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1Department of Neuroscience and Oral Physiology, Osaka University Graduate School of Dentistry, Osaka; 2Department of Physiology, School of Medicine, Kurume University, Fukuoka; 3Department of Oral Functional Science (Physiology), Graduate School of Dental Medicine, Hokkaido University, Hokkaido; 4Department of System Neuroscience, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, Tokyo; 5The Research Institute of Personalized Health Science, Health Sciences University of Hokkaido, Hokkaido, Japan; and 6Department of Oral Anatomy, School of Dentistry, Kyungpook National University, Daegu, South Korea
Submitted 17 February 2006; accepted in final form 14 April 2006
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ABSTRACT |
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0.5 mM) eliminated the amplitude difference. Furthermore, 4-AP dramatically shortened the delay in spike initiation without affecting the spike-backpropagation time in the stem axon, whereas it substantially prolonged the refractory period of S-spikes arising from axonal-spike invasion without significantly affecting that of presumed axonal spikes. These observations suggest that 4-AP–sensitive K+ currents exert two opposing effects on S-spikes depending on their origins: suppression of spike initiation and facilitation of axonal-spike invasion at higher frequencies. Consistent with these findings, strong immunoreactivities for Kv1.1 and Kv1.6, among 4-AP–sensitive and low-voltage–activated Kv1 family examined, were detected in the soma but not in the stem axon of MTN neurons. |
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INTRODUCTION |
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). Recently, the spike-initiation site has directly been demonstrated to exist in the axon using dual patch-clamp recordings, as revealed by spike-backpropagation (Stuart and Sakmann 1994). More recently, it was also reported that low-voltage–activated Na+ channels responsible for the spike initiation were localized in the axon far beyond the IS (Colbert and Johnston 1996; Colbert and Pan 2002). Further, it was proposed that the spike-initiation site is not necessarily a discrete site but rather a region typically expanding along the axon over 200–300 µm, and as long as a few millimeters in the most extreme case of myelinated axons (Colbert and Pan 2002; Gogan et al. 1983).
In primary sensory neurons (PSNs), such a spike-backpropagation has not been addressed. This is because impulses exclusively originate from the sensory ending of the peripheral axon, but not from the soma or IS of PSNs, which are located in the dorsal root ganglia (DRG) and are considered to receive no synaptic inputs, with the exception of a very small number of DRG neurons (Kayahara et al. 1981). Under physiological conditions, series of afferent impulses arising from the sensory ending propagate along the peripheral axon to the junction with the central and the stem axon, and thereafter propagate along the two axons separately to the soma and the presynaptic terminal. Similar to antidromic spikes, afferent impulses invade the soma through the stem axon, suggesting that the stem axon may be specialized to safely conduct axonal spikes "retrogradely" to the soma. However, it is also known that PSNs can generate spikes under pathological conditions such as neuropathic pain (Amir et al. 1999; Wall and Devor 1983) or in limited physiological conditions (Sengupta et al. 2004).
PSNs in the mesencephalic trigeminal nucleus (MTN), which supply periodontal mechanoreceptors or jaw-closer muscle spindles, are unique in receiving synaptic inputs (Hinrichsen and Larramendi 1970; Liem et al. 1992) onto various types of receptors in the soma (Copray et al. 1990; Ishii and Kang 2002; Lazarov 2002), thereby initiating spikes (Verdier et al. 2004). This is because their somata are exceptional in being located in the brain stem. MTN neurons are also unique in having a very long myelinated stem axon expanding over a few millimeters, which bifurcates into the peripheral and central axons (Shigenaga et al. 1988a,b), thereby forming the three-way junction.
Thus these unique PSNs in the MTN would display two kinds of spikes, one resulting from invasion of the impulse arising from the sensory ending and the other being triggered by somatic inputs. Three important questions then arise. 1) Where are spikes initiated in MTN neurons with specialized stem axons to secure the spike invasion? Is spike-backpropagation seen at spike initiation in MTN neurons? 2) Are there any differences between the two spikes recorded in the soma of MTN neurons? 3) If there are, would such differences reflect which one of the two kinds of impulses is forwarded to target synapses through the central axon by the three-way junction?
In the present study, we addressed these questions in MTN neurons by using single and dual patch-clamp recording methods together with an immunohistochemical technique.
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METHODS |
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Coronal slices (thickness: 200–250 µm) were made from the brain stem of Wistar rats (12–18 days postnatal). The standard extracellular solution had the following composition (in mM): 124 NaCl, 3 KCl, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 glucose. The internal solution had the following ionic composition (in mM): 123 K-gluconate, 18 KCl, 10 NaCl, 2 MgCl2, 2 ATP-Na2, 0.3 GTP, 0.2 EGTA, and 10 HEPES; pH 7.4 adjusted with KOH for whole cell recordings. The liquid junction potential between the internal solution for the whole cell recording (negative) and the standard extracellular solution was 10 mV. Membrane potential values given in the text were corrected for the junction potentials. 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 an Axopatch 1D, 200A, and 200B and an Axoclamp 2B (Axon Instruments, Foster City, CA) single or dual whole cell recordings were made on MTN neurons whose oval cell bodies were clearly visible under Nomarski optics (BX-50WI, Olympus, Tokyo, Japan). Lucifer yellow CH (0.1%; Sigma-Aldrich, St. Louis, MO) was included in the internal solution to clearly visualize the location of the axon hillock from which the stem axon protrudes. Two patch pipettes for simultaneous whole cell recordings were pulled using the same parameter configuration (P-97, Sutter Instruments, Novato, CA) to result in similar pipette resistances of 4–6 M
. The mean values of pipette resistance and capacitance were 5.5 M and 5.0 pF, respectively. The series resistance was usually <10 M and recordings in which the series resistance was >10 M were not included in the analysis. Therefore the Axopatch 200A/B was used in the normal current-clamp mode; consequently, there was no virtual difference in the current-clamp performance between the Axopatch 1D and 200A/B (Magistretti et al. 1996) (see Supplementary Material1 ). The time constants given by the product of the series resistance and pipette shunting capacitance should range between 30 and 50 µs, which should act as a low-pass filter but without distorting the action potential that displayed a relatively slower time course compared with the time constant (see Supplementary material). Because the series resistance compensation was disabled in the current-clamp mode of the Axopatch 1D and 200A or not used in the Axopatch 200B, only the recordings that showed no sign of an apparent bridge imbalance were included in the analysis. If the bridge balance is adequate, the electrotonic voltage responses should linearly increase at the offset of current pulses but not at the onset of current pulses with increasing current intensities, and the membrane potential at the onset of current pulses should remain at the holding potential. Moreover, because spikes were always triggered after the offset of current pulses, the artificial potential change created by inappropriate bridge balance should not affect the spike height.
Most of the recordings were made at room temperature (21–24°C). In some MTN neurons (n = 28), the effects of temperature on the peak level, the half duration, and the maximum rate of rise of spikes were examined at temperatures between 22 and 34°C (see Fig. 9 in Supplemental material) using a temperature control system (DTC-200, DIA Medical, Tokyo, Japan). The seal resistance was usually >5 G. Recordings 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 1332A, Axon Instruments), and stored on a computer hard disk. Under the cell-attached voltage-clamp mode, the patch potential was held at –70 mV to depolarize the patch membrane to about 0 mV for inactivation of the Na+ channels in the patch membrane. After the spike generation in this condition, only the capacitative current can be recorded by the patch electrode. 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 from the soma. 4-Aminopyridine (4-AP, Sigma-Aldrich) was bath-applied at concentrations of 0.2–0.5 mM. Data given in the text are presented as means ± SD unless otherwise indicated, and statistical significance was assessed using the two-tailed t-test and one-way ANOVA.
Immunohistochemistry
Adult Wistar rats of either sex were deeply anesthetized with sodium pentobarbital (50–60 mg/kg, administered intraperitoneally) and perfused transcardially with a 4% paraformaldehyde and 0.2% picric acid fixative dissolved in 0.1 M phosphate buffer (pH 7.3). The brains were immediately removed from the skull, postfixed with the same fresh fixative overnight at 4°C, and cryoprotected with 25% sucrose in 0.1 M phosphate buffer at 4°C. Coronal sections (thickness: 30 µm) were then cut serially on a freezing microtome and divided into six groups. Every sixth section through the MTN was processed for immunohistochemical staining with rabbit antibodies against 10 subtypes of K+ channels: Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv3.2, and Kv3.4 (Alomone, Jerusalem, Israel). After washes in 0.1 M phosphate-buffered saline (PBS; pH 7.3) containing 1% skimmed milk, each group of sections 180 µm apart was incubated overnight with one of the primary antibodies (1:500 dilution) in 0.1 M PBS containing 1% normal horse serum (NHS), 0.05% NaN3, and 0.25% carrageenan with or without Triton X-100 (0.3%). The optimal dilution for these primary antibodies was determined as 1:500 by testing the dilution ranging between 1:100 and 1:1,000. Subsequently, the sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:200 dilution; Vector Labs, Burlingame, CA) in the same fresh NHS–PBS incubation medium for 2 h, followed by ABC Elite (1:100 dilution; Vector Labs) in 0.1 M PBS for 1 h. Finally, the sections were reacted for 10 min in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.04% diaminobenzidine tetrahydrochloride (Dojindo, Kumamoto, Japan), 0.04% NiCl2, and 0.002% H2O2. All immunohistochemical procedures were carried out at room temperature, and the sections were rinsed three times (10 min each) in 0.1 M PBS between each of the steps. In control experiments, each of the primary antibodies was preabsorbed with the corresponding peptide and, also, replaced with normal IgG.
Double fluorescence histochemistry
In adult Wistar rats tetramethylrhodamine dextran amine (RDA, 10% solution; MW 3,000; Molecular Probes, Eugene, OR) was applied to the masseteric nerve. After survival periods of 5–7 days, the rats underwent perfusion-fixation as described above. Coronal sections (thickness: 30 µm) through the MTN were obtained from the brains and incubated overnight with rabbit antibodies against Kv1.1, Kv1.6, and Kv2.1 (1:500 dilution). Subsequently, the sections were incubated with the biotinylated goat anti-rabbit IgG antibody (1:200) for 2 h, followed by streptavidin-HRP (1:100; NEN, Boston, MA) for 1 h and then FITC-tyramide (1:100; NEN) for 10 min. The sections were mounted onto gelatin-coated glass slides, air-dried, coverslipped with ProLong Antifade Kit (Molecular Probes), and then observed with a confocal laser-scanning microscope (TCS SP, Leica, Tokyo, Japan); two laser systems providing excitation light of 488-nm wavelength (emission 510–525 nm) and that of 543-nm wavelength (emission 
570 nm) were used for FITC and RDA labeling, respectively. Digital images taken with the confocal microscopy were processed using Photoshop 7.0 (Adobe, San Jose, CA).
To ensure that the difference in the current-clamp performance between the Axoclamp 2B and Axopatch series of amplifiers does not affect the present results, data (partly illustrated in Fig. 9, Supplementary material) were obtained by using Axoclamp 2B. Effects of temperature on the spikes were also examined.
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RESULTS |
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Whole cell current-clamp (Fig. 1B) and cell-attached voltage-clamp recordings (Fig. 1C) were simultaneously obtained from the soma (S) and axon hillock (AH) of the same MTN neuron (Fig. 1A), respectively. In response to injection of a current pulse (* in Fig. 1B) into the soma at the resting membrane potential of –55 mV, an action potential was triggered during the decay phase of the electrotonic response (Fig. 1Ba). The temporal derivative of the spike potential (Fig. 1Bb) represents the rate of charging of the soma membrane, which is proportional to the capacitative transient current across the soma membrane (Magee and Johnston 1995). The simultaneously obtained cell-attached voltage-clamp recording at –70 mV (Fig. 1Cb) revealed two sequential negative peaks of capacitative current (Fig. 1Ca; see METHODS) in response to injection of a current pulse through the soma pipette. Because the latter was invariably larger than the former and slightly preceded the peak of the somatic capacitative current (S) (refer to vertical dotted line in Fig. 1, Bb and Ca), the latter would reflect the spike generated at the AH, from which the cell-attached recording was made. Then, the former capacitative current is most likely to have been generated in the patch membrane, presumably after the IS-spike generation (Colbert and Johnston 1996). Thus it is likely that, in response to the injection of current pulses through the soma pipette, the IS-spike was generated first, and this in turn backpropagated to sequentially trigger AH- and S-spikes. Similar observations were obtained from five paired recordings made on the soma and AH of MTN neurons. The time difference between the presumed IS and AH components of the capacitative currents recorded from the AH in response to the injection of current pulses through the soma pipette was 0.18 ± 0.03 ms (n = 5).
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Similar dual whole cell current-clamp recordings were made from the soma and AH (Fig. 2, Aa and Ab, respectively) of another MTN neuron (Fig. 2Ad). When short current pulses were applied through the soma pipette at a hyperpolarized membrane potential of –91 mV, S-spikes were triggered during the decay phase of the electrotonic responses (black traces in Fig. 2Aa), whereas AH-spikes were invariably triggered from the peak of the underlying subthreshold depolarization evoked at –85 mV whenever it reached the spike threshold (red traces in Fig. 2Ab). These observations indicate that the soma membrane is electrotonically more separated from the spike-initiation site than is the AH membrane. Nevertheless, the peak level of the S-spikes (+29.4 ± 8.3 mV) was significantly higher (P < 0.02, n = 5) than that of the AH-spikes (+18.1 ± 5.1 mV; compare the black and red horizontal dotted lines in Fig. 2Ab) when evoked at similar holding potentials (–82.8 ± 8.3 and –80.9 ± 7.1 mV for S- and AH-spikes, respectively). Furthermore, the maximum rate of rise of the S-spikes (325 ± 39 V/s) was also significantly (P < 0.004, n = 5) larger than that of the AH-spikes (259 ± 29 V/s). These features of backpropagation are in contrast to those of cortical pyramidal neurons (Stuart and Sakmann 1994; Stuart et al. 1997).
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This was not expected because the membrane potential is assumed to be isopotential anywhere in a round cell body. This electrotonic separation between the center of the soma and the AH was also evident from the amplitude attenuation (56 ± 6%, n = 5) and the lag time (0.45 ± 0.08 ms, n = 5) between the two peaks of subthreshold depolarizations in the soma and the AH (compare downward and upward arrowheads in Fig. 2, Aa and Ab). Thus the transient aniso-potentiality between the soma and AH was observed not only during the fast action potentials but also during the slow subthreshold responses. This indicates that the aniso-potentiality is not brought about artificially by the low-pass filtering, but likely by an unusually high internal resistance (Ri) between the soma and AH. To quantify the electrotonic separation, we calculated Ri by simulating the short-lived aniso-potentiality during the subthreshold responses (Fig. 2Bb). Ri was found to be unusually high (see DISCUSSION).
Furthermore, the simultaneous recordings disclosed two crucial differences between the S- and AH-spikes. Consistent with the observation shown in Fig. 1, B and C, the generation of an S-spike was actually preceded by that of an AH-spike (black and red open arrowheads, respectively, in Fig. 2Ac) and the spike duration at half-amplitude was significantly longer (P < 0.05, n = 8) in AH-spikes (0.80 ± 0.18 ms) than in S-spikes (0.71 ± 0.13 ms), as revealed in the superimposed traces of S- and AH-spikes (black and red traces, respectively, in Fig. 2Ac). Such a difference in the timing of the spike generation is also clearly seen in the temporal derivative traces (compare red and black arrows in the top traces in Fig. 2Ac). The time difference between the two positive peaks, reflecting the AH- and S-spikes, was 0.04 ± 0.01 ms in eight MTN neurons. Taken together, these data suggested that a spike was first initiated at the IS or the stem axon in response to the injection of current pulses at the soma, and then backpropagated to sequentially cause the AH- and S-spikes. Therefore by adding the backpropagation time from the spike-initiation site to AH (0.18 ± 0.03 ms, n = 5) to that from the AH to soma (0.04 ± 0.01 ms, n = 8), the total backpropagation time can be estimated as 0.22 ms.
Because the amplitude of the S-spikes, but not that of the AH-spikes, decreased with an increase in the current-pulse intensity (Fig. 2Aa), a rapidly activating K+ current involved in shaping spikes may be larger in the soma than in the AH. Therefore effects of 4-AP on the spike duration difference were examined. As shown in Fig. 2, Ca and Cb, the difference in the duration between the S- and AH-spikes was completely abolished by applying 4-AP (n = 3) (black and red traces, respectively), leaving the spike amplitude difference almost unchanged. Thus it is likely that a 4-AP–sensitive K+ current involved in shaping spikes was larger in the soma than in the AH. This possibility was further investigated in the next series of experiments.
Involvement of 4-AP–sensitive K+ current in shaping S-spikes
Whole cell current-clamp recordings were made from either the soma or the AH alone in the following series of experiments. The resting membrane potential was –67.8 ± 4.7 mV (n = 76) in the soma and –70.0 ± 5.1 mV (n = 13) in the AH. Similar to that seen in Fig. 2Aa, the S-spikes decreased in amplitude with an increase in the membrane depolarization level reached at the current-pulse end at a holding potential of –75 mV (Fig. 3Ac), but not at –60 mV (Fig. 3Ab). When the peak potential of the S-spikes (p in Fig. 3Ab) was plotted against the membrane depolarization level reached at the current-pulse end (v in Fig. 3Ab), p decreased with an increase in v for the S-spikes evoked at –75 mV (filled circles in Fig. 3Af), whereas p remained almost constant for those at –60 mV (open circles in Fig. 3Af). The difference in p between the S-spikes evoked at –60 and those at –75 mV became apparent at v > –50 mV, and increased with an increase in v (Fig. 3Af). The S-spikes evoked at –75 mV were also shorter in duration than those at –60 mV, as revealed by superimposed traces of the two respective S-spikes shown with asterisks in Fig. 3, Ac and Ab (compare continuous and interrupted traces in Fig. 3Ad). Furthermore, the application of 0.5 mM 4-AP increased the p values of the respective S-spikes evoked with different v values to the same level, abolishing the difference in the spike amplitude (refer to horizontal dotted line in Fig. 3Ae; see also filled squares in Fig. 3Af). Thus a 4-AP–sensitive K+ current that is largely inactivated at –60 mV and activated at potentials > –50 mV is likely to be involved in modulating the amplitude and duration of the S-spikes.
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It was also noticed that the delay in evoking S-spikes (i.e., the spike initiation delay plus the spike-backpropagation delay) was shortened by 4-AP (Fig. 3Ae), suggesting the involvement of K+ currents in increasing the apparent electrotonic distance from the soma to the spike-initiation site. As shown in Fig. 3C, when the time to the maximum rate of rise (MRR) of the S-spikes measured from the timing of the current-pulse offset (t in Fig. 3Ab) was plotted against v, t decreased nonlinearly with an increase in v. The t in evoking the S-spikes from –75 mV (filled circles) was larger than that from –60 mV (open circles), when compared at the same value of v. This difference became apparent at v > –50 mV and increased with an increase in v (Fig. 3, Ad and C). Thus the K+ current that largely inactivates at –60 mV and is active at potentials > –50 mV is likely to be involved in causing the delay in evoking S-spikes at –75 mV. In fact, the v–t relationship as a measure of the delay in evoking S-spikes was shifted negatively by applying 4-AP, as illustrated in Fig. 3C (compare filled squares with filled circles). In the v–t relationships obtained from five MTN neurons examined at holding potentials between –80 and –75 mV, the t value at the same v ranging between –50 and –30 mV was significantly shortened by 4-AP from 1.45 ± 0.23 to 0.76 ± 0.12 ms (n = 5, P < 0.005).
The voltage dependencies of K+ current shaping the S-spikes were very similar to those causing a delay in evoking the S-spikes (Fig. 3, Af and C) and the shortening of the voltage-dependent delay in the evoking S-spikes by 4-AP was always accompanied by the elimination of the voltage-dependent shaping of the S-spikes (Fig. 3Ae). Therefore the K+ current characterized by 1) sensitivity to 0.5 mM 4-AP, 2) large steady-state inactivation at –60 mV, and 3) activation at potentials > –50 mV is likely to be involved in shaping the S-spikes as well as in elongating the electrotonic distance from the soma to the spike-initiation site. This presumed 4-AP–sensitive K+ current is hereafter termed IK4-AP.
On the other hand, when whole cell recordings were obtained from the AH (Fig. 3Ba), the AH-spike amplitude did not decrease with an increase in the current-pulse intensity even at a holding potential of –77 mV (continuous traces in Fig. 3Bb). Furthermore, 4-AP did not affect the delay in evoking the AH-spikes while increasing the spike amplitude similarly (interrupted traces in Fig. 3Bb). Subsequently, the v–t relationship of the AH-spikes was not shifted negatively by 4-AP (compare open and filled triangles in Fig. 3C). These observations suggest that the presumed IK4-AP was not involved in causing the delay in evoking the AH-spikes. Similar observations were obtained in whole cell recordings made from the AH of the five MTN neurons examined at holding potentials between –90 and –75 mV. There was no significant difference (n = 5, P > 0.1) in the t value at the same v ranging from –60 to –40 mV, between the AH-spikes obtained before and after the application of 4-AP (0.57 ± 0.16 and 0.54 ± 0.15 ms, respectively).
Because 4-AP shortened the delay in evoking S-spikes by 0.69 ± 0.25 ms (n = 5), while leaving that in evoking AH-spikes almost unchanged (0.04 ± 0.03 ms, n = 5), 4-AP likely shortens the electrotonic distance from the soma to the spike-initiation site without significantly affecting that from the AH to the spike-initiation site (see DISCUSSION). These observations strongly suggest that the distribution of 4-AP–sensitive K+ channels differs between the soma and stem axon.
Input-dependent difference in the amplitude of S-spikes
In the same paired recordings as shown in Fig. 2A, a current-pulse injection into the AH caused a larger S-spike (continuous trace in Fig. 4Aa) in comparison with that evoked by a current-pulse injection into the soma (interrupted trace in Fig. 4Aa). The amplitude of the S-spikes triggered by the AH injection (97.4 ± 11.6 mV) was invariably and significantly (P < 0.02, n = 5) larger than that of the S-spikes triggered by soma injection (92.9 ± 10.4 mV), when compared at the same holding potential ranging between –73 and –91 mV (Fig. 4Aa). Such an input-dependent difference in the amplitude of S-spikes may indicate an input-dependent differential involvement of the presumed IK4-AP. In contrast, there was no significant difference (P > 0.7, n = 5) in the amplitude between AH-spikes evoked by current-pulse injections into the AH and those by current-pulse injections into the soma (81.8 ± 8.8 and 81.6 ± 9.3 mV, respectively; compare continuous and interrupted traces in Fig. 4Ab), when compared at the same holding potential ranging between –70 and –85 mV.
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Differential effects of 4-AP on init- and inv-S-spikes
As seen in Fig. 4Ba, recording and stimulating electrodes were placed on the soma and on the stem axon of an MTN neuron, respectively. A spike was evoked almost in an all-or-nothing manner by stimulation of the stem axon with a threshold intensity of 1.0 µA, and was identified as an inv-S-spike based on the abrupt rising phase of the spike at a latency of 0.25 ms (Fig. 4Bb).
Such S-spikes generated by stimulation of the stem axon were compared with those initiated by injection of current pulses in the same MTN neurons. When a current pulse injection was applied in combination with stimulation of the stem axon at –61 mV, there was no distinctive difference in the amplitude between the init- and inv-S-spikes (Fig. 4Bc). However, when the combined stimulation was applied at –76 mV, the amplitude of the init-S-spike (* in Fig. 4Bc) was smaller than that of the inv-S-spike (** in Fig. 4Bc). With membrane hyperpolarization, the peak potential level of the init-S-spikes (filled circles in Fig. 4Bd) decreased substantially, whereas that of the inv-S-spikes (open circles in Fig. 4Bd) decreased only slightly. Thus the difference in the amplitude between the init- and inv-S-spikes emerged voltage dependently. When inv- and init-S-spikes were evoked at the same holding potential ranging between –80 and –75 mV, the amplitude of the inv-S-spikes (99.0 ± 8.0 mV) was invariably and significantly (P < 0.01, n = 5) larger than that of init-S-spikes (89.2 ± 7.0 mV). However, the differences in the amplitude between the inv- and init-S-spikes at various holding potentials were completely eliminated by applying 4-AP (open and filled triangles in Fig. 4, Bd, Ca, and Cb). These observations clearly indicate that the involvement of the presumed IK4-AP in modulating the spike amplitude is larger in the init-S-spike than in the inv-S-spike. This may be consistent with the observation that the amplitude of the S-spike evoked by injection of a current pulse at the AH, which may be comparable to an inv-S-spike, is larger than that evoked by injection of a current pulse into the soma as the init-S-spike (Fig. 4A).
Consistent with that seen in Fig. 3, Ae and C, 4-AP shortened the delay in spike initiation, as revealed in the superimposed traces in Fig. 4Cc. By contrast, the delay or latency to spike invasion seemed not to be shortened as revealed in the superimposed traces, obtained after subtraction of the nothing responses from the inv-S-spikes in the respective conditions (Fig. 4Cd). There was no significant (P > 0.1, n = 5) change in the latency to the abrupt rising of the inv-S-spikes obtained before and after the application of 4-AP (0.20 ± 0.03 and 0.23 ± 0.05 ms, respectively). These observations suggest another aspect of the differential involvement of the presumed IK4-AP between spike initiation and invasion. 4-AP might have shortened the electrotonic separation from the soma to the spike-initiation site while leaving the length constant in the stem axon almost unchanged. This is consistent with the differential effects of 4-AP on the delay in evoking S- and AH-spikes (Fig. 3). Provided that the spike-initiation site is located in the stem axon, the shortening of the delay by 4-AP would largely result from the electrotonic separation between the soma and AH, but not from the electrotonic separation between the AH and the spike-initiation site. The validity of these notions would strongly depend on the presence or absence of 4-AP–sensitive K+ channels in the soma and stem axon, respectively. This issue was further examined in the next experiment.
Differential effects of 4-AP on the refractory period between inv-S-spikes and axonal spikes
Because 4-AP prolonged the duration of S-spikes, the refractory period of S-spikes should also be prolonged. Then, the sensitivity of axonal spikes to 4-AP can be indirectly assessed by examining its effects on the refractory period of inv-S-spikes. As shown in Fig. 5Aa, when the interstimulus interval (ISI) of paired stimulation applied to the stem axon was decreased, the second inv-S-spike displayed only a slight decrease in amplitude but disappeared suddenly at 3.1 ms ISI (open arrowhead in Fig. 5Aa), which was also illustrated by plotting the relative amplitude (RA) of the second to the first S-spike against the ISI (minimum RA = 0.96; open circles and arrow in Fig. 5Ca). Because the failure of the second S-spike occurred without a distinct attenuation of amplitude, the failure should have occurred at the stimulated site of the stem axon. Therefore the possible refractory period of the impulse caused at the stimulated site of the stem axon (about 3.1 ms) should be longer than that of the inv-S-spike itself. As seen in Fig. 5Ab, when 4-AP was applied, the second S-spike displayed a progressive attenuation of its amplitude with a decrease in the ISI to 3.6 ms (minimum RA = 0.75; filled circles and arrow in Fig. 5Ca), which led to the failure of the second S-spike at 3.5 ms ISI (filled arrowhead in Fig. 5Ab). However, it was not clear whether the failure of the second inv-S-spike occurred as a result of the refractoriness of the S-spike itself or of that of the impulses evoked at the stem axon. To distinguish between the two possibilities, paired current pulses were injected into the soma in the presence of 4-AP. When the interval between the two current pulses was decreased in the presence of 4-AP, the minimum RA of the init-S-spikes (0.67, Fig. 5B; see also open triangles in Fig. 5Cb) was smaller than that of the inv-S-spikes (0.75; compare Fig. 5, Ab and B and also Ca and Cb). Therefore the failure of the second inv-S-spike seen in Fig. 5Ab would be a result of the refractoriness of the impulse evoked at the stimulated site, but not of the refractoriness of the inv-S-spike itself. Thus 4-AP prolonged the possible refractory period of the presumed impulse in the stem axon from 3.1 to 3.5 ms (Fig. 5, Aa, Ab, and Ca).
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), K+ currents (Catacuzzeno et al. 2003), and L-type Ca2+ current (Workman et al. 1999), and also a similar modulation of the proportion of the two time constants of recovery from inactivation in an L-type Ca2+ current was reported in heart myocytes (Workman et al. 1999). With a decrease in the interval of paired current pulses, especially beyond 4.0 ms in the presence of 4-AP, the RA of the init-S-spikes decreased to <0.70, leading to a failure of the second init-S-spike (Fig. 5B and open triangles in Fig. 5Cb), whereas the interpeak interval remained almost constant at 4.0 ms (open triangles in Fig. 5Cc). Therefore the absolute refractory period of the init-S-spikes in the presence of 4-AP can be regarded as such an interpeak interval that remains almost unchanged in spite of decreases in the interpulse interval. At this minimum interpeak interval (i.e., the absolute refractory period), the RA was <0.70. Subsequently, the minimum interpeak intervals of 3.3 and 4.2 ms between the paired inv-S-spikes obtained before and after 4-AP application, respectively, can be regarded as the absolute refractory periods of presumed impulses in the stem axon that would cause the RA of 0.70 (open and filled diamonds, respectively, in Fig. 5Cb). Using the same two time constants but assuming the proportion of the two components arbitrarily between the two values of proportions for the inv-S-spikes in the absence and presence of 4-AP, the attenuation curves were reconstructed for the two refractory periods of the presumed impulse in the stem axon in the absence and presence of 4-AP (interrupted lines attached to open and filled diamonds, respectively, in Fig. 5Cb). Thus from the regression analyses, the attenuation curve of the presumed axonal spikes seemed to be less affected by 4-AP than that of the S-spikes (see Fig. 5Cb).
Similar differential effects of 4-AP on the refractory period between an S-spike and a presumed impulse in the stem axon were observed in four MTN neurons examined. Because of the presumed IK4-AP, the absolute refractory period of an inv-S-spike was invariably shorter than that of the presumed impulse in the stem axon (3.15 ± 0.43 ms, n = 4) as revealed by the large minimum RA (0.92 ± 0.04, n = 4) of the inv-S-spikes. Indeed, 4-AP prolonged the refractory period of the inv-S-spike to a degree similar to that of the presumed impulse in the stem axon (4.05 ± 0.27 ms, n = 4), as revealed by the small minimum RA (0.71 ± 0.05, n = 4) of the inv-S-spikes. This observation also indicates that there was a prolongation of the refractory period of the presumed impulses in the stem axon from 3.15 ± 0.43 to 4.05 ± 0.27 ms (n = 4), which would be much smaller than that of the refractory period of the inv-S-spikes in view of the large change in RA. This smaller prolongation, however, may not be necessarily caused by a direct effect of 4-AP. The broadening of inv-S-spikes by 4-AP could indirectly cause a prolongation of the refractory period of the presumed impulses in the stem axon as a result of the electrotonic continuity between the soma and the stem axon.
These electrophysiological observations strongly suggested that the distribution of 4-AP–sensitive K+ channels differs between the soma and stem axon of MTN neurons. Therefore in the next experiment, the distribution of K+ channels was directly investigated using an immunohistochemical technique.
Differential subcellular localization of 4-AP–sensitive versus –insensitive K+ channels
Compared with the Kv1 family, the Kv4 family is known to be less sensitive to 4-AP (IC50 of 4-AP = 1.5 to 10 mM; Coetzee et al. 1999; Faivre et al. 1999; Fiset et al. 1997; Tseng et al. 1996). Because a low concentration of 4-AP (0.5 mM) completely abolished the voltage-dependent modulations of init-S-spikes (Figs. 2C, 3, and 4C), it is unlikely that the Kv4 family is involved in these voltage-dependent modulations of S-spikes. Therefore the following 10 subtypes of K+ channels that are sensitive to relatively lower concentrations of 4-AP (Coetzee et al. 1999; North 1995) were examined in the present immunohistochemical analysis: Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, Kv2.1, Kv3.1, Kv3.2, and Kv3.4. Of these 10 subtypes, three subtypes—Kv1.1, Kv1.6, and Kv2.1—were predominantly expressed in MTN neurons. Kv1.1 (Fig. 6, A and B) and Kv1.6 (Fig. 6, C and D), which are highly sensitive to 4-AP with IC50 values of 0.16–1.0 (Christie et al. 1989; Stuhmer et al. 1988, 1989) and 0.3–1.5 mM (Grupe et al. 1990; Kirsch et al. 1991), respectively, were localized in the somata alone. A large number of such immunostained neurons were observed throughout the entire rostrocaudal extent of the MTN.
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; Belevych et al. 2002; Smirnov et al. 2002) and is relatively high voltage activated (activation threshold 
–30 to –20 mV, half-maximal activation at potentials +10 mV; Coetzee et al. 1999) in comparison with the Kv1 family, was distributed not only in the somata but also in the stem axons of MTN neurons (filled arrowheads in Fig. 6, E and F). These Kv2.1-positive neurons were found mainly at the caudal level of the MTN. In addition, weak immunostaining with anti-Kv1.2, -Kv1.3, -Kv1.5, -Kv3.2, and -Kv3.4 antibodies occurred only in the somata of MTN neurons. No MTN neurons were immunostained with anti-Kv1.4 or -Kv3.1 antibody.
Consistent with these observations, the present double fluorescence histochemistry revealed that only the somata, but not the stem axon, of MTN neurons retrogradely labeled with RDA were immunostained for Kv1.1 (Fig. 7A) and Kv1.6 (Fig. 7B), whereas both the somata and the stem axons of the RDA-labeled MTN neurons were immunostained for Kv2.1 (Fig. 7C). The immunoreactivity for each Kv subtype in the somatic region was located not only along the plasma membrane but also in the cytoplasm, probably in the endoplasmic reticulum, as previously reported in nodose ganglion neurons (Glazebrook et al. 2002). Of particular interest was that the AH region of MTN neurons was virtually devoid of Kv1.1 or Kv1.6 immunoreactivity (asterisks in Fig. 7, Ab and Bc), consistent with the diminished involvement of the presumed IK4-AP in AH-spikes in comparison with S-spikes (see Fig. 2Ac and compare Fig. 3, Ae and Bb).
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; Stuhmer et al. 1989; Swanson et al. 1990) and relatively low-concentration 4-AP–sensitive Kv1 family. |
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DISCUSSION |
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Irrespective of patch-clamp amplifiers, current follower (Axopatch series, Fig. 4, B and C) or voltage follower (Axoclamp 2B; see Fig. 9, A and B in the Supplementary material), the amplitude of the inv-S-spike was invariably and significantly larger than that of the init-S-spike, when compared in the same MTN neuron. Furthermore, the spike amplitude difference was abolished by 4-AP application (Fig. 4, Bd and C). Therefore it was strongly suggested that 4-AP–sensitive K+ channels were involved in differentially shaping the S-spikes, in a manner dependent on whether the S-spike was initiated (backpropagated) or generated by the invasion of axonal spikes.
The K+ current that was previously identified as having half-maximal activation and inactivation at –34.7 and –62.0 mV, respectively, in MTN neurons was sensitive to a low concentration (0.5 mM) of 4-AP (Del Negro and Chandler 1997). Because the activation of Na+ current is much faster than that of 4-AP–sensitive K+ currents (Patlak 1991; Rudy 1988), the involvement of the presumed IK4-AP would be smaller when an S-spike is evoked by the rapid invasion of an axonal spike than when an S-spike was activated with a delay during the decay phase of the depolarizing response to current pulses into the soma (Fig. 4Ca). The electrotonic separation of the soma from the spike-initiation site may allow time for backpropagated S-spikes to be shaped by the presumed IK4-AP. Thus as a result of the differential involvement of the presumed IK4-AP, the amplitude and duration of the init-S-spikes were distinct from those of inv-S-spikes. 4-AP shortened the delay in evoking S-spikes without affecting the delay in evoking AH-spikes (Fig. 3), indicating that the presumed IK4-AP increases the delay in the initiation of S-spikes without increasing the backpropagation time from the spike-initiation site in the stem axon to the AH. Consistent with this observation, 4-AP did not affect the latency of inv-S-spikes (Fig. 4Cd) and the refractory period of presumed axonal spikes was not notably prolonged by 4-AP (Fig. 5C). These observations strongly suggest the differential distribution of 4-AP–sensitive K+ channels in the soma and stem axon.
Kinetics of IK4-AP versus that of Kv1.1 and Kv1.6
The present immunohistochemical examinations demonstrated that Kv1.1, Kv1.6, and Kv2.1 channel subunits were localized in the somata of MTN neurons, whereas only the Kv2.1 subunit was expressed in the stem axon (Figs. 6 and 7). The possible inactivation kinetics of the presumed IK4-AP seen in the present study (Figs. 3, Ab, Ac, Af, and C and 4, Bc and Bd) seems to be inconsistent with that of a possible K+ current if these subunits form homomultimeric channels. The homomultimeric channels composed of these Kv subunits display little time-dependent and voltage-dependent inactivation even at potentials > –40 mV, as reported in an expression system (Christie et al. 1989; Stuhmer et al. 1989). However, the inactivation kinetics of those Kv channels can be enhanced and their voltage dependency may be shifted negatively by various mechanisms, including the formation of heteromultimeric channels (Isacoff et al. 1990; Ruppersberg et al. 1990), the coexpression with Kv
subunit (Heinemann et al. 1996; Rettig et al. 1994), and the posttranslational processing of Kv
/ subunit protein (Kwak et al. 1999; Nitabach et al. 2002). Therefore the electrophysiological findings in this study are not necessarily inconsistent with the inactivation kinetics of these Kv channels reported previously. Furthermore, the presumed IK4-AP was activated from potentials as low as –50 to –40 mV (Figs. 3 and 4) and was sensitive to a low concentration of 4-AP (0.5 mM). These properties of the presumed IK4-AP were consistent with those of the homomultimeric channels of these Kv subunits (Christie et al. 1989; Swanson et al. 1990). The presumed IK4-AP is likely to correspond to the slow transient outward current previously isolated in MTN neurons, which was largely inactivated at –60 mV and was largely sensitive to 0.5 mM 4-AP (Del Negro and Chandler 1997).
Functional differences among the peripheral, stem, and central axons
In a simulation study (Amir and Devor 2003), it was argued that the excitability in neither the IS nor the soma affects the through-conduction from the peripheral to the central axon. Nevertheless, whenever an impulse arising from the peripheral axon failed to invade the stem axon, it also failed to invade the central axon, as demonstrated in frog DRG neurons (Stoney 1990). Taken together with the present findings on the spike initiation mechanism in the stem axon, it is likely that impulses arising from the peripheral axon trigger impulses in the stem axon before they reach the branching point, and then invade into the soma and simultaneously propagate forward through the central axon (Fig. 8Aa). If this is the case, the Na+ channel density of the axonal membrane around the three-way junction would be relatively low. Then, without triggering impulse in the stem axon, there would be no through-conduction from the peripheral to the central axon. Indeed, as reported previously, the central axon of MTN neurons displayed a pause of impulse activity whenever soma spiking was inhibited during the jaw-opening phase (Kolta et al. 1995; Westberg et al. 2000). Taken together, it is most likely that no impulses would be seen in the central axon whenever impulses arising from the muscle spindles fail to invade the stem axon and/or soma, as originally suggested by a series of previous studies (Kolta et al. 1995; Westberg et al. 2000). On the other hand, when a spike was initiated in the stem axon in response to synaptic action, it would invade the central axon and also propagate backward into the soma (Fig. 8Ab).
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Voltage-dependent switching between two functional modes presumed in MTN neurons
Masticatory movement has been considered to be mediated by the central pattern generator (CPG), from which MTN neurons receive synaptic inputs and thereby act not only as PSNs but also as interneurons independent of the activities of the peripheral receptors (Fig. 8B). In addition, it is well established that the central axon of MTN neurons can act independently of the activities of somata through the mechanism of 
-aminobutyric acid (GABA)–mediated primary-afferent depolarization (Kolta et al. 1995; Verdier et al. 2003; Westberg et al. 2000). Thus MTN neurons can display multiple functional modes.
At depolarized membrane potentials in the somata (Fig. 8Ba) where 4-AP–sensitive K+ currents might be largely inactivated, MTN neurons display oscillatory firing activity either spontaneously (Pedroarena et al. 1999; Wu et al. 2001, 2005) or triggered synaptically (Verdier et al. 2004; Yamuy et al. 2000). In contrast, at hyperpolarized membrane potentials in the soma of MTN neurons (Fig. 8Bb), where the inactivation of 4-AP–sensitive K+ currents is largely removed, 4-AP–sensitive K+ currents would facilitate spike invasion arising from sensory endings (Fig. 8Ca), but prevent MTN neurons from initiating spikes in response to fast synaptic inputs (Fig. 8Cb). However, as suggested by previous studies (Kolta et al. 1995; Westberg et al. 2000), a further hyperpolarization in the soma might prevent the spike-initiation site from being activated by impulses arising from the peripheral axon, provided that the safety factor for the saltatory conduction from the peripheral axon to the spike-initiation site in the stem axon is relatively low in consequence of the low Na+ channel density around the three-way junction.
Thus MTN neurons can act either as PSNs or as interneurons depending on the membrane potential levels (Fig. 8B). Our study would provide a rationale for exploring the slow modulatory actions underlying the membrane depolarization and hyperpolarization, which have not yet been clarified. Serotonin and dopamine receptors expressed in MTN neurons (5-HT1/2: Kolta et al. 1993; Lazarov 2002; D2: Lazarov 2002; Lazarov et al. 1998) may be strong candidates for inducing the slow membrane depolarization (Beique et al. 2004; Zhang 2003) and hyperpolarization (Einhorn et al. 1991; Lacey et al. 1987), respectively.
Electrotonic separation in round somata of MTN neurons
In a neuron having a round cell body, the membrane potential across the soma area is considered to be isopotential, as has been assumed in the equivalent cylinder model with a single lumped soma compartment (Segev 1992). However, the round cell body of MTN neurons was not isopotential during subthreshold slow depolarizations or during fast spiking (Figs. 2, A and B and 4A). Although this aniso-potentiality was short-lived, both the amplitude attenuation (56 ± 6%, n = 5) and the lag time (0.45 ± 0.08 ms, n = 5) between the two peaks of subthreshold depolarizations in the soma and the AH (compare downward and upward arrowheads in Fig. 2, Aa and Ab) clearly indicate the unusual electrotonic separation within the spherical soma. The electrotonic separation is inversely proportional to the length constant (
). Therefore the internal resistance (Ri) between the two sites was estimated from the following simultaneous differential equations for the two-compartment model
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Because the cell size varies from neuron to neuron, we estimated Rm-AH/Ri and Ri · CAH, rather than the absolute value of Ri. For simplification, the two compartments of the soma and AH were assumed to have the same capacitance and membrane resistance. Rm-AH/Ri and Ri · CAH were estimated to be approximately 4 and 0.75 ms, respectively (Fig. 2Bb). Even if Rm-AH is as low as 15 M, which was estimated by using 1-ms current pulses (Fig. 4Ab) and thereby reflects only a small fraction of Rm-AH, Ri is calculated to be 3.7 M. This is >150-fold larger than that of the internal resistance (21 k) between the two points separated by 30 µm in an axon having a 30-µm diameter (50 · cm; Katz 1966).
Because of this unusual electrotonic separation, MTN neurons having round somata could display the backpropagation of an IS-spike to the soma through the AH. The nature of the active backpropagation is well reflected in the amplitude and the rate of rise of backpropagated S-spikes, which were larger than those of the preceding AH-spike (Fig. 2, Aa and Ab; see also top traces in Fig. 2Ac), unlike the case of cortical pyramidal cells (Stuart and Sakmann 1994).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The Supplementary Material for this article is available online at http://jn.physiology.org/cgi/content/full/00176.2006/DC1.
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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