Role of Calcium Conductances on Spike Afterpotentials in Rat Trigeminal Motoneurons

Masayuki Kobayashi1, Tomio Inoue1, Ryuji Matsuo1, Yuji Masuda1, Osamu Hidaka1, Youngnam Kang2, and Toshifumi Morimoto1

1 Department of Oral Physiology, Faculty of Dentistry, Osaka University, Suita, Osaka 565; and 2 Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Kobayashi, Masayuki, Tomio Inoue, Ryuji Matsuo, Yuji Masuda, Osamu Hidaka, Youngnam Kang, and Toshifumi Morimoto. Role of calcium conductances on spike afterpotentials in rat trigeminal motoneurons. J. Neurophysiol. 77: 3273-3283, 1997. Intracellular recordings were obtained from rat trigeminal motoneurons in slice preparations to investigate the role of calcium conductances in the depolarizing and hyperpolarizing spike afterpotential (ADP and mAHP, respectively). The mAHP was suppressed by bath application of 1 µM apamin, 2 mM Mn2+, and 2 mM Co2+, and also by intracellular injection of ethylene glycol-bis(b-aminoethylenether)-N,N,N',N'-tetraacetic acid (EGTA), suggesting that the potassium conductance generating the mAHP is activated by Ca2+ influx. Mn2+ (2 mM) or Cd2+ (500 µM) reduced the ADP, whereas the ADP amplitude was increased by raising extracellular Ca2+ concentration from 2 to 8 mM by bath application of Ba2+ (0.5-5 mM) and by intracellular injection of EGTA. This would suggest that Ca2+ itself is likely to be the charge carrier generating the ADP. Focal application of omega -conotoxin GVIA (10-30 µM) suppressed the mAHP and enhanced the ADP, whereas focal application of omega -agatoxin IVA (10-100 µM) reduced the ADP amplitude without apparent effects on the mAHP. We conclude that Ca2+ influx through omega -agatoxin IVA-sensitive calcium channels is at least in part responsible for the generation of the ADP and that Ca2+ influx through omega -conotoxin GVIA-sensitive calcium channels contributes to the generation of the mAHP. Because of the selective suppression of the ADP and mAHP by omega -agatoxin IVA and omega -conotoxin GVIA, respectively, it is suggested that both calcium channels are separated geometrically in rat trigeminal motoneurons.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The action potential is followed by afterdepolarization(ADP) and medium-duration afterhyperpolarization (mAHP)in spinal (Granit et al. 1963; Harada and Takahashi 1983; Walton and Fulton 1986), hypoglossal (Viana et al. 1993b), and guinea pig trigeminal (Chandler et al. 1994) motoneurons. Additional spikes can be triggered from the ADP when it is enhanced (Viana et al. 1993a), whereas the mAHP is an important factor for slowing the rate of firing (Hille 1992). Thus the potential balance between the ADP and mAHP seems to determine the firing pattern of trigeminal motoneurons (TMNs) and affects the quantitative relationship between the input to TMNs and the final motor output of the muscles.

Voltage-gated calcium channels have been classified into at least five types referred to as the L, N, P, Q, and T types (Bean 1989; Hess 1990; Llinas et al. 1989; Mintz et al. 1992; Randall and Tsien 1995), and multiple types of calcium channels coexist in a single neuron (Bean 1989; Miller 1987). It may be possible that different types of calcium channels participate in distinct physiological functions (Christie et al. 1995). Contribution of calcium currents to generating the ADP has been reported in spinal (Harada and Takahashi 1983; Walton and Fulton 1986) and hypoglossal (Umemiya and Berger 1994; Viana et al. 1993a) motoneurons. Furthermore, calcium-activated potassium current has been demonstrated to be involved in generating the mAHP in guinea pig TMNs (Chandler et al. 1994) as well as other motoneurons (Barrett and Barrett 1976; Nishimura et al. 1989; Sah and McLachlan 1992; Viana et al. 1993b; Walton and Fulton 1986; Zhang and Krnjevic 1987). Therefore Ca2+ influx may cause both the ADP and mAHP simultaneously. However, it is not clear whether the generation of the ADP and the mAHP were differentially regulated by distinct types of calcium channels in single neurons.

In the present study, differential regulations of the ADP and mAHP were investigated in rat TMN in vitro slice preparations by the use of the intracellular recording method. We demonstrate that Ca2+ influx through omega -agatoxin IVA (omega -Aga-IVA)-sensitive channels is responsible for the generation of the ADP, whereas Ca2+ influx through omega -conotoxin GVIA (omega -CTx-GVIA)-sensitive calcium channels activates calcium-activated potassium currents responsible for the mAHP. These results suggest that each specific type of calcium conductance may participate in distinct physiological functions in TMNs. Preliminary results of this study were reported previously in abstract form (Kobayashi et al. 1995).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Subjects

Seventy-nine Sprague-Dawley rats (3-6 wk old) were used for slice preparations. To identify the recorded cell as a TMN histologically, we employed the fluorescence double-labeling technique (cf. Viana et al. 1990) in the first series of the experiments (20 of the 79 animals). Those 20 animals were anesthetized with ketamine HCl (150 mg/kg im) and chlorpromazine HCl (12.5 mg/kg im), and both sides of the masseter nerve were dissected. A small amount of dextran-tetramethylrhodamine-lysine (DRL; molecular weight 10,000, Molecular Probes) in crystal was applied to the central cut ends of the nerve for 30 min, and then the cut ends were rinsed with saline. After 2-5 days the 20 animals were reanesthetized for slice preparation. Biocytin (Sigma), with which the recording microelectrode had been filled, was injected into the recorded neurons (n = 40 neurons), and biocytin-injected neurons were visualized with fluorescein isothiocyanate (FITC). Of the 40 neurons, 15 were also labeled by DRL (Fig. 1), indicating that these neurons were masseter motoneurons. The remaining 25 neurons were also visualized with horseradish peroxidase (HRP), and all of the 25 neurons were located within the trigeminal motor nucleus. It is most likely that the neurons within the trigeminal motor nucleus are TMNs, because interneurons are very few in the trigeminal motor nucleus (Sessle 1977). Therefore no histological efforts were made to identify the recorded neurons in the second series of experiments (n = 108 neurons).


View larger version (42K):
[in this window]
[in a new window]
 
FIG. 1. Photomicrographs of masseter motoneurons labeled retrogradely with dextran-tetramethylrhodamine-lysine (DRL; A) and of a motoneuron labeled by intracellular injection of biocytin (B). Arrows: double-labeled masseter motoneuron. Bar: 100 µm.

Slice preparation

Animals were anesthetized with ketamine HCl (150 mg/kg im) after an injection of chlorpromazine HCl (12.5 mg/kg im). After decapitation, the skull was removed and the brain was excised rapidly. The brain was placed in cold modified artificial cerebrospinal fluid (M-ACSF; see below for composition) and sectioned at the intercollicular level and at the obex. The rostral side of the brain stem was glued onto the stage of a microslicer (DTK-1500, Dosaka) with cyanoacrylate. Transverse slices (450 µm) were cut in cold M-ACSF. The slices were transferred to a holding chamber containing normal artificial cerebrospinal fluid (N-ACSF; see below for composition) maintained at room temperature.

Solutions

The composition of the N-ACSF was (in mM) 130 NaCl, 3 KCl, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, and 10 D-glucose. The M-ACSF was made from N-ACSF by replacing 130 mM NaCl with 260 mM sucrose. The N-ACSF and the M-ACSF were bubbled with a mixture of 95% O2-5% CO2, and the pH of these solutions was 7.35-7.40. Viable motoneurons were obtained by the use of M-ACSF during slice preparation (Aghajanian and Rasmussen 1989; Chandler et al. 1994). In some experiments the Ca2+ concentration was raised to 6-8 mM. When Mn2+, Co2+, Ba2+, or Cd2+ was added to the ACSF, 2 mM CaCl2 was replaced with 2 mM MnCl2, CoCl2, or BaCl2, or 500 µM CdCl2, respectively, and NaH2PO4 was omitted to avoid precipitation. For anion substitution experiments, Cl- was replaced with equimolar amounts of isethionate and the bath was grounded through an agar-KCl bridge. The following drugs were added directly to the perfusate: tetrodotoxin (1 µM) (Wako), tetraethylammonium chloride (TEA, 1-10 mM replacing equimolar NaCl), 4-aminopyridine (4-AP, 0.5 mM), charybdotoxin (ChTX, 10-30 nM) (Peptide), NiCl2 (0.5 mM), and nifedipine (10-20 µM dissolved in absolute ethanol). Apamin (10-20 µM) (Peptide), omega -CTx-GVIA (10-30 µM) (Peptide), and omega -Aga-IVA (10-100 µM) (Peptide) were each dissolved in N-ACSF and applied to the surface of a slice by pressure as microdroplets from a micropipette. All drugs were obtained from Nakarai tesque (Kyoto, Japan) unless otherwise specified.

Recording

After 2-12 h of incubation in the holding chamber, slices were transferred to an interface-type chamber. The recording chamber was continuously perfused with N-ACSF at a rate of 1-1.5 ml/min, and humidified 95% O2-5% CO2 flowed over the slice. All experiments were performed at 32 ± 1°C. Intracellular recordings were obtained with glass microelectrodes (1.2-1.5 mm OD) (Sutter Instruments). In the first series of experiments, the microelectrode was filled with 1% biocytin in 1 M KCl and 0.05 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.6) for intracellular staining. In the second series of experiments, the microelectrode was filled with 1 M KCl and 0.05 M Tris buffer (pH 7.6). For intracellular injection of ethylene glycol-bis(b-aminoethylenether)-N,N,N',N'-tetraacetic acid (EGTA) (Sigma), a calcium chelator, the microelectrode was filled with 0.25 M EGTA and 1 M KCl. The DC resistance of the microelectrodes ranged from 30 to 130 MOmega . The amplifier, an Axoclamp 2B (Axon Instruments), was used in either bridge or discontinuous current-clamp mode. During discontinuous current-clamp recordings, a 2- to 5-kHz sampling rate was employed at a 30% duty cycle. The head stage output was monitored on a separate oscilloscope to ensure proper capacitance adjustment and adequate settling of the microelectrode. Membrane potential and current were digitized and stored on a computer hard disk with the use of software (Clampex, Axon Instruments) through an A-D converter.

Data are presented as means ± SE. Comparisons of data before and after the drug application were based on Student's paired t-tests. The Mann-Whitney U test was used when the difference of the variances was great. The Friedman test was performed for comparison of data on the amplitude of ADPs during repetitive firing before and after the drug application. The level of P < 0.05 was assumed as significant.

Histology

After recordings, the slices obtained from the DRL-injected animals were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 12-24 h, and were then transferred to 25% sucrose in 0.02 M phosphate-buffered saline for 12-24 h. The slices were frozen, sectioned at 25-30 µm with a cryostat, and thaw-mounted onto poly-L-lysine-subbed glass slide. The sections were incubated with FITC-conjugated streptavidin (1:100) (Amersham) for 10 min, examined with a fluorescein microscope equipped with an appropriate excitation filter (B2 filter for FITC; G filter for DRL), and photographed. To examine whether biocytin-injected neurons were localized in the trigeminal motor nucleus, sections were reincubated with HRP-conjugated streptavidin (1:500) (Dako) for 60 min at room temperature, and HRP was developed with 3,3'-diaminobenzidine and 0.003% H2O2 in 0.5 M Tris-HCl buffer (pH 7.4) with nickel ammonium sulfate intensification. Sections were counterstained with neutral red.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

General properties

Through the stereomicroscope, we could see the trigeminal motor nucleus as a pale gray area medial to the trigeminal sensory nucleus. We placed the recording electrode in this area. The present study is based on recordings from 148 neurons that had stable resting potentials more negative than -55 mV (-67.2 ± 0.5 mV; n = 148) and displayed spikes >70 mV (measured from resting membrane potential to the spike peak). Input resistance, calculated from the relationship between injected current intensity (-0.2 to -0.5 nA) and the steady-state (>200 ms) voltage response, was 13.4 ± 0.7 MOmega (n = 141). Rheobase, measured as the minimum current (duration 300 ms) necessary to evoke a single action potential 100% of the time in response to at least five consecutive stimuli, was 1.5 ± 0.1 nA (n = 94). No neurons fired spontaneously. Single action potentials were elicited by an injection of brief (2- to 3-ms) depolarizing current pulses. Action potential amplitude was 94.8 ± 0.8 mV(n = 148), and duration was 0.7 ± 0.01 ms (n = 148; measured 10 mV positive to threshold).

Action potentials were followed by a biphasic afterhyperpolarization (AHP), consisting of the fast AHP (fAHP) and mAHP as shown in Fig. 2A. The mAHP amplitude, measured from resting potential to the most negative peak (Fig. 2A, open arrowhead), was 4.7 ± 0.2 mV (n = 148). The half-decay time of the mAHP was 25.4 ± 0.7 ms (n = 148). In 132 (89%) of the 148 neurons, these two AHPs were separated by a depolarizing waveform, the ADP. The amplitude of the ADP, measured from the resting potential to its peak (Fig. 2A, filled arrowhead), was 10.6 ± 0.5 mV (n = 132). In response to injection of subthreshold depolarizing current pulses, the ADP was rarely observed without generation of action potentials. The remaining 16 neurons did not display a prominent ADP between the fAHP and the mAHP, and were not included in the analysis of the ADP. As shown in Fig. 2B, there is a significant negative correlation between the amplitude of the mAHP and that of the ADP (r = -0.56, P < 0.001).


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 2. Properties of action potential and afterpotentials of trigeminal motoneurons of rat. A: action potential evoked from resting potential (-59 mV) by injection of 2-ms depolarizing current pulse (bottom trace) is followed by a fast afterhyperpolarization (fAHP), an afterdepolarization (ADP), and a medium-duration afterhyperpolarization (mAHP). B: amplitude of ADP plotted against that of mAHP at resting membrane potential. C: superimposed traces of action potentials before and after bath application of 1 and 10 mM tetraethylammonium chloride (TEA). TEA prolongs action potential duration and blocks fAHP. Dotted line: resting membrane potential (-70 mV). D: superimposed traces of action potentials before and after bath application of 500 µM 4-aminopyridine (4-AP). Prolongation of action potential duration is observed. Resting membrane potential: -70 mV (dotted line).

Ionic basis for the action potential

Bath application of 1 and 10 mM TEA invariably increased the spike duration by 118 ± 19% (n = 6) and 497 ± 66% (n = 5), respectively, without affecting rising phase of the action potential (Fig. 2C). By contrast, 1 mM TEA increased the amplitude and the half-decay time of the mAHP by 66 ± 17% (n = 6) and 23 ± 6% (n = 6), respectively, whereas 10 mM TEA reduced the mAHP amplitude by 34 ± 5% in four of five neurons tested and prolonged the half-decay time of the mAHP by 32 ± 12% (n = 5). These effects of TEA were reversible. Similarly to the effect of 1 mM TEA, bath application of 500 µM 4-AP increased the spike duration in all neurons tested by 141 ± 10% (n = 4) without affecting rising phase of the action potential (Fig. 2D). However, we could not analyze the effect of 4-AP on the ADP and mAHP because of a substantial increase in spontaneous synaptic potentials. No consistent effects on the resting membrane potential were observed after 4-AP application. These results suggest that TEA-sensitive, voltage-dependent, delayed-rectifier-type potassium currents and 4-AP-sensitive currents are involved in generating spike repolarization in rat TMNs as has been reported in TMNs of the guinea pig (Chandler et al. 1994), facial motoneurons (Nishimura et al. 1989), and hypoglossal motoneurons (Viana et al. 1993b).

Calcium-activated potassium channels have been suggested to be involved in the generation of spike repolarization in rat spinal (Takahashi 1990), hypoglossal (Umemiya and Berger 1994), and vagal (Sah and McLachlan 1992) motoneurons. To examine the extent to which calcium-activated potassium currents are involved in spike repolarization, effects of apamin, a selective blocker for small-conductance calcium-activated potassium channels (SK channels) (Blatz and Magleby 1986; Pennefather et al. 1985), or ChTX, a selective blocker for large-conductance calcium-activated potassium channels (Blatz and Magleby 1987; Latorre et al. 1989), were examined. Microdroplet application of 10-20 µM apamin to the surface of the slice or bath application of 10-30 nM ChTX had almost no effects on the spike duration (Fig. 3, insets in A and B), suggesting that calcium-activated potassium channels are not essential in spike repolarization of TMNs in young adult rats. A similar conclusion has been reached in TMNs (Chandler et al. 1994; Kim and Chandler 1995) and facial motoneurons (Nishimura et al. 1989) of the guinea pig.


View larger version (9K):
[in this window]
[in a new window]
 
FIG. 3. Effects of blockers for calcium-activated potassium channels on afterpotentials. A: superimposed traces of action potentials before and after microdroplet application of 10 µM apamin on surface of slice. Apamin blocks mAHP completely. Dotted line: resting membrane potential (-70 mV). Inset: duration of action potential is little affected by apamin. B: superimposed traces before and after bath application of 30 nM charybdotoxin (ChTX; resting membrane potential is -75 mV). ChTX has little effect on afterpotentials. Inset: ChTX has little effect on duration of action potential.

Ionic basis for the mAHP and ADP

To determine the calcium dependence of the mAHP, the following experiments were performed. First, replacement of extracellular Ca2+ concentration ([Ca2+]o; 2 mM) with the inorganic calcium channel blocker Mn2+ (2 mM) and Co2+ (2 mM) reduced the mAHP amplitude by 88 ± 8% (n = 5) and 92 ± 4% (n = 5), respectively, 30-60 min after substitution (Fig. 4A). Second, an intracellular injection of EGTA also depressed the mAHP. In five neurons tested, the mAHP amplitude was invariably and completely suppressed 10-40 min after impalement of the microelectrode containing 0.25 M EGTA into the neuron (Fig. 4B). Third, application of apamin to the surface of the slice (10-20 µM, n = 15) or to the perfusate (0.5 or 1 µM, n = 2) almost completely depressed the mAHP in all the neurons tested. As shown in Fig. 3A, the mAHP amplitude was reduced by 98 ± 2% (n = 17) without any appreciable changes in the height and duration of action potential and input resistance. On the other hand, bath application of ChTX (10-30 nM) had little effect on the ADP and mAHP (n = 8; Fig. 3B). These results indicate that the mAHP is calcium dependent and that SK channels are likely responsible for generating the mAHP.


View larger version (17K):
[in this window]
[in a new window]
 
FIG. 4. Calcium-dependent generation of mAHP and ADP. A: superimposed traces of action potentials before and after bath application of 2 mM Mn2+ (replacing extracellular Ca2+ with Mn2+). mAHP is blocked by application of Mn2+. Inset: ADP is also suppressed by application of Mn2+. Dotted line: resting membrane potential (-64 mV). B: progressive suppression of mAHP and progressive enhancement of ADP are observed with ethylene glycol-bis(b-aminoethylenether)-N,N,N',N'-tetraacetic acid (EGTA)-containing electrode. Resting membrane potential: -68 mV (dotted line). Ca: superimposed traces of action potentials before and after bath application of 1 and 2 mM Ba2+ (0 Ca2+). Suppression of mAHP and enhancement of ADP are observed. After application of 2 mM Ba2+, an additional spike was triggered from ADP. Resting membrane potential: -75 mV (dotted line). Cb: injection of short (2-ms) depolarizing current pulse induces burst firing emerging from an enhanced ADP in same neuron as in Ca after bath application of 5 mM Ba2+ (0 Ca2+). Resting membrane potential: -75 mV (dotted line). With increasing extracellular Ba2+ concentration ([Ba2+]o), ADP progressively augments, leading to a generation of a burst firing.

Calcium dependence of the ADP was also examined. To quantitatively evaluate the amplitude of the ADP that is generated in association with action potentials, the membrane potential response evoked by a short pulse with an intensity just below the threshold to elicit the action potentials was digitally subtracted from the threshold response with an action potential (Fig. 5, A and B). The effect of apamin on the ADP was evaluated by the use of this digitally calculated waveform in the 15 neurons in which apamin decreased the mAHP amplitude by >90%. Apamin application did not change the peak potential level and the peak time of the fAHP in the 15 neurons examined, but delayed the peak time of the ADP by 0.6 ± 0.1 ms (n = 15; Fig. 5B). Therefore the ADP amplitude was measured at the respective peak times of ADPs in control (upward filled arrow) and after apamin application (open arrow) from the baseline potential, as shown in Fig. 5B. After apamin application, the ADP amplitude at the former and latter times increased by 1.0 ± 0.3 mV (n = 15; 12 ± 4% of control) and 2.3 ± 0.3 mV (n = 15; 32 ± 6% of control), respectively. These results suggest that apamin-sensitive potassium conductances responsible for the generation of the mAHP become active slightly before the ADP peak and become maximum around the peak of the mAHP. Therefore the ADP is likely to be curtailed by the early phase of the mAHP. In later analysis, the ADP amplitude was measured at the time of the ADP peak in control by the use of such digitally calculated waveform. Then effects of Mn2+, Cd2+, or high-Ca2+ Ringer solution on the ADP were investigated after suppression of the mAHP by apamin. These inorganic calcium channel blockers invariably reduced the ADP amplitude by 42 ± 9% (n = 6) in the presence of apamin, without any appreciable changes in the peak time of the ADP (Fig. 5C). Raising [Ca2+]o from 2 to 6-8 mM invariably increased the ADP amplitude by 25 ± 10% in the presence of apamin (n = 3; Fig. 5D). These results indicate that the currents underlying the ADP are at least in part calcium dependent. An intracellular injection of EGTA abolished the mAHP and also increased the ADP amplitude by 149 ± 54% (n = 5; Fig. 4B). In contrast to effects of apamin on the ADP, the peak potential of the fAHP was depolarized by EGTA. Furthermore, bath application of 0.5-5 mM Ba2+ with no added Ca2+ enhanced the ADP dose dependently (Fig. 4, Ca and Cb). Because Ba2+ blocks K+ channels (Hille 1992), bath application of Ba2+ might cause a plateau-type potential, as shown in Fig. 4Cb. However, 10 mM TEA and 500 µM 4-AP, which block many K+ channels, did not produce a plateau-type potential but prolonged and enhanced the mAHP in the present study. Furthermore, apamin did not produce any plateau-type potentials. Thus effects of Ba2+ may be different from those of K+ channel blockers but somewhat similar to those of intracellular injection of EGTA, in which the peak level of fAHP was commonly depolarized. Therefore it can be assumed that removing Ca2+-dependent inactivation of Ca2+ currents by Ba2+ or EGTA might have caused enhancements of ADP. These observations suggest that Ca2+ itself is likely to be the charge carrier generating the ADP, rather than to trigger a calcium-dependent process.


View larger version (17K):
[in this window]
[in a new window]
 
FIG. 5. Factors affecting amplitude of ADP. A: superimposed traces of action potentials before (Control) and after (Apamin) microdroplet application of 20 µM apamin. Subthreshold response (Subthreshold) before apamin application is also superimposed. Dotted line: resting membrane potential (-70 mV). B: same neuron in A at faster sweep and higher gain. Subthreshold responses before and after apamin application were digitally subtracted from action potentials before and after apamin application shown in A. ADP amplitude was measured at respective peak times of ADPs before (filled arrow) and after (open arrow) apamin application from baseline potential. C: 500 µM Cd2+ suppresses ADP in presence of apamin. Potential level at peak of fAHP is hyperpolarized. Dotted line: resting membrane potential (-68 mV). Inset: digitally subtracted waveforms before and after Cd2+ application in presence of apamin. D: high-Ca2+ (8 mM) Ringer solution enhances ADP and depolarizes peak of fAHP in presence of apamin (resting membrane potential: -70 mV).

Effects of selective calcium channel blockers on the ADP and mAHP

We further examined the effects of selective blockers of high-voltage-activated (HVA) calcium currents on the ADP and mAHP. Microdroplet application of omega -CTx-GVIA (10-30 µM), an N-type calcium channel blocker, strongly decreased the mAHP amplitude by 87 ± 9% (n = 5), whereas omega -CTx-GVIA increased the amplitude of both the fAHP and ADP by 21 ± 9% and 33 ± 5%, respectively (n = 5; Fig. 6A). Such an increment of the ADP by omega -CTx-GVIA is significantly larger than that by apamin (P < 0.01). On the other hand, omega -Aga-IVA (10-100 µM), a potent blocker of P-type calcium channels, decreased the ADP amplitude by 37 ± 7% (n = 5), but did not alter apparently the peak mAHP amplitude (Fig. 6B). We also applied omega -Aga-IVA in combination with apamin. omega -Aga-IVA (100 µM) reduced the ADP amplitude by 37 ± 11% (n = 6) in the presence of apamin (Fig. 6C). Neither the ADP nor mAHP was apparently affected by nifedipine (10-20 µM), a selective blocker of L-type calcium channels (n = 3; data not shown).


View larger version (17K):
[in this window]
[in a new window]
 
FIG. 6. Effects of selective calcium channel blockers on afterpotentials. A: superimposed traces of action potentials (truncated) before (Control) and after (omega -CTx-GVIA) microdroplet application of omega -conotoxin GVIA (omega -CTx-GVIA) (20 µM) on surface of slice. Dotted line: resting membrane potential (-68 mV). B: superimposed traces of action potentials before (Control) and after (omega -Aga-IVA) microdroplet application of 100 µM omega -agatoxin IVA (omega -Aga-IVA) (resting membrane potential: -68 mV). C: superimposed traces of action potentials before (Apamin) and after (Apamin + omega -Aga-IVA) microdroplet application of 100 µM omega -Aga-IVA in presence of apamin. Resting membrane potential: -65 mV. D: superimposed traces of action potentials before (Control) and after (Ni2+) bath application of 500 µM Ni2+ (resting membrane potential: -68 mV). E: superimposed traces of action potentials before (Apamin) and after (Apamin + Ni2+) bath application of 500 µM Ni2+ in presence of apamin (resting membrane potential: -77 mV). F: superimposed traces of action potentials before (Apamin) and after bath application of 500 µM Ni2+ (Apamin + Ni2+), and after additional application of 100 µM omega -Aga-IVA (Apamin + Ni2+ + omega -Aga-IVA) in presence of apamin. Subthreshold membrane potential was subtracted digitally from action potentials.

We also examined the effect of blockers of low-voltage-activated calcium currents on the ADP and mAHP. Ni2+ has been reported to block low-voltage-activated calcium currents in spinal (McCobb et al. 1989) and hypoglossal (Viana et al. 1993a) motoneurons, although it has been shown to reduce HVA calcium currents as well (Randall and Tsien 1995; Zhang et al. 1993). Addition of 500 µM Ni2+ to perfusate containing 2 mM Ca2+ decreased the ADP amplitude by 37 ± 12%, whereas Ni2+ did not significantly affect the amplitude of the mAHP (n = 4) (Fig. 6D). Next we examined the selective effects of Ni2+ on the ADP during blockade of the mAHP by apamin in five neurons. Ni2+ (500 µM) reduced the ADP amplitude in all of the five neurons by 33 ± 3% in the presence of apamin (Fig. 6E). Both omega -Aga-IVA-sensitive and Ni2+-sensitive currents were likely involved in the generation of the ADP, because effects of omega -Aga-IVA and Ni2+ on the ADP were additive in the presence of apamin (Fig. 6F). However, there was a difference between the effects of Ni2+ and omega -Aga-IVA on the ADP when applied during repetitive firing, as described in the next section.

Differential effects of calcium channel blockers on the spike afterpotentials during repetitive firing

Trains of spikes were elicited by intracellular injections of long (1-s) depolarizing current pulses (Fig. 7). The ADP was observed following individual action potentials. In 67 of 71 neurons examined, the amplitude of the first ADP (arrowhead) was larger than those of the remaining ADPs in spike trains (Fig. 7, A, left and B, left). An application of omega -Aga-IVA hyperpolarized the peak potentials of the first and the remaining ADPs (Fig. 7A, compare left and right). On the other hand, Ni2+ hyperpolarized the peak potentials of the first ADP but did not hyperpolarize that of the remaining ADPs markedly (Fig. 7B, compare left and right). The amplitude of ADPs measured from the baseline potential became smaller successively and reached a steady level by the 6th-10th ADPs. Because the peak level of the ADPs varied from spike train to spike train depending on the intensity of injected current pulses, the amplitude of respective ADPs was normalized to the mean amplitude of the 6th-10th ADPs and was plotted against the order of ADPs in respective spike trains obtained by injections of current pulses with varying intensities (Fig. 8A). The normalized amplitude of ADPs did not significantly differ in terms of the current intensity (Friedman test, P > 0.1), whereas it significantly differed in terms of order of the ADPs (Friedman test, P < 0.001). Thus, regardless of the current intensity, the profile of successive changes in the normalized amplitude of ADPs was almost stereotyped. In terms of the profile of successive changes in the normalized amplitude of the ADP, effects of omega -Aga-IVA or Ni2+ on individual ADPs in spike trains were further analyzed. Figure 8, B and C, shows the effects of omega -Aga-IVA and Ni2+, respectively. Open and filled circles indicate the mean of the normalized amplitude of ADPs evoked by injections of current pulses with varying intensities before and after application of omega -Aga-IVA, respectively. Open and filled triangles indicate those values before and after application of Ni2+, respectively. The normalized amplitude of neither the first nor remaining ADPs changed after application of omega -Aga-IVA (Fig. 8B). However, Ni2+ significantly decreased the normalized amplitude of the first ADP (Mann-Whitney U-test, P < 0.01) without altering the normalized amplitude of remaining ADPs. These findings suggest that omega -Aga-IVA attenuates all ADPs whereas Ni2+ mainly decreases thefirst ADP.


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 7. Effects of omega -Aga-IVA and Ni2+ on individual ADPs during spike trains. A: trains of spikes before (left) and after (right) application of omega -Aga-IVA, in response to injections of 1-s depolarizing current pulses of 1.8 nA. After omega -Aga-IVA application, a depolarizing DC was injected to keep original membrane potential (bottom dotted line; -68 mV). B: trains of spikes before (left) and after (right) application of Ni2+, in response to injections of 1-s depolarizing current pulses of 3.0 and 2.2 nA, respectively. After Ni2+ application, a hyperpolarizing DC was injected to keep original membrane potential (bottom dotted line; -69 mV). Arrowheads: 1st ADPs in spike trains. Top and middle dotted lines in A and B: peak level of 1st ADP and level of mean amplitude of 6th-10th ADPs in each spike trains, respectively. Note that both 1st and remaining ADPs were suppressed after omega -Aga-IVA application, whereas Ni2+ suppressed 1st ADP but had little effect on remaining ADPs.


View larger version (10K):
[in this window]
[in a new window]
 
FIG. 8. Effects of omega -Aga-IVA and Ni2+ on normalized amplitude of individual ADPs during repetitive firing. A: plots of normalized amplitude of ADP vs. order of ADPs in respective spike trains obtained by injections of current pulses with varying intensities (2.6-3.6 nA). Amplitude of individual ADPs was normalized to mean amplitude of 6th-10th ADPs. Note that profile of successive changes in normalized amplitude of ADPs was almost stereotyped regardless of current intensity. B and C: plots of mean normalized amplitude of ADP vs. order of ADPs before and after application of omega -Aga-IVA (B) or Ni2+ (C). Open and filled circles: mean normalized amplitude before and after application of omega -Aga-IVA, respectively. Open and filled triangles: values before and after application of Ni2+, respectively. Each symbol and error bar indicate mean and SE values calculated from normalized amplitude obtained by current pulses with 6 different intensities of 1.5-2.0 nA (before omega -Aga-IVA application), 1.3-1.8 nA (after omega -Aga-IVA application), 2.4-3.4 nA (before Ni2+ application), and 2.0-3.0 nA (after Ni2+ application). Note that omega -Aga-IVA had little effect on profile of successive changes in normalized amplitude of ADPs, whereas Ni2+ altered that profile by reducing normalized amplitude of 1st ADP.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Calcium conductances underlying the mAHP and ADP

Umemiya and Berger (1994) reported that the mAHP was attenuated by either omega -CTx-GVIA or omega -Aga-IVA but not by nimodipine in neonatal rat hypoglossal motoneurons. In the present study, omega -CTx-GVIA almost abolished the mAHP and neither omega -Aga-IVA (microdroplet application as high as 100 µM) nor nifedipine apparently affected the mAHP. Therefore N-type calcium channels are most likely responsible for activating the calcium-activated potassium conductance underlying the mAHP conductance in rat TMNs. Because omega -Aga-IVA reduced the ADP almost selectively without marked effects on the mAHP, the discrepancy of the effects of omega -Aga-IVA on the mAHP between the two studies might not be due to the difference in the doses of omega -Aga-IVA but might be related to age or origin of the motoneurons.

In the presence of apamin application, inorganic calcium channel blockers reduced the ADP amplitude and high-Ca2+ Ringer increased the ADP amplitude in the rat TMNs. These results indicate that the currents underlying the ADP are at least in part calcium dependent. Such calcium dependence of the ADP is in accord with the results from motoneurons (Harada and Takahashi 1983; Viana et al. 1993a; Walton and Fulton 1986), cortical neurons (Connors et al. 1982; Higashi et al. 1993), thalamic neurons (Bal and McCormick 1993), and sympathetic preganglionic neurons (Yoshimura et al. 1987).

It has been also suggested that calcium-dependent nonselective cation currents (Bal and McCormick 1993; Swandulla and Lux 1985) or calcium-dependent chloride currents (Higashi et al. 1993; Sanchez-Vives and Gallego 1994) might be responsible for the ADP. However, Cl- is less likely to be involved in generating the ADP, because reducing [Cl-]o from 140 to 15-75 mM had little effect on the ADP (n = 4, data not shown). Because chelation of intracellular free calcium by intracellular injection of EGTA and substitution of extracellular Ca2+ with Ba2+ did not decrease the ADP amplitude but increased it, it is likely that at least some fraction of the ADP is directly generated by calcium currents. The increment of the ADP by EGTA and Ba2+ is probably due to removal of the calcium-dependent inactivation of calcium current. However, it is possible that calcium-independent inward currents such as nonselective cation current and sodium current are also involved in producing the ADP (Alzheimer 1994; Azouz et al. 1996), because inorganic calcium channel blockers failed to abolish the ADP.

We tried to determine which type of specific calcium conductance contributes to the generation of the ADP. During repetitive firing in response to injection of long (1-s) depolarizing current pulses, the first ADP was larger than the remaining ADPs in the spike train, implying that some components of the ADP might be inactivating. Ni2+ largely suppressed the first ADP but had little effect on the remaining ADPs. These results suggest that Ni2+-sensitive rapidly inactivating currents might be involved in the first ADP, but did not contribute to the generation of the remaining ADPs. In contrast to the effect of Ni2+, application of 10-100 µM omega -Aga-IVA reduced not only the first ADP but also the remaining ADPs to the same extent during repetitive firing. Furthermore, omega -Aga-IVA decreased the ADP even after application of apamin and Ni2+. Although this dose (10-100 µM) was relatively high, the effects of omega -Aga-IVA did not seem to be nonspecific, because the mAHP appeared to be insensitive to omega -Aga-IVA. Therefore it is likely that omega -Aga-IVA-sensitive HVA calcium currents mainly contribute to the generation of calcium-dependent component of the ADP. Nevertheless, the mean peak value of the ADP was around -56 mV, lower than the threshold of the HVA calcium currents in other neurons (Hille 1992). There are two possible explanations for this finding. First, the calcium currents responsible for the generation of the ADP might occur in dendritic site and the potential might be attenuated during electrotonic spread from the dendritic site to the soma. Second, the intermediate threshold calcium currents, as has been observed in neurons that display pacemaker activity (Alonso and Llinas 1992; Kang and Kitai 1993; Onimaru et al. 1996), might be involved in generation of the ADP. In contrast to those studies, subthreshold depolarizing currents rarely evoked the ADP in the present study. Thus it is more likely that omega -Aga-IVA-sensitive HVA calcium currents generated at the dendritic site are responsible for the ADP.

Differential contribution of calcium currents to the ADP and mAHP

omega -CTx-GVIA reduced the mAHP and enhanced the ADP, whereas omega -Aga-IVA reduced the ADP without marked effects on the mAHP. Thus calcium currents contributing to the ADP and mAHP could be different. However, two important questions now arise. Why didn't omega -Aga-IVA reduce the mAHP? Why didn't omega -CTx-GVIA reduce the ADP but enhance it?

It has been reported that distinct types of calcium channels are differentially distributed in neurons (Christie et al. 1995; Elliott et al. 1995), and that some types of calcium channels and calcium-activated potassium channels are located close to each other (Robitaille and Charlton 1992; Westenbroek et al. 1992). Because omega -CTx-GVIA reduced the mAHP whereas omega -Aga-IVA apparently did not reduce the mAHP in the present study, it is likely that SK channels are located closer to omega -CTx-GVIA-sensitive calcium channels than to omega -Aga-IVA-sensitive ones. Such colocalization of SK channels and omega -CTx-GVIA-sensitive calcium channels was also suggested in hypoglossal (Viana et al. 1993a) and vagal (Sah 1995) motoneurons. If these channels are closely located, Ca2+ influx through omega -CTx-GVIA-sensitive calcium channels during an action potential may activate the considerable number of SK channels. Because EGTA and Ba2+ enhanced the ADP markedly, omega -Aga-IVA-sensitive calcium channels might inactivate calcium dependently. If this is the case, the amount of Ca2+ influx through omega -Aga-IVA-sensitive channels in dendrites might be relatively small because of the inactivation. And the amount of intracellular Ca2+ of omega -Aga-IVA-sensitive channels origin might become smaller in association with its diffusion through dendrites to somatic SK channels, or it might be buffered (Lancaster and Pennefather 1987) before activation of SK channels. Thus Ca2+ influx through omega -Aga-IVA-sensitive channels might activate only a small number of SK channels. This may explain why omega -Aga-IVA did not reduce the mAHP. And it is likely that both omega -CTx-GVIA-sensitive and omega -Aga-IVA-sensitive calcium channels may be geometrically segregated to some extent.

Because omega -CTx-GVIA abolished the mAHP and enhanced the ADP, it is likely that omega -CTx-GVIA indirectly increased the ADP amplitude by suppressing the mAHP, as was the case with apamin. However, in contrast to the case of apamin, the peak level of the fAHP where mAHP is not involved was depolarized by omega -CTx-GVIA (Fig. 6A) and the enhancement of the ADP by omega -CTx-GVIA was larger than that of apamin. These observations indicate the presence of an additional cause. If Ca2+ influx through omega -CTx-GVIA-sensitive calcium channels contributes to the inactivation of omega -Aga-IVA-sensitive calcium channels, application of omega -CTx-GVIA may lead to direct enhancement of the ADP.

Role of afterpotentials for firing patterns

The suppression of the mAHP by calcium channel blockers or apamin greatly increased the firing frequency of repetitive firing induced by constant current injection (data not shown), as reported in other cranial motoneurons (Chandler et al. 1994; Nishimura et al. 1989; Sah and McLachlan 1992; Viana et al. 1993b), spinal motoneurons (Walton and Fulton 1986), and cortical neurons (Schwindt et al. 1988). When the ADP was enhanced by Ba2+ application or by attenuation of the mAHP, additional action potentials could be triggered from the several ADPs in the initial phase of a train of firing in response to long depolarizing current pulses and the interval between the first and second spikes could be greatly decreased (data not shown). Thus repetitive firing patterns were greatly affected by alterations of the ADP and mAHP. Burke et al. (1970) demonstrated that the insertion of a single extra action potential in a low-frequency spike train of a spinal motoneuron can cause marked, long-lasting tension enhancement produced by the muscle fibers that are innervated by the motoneuron. Therefore not only the average firing rate of a motoneuron but also the pattern of firing seems to alter the amount of force produced by the muscle fibers. It has been also reported that some neuromodulators, such as serotonin and noradrenaline, affect the mAHP and/or the ADP in cranial and spinal motoneurons (Berger et al. 1992; Parkis et al. 1995; Takahashi and Berger 1990). It is likely that those neuromodulators may regulate changes in muscle tension by affecting these spike afterpotentials.

    ACKNOWLEDGEMENTS

  We thank Prof. S. H. Chandler for critical reading of this manuscript. We also acknowledge Dr. S. Wakisaka for useful suggestions on the histological study.

  This study was supported by Grants-in-Aid for Scientific Research (07838018, 07457439, and 07407053) from the Japanese Ministry of Education, Science and Culture.

    FOOTNOTES

  Address for reprint requests: T. Inoue, Dept. of Oral Physiology, Faculty of Dentistry, Osaka University, Yamadaoka 1-8, Suita, Osaka 565, Japan.

  Received 21 September 1996; accepted in final form 24 February 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society