Contribution of Nav1.8 Sodium Channels to Action Potential Electrogenesis in DRG Neurons

Muthukrishnan Renganathan, Theodore R. Cummins, and Stephen G. Waxman

Department of Neurology and Paralyzed Veterans of America/Eastern Paralyzed Veterans Association Neuroscience Research Center, Yale University School of Medicine, New Haven 06510; and Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare Center, West Haven, Connecticut 06516


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Renganathan, Muthukrishnan, Theodore R. Cummins, and Stephen G. Waxman. Contribution of Nav1.8 Sodium Channels to Action Potential Electrogenesis in DRG Neurons. J. Neurophysiol. 86: 629-640, 2001. C-type dorsal root ganglion (DRG) neurons can generate tetrodotoxin-resistant (TTX-R) sodium-dependent action potentials. However, multiple sodium channels are expressed in these neurons, and the molecular identity of the TTX-R sodium channels that contribute to action potential production in these neurons has not been established. In this study, we used current-clamp recordings to compare action potential electrogenesis in Nav1.8 (+/+) and (-/-) small DRG neurons maintained for 2-8 h in vitro to examine the role of sodium channel Nav1.8 (alpha -SNS) in action potential electrogenesis. Although there was no significant difference in resting membrane potential, input resistance, current threshold, or voltage threshold in Nav1.8 (+/+) and (-/-) DRG neurons, there were significant differences in action potential electrogenesis. Most Nav1.8 (+/+) neurons generate all-or-none action potentials, whereas most of Nav1.8 (-/-) neurons produce smaller graded responses. The peak of the response was significantly reduced in Nav1.8 (-/-) neurons [31.5 ± 2.2 (SE) mV] compared with Nav1.8 (+/+) neurons (55.0 ± 4.3 mV). The maximum rise slope was 84.7 ± 11.2 mV/ms in Nav1.8 (+/+) neurons, significantly faster than in Nav1.8 (-/-) neurons where it was 47.2 ± 1.3 mV/ms. Calculations based on the action potential overshoot in Nav1.8 (+/+) and (-/-) neurons, following blockade of Ca2+ currents, indicate that Nav1.8 contributes a substantial fraction (80-90%) of the inward membrane current that flows during the rising phase of the action potential. We found that fast TTX-sensitive Na+ channels can produce all-or-none action potentials in some Nav1.8 (-/-) neurons but, presumably as a result of steady-state inactivation of these channels, electrogenesis in Nav1.8 (-/-) neurons is more sensitive to membrane depolarization than in Nav1.8 (+/+) neurons, and, in the absence of Nav1.8, is attenuated with even modest depolarization. These observations indicate that Nav1.8 contributes substantially to action potential electrogenesis in C-type DRG neurons.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dorsal root ganglia (DRG) neurons, particularly small C-type DRG neurons, as well as other sensory neurons such as trigeminal and nodose neurons, are unusual in that they are capable of generating tetrodotoxin-resistant (TTX-R) action potentials (Yoshida et al. 1978). Studies using sharp microelectrodes have shown that, in some DRG neurons, these TTX-R action potentials are sodium dependent (Matsuda et al. 1978), suggesting that they might be produced by TTX-R Na+ channels. More recently, patch-clamp studies have demonstrated that DRG neurons are unique in expressing TTX-R as well as classical fast TTX-sensitive (TTX-S), Na+ currents (Caffrey et al. 1992; Cummins et al. 1999; Elliott and Elliott 1993; Kostyuk et al. 1981; Rizzo et al. 1994; Roy and Narahashi 1992). Electrophysiological studies have indicated that one or more TTX-R sodium channels can support action potential conduction in the unmyelinated C fibers that arise from small DRG neurons (Jeftinija 1994; Quasthoff et al. 1995)

It is now clear from studies at the molecular level that DRG neurons express multiple sodium channel isotypes (Black et al. 1996). Two neuronal TTX-R sodium channels, Nav1.8 (SNS) (Akopian et al. 1996; Sangameswaran et al. 1996; see Goldin et al. 2001, with respect to nomenclature) and Nav1.9 (NaN) (Dib-Hajj et al. 1998), both expressed in small DRG neurons, have been cloned; but the molecular identity of the sodium channel that produces TTX-R sodium-dependent action potentials has thus far not been determined. Behavioral observations in transgenic Nav1.8-null mice suggest a role of Nav1.8 in inflammatory pain (Akopian et al. 1999), and altered pain behavior after administration of Nav1.8 antisense DNA is consistent with a role in pain (Gold 1999; Porreca et al. 1999). However, the role of Nav1.8 in action potential electrogenesis has not been examined. In this study, we compared small DRG neurons isolated from transgenic Nav1.8 (+/+) and (-/-) mice (Akopian et al. 1999) to examine the contribution of Nav1.8 to electrogenesis. Our results identify Nav1.8 as a TTX-R sodium channel that contributes significantly to the production of sodium-dependent action potentials in C-type DRG neurons.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care

A breeding pair of Nav1.8 (+/-) [SNS (+/-)] mice (Akopian et al. 1999) was generously provided by Prof. John Wood, and a colony of Nav1.8 (+/+) and Nav1.8 (-/-) animals were raised from this pair. Three-month-old Nav1.8 (+/+) and Nav1.8 (-/-) mice (25-g weight) from the colony were used in this study. Animals were fed ad libitum and housed in a pathogen-free area at the Veterans Affairs Medical Center, West Haven. All animals were individually genotyped prior to death. Animal care and surgical procedures followed a protocol approved by the Animal Care and Use Committee of Yale University.

Culture of dorsal root ganglia (DRG) neurons

Mice were rendered unconscious by exposure to rising concentrations of CO2 and decapitated. L4 and L5 lumbar DRG were freed from their connective tissue sheaths in sterile calcium-free saline solution. Cell cultures were prepared as previously described (Renganathan et al. 2000b). Briefly, the L4 and L5 DRG ganglia were harvested, treated with collagenase and papain, and dissociated in DMEM and Ham's F12 medium supplemented with 10% fetal bovine serum. The DRG neurons were plated on polyornithine and laminin-coated glass coverslips. Neurons were placed in a 5% CO2-95% O2 incubator at 37°C and, 1 h after isolation, were fed with fresh culture medium. DRG neurons were studied using patch clamp 2-8 h after isolation because at this time in culture, neurons had not yet sprouted neurites and yielded a better seal and a higher yield of high-quality recordings. Only C-type DRG neurons (20-25 µm diam) were used in this study.

Electrophysiological recordings

Coverslips were mounted in a series 20 recording/perfusion chamber (Warner Instruments, Hamden, CT) positioned on the stage of a Nikon Diaphot microscope (Nikon) and were continuously perfused with the bath-external solution (see following text) with a push-pull syringe pump (WPI, Saratoga, FL). Cells were current-clamped via the whole cell configuration of the patch clamp with an Axopatch-200B amplifier (Axon Instruments, Foster City, CA) using standard techniques. Micropipettes were pulled from borosilicate glasses (Boralex) with a Flaming Brown micropipette puller (P80, Sutter Instrument, Novato, CA) and polished on a microforge (Narishige, Tokyo) to obtain electrode resistances ranging from 1 to 2.5 MOmega . The pipette solution contained (in mM) 140 KCl, 0.5 EGTA, 5 MgATP, and 5 HEPES, pH 7.3 with KOH. To determine the contribution of Na+ currents produced by Nav1.8 to peak action potential amplitude, 100 µM CdCl2 was used to block Ca2+ currents, and in some experiments, 5 mM Na-HEPES was used instead of HEPES to elevate the intracellular Na+ concentration to 5 mM. The following bath solution was used (in mM): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, pH 7.3. The pipette potential was zeroed before seal formation. Liquid junction potentials were <= 4 mV and were not corrected. Whole cell membrane voltage changes were filtered at 5 kHz and acquired at 20 kHz by a computer using pCLAMP 8.1 software (Axon Instruments). A Digidata 1200B interface (Axon Instruments) was used for A-D conversion, and the data were stored on compact disk and analyzed using pCLAMP 8.1 software (Axon Instruments). Experiments were carried out at a room temperature of 22-25°C.

As shown in Fig. 4, action potential characteristics were affected by resting potential. Therefore only cells with a stable resting potential up to -45 mV were used in this study. Action potentials were detected in 100% of cells in response to suprathreshold current injections. Input resistance was calculated by recording voltage changes by injection of -10-pA hyperpolarizing currents. Action potential threshold was measured at the beginning of the sharp upward rise of the depolarizing phase of the action potential. Current threshold was determined by a series of depolarizing currents from 0 to 250 pA in 10-pA step increments. The waveform characteristics of the action potentials recorded from Nav1.8 (+/+) and (-/-) small DRG neurons, i.e., maximum rise slope, maximum fall slope, rise time, decay time, action potential duration, were determined using Clampfit 8.1 software (Axon Instruments). Repetitive firing of action potentials was measured by recording voltage changes in response to sustained (1 s) injection of depolarizing currents.

Statistics

All results are expressed as means ± SE. Statistical significance was evaluated using Student's t-test. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The principal aim of this study was to investigate the role of Nav1.8 channels in action potential electrogenesis. Because the slow TTX-R Na+ currents produced by Nav1.8 Na+ channels are present in >90% of wild-type small C-type DRG neurons (Cummins and Waxman 1997; Cummins et al. 1999) but are absent in C-type neurons from Nav1.8 (-/-) mice (Akopian et al. 1999; Cummins et al. 1999), we compared C-type DRG neurons isolated from Nav1.8 (+/+) wild-type and (-/-) null mice in this study.

C-type DRG neurons can generate TTX-R action potentials

Nav1.8 (+/+) C-type DRG neurons generated action potentials with long duration (mean duration 3.87 ± 0.29 ms measured at 0 mV) and peaking at about +55 mV (Fig. 1A). Digital differentiation revealed two peaks in the falling phase of somatic action potential (Fig. 1A, inset), indicating the presence of inflection in most cells studied. The long action potential duration and inflection have been associated with small neurons within intact DRGs (Harper and Lawson 1985; Villiere and McLachlan 1996; Waddell and Lawson 1990), and their presence suggests that isolation did not alter fundamental electrophysiological features of the sensory neurons examined in our study. Consistent with earlier sharp microelectrode recordings in mouse DRG neurons (Matsuda et al. 1978), action potentials were still evoked in four of five small neurons when 250 nM TTX was added to the bath solution, and the peak action potential amplitude did not decrease on addition of TTX (Fig. 1B). The inflection in the falling phase was still observed in the presence of TTX (Fig. 1B, inset), demonstrating that inflections can occur in the absence of TTX-S Na+ currents. Further, repetitive firing of action potentials was observed in the presence of 250 nM TTX (Fig. 1C). These results demonstrate the presence of TTX-R action potentials in C-type neurons.



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Fig. 1. Nav1.8 (+/+) C-type dorsal root ganglia (DRG) neurons can generate TTX-resistant (TTX-R) action potentials. A: action potentials were evoked at 10-, 20-, and 30-pA current injections. Inset: the digitally differentiated traces (rate of membrane potential change) evoked by a 30-pA current injection. The voltage change due to -10-pA current injection was used to evoke voltage responses and to estimate the input resistance of the neuron. B: when exposed to 250 nM TTX, the same neuron still produced action potentials. Action potentials were generated by 40-, 50-, and 80-pA current injections. Inset: the rate of membrane potential change due to 40-pA current injection. C: the same neuron, in the presence of 250 nM TTX, generated sustained repetitive action potential firing in response to injection of 300-pA current for 1 s.

Resting potential, input resistance, action potential threshold, and current threshold are unchanged in Nav1.8 (-/-) DRG neurons

The mean resting membrane potential was -52.9 ± 1.2 mV (n = 32) in Nav1.8 (+/+) C-type neurons and -53.7 ± 1.5 mV (n = 42) in Nav1.8 (-/-) DRG neurons (P > 0.05, Student's t-test, Fig. 2A). The input resistance for Nav1.8 (+/+) DRG neurons was 521.3 ± 43.6 MOmega (n = 32) and for Nav1.8 (-/-) DRG neurons was 596.9 ± 37.8 MOmega (n = 42; Fig. 2B). No spontaneous firing of action potentials was observed either in Nav1.8 (+/+) or Nav1.8 (-/-) DRG neurons at resting membrane potential. On injection of depolarizing current, all Nav1.8 (+/+) and (-/-) neurons generated overshooting responses. Action potential threshold for Nav1.8 (+/+) and (-/-) DRG neurons was -26.9 ± 1.5 mV (n = 32) and -26.8 ± 1.7 mV (n = 42), respectively (Fig. 2C). The current threshold required to elicit action potentials for Nav1.8 (+/+) and (-/-) neurons was 68.2 ± 11.9 pA (n = 32) and 65.0 ± 19.8 pA (n = 42), respectively (Fig. 2D).



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Fig. 2. Passive and active membrane properties in Nav1.8 (+/+) and (-/-) neurons. A: resting potentials in Nav1.8 (+/+) (n = 32) and (-/-) (n = 42) neurons measured 3-4 min after breaking into whole cell configuration. B: input resistance determined from the voltage change associated with a -10-pA current injection is similar in Nav1.8 (+/+) and (-/-) neurons. C: action potential threshold was measured at the beginning of the sharp upward rise of the depolarizing phase of the action potential. In Nav1.8 (+/+) and (-/-) neurons that generated graded action potential responses, threshold was measured in action potentials that had peak amplitude of >0 mV. D: current threshold was determined by a series of depolarizing current injections from 0 to 250 pA in 10-pA step increments and was defined as the current that evoked an action potential with peak amplitude more than 0 mV. E: peak of the action potential amplitude in Nav1.8 (+/+) and (-/-) neurons, measured from responses that had peak of >0 mV. *, statistically significant difference (P < 0.05) between Nav1.8 (+/+) and (-/-) neurons.

Action potential characteristics are different in Nav1.8 (-/-) DRG neurons

Action potentials elicited from Nav1.8 (+/+) and (-/-) C-type neurons peaked at +55.0 ± 4.3 mV (n = 32 from 7 different preparations) and +31.5 ± 2.2 mV (n = 42 from 7 different preparations), respectively (Fig. 2E). The decrease in action potential amplitude in Nav1.8 (-/-) C-type DRG neuron is statistically significant (P < 0.0001). These results demonstrate that TTX-R Na+ currents produced by Nav1.8 Na+ channels contribute to action potential overshoot in small C-type DRG neurons.

The action potential characteristics of Nav1.8 (+/+) and (-/-) neurons are summarized in Table 1. The rate of depolarization is 80% faster in Nav1.8 (+/+) neurons than Nav1.8 (-/-) neurons, which is reflected in a smaller 10-90% rise time for Nav1.8 (+/+) neurons. These results suggest that a current component that contributes to the rate of depolarization is missing in Nav1.8 (-/-) neurons. Other parameters, including rate of repolarization, decay time, and half-width duration in Nav1.8 (+/+) and (-/-) neurons, did not show statistically significant differences, although half-width was 13% larger in Nav1.8 (-/-) neurons. Because action potential amplitude is smaller in Nav1.8 (-/-) neurons, we also determined the action potential duration at 0 mV and found no significant difference between Nav1.8 (+/+) and (-/-) neurons.


                              
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Table 1. Action potential parameters in Nav1.8 (+/+) and (-/-) neurons

Most Nav1.8 (+/+) neurons produce all-or-none overshooting action potentials

Most Nav1.8 (+/+) small DRG neurons evoked all-or-none action potentials (Fig. 3A). That is, on injection of depolarizing current, these neurons displayed action potentials with amplitude of ~115 mV (-55-60 mV), and on further depolarization, the action amplitude did not increase. This pattern of all-or-none action potential was observed in 84% (27 of 32) of the DRG neurons. Mean resting potential in this subgroup of neurons was -55.5 ± 0.9 mV. The action potential amplitude in these neurons peaked at +59.2 ± 3.9 mV. In contrast, in 5 of 32 small DRG neurons, depolarization evoked smaller graded responses (Fig. 3B), which tended to peak at an average potential of +26.7 ± 0.9 mV. With graded depolarization, these neurons initially generated responses with a peak close to 0 mV, which increased to about +30 mV on further depolarization. These graded responses were observed in neurons with resting potentials of -48.2 ± 1.2 mV, where most of the TTX-S fast Na+ channels in these neurons are inactive (Cummins and Waxman 1997; Renganathan et al. 2000a) and only a few channels are able to participate in the generation of action potentials. Figure 4, A and B, shows a cell that initially exhibited a depolarized resting potential, close to -30 mV, and small graded responses to depolarizing stimuli immediately after whole cell configuration was established. After 4 min, resting potential shifted to -60 mV and large, overshooting (+68 mV) action potentials were seen. These observations suggest that in some Nav1.8 (+/+) neurons, TTX-S fast Na+ channels can produce all-or-none action potentials but, presumably as a result of steady-state inactivation of these channels, electrogenesis in these neurons is more sensitive to membrane depolarization.



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Fig. 3. Nav1.8 (+/+) neurons generate all-or-none action potential. A: 84% of SNS (+/+) neurons elicit all-or-none action potentials on stimulation with depolarizing currents. The resting potential of these neurons varied from -80 to -50 mV (mean, -55.48 ± 0.89 mV). Action potentials were generated by depolarizing current injections from -10 to 250 pA. For clarity, traces generated by -10-, 0-, 20-, 40-, 60-, 70-, 80-, 90-, 100-, 110-, 150-, and 250-pA current injection only are shown. B: 15% of Nav1.8 (+/+) neurons, which had a mean resting potential of -48.24 ± 1.22 mV, generated smaller graded responses. To avoid overcrowding, traces generated by -10-, 0-, 10-, 20-, 80-, 130-, and 250-pA current injections are shown. Most Nav1.8 (-/-) neurons generate smaller graded responses. C: 80% of Nav1.8 (-/-) neurons generated smaller graded responses. The resting potential of these neurons varied from -80 to -50 mV (mean, -49.48 ± 0.87 mV). For clarity, traces generated by -10-, 0-, 10-, 20-, 30-, 60-, 80-, 100-, 120-, 140-, 180-, and 240-pA current injections are shown. D: 20% of SNS (-/-) neurons that had a resting potential between -70 and -60 mV (mean, -65.07 ± 0.87 mV) elicited all-or-none action potentials. For clarity, traces generated by -10-, 20-, 30-, 50-, 70-, 80-, 90-, 100-, 120-, 160-, and 220-pA current injections are shown.



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Fig. 4. Resting potential is critical for action potential overshoot in Nav1.8 (+/+) and (-/-) neurons. A: Nav1.8 (+/+) neuron immediately after establishing the whole cell configuration had a resting potential of -30 mV and depolarizing current injections from -10 to 140 pA in 10-pA step increments evoked smaller graded responses. B: after 4 min, the same neuron had a resting potential of -60 mV, and depolarizing current injections from -10 to 150 pA in 10-pA increments evoked all-or-none action potential overshoot. C: Nav1.8 (-/-) neuron had a resting potential of -70 mV 4 min after establishing the whole cell configuration, and depolarizing current injections from 0 to 250 pA in 10-step increments evoked all-or-none action potential overshoot. For clarity, traces evoked by 0, 20, 30, 50, 80, 90, 100, 110, 120, 140, 150, 170 190, 210, 230, and 250 pA are shown. Current threshold to elicit action potential for this neuron was 100 pA. D: 80 pA was injected to depolarize the resting potential from -70 to -55 mV. Further depolarization from 100 to 310 pA in 10-pA step increments evoked graded responses in this neuron. For clarity, traces evoked by 100, 110, 120,130, 140, 150, 160, 170, 180, 200, 220, 240, 260, 280, and 310 pA are shown.

Most Nav1.8 (-/-) neurons produce small graded responses

Thirty-two of 42 small DRG neurons isolated from Nav1.8 (-/-) null mice produced small graded responses in response to depolarizing stimuli when studied immediately after establishing whole cell configuration (Fig. 3C), unlike the pattern of all-or-none action potentials observed in most Nav1.8 (+/+) neurons. The mean resting potential in this majority of the neurons was -49.5 ± 0.9 mV. The graded responses in Nav1.8 (-/-) neurons peaked at a maximum of +24.6 ± 1.4 mV. These results demonstrate that in the absence of slow TTX-R Na+ currents produced by Nav1.8, the ability of most Nav1.8 (-/-) neurons to generate overshooting all-or-none action potentials is impaired.

In 10 of 42 small Nav1.8 (-/-) DRG neurons studied immediately after establishing whole cell configuration, depolarizing stimuli evoked all-or-none action potentials that peaked at 45.9 ± 1.2 mV (Fig. 3D), significantly higher than observed in the other 32 Nav1.8 (-/-) neurons (P < 0.0001). The larger action potential amplitude and all-or-none behavior in this population of Nav1.8 (-/-) neurons may reflect their relatively hyperpolarized resting potentials (-65.1 ± 0.9 mV for this subgroup of neurons) compared with the other 32 neurons, which as noted in the preceding text, had resting potential amplitude of -49.5 ± 0.9 mV. At the more hyperpolarized resting potential, ~50% of the fast TTX-S Na+ channels would be expected to be available for activation (see Cummins and Waxman 1997) and therefore able to contribute to the all-or-none action potential overshoot. We tested this hypothesis in a Nav1.8 (-/-) neuron whose resting potential was -70 mV immediately after establishing whole cell configuration. At this resting potential (Fig. 4C), this cell generated all-or-none action potentials with a peak at +55 mV. When we depolarized the cell and held it at a resting potential of -50 mV, the cell generated smaller graded responses (Fig. 4D).

TTX-S Na+ channels produce all-or-none action potentials in Nav1.8 (-/-) neurons

To determine whether TTX-S Na+ channels produce all-or-none action potentials in Nav1.8 (-/-) neurons, Nav1.8 (-/-) neurons that generated all-or-none action potentials were exposed to 250 nM TTX. Figure 5 shows recordings from a representative Nav1.8 (-/-) neuron and illustrates action potential generation in the absence of TTX (Fig. 5A) and the failure of action potential electrogenesis in the presence of 250 nM TTX (Fig. 5B). In the absence of TTX, the neuron produced action potentials with peak at +60 mV, and on addition of TTX, the neuron produced subthreshold membrane depolarizations but did not generate action potentials on depolarizing current injection. Similar results were obtained in 3/3 Nav1.8 (-/-) neurons exposed to 250 nM TTX. These results indicate that TTX-S Na+ channels can produce all-or-none action potentials in Nav1.8 (-/-) neurons.



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Fig. 5. TTX-S Na+ channels produce all-or-none action potentials in Nav1.8 (-/-) neurons. All-or-none action potentials are produced in a typical Nav1.8 (-/-) neuron (A) but was abolished by 250 nM TTX (B). Smaller graded responses in Nav1.8 (-/-) neurons are not Na+ action potentials. Generation of graded responses in Nav1.8 (-/-) (C) neuron was not prevented by 250 nM TTX (D).

Inflected responses can occur in the absence of Nav1.8 Na+ channels

Action potentials in C-type DRG neurons have been shown to have a shoulder (inflection) on the falling phase (Gold et al. 1996a; Harper and Lawson 1985; Villiere and McLachlan 1996; Waddell and Lawson 1990). C-cell action potentials have been suggested to be TTX resistant (Villiere and McLachlan 1996), while all those without inflections have been reported to be TTX sensitive (Villiere and McLachlan 1996; Waddell and Lawson 1990). Axotomized cutaneous neurons display a reduction in TTX-R Na+ current and an increase in TTX-S Na+ current with a reduction in the number of neurons that had inflections on the falling phase of the action potential (Oyelese et al. 1997). Taken together, these results might be interpreted as suggesting that the presence of Nav1.8 Na+ currents endows DRG neurons with inflections on the falling phase of the action potential. However, we found that most (37 of 42) Nav1.8 (-/-) neurons displayed an inflection or hump on the falling phase, and only a few (5 of 42) neurons had simple, uninflected falling phase of the action potential. A broad plateau or hump on the falling phase was observed in 7 of 42 Nav1.8 (-/-) neurons (Fig. 6), while less pronounced inflections in the falling phase was observed in 30 of 42 Nav1.8 (-/-) neurons (Fig. 8, A and B). These results demonstrate that Nav1.8 is not required for an inflection in the falling phase of the somatic action potential.



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Fig. 6. Inflection in the repolarization phase of the action potential can occur in the absence of Nav1.8 channels. Ninety percent of Nav1.8 (-/-) neurons showed an inflection in the falling phase. Of these neurons, 20% showed a broad plateau or hump in the falling phase as shown in the Fig. 1, inset, displays differentiated record showing the rate of membrane potential change (dupsilon /dt) taken from the trace indicated left-arrow .

Ca2+ channels can contribute to inflection of the action potential in Nav1.8 (+/+) neurons

A majority of Nav1.8 (+/+) neurons (24/29) showed inflections on the falling phase of the action potential. To determine whether Ca2+ channels contribute to inflection on the falling phase of the action potential, Nav1.8 (+/+) neurons were depolarized to evoke action potentials in the absence and presence of 100 µM Cd2+, a concentration that effectively blocks Ca2+ currents in these neurons. Figure 7A illustrates an action potential evoked from a typical Nav1.8 (+/+) neuron in the absence of 100 µM Cd2+. Its derivative, which indicates the corresponding rate of depolarization, is given in Fig. 7B. The presence of two peaks on the falling phase of the derivative is due to the presence of inflection on the repolarization phase of action potential. The same neuron, when exposed to 100 µM Cd2+, generates action potentials with similar peak amplitude (Fig. 7C), but the falling phase depicts only one peak (Fig. 7D). Similar results were obtained in 4/5 neurons studied. These results demonstrate that the inflection on the falling phase of the action potential can be produced by calcium currents in Nav1.8 (+/+) neurons. In one neuron, Cd2+ did not eliminate the inflection on the falling phase, suggesting that inflection can occur in the absence of calcium currents in some DRG neurons. Peak action potential amplitude was not significantly decreased in Nav1.8 (+/+) neurons by exposure to Cd2+. The action potential in the absence and presence of Cd2+ peaked at 59.6 ± 1.8 and 56.7 ± 4.3 mV, respectively (P < 0.05). The rate of depolarization, repolarization and half-width duration were not significantly different in the presence of Cd2+ in Nav1.8 (+/+) neurons (Table 2).



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Fig. 7. Calcium channels can contribute to inflections in the falling phase of the action potential in Nav1.8 (+/+) neurons. A: all-or-none action potentials were elicited with depolarizing current stimuli from -10 to 250 pA at 10-pA increments. For clarity, traces generated by current injections from -10, 0, 10, 20, 30, 40, 50, 60, 80, 120, 140 and 220 pA are shown. B: the rate of membrane potential change (dupsilon /dt) from the traces illustrated in A is shown. Two peaks on the repolarizing phase of the action potentials are present in all traces. C and D: in 4 of 5 Nav1.8 (+/+) neurons, 100 µM Cd2+ (added via the bath solution) abolished the 2nd peak (D) without decreasing the peak action potential amplitude (C). For clarity, traces generated by current injections from -10, 0, 10, 20, 30, 40, 50, 60, 80, 140 and 220 pA are shown. The rate of membrane potential change (dupsilon /dt) was obtained from traces illustrated in C.


                              
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Table 2. Action potential parameters in Nav1.8 (+/+) and (-/-) neurons in the absence and presence of 100 µM Cd2+

Ca2+ channels produce overshooting action potentials in some Nav1.8 (-/-) neurons

Responses were elicited from Nav1.8 (-/-) neurons (n = 10) in the absence and presence of 100 µM Cd2+ to determine whether Ca2+ channels contribute to action potential electrogenesis. Action potentials elicited from a Nav1.8 (-/-) neuron in the absence of Cd2+ are shown in Fig. 8A. On exposure to 100 µM Cd2+, the same neuron elicited graded responses with smaller amplitude and no clear threshold (Fig. 8B). In 8/10 neurons studied, Cd2+ had this effect. The response in the absence and presence of Cd2+ peaked at 27.5 ± 2.5 and 5.3 ± 3.2 mV (n = 8, P < 0.0001), respectively. The rate of depolarization and repolarization, but not the half-width duration, were significantly reduced in the presence Cd2+ in SNS (-/-) neurons (Table 2). These results suggest that Ca2+ currents can contribute significantly to electrogenesis in Nav1.8 (-/-) neurons.



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Fig. 8. Calcium channels produce overshooting action potentials in some Nav1.8 (-/-) neurons. A: action potentials were elicited by depolarizing current injections from -10 to 250 pA at 10-pA increments. B: 100 µM Cd2+ decreased response amplitude in all Nav1.8 (-/-) neurons (n = 8). For clarity, traces generated by current injections of -10, 10, 30, 60, 130, 140, 230 and 250 pA are shown in A and B.

Temporal patterns of action potential generation are different in Nav1.8 (-/-) and Nav1.8 (+/+) neurons

Although injection of sustained depolarization currents evoked repetitive firing in ~40% of Nav1.8 (+/+) and (-/-) neurons, the pattern of firing was different. Repetitive firing from a typical Nav1.8 (+/+) neuron is shown in Fig. 9, A and B, while firing of a representative Nav1.8 (-/-) neuron is shown in Fig. 9, C and D. The current protocols that elicited these responses are illustrated in Fig. 9, E and F. The Nav1.8 (+/+) neuron generated repetitive firing of action potentials at a frequency of 9 Hz (Fig. 9A) on injection of 75-pA current for 1 s. With a stronger depolarizing current injection (150 pA), the same neuron evoked action potentials at a higher frequency (14 Hz; Fig. 9B). Repetitive firing of Nav1.8 (-/-) neurons was not as robust as in Nav1.8 (+/+) neurons; the number of spikes was smaller, and they either occurred in bursts (Fig. 9C) or the spike train aborted after firing a few spikes (Fig. 9D). In addition, the spike amplitudes were smaller and the later spikes tended to fall off in amplitude in Nav1.8 (-/-) neurons. In Nav1.8 (+/+) and (-/-) neurons, the peak amplitude for the first spike was 63.4 ± 1.3 mV (n = 8) and 22.5 ± 3.6 mV (n = 13, P < 0.003), respectively. The peak amplitude for the fourth spike in Nav1.8 (+/+) and (-/-) neurons was 59.1 ± 0.9 and 0.6 ± 3.1 mV, respectively (P < 0.00001). The action potential amplitude for the fourth spike displayed a small but statistically significant reduction (4%) compared with the first spike in Nav1.8 (+/+) neurons, but the decrease was much more dramatic (30%) in Nav1.8 (-/-) neurons. These results suggest that Nav1.8 contributes to secure repetitive firing in DRG neurons.



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Fig. 9. Nav1.8 (+/+) neurons show robust and sustained repetitive firing. A: Nav1.8 (+/+) neuron produced a continuous and sustained repetitive firing on injecting depolarizing stimuli of 75 pA (A) and 150 pA (B). Nav1.8 (-/-) neurons displayed intermittent action potentials (C) or failed to sustain high-frequency firing (D). The current pulse protocols used to elicit action potential firing in A and C and in B and D are shown in E and F, respectively.


    DISCUSSION
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In this study, we compared action potentials generated by small DRG neurons from Nav1.8 (+/+) and (-/-) mice to investigate the role of Nav1.8 channels in action potential electrogenesis in these neurons. Our experiments did not reveal significant differences in resting membrane potential, input resistance, current threshold, or voltage threshold in Nav1.8 (+/+) and (-/-) DRG neurons. However, there were major differences in action potential electrogenesis between Nav1.8 (+/+) and (-/-) DRG neurons. The peak action potential response was significantly reduced and the rate of depolarization was substantially slower in Nav1.8 (-/-) neurons compared with Nav1.8 (+/+) neurons. We also observed different temporal patterns of spike generation in Nav1.8 (+/+) and (-/-) neurons with robust and continuous firing in response to sustained depolarization in Nav1.8 (+/+) neurons while frequency-related failure was observed in Nav1.8 (-/-) neurons. These results indicate that Nav1.8 TTX-R Na+ channels can play an important role in generating and maintaining the action potential in C-type DRG neurons in contrast to Nav1.9 TTX-R Na+ channels, which are thought to contribute to modulating resting potential (Herzog et al. 2001).

Nav1.8 mRNA (Dib-Hajj et al. 1996) and protein (Sleeper et al. 2000) as well as the slowly inactivating TTX-R Na+ currents attributable to Nav1.8 (Cummins and Waxman 1997) are all downregulated in DRG neurons following axotomy within the sciatic nerve. The absence of significant differences in resting membrane potential and input resistance in Nav1.8 (+/+) and (-/-) neurons is similar to the results obtained in DRG neurons isolated from control uninjured and axotomized neurons (Zhang et al. 1997). The absence of difference in somatic action potential threshold observed between Nav1.8 (+/+) and (-/-) DRG neurons, however, is different from the results obtained in axotomized neurons (Zhang et al. 1997, 1999), where a reduction in action potential threshold was seen. In electrophysiological studies that measured dorsal root compound action potentials in response to sciatic nerve stimulation, Akopian et al. (1999) observed a small (~20% for maximal compound action potential) decrease in C-fiber threshold in Nav1.8 (-/-) compared with Nav1.8 (+/+) mice. The disparity between this result and the findings in the present study may reflect a difference in channel expression in neuronal somata compared with axons. Previous studies have also demonstrated an upregulation of mRNA for Nav1.7 that encodes a TTX-S Na+ channel (Akopian et al. 1999) and TTX-S fast Na+ currents (Akopian et al. 1999; Renganathan et al. 2000b) in Nav1.8 (-/-) neurons. These compensatory changes may maintain or even lower the threshold of these neurons.

We found that TTX-S Na+ channels can produce all-or-none action potentials in some Nav1.8 (-/-) neurons. The Nav1.8 (-/-) neurons that generated all-or-none action potentials had a more hyperpolarized resting potential than the neurons that generated only graded responses. This indicates that electrogenesis in Nav1.8 (-/-) neurons is more sensitive to resting potential than is electrogenesis in Nav1.8 (+/+) neurons. As noted in the following text, this may be due to the relatively hyperpolarized voltage dependence of steady-state inactivation of the TTX-S channels that are expressed in DRG neurons (see e.g., Cummins and Waxman 1997; Cummins et al. 1998).

Nav1.8 (-/-) and action potential inflection

We observed that Nav1.8 (-/-) neurons generate action potentials with inflections on the falling phase. Thus Nav1.8 is not required for these inflections. Na+ channels other than Nav1.8 (Black et al. 1996), or Ca2+ channels (Baccei and Kocsis 2000) and K+ channels (Gold et al. 1996b) in small DRG neurons may in theory contribute to these inflections. We found that in some neurons, Ca2+ channels can contribute to inflections in the action potential. While inflections in the action potential have been used to classify DRG neurons into distinct groups (Gold et al. 1996b; Harper and Lawson 1985; Villiere and McLachlan 1996; Waddell and Lawson 1990), our results indicate that the presence of inflections per se cannot be attributed to the activity of any single channel isotype.

Different action potential characteristics in Nav1.8 (-/-) DRG neurons

Action potentials in Nav1.8 (-/-) neurons displayed a significantly lower peak, (+31.5 ± 2.2 mV) compared with Nav1.8 (+/+) neurons (+55.0 ± 4.3 mV; P < 0.0001). One explanation for this is that Nav1.8 channels in Nav1.8 (+/+) cells are open at the peak of the action potential, adding to the total PNa. A slower rate of membrane potential depolarization in Nav1.8 (-/-) neurons (Table 1) further suggests that Nav1.8 channels contribute to the magnitude of the action potential in Nav1.8 (+/+) neurons.

Peak action potential amplitude determined in the absence of Ca2+ channel activity, i.e., in the presence of 100 µM Cd2+, can be used to estimate the ratio of PNa (Na+ channel permeability) in Nav1.8 (-/-) and (+/+) neurons, i.e., the ratio of PNa(-/-) to PNa(+/+). According to the Goldman-Hodgkin-Katz equation
<IT>V</IT><SUB><IT>m</IT>(<IT>+/+</IT>)</SUB><IT>=</IT><FR><NU><IT>E</IT><SUB><IT>Na</IT></SUB><IT>P</IT><SUB><IT>Na</IT>(<IT>+/+</IT>)</SUB><IT>+</IT><IT>E</IT><SUB><IT>k</IT></SUB><IT>P</IT><SUB><IT>k</IT></SUB></NU><DE><IT>P</IT><SUB><IT>Na</IT>(<IT>+/+</IT>)</SUB><IT>+</IT><IT>P</IT><SUB><IT>k</IT></SUB></DE></FR>
and
<IT>V</IT><SUB><IT>m</IT>(<IT>−/−</IT>)</SUB><IT>=</IT><FR><NU><IT>E</IT><SUB><IT>Na</IT></SUB><IT>P</IT><SUB><IT>Na</IT>(<IT>−/−</IT>)</SUB><IT>+</IT><IT>E</IT><SUB><IT>k</IT></SUB><IT>P</IT><SUB><IT>k</IT></SUB></NU><DE><IT>P</IT><SUB><IT>Na</IT>(<IT>−/−</IT>)</SUB><IT>+</IT><IT>P</IT><SUB><IT>k</IT></SUB></DE></FR>
For both Nav1.8 (+/+) and Nav1.8 (-/-)
<IT>P</IT><SUB><IT>Na</IT></SUB><IT>V</IT><SUB><IT>m</IT></SUB><IT>+</IT><IT>P</IT><SUB><IT>K</IT></SUB><IT>V</IT><SUB><IT>m</IT></SUB><IT>=</IT><IT>E</IT><SUB><IT>Na</IT></SUB><IT>P</IT><SUB><IT>Na</IT></SUB><IT>+</IT><IT>E</IT><SUB><IT>K</IT></SUB><IT>P</IT><SUB><IT>K</IT></SUB>
so that
<IT>P</IT><SUB><IT>Na</IT></SUB><IT>=</IT><FR><NU><IT>P</IT><SUB><IT>K</IT></SUB>(<IT>E</IT><SUB><IT>K</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>m</IT></SUB>)</NU><DE><IT>V</IT><SUB><IT>m</IT></SUB><IT>−</IT><IT>E</IT><SUB><IT>Na</IT></SUB></DE></FR>
Thus
<IT>P</IT><SUB><IT>Na</IT>(<IT>+/+</IT>)</SUB><IT>=</IT><FR><NU><IT>P</IT><SUB><IT>K</IT></SUB>(<IT>E</IT><SUB><IT>K</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>m</IT>(<IT>+/+</IT>)</SUB>)</NU><DE><IT>V</IT><SUB><IT>m</IT>(<IT>+/+</IT>)</SUB><IT>−</IT><IT>E</IT><SUB><IT>Na</IT></SUB></DE></FR>

<IT>P</IT><SUB><IT>Na</IT>(<IT>−/−</IT>)</SUB><IT>=</IT><FR><NU><IT>P</IT><SUB><IT>K</IT></SUB>(<IT>E</IT><SUB><IT>K</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>m</IT>(<IT>−/−</IT>)</SUB>)</NU><DE><IT>V</IT><SUB><IT>m</IT>(<IT>−/−</IT>)</SUB><IT>−</IT><IT>E</IT><SUB><IT>Na</IT></SUB></DE></FR>
Therefore if we assume that EK, PK, and ENa are equal in Nav1.8 (+/+) and (-/-) neurons, then
<FR><NU><IT>P</IT><SUB><IT>Na</IT>(<IT>−/−</IT>)</SUB></NU><DE><IT>P</IT><SUB><IT>Na</IT>(<IT>+/+</IT>)</SUB></DE></FR><IT>=</IT><FR><NU>(<IT>E</IT><SUB><IT>K</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>m</IT>(<IT>−/−</IT>)</SUB>)</NU><DE>(<IT>E</IT><SUB><IT>K</IT></SUB><IT>−</IT><IT>V</IT><SUB><IT>m</IT>(<IT>+/+</IT>)</SUB>)</DE></FR> <FR><NU>(<IT>V</IT><SUB><IT>m</IT>(<IT>+/+</IT>)</SUB><IT>−</IT><IT>E</IT><SUB><IT>Na</IT></SUB>)</NU><DE>(<IT>V</IT><SUB><IT>m</IT>(<IT>−/−</IT>)</SUB><IT>−</IT><IT>E</IT><SUB><IT>Na</IT></SUB>)</DE></FR>
where Vm(+/+) = peak action potential overshoot in the presence of Cd2+ in Nav1.8 (+/+) neuron, Vm(-/-) = peak action potential overshoot in the presence of Cd2+ in Nav1.8 (-/-) neuron, EK = K+ reversal potential, and ENa = Na+ reversal potential.

Under our experimental conditions, the reversal potentials for K+ and Na+ ions are -98.0 and +85.0 mV, respectively. The peak action potential overshoots in Nav1.8 (+/+) and (-/-) neurons in the presence of Cd2+ are 56.7 ± 4.3 mV (n = 8) and 5.3 ± 3.2 mV (n = 8), respectively. These values yield
<FR><NU><IT>P</IT><SUB><IT>Na</IT>(<IT>−/−</IT>)</SUB></NU><DE><IT>P</IT><SUB><IT>Na</IT>(<IT>+/+</IT>)</SUB></DE></FR><IT>=0.22</IT>
However, the K+ channel permeability (PK) at the peak of the action potential might be different in Nav1.8 (+/+) and (-/-) neurons, as a result of different membrane potentials at the peak of the action potential. Using the data of Safronov et al. (1996) for DRG neurons, the permeability of type-A K+ channels and slowly inactivating K+ channels are 70 and 50% at the action potential peak in Nav1.8 (-/-) neurons, compared with the action potential peak in Nav1.8 (+/+) neurons, and the permeability of the delayed rectifier K+ channels is similar at the peaks of the action potential in Nav1.8 (-/-) and (+/+) neurons. Using corrected values of PK (60%)
<FR><NU><IT>P</IT><SUB><IT>Na</IT>(<IT>−/−</IT>)</SUB></NU><DE><IT>P</IT><SUB><IT>Na</IT>(<IT>+/+</IT>)</SUB></DE></FR><IT>=0.13</IT>
was obtained. In both cases, Nav1.8 channels contribute a substantial fraction (0.8-0.9) of the inward membrane current that flows during the peak of the action potential amplitude, assuming that other sodium channel isotypes are not expressed at increased levels in Nav1.8 (-/-) neurons. Up-regulation of other sodium channel isotypes (Akopian et al. 1999) would imply that Nav1.8 channels contribute an even higher fraction.

The present results indicate that Nav1.8 participates in action potential electrogenesis in the cell bodies of small DRG neurons. Although we have no direct evidence concerning the role of Nav1.8 within the axons of these neurons, immunocytochemical studies demonstrate the presence of Nav1.8 within the superficial laminae of the spinal cord where the central projections of the C-type neurons terminate (Novakovic et al. 1998). Scroggs and Fox (1992) used action potential waveforms as stimuli and found that activation of N-type calcium channels is highly dependent on action potential shape and amplitude. The effects of nerve injury on Nav1.8 expression in the axon terminals of DRG neurons have not been studied, but it is known that axotomy produces long-lasting decreases in expression of Nav1.8 mRNA within DRG neurons (Dib-Hajj et al. 1996). We have demonstrated (Fig. 3 and Table 1) that in C-type DRG neurons that lack Nav1.8, amplitudes of action potentials are decreased. Moreover action potential amplitude in Nav1.8 (-/-) neurons is especially sensitive to resting potential (Fig. 4), consistent with the voltage dependence of steady-state inactivation of the TTX-S Na+ channels in DRG neurons (Cummins and Waxman 1997; Cummins et al. 1998). This raises the question of whether action potential waveform at the central terminals of C-type DRG neurons is altered with a resultant change in the efficacy of transmission to second-order sensory neurons as a result of reduced Nav1.8 transcription after nerve injury.

In addition, we observed different temporal patterns of action potential generation in Nav1.8 (+/+) and (-/-) neurons. Nav1.8 Na+ channels exhibit rapid recovery from inactivation (Cummins and Waxman 1997; Elliott and Elliott 1993); this appears to be due, at least in part, to the presence of a specific tripeptide insertion in the D4S3-S4 linker (Dib-Hajj et al. 1997). This rapid recovery from inactivation, coupled with the relatively depolarized voltage dependence of steady-state inactivation observed for Nav1.8 Na+ channels (Akopian et al. 1996), might allow neurons that express Nav1.8 channels to sustain repetitive firing at depolarized membrane potentials. Upregulation of Nav1.8 expression has been observed in cerebellar Purkinje cells in two experimental models of multiple sclerosis (Black et al. 1999, 2000) and in multiple sclerosis (Black et al. 2000). Although the physiological concomitants of abnormal Nav1.8 expression in Purkinje cells have not yet been directly studied, Black et al. (2000) speculated that the abnormal expression of Nav1.8 could alter the pattern of electrogenesis, thereby contributing to clinical abnormalities such as cerebellar ataxia. We observed different patterns of action potential electrogenesis, in response to the same stimulus, in Nav1.8 (+/+) and (-/-) DRG neurons. While compensatory changes in the expression of other channels in the Nav1.8 (-/-) neurons (Akopian et al. 1999) may also contribute to these differences, the altered pattern of action potential generation in Nav1.8 (+/+) as compared with Nav1.8 (-/-) DRG neurons is consistent with the idea that Nav1.8 expression can modify the pattern of firing in neurons where it is expressed.


    ACKNOWLEDGMENTS

A breeding pair of Nav1.8 (+/-) [SNS (+/-)] mice was kindly provided by Prof. J. N. Wood, University College London. We thank Drs. Joel Black and Sulayman Dib-Hajj for establishing and genotyping the colony of Nav1.8 (-/-) mice and B. Toftness for help with figures.

This work was supported in part by grants from the Medical Research Service and Rehabilitation Research Service, Department of Veterans Affairs and from the National Multiple Sclerosis Society. We also thank the Eastern Paralyzed Veterans Association and the Paralyzed Veterans of America for support, including a grant which supports the Yale-London collaboration.


    FOOTNOTES

Address for reprint requests: S. G. Waxman, Dept. of Neurology, LCI 707, Yale Medical School, 333 Cedar St., New Haven, CT 06510 (E-mail: stephen.waxman{at}yale.edu).

Received 8 January 2001; accepted in final form 23 April 2001.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society