Voltage-Dependent Sodium and Calcium Currents in Acutely Isolated Adult Rat Trigeminal Root Ganglion Neurons

Hyung-Chan Kim1,2 and Man-Kyo Chung1

 1Department of Oral Physiology and Institute of Oral Biology, School of Dentistry, and  2East-West Medical Research Institute, Kyung Hee University, Seoul 130-701, Korea


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kim, Hyung-Chan and Man-Kyo Chung. Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons. Voltage-dependent sodium (INa) and calcium (ICa) currents in small (<30 µM) neurons from adult rat trigeminal root ganglia were characterized with a standard whole cell patch-clamp technique. Two types of INa showing different sensitivity to tetrodotoxin (TTX) were recorded, which showed marked differences in their activating and inactivating time courses. The activation and the steady-state inactivation kinetics of TTX-resistant INa were more depolarized by about +20 and +30 mV, respectively, than those of TTX-sensitive INa. Voltage-dependent ICa was recorded under the condition that suppressed sodium and potassium currents with 10 mM Ca2+ as a charge carrier. Depolarizing step pulses from a holding potential of -80 mV evoked two distinct inward ICa, low-voltage activated (LVA) and high-voltage activated (HVA) ICa. LVA ICa was first observed at -60 to -50 mV and reached a peak at about -30 mV. Amiloride (0.5 mM) suppressed ~60% of the LVA ICa, whereas ~10% of HVA ICa was inhibited by the same concentration of the amiloride. LVA ICa was far less affected by the presence of external Cd2+ or the replacement of Ca2+ by 10 Ba2+ than HVA ICa. The omega -conotoxin GVIA (omega -CgTx), an N-type ICa blocker, suppressed ~65% of the whole cell HVA ICa at the concentration of 1 µM. The omega -CgTx-resistant HVA ICa was sensitive to nifedipine (10 µM), a dihydropyridine (DHP) calcium channel antagonist, which produced an additional blockade by ~25% of the drug-free control (~70% of the omega -CgTx-resistant ICa). The combination of 10 µM nifedipine and 1 µM omega -CgTx left ~13% of the drug-free control ICa unblocked. The DHP agonist S(-)-BayK8644 (5 µM) shifted the activation of the HVA ICa to more negative potentials and increased its maximal amplitude. Additionally, S(-)-BayK8644 caused the appearance of a slowed component of the tail current. These results clearly demonstrate that the presence of two types of sodium channels, TTX sensitive and resistant, and three types of calcium channels, T, L, and N type, in the small-sized adult rat trigeminal ganglion neurons.


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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Dorsal root ganglia (DRG) neurons transmit various sensory information such as touch, pressure, pain, and temperature from the peripheral region to the CNS. Similarly, a pair of trigeminal root ganglia (TRG) is responsible for the sensory inputs from the oromaxillofacial region to which the trigeminal nerve is innervated.

Intracellular recordings in the mammalian TRG neurons have shown that three types of action potential exist: 1) fast spikes, most of which were completely blocked by the external application of tetrodotoxin (TTX), 2) Co2+- and TTX-resistant humped spikes (Puil et al. 1986), and 3) slowly decaying TTX-resistant and Cd2+-sensitive action potentials (Galdzicki et al. 1990). These reports suggest that voltage-dependent sodium current (INa) and calcium current (ICa) may be involved in the electrical activities of the TRG neurons. However, high-voltage activated ICa only was reported in the rat (Nah and McCleskey 1994) and chicken TRG neurons (Galdzicki et al. 1990). Recently, TRG neurons were used in the study to elucidate the mechanisms of a chemosensory transduction (Liu and Simon 1996) and a nociception evoked by protons (Pidoplichko 1992). However, there was little effort to investigate further electrophysiological characteristics of TRG neurons, which may be due to the assumption that the electrophysiological characteristics of TRG neurons would be identical to those described for DRG neurons.

The electrophysiological properties of the primary afferent neurons of DRG in various species were investigated in detail, not only because they are simple and accessible model neurons but also because there are some similarities between the properties of the somata and those of their central and peripheral terminals (Bellmonte and Gallego 1983; Jeftinija 1994; Scroggs and Fox 1992). The sensory neurons of mammalian DRG are known to express various types of voltage-dependent ion channels: 1) at least two types of INa distinguished by their sensitivity to TTX (Bossu and Feltz 1984; Caffrey et al. 1992; Elliott and Elliott 1993; Fedulova et al. 1991; Kostyuk et al. 1981; McLean et al. 1988; Ogata and Tatebayashi 1993; Ogata and Tatebayashi 1992a,b; Roy and Narahashi 1992; Schwartz et al. 1990), 2) T, L, N, and P types of ICa showing distinct pharmacological and kinetic properties (Mintz and Bean 1992; Scroggs and Fox 1991, 1992), and 3) a number of potassium currents (IK) (Gold et al. 1996). In addition, the distribution of these numerous ion channels in DRG neurons, according to the size of cells and developmental stages, was widely studied to elucidate the developmental and functional role of each type of ion channels in the modulation of specific sensory modalities (Roy and Narahashi 1992; Schwartz et al. 1990; Scroggs and Fox 1991, 1992).

The main goals of this study were to survey the ranges of voltage-dependent INa and ICa in adult rat TRG neurons and to compare them with those of DRG neurons as a part of an attempt to understand their role in the processing of sensory information, especially pain, from oromaxillofacial region. The results show that two types of INa distinguished by their TTX sensitivity and T-, L-, and N-type ICa are present in the adult rat TRG neurons.


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INTRODUCTION
METHODS
RESULTS
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Preparation of rat TRG neurons

Rat TRG neurons were prepared by the method described by Liu and Simon (1996) as follows. After the decapitation of the ketamine-anesthesized (100-125 mg/kg im) adult Sprague-Dawley rat (100-150 g), a pair of trigeminal ganglia were dissected and washed several times in cold (4°C) modified Hanks' balanced salt solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 0.3 KH2PO4, 4 NaHCO3, 0.3 Na2HPO4, 5.6 D-glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.3. They were incubated for 40 min at 37°C in HBSS containing 1 mg/ml of type XI collagenase, triturated with flamed Pasteur pipettes, and finally incubated at 37°C for 6 min with 1 mg/ml of type IV DNase I. Then they were retriturated and washed/centrifuged three times in Dulbecco's modified Eagle medium (DMEM) and maintained in room temperature. Cells were used in electrophysiological recording within 6 h.

Recording solutions and drugs

The composition of internal solution used for the isolation of INa was (in mM) 110 CsF, 10 NaCl, 0.1 CaCl2, 10 HEPES, and 11 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 7.3 adjusted with CsOH. The external solution used for INa contained (in mM) 60 choline-Cl, 60 NaCl, 20 tetraethylammonium (TEA)-Cl, 5 KCl, 5 MgCl2, 2 CaCl2, 10 HEPES, and 20 glucose, pH 7.4 adjusted with NaOH. 0.1 mM CdCl2 was added to this solution to suppress ICa. The internal solution used for ICa consisted of (in mM) 100 Cs-methanesulfonate (Cs-MeSO3), 0.1 CaCl2, 20 TEA-Cl, 10 HEPES, 11 EGTA, and 2 Mg-ATP, pH 7.3 adjusted with CsOH. The external solution used for ICa consisted of (in mM) 140 choline-Cl, 2 MgCl2, 10 CaCl2, 10 HEPES, and 20 glucose, pH 7.4 adjusted with CsOH. All the enzymes and chemicals were purchased from Sigma, except DMEM and methanesulfonic acid, which was purchased from Gibco Laboratory and Fluka, respectively. TTX (Research Biochemical International) was dissolved first in ethyl alcohol and then diluted in the external solution before use. A stock solution of amiloride-HCl, nifedipine (Sigma), and S(-)BayK8644 (Research Biochemical International) was made by dissolving them in dimethyl sulfoxide. The omega -conotoxin GVIA (omega -CgTx, Research Biochemical International) was stored as a stock solution of 1 mM in distilled water. All experiments with dihydropyridine (DHP) drugs were carried out in dim light.

Electrophysiology

The isolated cells were plated onto a polyethyleneimine-coated glass coverslip in a recording chamber (100-µl volume) for superfusion (3 ml/min) with control and drug-containing solutions. Patch-clamp pipettes were manufactured from soda lime glass capillaries with a two-stage vertical pipette puller (L/M-3P-A, List Electronics) and fire-polished with a microforge (MF-83, Narishige). Because we had large currents, low-resistance pipettes (~0.7-1.5 MOmega ) were employed to reduce the voltage drop across the series resistance, which was compensated 70-75% usually. We gave up further recordings when the resultant series resistance was >3 MOmega after compensations. Whole cell patch-clamp recordings were performed with an Axopatch 200A patch-clamp amplifier (Axon Instruments). Stimulus application and data acquisition were controlled by an IBM-compatible personal computer in conjunction with a Labmaster (PP-50) DMA interface (Warner Instrument). The data were low-pass filtered at 2 kHz (-3 dB) with the amplifier's Bessel filter and digitized every 20 µs (INa) or 50 µs (ICa). All data collection and analysis were carried out with pCLAMP 6.0 software (Axon Instruments).

Neuron diameter was measured with an eyepiece micrometer under phase contrast illumination and categorized into three groups: small (20-27 µm), medium (33-38 µm), and large (45-51 µm) cells, according to the classification of Scroggs and Fox (1992a). We recorded voltage-dependent ion currents primarily in the small cells of 15-30 µm in diameters without or with short processes because 1) they are known to be related to pain sensation (Harper and Lawson 1985a), 2) the amplitude of current recorded from the medium or large cells was so great that a serious series resistance error and the saturation of an amplifier could not be avoided under the ionic condition in this experiment (i.e., 10 Ca2+ or 60 Na+ in external bathing solution), and 3) the complete compensation of cell membrane capacitances was difficult frequently and the signs of poor space-clamp condition such as a slow deactivating current used to be detected in some medium and large cells or cells with processes.

All experiments were carried out at room temperature (22-25°C).

Data analysis

The peak conductance (G) of INa or ICa at each potential was calculated from the corresponding peak current by using the equation
<IT>G</IT><IT>=</IT><IT>I</IT><IT>/</IT>(<IT>E</IT><IT>−</IT><IT>E</IT><SUB><IT>Rev</IT></SUB>)
where ERev is the reversal potential of INa or ICa, I is the peak current amplitude of INa or ICa, and E is the membrane potential.

Normalized peak conductance (G/Gmax) and the data describing the fractional decrease in the peak current during the steady-state inactivation (I/Imax) were fitted with a Boltzmann function
<IT>I</IT><IT>/</IT><IT>I</IT><SUB><IT>max</IT></SUB><IT> or </IT><IT>G</IT><IT>/</IT><IT>G</IT><SUB><IT>max</IT></SUB><IT>=1+exp</IT>(<IT>V</IT><SUB><IT>1/2</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>m</IT></SUB>)<IT>/</IT><IT>k</IT>
where V1/2 is the membrane potential at which 50% inactivation of the current is observed, Vm is the prepulse membrane potential, and k is the slope of the function. All data are expressed as means ± SE (n = number of observations) for at least three measurements, unless otherwise stated.


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TTX-s and TTX-r INa

We tried to isolate INa from the other kinds of voltage-dependent ionic currents in whole cell patch-clamp recordings by 1) suppressing IK with the use of the equimolar replacement of K+ with Cs+ in the internal solution and by the addition of 20 mM TEA in the external solution, 2) suppressing ICa by 0.1 mM Cd2+ in the external solution and 5 mM Mg2+ in the internal and external solutions, and 3) decreasing the concentration of Na+ in the external solution to 60 mM by the isomolar substitution of choline to prevent the saturation of the amplifier by large INa. Inward current recorded under this ionic condition was reduced as expected by the partial replacement of the external Na+ by choline (Fig. 1A). The observed potential for zero current flow was 46.7 ± 0.5 mV (n = 30), as expected for a sodium-selective channel, and moved 11.3 ± 0.3 mV (n = 3) in the hyperpolarizing direction when the external Na+ concentration was reduced from 60 to 40 mM (Fig. 1B), which is identical to the predicted value by the Nernst equation for the sodium-selective channel. In addition, when the Na+ in the bathing solution was totally removed, the current traces became much smaller and the direction of the current became outward (n = 3, not shown). These observations indicate that the inward current is the voltage dependent INa.



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Fig. 1. Inward current component carried by Na+. A: current traces evoked by the depolarizing pulse to -10 mV from a Vh of -70 mV. External solution contained (in mM) 60 choline-Cl, 60 NaCl (or 80 choline-Cl and 40 NaCl), 20 tetraethylammonium (TEA)-Cl, 5 KCl, 5 MgCl2, 2 CaCl2, 0.1 CdCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 20 glucose, pH 7.4. The composition of internal solution was (in mM) 10 CsF, 10 NaCl, 0.1 CaCl2, 10 HEPES, and 11 bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 7.3. B: peak I-V relationships obtained from the current traces in A. Note the significant reduction in the amplitude of the inward current and the shift of Erev in a negative direction in the external solution with 40 Na+.

Figure 2A shows typical INa recorded from the acutely isolated rat TRG neuron under the ionic condition described above. INa was first observed at the potential of -40 to -30 mV and peaked at -20 to +10 mV (Fig. 2A). The current appeared to be composed of two components of the voltage-dependent INa distinguished by their sensitivity to TTX. In most cases, 0.1 µM TTX failed to inhibit INa completely (Fig. 2Ab). The component that was resistant to 0.1 µM TTX was not also significantly inhibited by 20 µM TTX (n = 2, not shown). TTX-r INa was observed in the >90% of the small TRG neurons <30 µm in diameter in the presence of 0.1 µM TTX. However, external application of 0.1 µM TTX suppressed most of the current in some preparations (Fig. 2B). This suggests that most of the small TRG neurons from adult rat may express TTX-r INa with or without TTX-s INa, which resulted in difficulties in direct observation of isolated TTX-s INa in small neurons.



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Fig. 2. Two types of INa; tetrodotoxin (TTX)-s and TTX-r INa. A: typical INa recorded from a rat TRG neuron before (a) and after (b) the application of 0.1 µM TTX. Current traces in a and b were evoked by the depolarizing step pulses from a Vh of -70 mV. TTX-sensitive component in c was obtained from the digital subtraction of the TTX-resistant component in b from the control current traces in a. Peak amplitudes of the control current, TTX-resistant component, and the subtracted TTX-sensitive component were plotted against the membrane potentials in d. Ba: superimposed current traces recorded from another rat TRG neuron. INa was evoked by the step pulse to -20 mV before and after the application of 0.1 µM TTX, which inhibited most of the inward current in this cell. Bb: magnitudes of currents shown in Ba were plotted against the membrane potentials.

TTX-s INa started to activate at the voltage of near -40 mV, which was less depolarized than those for TTX-r INa. The peak voltage for TTX-s INa was also slightly hyperpolarized to that for TTX-r INa. The maximal current density of the TTX-r INa was quite various from cell to cell. The maximal TTX-r INa was elicited at a test potential of 0 or +10 mV (Fig. 2Ad), and the current density of TTX-r INa elicited by voltage steps to +10 mV from a holding potential (Vh) of -70 mV was -268.5 ± 20.6 pA/pF (n = 20). The peak current density of the TTX-s INa was -356.5 ± 56.0 pA/pF (n = 7). Figure 3 illustrates that TTX-resistant component of INa activated and decayed more slowly than TTX-s INa by ~10- and 7-fold, respectively, at the voltage of +10 mV. These results indicate that at least two types of INa, i.e., TTX-s and TTX-r INa showing different sensitivity to TTX, activation range, and distinct activating and inactivating time courses, are expressed in the small neurons from rat TRG.



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Fig. 3. Rise and decay time for TTX-s and TTX-r INa. An example of the TTX-s INa in A and TTX-r INa in B. TTX-r INa was recorded in the presence of 0.1 µM TTX. Note the distinct differences in the onset and the decay of current; 10-90% rise time and 10-90% decay time were averaged and plotted against the membrane potentials in C and D, respectively. Data were obtained from 6 and 3 cells for TTX-r and TTX-s INa, respectively.

Voltage dependence of INa

We investigated the voltage dependence of the activation and the steady-state inactivation of INa in the presence of 2 mM Ca2+, 5 mM Mg2+, and 0.1 mM Cd2+ in the external bathing solution. For the evaluation of the steady-state inactivation properties, 1-s conditioning pulses to various potentials ranging from -100 mV to 0 mV were followed by a constant step pulse to +10 mV. Current traces were not leak-subtracted, and the current amplitude was measured relative to the plateau phase near the end of the test pulse. Figure 4A shows the steady-state inactivation curves of a cell expressing TTX-s and TTX-r INa. The availability curve (Fig. 4D, ) of the INa from the cell had an inflection because of the presence of both TTX-s and TTX-r sodium channels. Isolated inactivation curves of TTX-r INa (Fig. 4, B and D, open circle ) and TTX-s INa (Fig. 4, C and D, ) were obtained with 0.1 µM TTX. TTX-r and TTX-s INa showed marked differences in their inactivating properties. A half-maximal potential (V1/2) for the inactivation of TTX-s INa was more hyperpolarized than that of TTX-r INa by approximately -30 mV (Fig. 4D). We evaluated the steady-state inactivation properties of the two types of INa with this protocol. Figure 5 shows that the mean value of V1/2 was -60.5 ± 1.6 mV for the TTX-s INa (Fig. 5, ) and -29.3 ± 1.1 mV for the TTX-r INa (Fig. 5, ), giving a mean difference in V1/2 of 31.1 ± 1.4 mV (n = 5). The slope of the steady-state inactivation curve was much steeper for the TTX-r INa (k = 4.9 ± 0.5 mV for the TTX-r and 7.2 ± 0.3 mV for TTX-s INa, n = 5), and the mean ratio of the k was 1.5:1 (n = 5). The peak sodium conductance for TTX-r INa was calculated as described in METHODS. The conductance curve for TTX-r INa (Fig. 5, open circle ) was obtained in the presence of 0.1 µM TTX, and a curve for TTX-s INa (Fig. 5, ) was derived from the cells showing virtually complete inhibition (i.e., >95%) of the INa by the 0.1 µM TTX. The V1/2 for the activation of TTX-s INa was approximately -20 mV more hyperpolarized than that of the TTX-r INa. The averaged V1/2 was -19.7 ± 2.2 mV for TTX-s INa and 0.1 ± 2.0 mV for the TTX-r INa. The slope of the peak conductance curve was slightly steeper for the TTX-s INa (k = 5.6 ± 0.5 mV for TTX-r INa and 4.1 ± 0.5 mV for TTX-s INa).



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Fig. 4. Inactivation properties of two types of INa. The availability of INa was evaluated in a partially TTX-sensitive cell. A: current traces evoked by the constant step pulses to +10 mV preceded by 1-s prepotentials from -100 to 0 mV with 10-mV increment. B: steady-state inactivation of TTX-r INa obtained from the same cell in A after the application of 0.1 µM TTX. Leaks were not subtracted in A and B. C: TTX-s INa isolated by the subtraction of TTX-r INa from the control current traces. D: plots of normalized current amplitudes measured at the peak of each current trace shown in A-C------ in open circle  and  were derived from the curve fit to Boltzmann function, which yielded V1/2 and k of -29.9 mV and 5.7 mV in TTX-r INa and -61.7 mV and 7.9 mV in TTX-s INa, respectively.



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Fig. 5. Voltage-dependent kinetics of INa. Midpoints of maximal activation and inactivation for TTX-s INa were less depolarized by about -30 and -10 mV, respectively, than those of TTX-r INa. Steady-state inactivation curves were derived from the experiment in Fig. 4, and normalized peak conductances were calculated as described in METHODS.

Whole cell ICa from adult rat TRG neurons

ICa was recorded from rat TRG neurons under conditions that 1) suppressed IK by the replacement of internal K+ with Cs+ and 20 mM TEA included in external solution, 2) suppressed INa with choline replacing external Na+, and 3) enhanced ICa with 10 mM Ca2+ as a charge carrier.

Figure 6 shows whole cell ICa obtained from a small-diameter neuron evoked by depolarizing steps to various test potentials from a Vh of -80 mV, which is thought to be comprised of multiple components of ICa. By applying depolarizing steps from a Vh at -80 mV, transient inactivating ICa was first observed at about -60 mV. The currents reached its peak in 5-30 ms after the onset of the step pulse and decayed rapidly in a single-exponential time course with a time constant of 15-30 ms. With further depolarization the peak amplitude increased smoothly and reached a plateau at about -30 mV, resulting in a distinct shoulder in I-V relationship (Fig. 6C). At the potential of about -30 to -20 mV, a step increase in current amplitude was observed, and the decay of the current became progressively less pronounced. When the cell was held at -40 mV, all of the decaying component of ICa could no longer be evoked over the whole range of test potentials, but sustained ICa was activated at the stronger depolarizations. The current showed little inactivation during the 80-ms step pulse (Fig. 6B) and was maximal at near -10 mV (Fig. 6C). These observations suggest the presence of two current components with different activation range, i.e., low-voltage activated (LVA) and high-voltage activated (HVA) ICa, in small-sized rat TRG neurons.



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Fig. 6. Three components of ICa observed in a rat trigeminal root gangion (TRG) neuron. A: current traces evoked by step pulses to test potentials from -80 to +40 mV from a Vh of -80 mV. The composition of the external solution was (in mM) 140 choline-Cl, 2 MgCl2, 10 CaCl2, 10 HEPES, and 20 glucose, pH 7.4, and the internal solution contained (in mM) 100 Cs-MeSO3, 0.1 CaCl2, 20 TEA-Cl, 10 HEPES, 11 EGTA, and 2 Mg-ATP, pH 7.3. B: current traces elicited from a Vh of -40 mV. Note the disappearance of decaying components shown in A. C: I-V plots derived from the current traces in A and B. Note the low-voltage activated (LVA) ICa was not induced by the Vh of -40 mV ().

HVA ICa was recorded in every cell investigated, whereas LVA ICa did not appear to be recorded uniformly. In some preparations, low-threshold transient LVA ICa was recorded prominently, whereas the sustained HVA ICa component was predominant Ca2+ entry pathway in some cells. These may indicate that the densities of the two types of the Ca2+ channels may be various from cell to cell.

Figure 7 shows the results obtained from a single cell expressing LVA and HVA ICa when a 10 mM Ca2+ containing medium was changed to the solution with equimolar Ba2+. With Ba2+ as a charge carrier the magnitude of the sustained ICa was enhanced almost twofold at 0 mV, and the I-V relationship was shifted ~10 mV to a hyperpolarizing direction. However, Ba2+ showed negligible effect on the LVA ICa, and in some cells the current amplitude was slightly decreased by the replacement of Ba2+. The effect of Ba2+ on ICa was completely reversible (not shown). These results support the notion that the whole cell ICa recorded in adult rat TRG neurons consists of independent types of ICa.



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Fig. 7. Enhancement of high-voltage activated (HVA) ICa by substitution of 10 Ba2+ for 10 Ca2+. A: examples of superimposed current traces enhanced by the equimolar substitution of Ca2+ with Ba2+. Traces marked by asterisk represent the current traces carried by Ba2+. B: peak I-V relationships in 10 mM Ca2+ and in 10 mM Ba2+. The peak HVA ICa nearly doubled in the amplitude in 10 mM Ba2+, whereas the magnitude of LVA ICa little changed or slightly decreased.

Effects of calcium channel blockers on ICa

Different types of Ca2+ channels show the differential sensitivities to some inorganic Ca2+ channel blockers (Fox et al. 1987). We tested the effects of several calcium channel blockers on ICa. As illustrated in Fig. 8 Cd2+ seemed to be more effective in blocking the HVA ICa. The addition of 50 µM Cd2+ to the bath virtually eliminated the HVA ICa, whereas the LVA component was comparatively less affected; 100 µM Cd2+ also did not abolish LVA ICa completely but suppressed HVA ICa by >90%. The inhibitory effects of Cd2+ were readily reversible (not shown). These results are similar to those reported by others regarding the differential sensitivity of LVA and HVA ICa in DRG neurons (Fox et al. 1987; Gross and McDonald 1987).



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Fig. 8. Preferential block of HVA ICa by Cd2+. A: superimposed current traces elicited by 80-ms step pulses to -30 mV (top traces) and 0 mV (bottom traces) from a Vh of -80 mV in the control medium and after superfusion with 50 and 100 µM Cd2+. Note that the blocking effects were considerably greater on the HVA ICa than on the LVA ICa. B: peak I-V relationships obtained from the complete sets of records exemplified in A. Note the incomplete block of the LVA ICa by 100 µM Cd2+.

Amiloride, a K+-sparing diuretic, was known to block LVA ICa effectively in many preparations (Fox et al. 1987; Scroggs and Fox 1991, 1992). Figure 9 illustrates the selective inhibitory effect of amiloride on LVA ICa evoked from a TRG neuron. LVA ICa elicited from Vh of -80 mV with a step pulse to -30 mV was reversibly inhibited by 0.5 mM amiloride (60.4 ± 5.4%, n = 3). However, 0.5 mM amiloride showed the smaller inhibitory effect on HVA ICa (12.6 ± 3.9%, n = 3). The inhibitory effect of amiloride on HVA ICa may be due to the incomplete inactivation of T-type ICa in our protocols (see Scroggs and Fox 1992).



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Fig. 9. Effects of amiloride on the LVA and HVA ICa in a rat TRG neuron. A: LVA and HVA ICa were evoked by step pulses to -30 and 0 mV respectively from a Vh of -70 mV at 0.1 Hz in a small rat TRG neuron before and after the application of 0.5 mM amiloride. The inhibitory effect of amiloride was far more pronounced in LVA ICa than in HVA ICa. Numbers represent the location in B at which the current traces were derived from. B: peak amplitudes of LVA and HVA ICa were plotted against time. Amiloride inhibited LVA ICa by ~65% immediately after superfusion at the concentration of 0.5 mM. However, HVA ICa was far less affected by the drug. The effects of amiloride were completely reversed after washout.

Nifedipine, a DHP calcium channel antagonist, has been shown to preferentially block the sustained component of ICa in many other preparations (Regan et al. 1991), and the synthetic toxin omega -conotoxin GVIA (omega -CgTx) is a potent blocker of a component of the HVA ICa that reflects the activation of N-type ICa in a variety of neurons (Fox et al. 1987; Scroggs and Fox 1991, 1992; Tsien et al. 1981). We tested the effects of omega -CgTx and nifedipine on the HVA ICa from cells in which the LVA ICa produced only a very small deflection in the I-V relationships. The HVA ICa was evoked by repetitive step pulses to +10 mV from a Vh of -80 mV every 10 s. After a baseline of five to seven traces was established in the drug-free condition, omega -CgTx (1 µM) was superfused over the neuron. The omega -CgTx blocked 62.3 ± 4.0% (n = 4) of the peak HVA ICa at the concentration of 1 µM (Fig. 10Aa). Subsequent superfusion of 10 µM nifedipine in the presence of 1 µM omega -CgTx blocked an additional 26.2 ± 1.6% (n = 4) of the drug-free control ICa, which indicates that the 69.5 ± 1.6% (n = 4) of omega -CgTx-resistant ICa was suppressed by 10 µM nifedipine. The combination of 1 µM omega -CgTx and 10 µM nifedipine left some ICa unblocked. On average, 13.0 ± 3.3% (n = 4) of the control ICa was resistant to the mixture of 10 µM nifedipine and 1 µM omega -CgTx in small rat TRG neurons (Fig. 10Ab). Figure 10B illustrates the representative current traces evoked by 400-ms step pulses to 10 mV and corresponding I-V curves after the application of 1 µM omega -CgTx and 10 µM nifedipine. Figure 10, Ba and Bb, show that the inactivating transient component of the HVA ICa was blocked by the application of 1 µM omega -CgTx. The current amplitudes of the digitally subtracted nifedipine-sensitive ICa and omega -CgTx-sensitive ICa were plotted against the membrane potentials, which revealed that the omega -CgTx-sensitive component was activated at more hyperpolarized potential than the nifedipine-sensitive component (Fig. 10, Bc and Bd). These results suggest that there are at least two distinct types of the HVA ICa showing different pharmacological properties in rat TRG neurons.



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Fig. 10. Blocking effects of nifedipine and omega -conotoxin GVIA (omega -CgTx) on HVA ICa. Aa: repetitive constant step pulses to 10 mV from a Vh of -80 mV were delivered to evoke the HVA ICa in small-sized rat TRG neurons. Superfusion with 1 µM omega -CgTx blocked ~65% of peak ICa. Additional application of 10 µM nifedipine inhibited the remained ICa by ~70%. Superimposed current traces in b were selected from continuous recordings in a at the locations indicated by corresponding numbers. Ba: from a Vh of -80 mV the HVA ICa was evoked by the 400-ms step pulse to 0 mV in the control solution (1), in the presence of 1 µM omega -CgTx (2), and after the additional application of 10 µM nifedipine (3). Bb: examples of the nifedipine-sensitive current component (2 and 3) and the omega -CgTx-sensitive current component (1 and 2) obtained by the digital subtraction of the currents in Ba. Bc: peak I-V relationships obtained from the current traces in Ba. Bd: nifedipine-sensitive and the omega -CgTx-sensitive ICa were plotted against the membrane potentials. The membrane potential for maximal omega -CgTx-sensitive ICa is less depolarized than that of the nifedipine-sensitive ICa.

Finally, the DHP calcium channel agonist S(-)-BayK8644, which increases current amplitude through L-type calcium channels by enhancing the probability of channel opening, was tested to confirm the presence of L-type calcium channel in rat TRG neurons. When S(-)-BayK8644 (5 µM) was superfused over rat TRG neurons held at -80 mV, the amplitude of HVA ICa was increased, which was greatest at weak test depolarizations compared with strong test potentials (Fig. 11, A and B). S(-)-BayK8644 also greatly increased the amplitude and decreased the rate of inactivation of the tail current (Fig. 11C). In addition, the calcium channel activation threshold and the peak of the I-V curve was shifted in the hyperpolarizing direction (Fig. 11D). These effects are all characteristics of DHP agonists that were previously described (Scroggs and Fox 1991). Similar results were obtained from other five cells.



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Fig. 11. Effects of S(-)-BayK8644 on HVA ICa. A: superimposed current traces elicited by 80-ms step pulses to -20 mV in Aa and +20 mV in Ab from a Vh of -80 mV in the control medium and after superfusion with 5 µM S(-)-BayK8644. Note the marked increase in current amplitude at a weak depolarization to -20 mV compared with +20 mV. B: S(-)-BayK8644 increased the amplitude and decreased the rate of inactivation of tail currents compared with control. C: peak I-V relationships obtained from the complete sets of records exemplified in A. Note the shift of the potential for the activation and peak of ICa to a hyperpolarizing direction.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Classification of primary afferent neurons of the mammalian DRG is based on the morphological and electrophysiological characteristics of neurons (Harper and Lawson 1985a,b). The cell size is known to be correlated with the axonal conduction velocity well; large neurons with myelinated Aalpha ,beta fibers are fast conducting, whereas small cells with myelinated Adelta and unmyelinated C fibers are slow conducting (Harper and Lawson 1985a). However, properties of the somatic action potentials are thought to be associated with the cell types at lesser extent: long action potential with inflected repolarization that is mainly recorded from the Abeta and C cells and short action potential without plateau during the falling phase recorded in Aalpha and Adelta cells (Harper and Lawson 1985b). Patch-clamp techniques were used to investigate the range and properties of ionic channels underlying these electrical activities in dissociated mammalian DRG neurons, and two types of INa were characterized; in general, TTX-s INa is found in large-diameter cells, and TTX-r INa is expressed in small-diameter cells (Caffrey et al. 1992; Elliott and Elliott 1993; Kostyuk et al. 1981; McLean et al. 1988; Ogata and Tatebayashi 1992a; Puil et al. 1986; Schwartz et al. 1990). Although it is generally accepted that TTX-r INa is found in the soma of small DRG neurons that give rise primarily to C-type afferent fibers, the frequency of recording TTX-r INa in small neurons is in disagreement among studies from the same type of cell. For example, Caffrey et al. (1992) reported that TTX-r INa was always observed in conjunction with TTX-s INa in small DRG neurons from adult rats, where as Elliott and Elliott (1993) suggested that only 45% of small cells express TTX-r INa from the same preparation. We recorded INa primarily from the small-sized TRG neurons ranging from 15 to 30 µm and observed TTX-r INa in every cell investigated in the presence of 0.1 µM TTX, in agreement to the report of Caffrey et al. (1992).

These two types of voltage-dependent INa from adult rat TRG neurons showed additional distinct characteristics as well as a distinct sensitivity to TTX. TTX-r INa showed slower kinetics of activation and inactivation (Figs. 2 and 3), more depolarized potential for the activation threshold (Fig. 2Ad, and the more depolarized voltage dependence of the activation and the steady-state inactivation (Figs. 4 and 5). These properties of TTX-r INa are similar to those described in DRG neurons (Fedulova et al. 1991; Kostyuk et al. 1981; McLean et al. 1988; Ogata and Tatebayashi 1993; Roy and Narahashi 1992).

Although the functional contribution of TTX-r INa to the electrophysiological activities of sensory neurons is not clear, it is suggested that TTX-r INa may contribute to the prolongation of the interspike interval by extending the inactivated state of TTX-s INa during sustained depolarization of the membrane potential (Bossu and Feltz 1984) or may be responsible for the slow adaptive properties and the generation of the long trains of action potentials because TTX-r INa shows the extremely slow inactivation process and the rapid repriming kinetics (Elliott and Elliott 1993; Ogata and Tatebayashi 1992b). Recent studies suggested that TTX-r INa may participate in the sensitization to noxious stimuli in small capsaicin-sensitive primary afferent neurons based on its modulation by pain-inducing agents (Cesare and McNaughton 1997). In addition, TTX-resistant Na+-dependent spikes are reported to be activated selectively in primary afferent fibers by elevated extracellular K+ concentration (Jeftinija 1994), which may be frequently occurred at the site of injury and depolarize the neuronal membrane potential. The more depolarized activation and steady-state inactivation kinetics of TTX-r INa than those of TTX-s INa in sensory neurons may contribute to the selective transmission of sensory signals by TTX-r INa under the condition depolarizing sensory neurons. It may be valuable for the further comprehension and better management of the oromaxillofacial pain to elucidate the association of TTX-r INa with the transmission of noxious signals from the maxillofacial region.

Rat DRG neurons are known to have several types of calcium channels (Scroggs and Fox 1991, 1992) that are similar to those reported in chick sensory neurons (Fox et al. 1987): 1) a transient (T-type) or LVA ICa, 2) a dihydropyridine-sensitive, sustained (L-type) or HVA ICa, and 3) N-type ICa showing kinetic properties between the T- and L-type ICa and a selective suppression by omega -CgTx. Recently, a fourth type, known as the P-type ICa, distinguished by its unique sensitivity to omega -agatoxin, also was described in the rat DRG neurons (Mintz and Bean 1992). We identified both LVA and HVA ICa from acutely isolated rat TRG neurons. LVA ICa was identified by the low threshold for the activation at about -60 to -50 mV with maximal current amplitude at the voltage of -30 mV, a fast rate of inactivation, and a shoulder in the I-V curve. LVA ICa recorded in rat TRG neurons showed several characteristics similar to those described for T-type ICa in other neuronal preparations (Fox et al. 1987; Scroggs and Fox 1991, 1992); 1) LVA ICa inactivated when the cell held at depolarized potential of about -40 mV (Fig. 1), 2) the channel for the current was found to be more permeable to Ca2+ than Ba2+ (Fig. 7), and 3) the current was more sensitive to amiloride and resistant to Cd2+ than HVA ICa (Figs. 8 and 9). HVA ICa was present in the virtually all of the rat TRG neurons investigated and typically activated at about -30 to -20 mV, with the maximal peak current occurring between 0 and 10 mV. HVA ICa was greatly suppressed by 0.1 mM Cd2+ (Fig. 8) and greatly enhanced by Ba2+ (Fig. 7), in agreement with other preparation (Fox et al. 1987). Several characteristics of HVA ICa indicate that it may have at least two components; 1) the decays of current traces at positive test potentials (Figs. 6A and 10B) were poorly fitted by single exponential (not shown), 2) when HVA ICa was elicited from a Vh of -40 mV, the amplitudes were reduced significantly and the ICa decayed little or very slowly (Fig. 6B), 3) the omega -CgTx-resistant current was largely inhibited by nifedipine (Fig. 10), and 4) DHP agonist S(-)-BayK8644 increased the amplitude of HVA ICa (Fig. 11). These observations strongly suggest the presence of at least two types of HVA ICa in acutely dissociated rat TRG neurons, resembling the DHP-sensitive sustained L-type ICa and omega -CgTx-sensitive N-type ICa described in other preparations (Fox et al. 1987; Gross and McDonald 1987; Regan et al. 1991; Scroggs and Fox 1991, 1992). When the HVA ICa was evoked from a Vh of -80 mV, the proportion of nifedipine/omega -CgTx-resistant ICa was relatively constant (10-15%), which was similar to the results from small-diameter rat DRG neurons (Scroggs and Fox 1992). This nifedipine/omega -CgTx-resistant ICa component may be interpreted as the incomplete block of L- or N-type ICa at those concentration of drugs or the presence of other types of HVA ICa components in rat TRG neurons.

The calcium channels are known to be involved in the pacemaker depolarization, Ca2+-dependent secretion, and the activation of Ca2+-dependent ionic conductance in neuronal preparations, although linking a specific type of ICa to a particular cellular process may be difficult (Tsien et al. 1988). It may be certain, however, that the subtypes of ICa contribute in different ways to the transmission of sensory signals of different modalities in primary afferent neurons. The subtypes of ICa are known to show diameter-dependent variation in acutely isolated rat DRG neurons, i.e., medium-diameter neurons had a large amount of T-type ICa, whereas significantly large proportion of the whole cell ICa was L type in the small cells (Scroggs and Fox 1991, 1992), which shaped action potentials differently and therefore may significantly affect the sensory transmission.

In conclusion, we identified the voltage-dependent TTX-r and TTX-s sodium currents, amiloride-sensitive T-type, DHP-sensitive L-type, and omega -CgTx-sensitive N-type calcium currents from acutely dissociated TRG neurons of adult rats. Although three types of proton-gated INa were identified in rat TRG neurons (Pidoplichko 1992) and TTX-r sodium channels were detected by in situ hybridization technique in the same preparation (Akopian et al. 1996), no direct observation of voltage-dependent INa was reported from TRG neurons in any species, as far as we know. In addition, T-type ICa, DHP-sensitive L-type, and omega -CgTx-sensitive N-type ICa were not reported in this preparation.


    ACKNOWLEDGMENTS

This work was supported in part by the Basic Medical Research Fund (L-42) in 1996 from the Ministry of Education, Republic of Korea.


    FOOTNOTES

Address for reprint requests: H.-C. Kim, Dept. of Oral Physiology, School of Dentistry, Kyung Hee University, #1, Heoki-dong, Dongdaemoon-ku, Seoul 130-701, Korea.

Received 17 November 1997; accepted in final form 4 August 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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