the S-spike (Fig. 2Ba). Due to this ramplike depolarization, S-spikes could be triggered with much longer delays. Such ramplike depolarization appeared to be mediated by INaP (Schwindt and Crill 1996; Stafstrom et al. 1985). Indeed, 10 nM TTX significantly attenuated the ramplike depolarization (Fig. 2B, a and b) and shifted the v–TMRR relationship positively by 8.7 ± 2.3 mV (n = 4, Fig. 2B, c and d) in the presence of 0.2–0.5 mM 4-AP, when the two v values, having almost the same TMRR measured before and after TTX application (0.59 ± 0.13 and 0.59 ± 0.12 ms, respectively), were compared. Similarly, 10 µM riluzole also shifted the v–TMRR relationship positively by 9.3 ± 1.8 mV (n = 4) in the presence of 0.2–0.5 mM 4-AP, when the two v values, having almost the same TMRR measured before and after riluzole application (0.59 ± 0.09 and 0.58 ± 0.09 ms, respectively), were compared. In these pooled data, MTN neurons in which the S-spike attenuated by >6% of its control amplitude by TTX or riluzole were not included. S-spikes were attenuated by only 4.7 ± 0.9 and 4.4 ± 0.6% of their control amplitudes by 10 nM TTX and 10 µM riluzole, respectively. Thus a stepwise increase in the threshold for or a prolongation of the delay in the activation of S-spikes without marked changes in amplitude was seen after the application of these blockers, suggesting an involvement of INaP in the initiation of S-spikes. In the next experiments, we performed a dual whole cell recording to examine which processes during the spike initiation and backpropagation were affected by TTX. Differential sensitivity to TTX between soma and axonal spikes
Simultaneous patch-clamp recordings were obtained from the soma and AH to investigate whether such an activation delay or threshold increase occurred at the cell body or at the stem axon where the axonal spike is initiated. S- and AH-spikes were evoked by each one of the three kinds of electrical stimulation applied every 10 s in the following order: current-pulse injection into the soma (S1), that into the AH (S2), and stimulation of the stem axon (S3), as schematically illustrated in Fig. 3A. As partly shown in Fig. 3Ba, during the perfusion of 50 nM TTX for 350 s, both the simultaneously recorded S- and AH-spikes evoked by current-pulse injection into the AH (and soma; figure not shown) gradually decreased in amplitude as well as in the maximum rate of rise (MRR) (Fig. 3E, a and b). Similarly, the S- and AH-spikes arising from the invasion of axonal spikes after stimulation of the stem axon decreased in amplitude (Fig. 3Bb). These decreases in amplitude and MRR were accompanied by the parallel increases in the activation delay of the AH- and S-spikes in response to injection of current pulses (*1 and *3 in Fig. 3Ba). Simultaneously, the spikes caused by axonal stimulation also dramatically increased in latency (*2 in Fig. 3Bb). Ten seconds after such a delayed activation in response to the current-pulse injection (*3 in Fig. 3Ba), even a stimulation of the stem axon with intensities up to 1.25-fold the threshold intensity failed to evoke "antidromic" S- and AH-spikes, leaving no apparent depolarization (*4 and *6 in Fig. 3Bb). After the failure of spike generation in the stem axon seen exactly 350 s after the TTX application, the current-pulse injection into the AH also failed to evoke spikes (*5 in Fig. 3Ba).
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Provided that there are no differences in the kinetics, density, and TTX sensitivity among Na+ channels distributed in the soma, the AH, and the stem axon, it is possible that the invasion of the axonal spike into the AH would fail most easily during the progress of the blockade of Na+ channels because the threshold increase would be the greatest due to the large capacitative load of the large cell body. If this is the case, the AH pipette should record some subthreshold response after the possible blockade of the spike invasion. However, there was no subthreshold response in the AH recording (see traces *4 and *6 in Fig. 3, Bb and Cb), indicating that the blockade of the axonal spike is not the result of invasion failure. Instead, the pronounced prolongation of the antidromic latency (see traces *2 in Fig. 3Bb) suggests that the 50 nM TTX first suppressed the activation of Na+ channels at the site to which the stimulation was applied, consequently causing a conduction delay and complete blockade of axonal spikes. In fact, this marked prolongation of the antidromic latency occurred simultaneously with the increase in TMRR, which sharply increased from 0.46 ± 0.13 to 1.03 ± 0.16 ms by 0.58 ± 0.11 ms (n = 5) just before the abolishment of the axonal spike (Fig. 3Ec). In spite of the lack of substantial differences in the peak amplitude between the spikes obtained in response to current-pulse injections just before and after the blockade of the axonal spike (compare traces *3 and *7 in Fig. 3, Ba and Ca), the current-pulse depolarization for evoking spikes has to be increased in a stepwise manner by 13.8 ± 1.3 mV (n = 5) immediately after the failure of spike generation in the stem axon (compare the voltage levels at the pulse end indicated by horizontal arrows in Fig. 3, Ba and Ca). This value may be slightly overestimated because these stronger depolarizations activated spikes with a shorter TMRR (2.39 ± 0.18 ms) than that of spikes seen just before the activation failure (2.63 ± 0.24 ms). This observation is consistent with a previous report, in which a similar threshold increase after abolishment of the axonal spikes with local application of TTX was observed in subicular pyramidal cells that display the spike initiation in the proximal axon in response to soma depolarization (Colbert and Johnston 1996). In view of such a spike-initiation mechanism, the present observations would indicate that the lower-threshold axonal spike is more sensitive to TTX than the higher-threshold S-spike. Therefore it is likely that the activation delay of the S-spike increased with the progress of the blockade of Na+ channels in the stem axon and the threshold for activation of the S-spike increased in a stepwise manner just after the complete blockade of the axonal spike initiation. This possibility was further analyzed in the next experiment.
Delayed spike initiation by TTX disclosed by dual patch-clamp recording from the soma and AH
The zero-crossing time and the rise time of the first time derivative of the S-spike were denoted as Ton-S and Tr-S, which represent the onset latency and the reciprocal of the rate of regenerative process of the S-spike, respectively (Fig. 4A). Then, the TMRR of the S-spike (TMRR-S) corresponds to the sum of Ton-S and Tr-S. In our previous study, the spike backpropagation in MTN neurons was well revealed by the simultaneous whole cell current-clamp and cell-attached voltage-clamp recordings from the soma and AH, respectively (see Fig. 1 in Saito et al. 2006). A depolarizing current-pulse injection into the soma first generated the axonal spike, which in turn backpropagated to sequentially trigger AH- and S-spikes (Fig. 4B, and also see Fig. 1 in Saito et al. 2006). Because the initiated axonal spike is well reflected in the onset of the S- or AH-spikes resulting from the electrotonic continuity between the stem axon and the soma, the Ton-S reflects the latency to the axonal spike generation (Ton-A 
Ton-AH Ton-S; Fig. 4, A and B), whereas the Tr-S primarily reflects the time for the regenerative process of the S-spike (
TS Tr-S; Fig. 4, A and B). The possible differential effects of TTX on the Na+ channels distributed across the soma and the stem axon were evaluated by analyzing these parameters: MRR, TMRR, TS, Ton, and Tr.
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TS) between the timing of the MRR of the AH-spike and that of the S-spike (TMRR-S – TMRR-AH) remained almost constant before the blockade of the axonal spikes (Fig. 4Cb), indicating that the spike-backpropagation time from the AH to the soma did not change appreciably in spite of the delayed emergence of S-spikes. This suggests that the delay occurred primarily at the stem axon. Therefore it is likely that the blockade of Na+ channels by TTX progressed first in the stem axon without profoundly affecting the soma and AH. This possibility was further explored by quantifying the respective processes during the spike initiation and its backpropagation.
When the threshold for evoking the axonal spike is increased by the progress of the blockade of Na+ channels in the stem axon (Hille 1968), the delay in the axonal spike generation would increase with an increase in the threshold for evoking axonal spikes. This is because the threshold increase and the spike generation delay occur in parallel when spikes are triggered from potentials changing along the rising envelope of the subthreshold underlying depolarization. Then, the Ton increase primarily reflects the threshold increase for evoking axonal spikes, consistent with the positive shift of the v–T relationship after the application of TTX and riluzole (Fig. 2). By contrast, the Tr increase may reflect the decrease in the rate of the regenerative process of spikes in the soma. Before the blockade of the axonal spike, the increase in TMRR was more accurately reflected in the increase in the Ton than in the Tr (Fig. 4C, c and d), suggesting an increase in the threshold for evoking the axonal spike with the progress of the blockade of Na+ channels in the stem axon (see DISCUSSION), which could lead to the failure of axonal spike generation. Therefore the increase in the TMRR before the blockade of axonal spikes is completely consistent with the simultaneous prolongation of the antidromic latency resulting from a decrease in the excitability in the stem axon. Following the application of 50 nM TTX, the Ton increased in S-spikes from 0.37 ± 0.27 to 0.83 ± 0.17 ms by 0.46 ± 0.09 ms (n = 5) and similarly increased in AH-spikes from 0.29 ± 0.22 to 0.60 ± 0.31 ms by 0.30 ± 0.12 ms (n = 5). There was no significant difference (P > 0.9) in the ratio of the Ton increase to the TMRR increase between S-spikes (83 ± 9%) and AH-spikes (83 ± 4%).
After the blockade of axonal spikes, the increase in TMRR was almost equally reflected in the increase in the Ton and the Tr (Fig. 4C, c and d). Because there is no spike backpropagation after blockade of axonal spikes, the parallel increases in the Ton and Tr may simply be explained by the threshold increase in the soma spike and the slowdown of the regenerative process in the generation of the soma spike, respectively, presumably following the progress of the blockade of Na+ channels expressed on the soma membrane with larger MRR decreases. Thus a large Ton increase accompanied by a small Tr increase occurred following an initial small MRR decrease until the complete blockade of the axonal spike, whereas smaller parallel increases in Ton and Tr occurred following a large MRR decrease after blockade of axonal spikes, suggesting a differential effect of TTX on the increase in the threshold between the axonal and S-spike. The Ton increase during a 10% decrease in MRR was more than twofold larger in the S-spike (0.21 ± 0.06 ms) recorded before than in that (0.09 ± 0.02 ms) recorded after the blockade of axonal spikes and/or the stepwise threshold increase, following application of 50 nM TTX in five MTN neurons examined. Thus it is likely that, in terms of the threshold increase measured as the Ton increase, the TTX sensitivity is more than twofold higher in Na+ channels, presumably expressed on the axon than in those expressed on the soma.
Differential effects of QX-314 on S- and AH-spikes
Because the axonal spike appeared to be most sensitive to a low concentration of TTX, the involvement of INaP in the initiation of spikes in the stem axon was further examined in the next experiments by using QX-314, to which INaP is more sensitive than the rapidly inactivating INa (Schwindt and Crill 1996; Stafstrom et al. 1985). Considering the nature of spike backpropagation, the attenuation of axonal spikes generated in the stem axon should be more accurately reflected in AH-spikes than in S-spikes. Therefore it was examined whether AH-spikes are more sensitive than S-spikes to QX-314. Dual whole cell recordings were made from the soma and AH of single MTN neurons; only one of the two patch pipettes contained 0.5 mM QX-314. When the degree of attenuation is simply larger in the spike recorded by the QX-314–containing pipette than in that simultaneously recorded by the QX-314–free pipette, it would not be possible to determine whether QX-314–sensitive INaP is highly expressed only at the site of the QX-314 pipette or evenly expressed across the two recording sites. To prove the higher expression of INaP on the AH, it must be examined under the condition of a decreasing concentration gradient of QX-314 from the soma to AH whether the degree of spike attenuation is greater in AH-spikes than that in S-spikes. Therefore we placed one pipette containing 0.5 mM QX-314 internal solution on the soma and the other pipette containing normal internal solution on the AH, whose patch membrane was ruptured first (see METHODS).
Alternate injections of current pulses into the soma and AH every 10 s were started under a presumed steady gradient of the QX-314 concentration reached 5–7 min after establishing the dual whole cell recordings (see METHODS). Intracellular injection of 0.5 mM QX-314 apparently did not affect the holding potential and the apparent membrane time constant. However, with repetition of the current-pulse injection, the AH-spike decreased in amplitude sharply whereas the amplitude of the S-spike remained almost constant, irrespective of the current-pulse injection site as seen in the superimposed traces (Fig. 5A, a and b) and in the plot of the normalized spike amplitude of the AH- and S-spikes against the time after rupture of the membrane patch of the QX-314–containing pipette (P < 0.001, ANOVA, Fig. 5Ba). Moreover, the normalized MRR of the respective spikes decreased more promptly in AH-spikes than in S-spikes (P < 0.001, ANOVA, Fig. 5Bb). In four MTN neurons examined (Fig. 5C, a and b), both the normalized peak amplitude and MRR of AH-spikes (peak amplitude, 0.79 ± 0.14; MRR, 0.48 ± 0.13) obtained 20 to 25 min after rupture of the membrane patch of the 0.5 mM QX-314–containing pipette were significantly (P < 0.05, ANOVA) smaller than those of S-spike (peak amplitude, 0.95 ± 0.02; MRR, 0.64 ± 0.12). Thus in spite of the decreasing concentration gradient of QX-314 from the soma toward the AH, the AH-spike was more appreciably attenuated than the S-spike. Similar observations were made in six MTN neurons, in which either 0.5 mM (n = 4) or 1 mM QX-314 (n = 2) was injected through the soma pipette. However, with the 5 mM QX-314–containing pipette (n = 5), both the S- and AH-spikes disappeared after a few abortive spikes were evoked, in response to current-pulse injections that caused depolarization to a level more positive than 0 mV (figure not shown), indicating no apparent contamination of Na+ spikes by Ca2+ spikes, as reported previously in MTN neurons (Yoshida and Oka 1998). Therefore the backpropagated S-spike is likely to be much less sensitive to QX-314 than the AH-spike. These observations indicate a larger involvement of INaP in the AH-spike than in the S-spike, presumably resulting from a larger reflection of axonal spikes in the AH-spike than in the S-spike.
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DISCUSSION |
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MTN neurons, having round somata, displayed the backpropagation of the axonal spike to the soma through the AH. The nature of the active backpropagation is well reflected in the amplitude and rate of rise of the backpropagated S-spikes, which are larger than those of the preceding AH-spikes (see Figs. 1 and 2 in Saito et al. 2006), differing from that seen in cortical pyramidal cells (Stuart and Sakmann 1994). This unusual backpropagation suggests that the threshold for evoking spikes is higher in the soma than that in the stem axon in spite of the abundant presence of Na+ channels in the soma membrane as revealed by a larger MRR in S-spikes than in AH-spikes. The application of Na+ channel blockers TTX, riluzole, and QX-314 commonly caused an appreciable activation delay in such spike backpropagation. The dual patch-clamp recordings disclosed that the activation delay was increased because of the blockade of Na+ channels in the stem axon.
How then did Na+ channel blockade in the stem axon increase the activation delay? Because of the electrotonic separation between the soma and the stem axon in MTN neurons, the membrane depolarization evoked in the soma membrane would spread into the stem axon, reaching the peak value 
0.5 ms after the peak of the soma depolarization as revealed in the dual whole cell recordings from the soma and AH (see Fig. 2B in Saito et al. 2006). Provided that axonal spikes are triggered from the threshold potentials shifting positively along the rising time course of the subthreshold depolarization in the stem axon resulting from a progressive blockade of Na+ channels in the stem axon, the degree of prolongation in the activation delay of S-spikes is dependent on the electrotonic distance between the soma and the spike-initiation zone and on the degree of the blockade of Na+ channels in the stem axon. In spinal motoneurons, a similar delay in the generation of low-voltage–activated Na+ current responsible for spike initiation in the initial segment was seen in response to a voltage command applied to the soma under a voltage-clamp condition (Araki and Terzuolo 1962). Thus the prolongation of the activation delay of S-spikes without changing its shape suggests a partial blockade of Na+ channels in the spike-initiation zone that is electrotonically remote from the soma.
Differential sensitivities to TTX and QX-314 between the transient and persistent Na+ currents
It was previously reported in CA1 hippocampal neurons that the IC50 values for the effect of TTX on the persistent and transient Na+ currents are approximately 9 and 37 nM, respectively, whereas 1 µM lidocaine reduced the INaP to 24% of the control, leaving the transient Na+ current almost unchanged (Hammarstrom and Gage 1998). In the present study, riluzole and 10 nM TTX increased the onset delay and the threshold of the S-spike without changing its shape (Fig. 2), suggesting that the blockade of INaP progressed selectively in the stem axon. During the perfusion of 50 nM TTX, with a decrease in the MRR, the onset delay increased twofold more sharply in S-spikes before the blockade of the axonal spike than in those evoked with stronger current pulses after the blockade, presumably reflecting the difference in the threshold increase between the possible axonal spike and S-spike (Fig. 4C, c and d). This may be consistent with the difference in the IC50 for TTX between the persistent and transient Na+ currents. Furthermore, the dual whole cell recordings using QX-314–containing and QX-314–free patch pipettes revealed that the MRR decrease with an increase in the activation delay was significantly greater in AH-spikes than in S-spikes (Fig. 5D). Considering the nature of spike backpropagation in MTN neurons, this observation strongly suggests that the presumed axonal spike, compared with S-spikes, is highly sensitive to QX-314. Thus the differential sensitivities to riluzole, TTX, and QX-314 between the possible axonal spike and the S-spike strongly suggest that MTN neurons express different Na+ channels in the stem axon and the soma and that the stem axon of MTN neurons expresses INaP.
Involvement of the transient versus persistent Na+ currents in spike initiation
It was previously reported in CA1 hippocampal pyramidal neurons that Na+ channels involved in the initiation of spikes are located in the axon, 30–60 µm away from the soma, and have an activation threshold lower by 7–8 mV than those of Na+ currents or spikes examined in the soma membrane (Colbert and Johnston 1996). A similar observation was also made in neocortical pyramidal neurons (Colbert and Pan 2002). Such a difference in the activation threshold of Na+ currents between the proximal axon and soma membrane is consistent with the results of a classical study on motoneurons under a voltage-clamp condition where IS and soma-dendritic spikes were mediated by two distinct Na+ currents having lower and higher activation thresholds, respectively (Araki and Terzuolo 1962). However, these Na+ currents having a lower activation threshold appeared to be transient, but not persistent.
The INaP is known to have an activation threshold lower by about 10 mV than the transient or fast inactivating one (Crill 1996; Stafstrom et al. 1985). The upregulation of INaP by PKC caused a decrease in the threshold for spike activation in neocortical pyramidal neurons (Astman et al. 1998), suggesting that INaP is involved in spike initiation. Furthermore, the local application of TTX to the axon of neocortical layer V pyramidal neurons was shown to abolish the INaP recorded in the soma, suggesting that INaP is primarily generated in the proximal axon of neocortical pyramidal layer V neurons (Astman et al. 2006). Because the axon including IS of retinal ganglion cells was found to densely express the sodium channel Nav1.6, this channel was assumed to be responsible for initiating impulses in the axon (Boiko et al. 2003). However, the kinetics of Nav1.6 examined in the recombinant expression system was distinct from that of the INaP (Dietrich et al. 1998; Smith et al. 1998). Nevertheless, it is of interest that in Purkinje neurons obtained from ataxic mice lacking the expression of Nav1.6, persistent and resurgent Na+ currents were dramatically reduced compared with transient Na+ current (Raman and Bean 1997; Raman et al. 1997). Taken together, as demonstrated in the present study, the spike initiation at the stem axon is likely to be mediated by the lower threshold and persistent type of Na+ channel that is highly sensitive to riluzole, TTX, and QX-314.
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Address for reprint requests and other correspondence: Y. Kang, Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565-0871, Japan (E-mail: kang{at}dent.osaka-u.ac.jp)
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