Tetrodotoxin-sensitive and -resistant Na+ channel currents in subsets of small sensory neurons of rats

Brain Research 1029 (2004) 251 – 258 www.elsevier.com/locate/brainres

Research report

Tetrodotoxin-sensitive and -resistant Na+ channel currents in subsets of small sensory neurons of rats Zi-Zhen Wu, Hui-Lin Pan* Department of Anesthesiology, The Pennsylvania State University College of Medicine, 500 University Drive, The Milton S. Hershey Medical Center, Hershey, PA 17033-0850, United States Accepted 28 September 2004 Available online 27 October 2004

Abstract Voltage-activated Na+ channels in the primary sensory neurons are important for generation of action potentials and regulation of neurotransmitter release. The Na+ channels expressed in different types of dorsal root ganglion (DRG) neurons are not fully known. In this study, we determined the possible difference in tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na+ channel currents between isolectin B4 (IB4)-positive and IB4-negative small DRG neurons. Whole-cell voltage- and current-clamp recordings were performed in acutely isolated DRG neurons labeled with and without IB4 conjugated to Alexa Fluor 594. The peak Na+ current density was significantly higher in IB4-negative than IB4-positive DRG neurons. While all the IB4-negative neurons had a prominent TTX-S Na+ current, the TTX-R Na+ current was present in most IB4-positive cells. Additionally, the evoked action potential had a higher activation threshold and a longer duration in IB4-positive than IB4-negative neurons. TTX had no effect on the evoked action potential in IB4-positive neurons, but it inhibited the action potential generation in about 50% IB4-negative neurons. This study provides complementary new information that there is a distinct difference in the expression level of TTX-S and TTX-R Na+ channels between IB4-negative than IB4-positive small-diameter DRG neurons. This difference in the density of TTX-R Na+ channels is responsible for the distinct membrane properties of these two types of nociceptive neurons. D 2004 Elsevier B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Sodium channels Keywords: Nociceptor; Dorsal root ganglia; Neurotrophin; Sodium channel

1. Introduction Dorsal root ganglion (DRG) neurons are functionally heterogenous and contain various neurotransmitters as well as ion channels. Small-diameter DRG neurons and associated Ay- and C-fiber afferents are generally considered as nociceptors since they are capable of detecting noxious stimuli and initiating pain sensation [17,28,31]. In response to noxious stimuli, these putative nociceptive neurons generate action potentials, which trigger neurotransmitter release from presynaptic afferent terminals to second-order * Corresponding author. Tel.: +1 717 531 8433; fax: +1 717 531 6221. E-mail address: [email protected] (H.-L. Pan). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.09.051

neurons in the spinal cord. A novel approach to the classification of DRG neurons is derived from the requirements of subsets of neurons for specific neurotrophins because DRG neurons subserving specific sensory modalities are supported by different neurotrophins [30]. Two broad classes of small DRG neurons have been classified by the growth factors that regulate them in adulthood and by the presence of neuropeptides [2,20–22]. The first class expresses TrkA receptors for nerve growth factor (NGF), depends on NGF for survival during postnatal development, and contains neuropeptides such as substance P and calcitonin gene-related peptide. The second class possesses receptors for glial-derived neurotophic factor (GDNF) and depends on GDNF for survival during postnatal maturation.

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These GDNF-responsive neurons are relatively dpeptide poorT but possess cell surface glyco-conjugates that bind the Griffonia simplicifolia isolectin B4 (IB4) [5,35]. Many IB4binding DRG neurons also express P2X3 receptors [8,16]. These neurochemical differences have led to a proposal that IB4-positive and IB4-negative small DRG neurons may be functionally distinct groups of nociceptors [30,31]. Although IB4-positive and IB4-negative DRG neurons clearly possess unique neurotransmitters and receptors [31,37,38], the different physiological roles played by these two types of presumptive nociceptors in acute and chronic pain conditions remain to be established. The voltageactivated Na+ channels, including both tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na+ channels, are important for generation of action potentials and nociceptive neurotransmission [3,7]. However, the different types of the voltage-activated Na+ channels in IB4-positive and IB4negative DRG neurons are not fully known. A previous study of mice has shown that IB4-positive neurons have higher densities of TTX-R Na+ currents than IB4-negative neurons [31]. Rat DRG small-diameter neurons are more commonly used in pain and analgesia studies. Because the species difference may exist in the expression of different types of Na+ channels in the DRG, it is important to further determine the difference of voltage-activated Na+ currents in IB4-positive and IB4-negative DRG neurons of rats. In the current study, we tested a hypothesis that the density of TTX-S and TTX-R Na+ channel currents is differentially present in IB4-positive and IB4-negative DRG neurons of rats, which accounts for the distinct firing properties of these two types of primary sensory neurons.

2. Materials and methods 2.1. Isolation of DRG neurons All procedures conformed to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine. Male Sprague–Dawley rats (5–6 weeks old, Harlan, Indianapolis, IN) were anesthetized with halothane and then rapidly decapitated. The thoracic and

lumbar segments of vertebrate column were dissected. The DRGs, together with the nerve roots, were quickly removed and transferred immediately into Dulbecco’s modified Eagle’s Medium (DMEM, Gibco, Carlsbad, CA). After removal of attached nerves and surrounding connective tissues, the DRGs were minced with fine spring scissors and the ganglion fragments were placed in a flask containing 5 ml of DMEM in which trypsin (type III, 0.5 mg/ml, Sigma, St. Louis, MO), collagenase (type I, 1 mg/ml, Sigma) and DNase (type I, 0.1 mg/ml, Sigma) had been dissolved. After incubation at 34 8C in a shaking water bath for 30 min, soybean trypsin inhibitor (type II-s, 1.25 mg/ml, Sigma) was then added to stop trypsin digestion. The cell suspension was centrifuged (500 rpm, 3 min) to remove the supernatant and replenished with DMEM. Cells were then plated onto a 35mm culture dish containing poly-l-lysine (50 Ag/ml) precoated coverslips and kept for at least 30 min before electrophysiological recordings. The neurons selected for recordings were 15–30 Am since IB4 primarily labels small DRG cells [38]. Approximately equal number of IB4positive and IB4-negative neurons was studied in one rat. Recordings were made within 10 h after dissociation to keep the experiment as similar to in vivo as possible and to minimize the space clamp error. 2.2. Electrophysiology recordings Patch electrodes with a resistance of ~2 MV were pulled from GC150TF-10 glass capillaries (i.d. 1.17 mm, o.d. 1.5 mm, Harvard Apparatus, Holliston, MA) using a micropipette puller (P-97 Sutter Instrument, Novato, CA) and firepolished (DMF1000, World Precision Instruments, Sarasota, FL). Neurons were visualized using a combination of epifluorescence illumination and differential interference contrast (20–40) optics on an inverted microscope (Olympus Optical, Tokyo, Japan). Images of IB4-positive and IB4-negative cells were taken with a CCD camera and displayed on a video monitor (Fig. 1). Neurons were patched in the whole-cell configuration and recorded at a holding potential of 90 mV using an EPC-10 amplifier (HEKA Instruments, Germany). Seals (1–10 GV) between the electrode and cell were established in a modified Tyrode solution containing (in mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose (pH 7.4 adjusted with NaOH,

Fig. 1. Identification of IB4-positive and IB4-negative DRG neurons. (A) DRG neurons viewed with differential interference contrast optics. (B) The same microscopic field viewed with fluorescence illumination. (C) Digitally merged images from A and B. The IB4-positive neuron is indicated by the arrow. Scale bar, 20 Am.

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osmolarity 320 mosM). After whole-cell configuration was established, the cell membrane capacitance and series resistance were electronically compensated. All experiments were performed at room temperature (~25 8C). Signals were filtered at 1 kHz, digitized at least 10 kHz, and acquired using Pulse program (HEKA). Immediately before recording, neurons were treated with IB4–Alexa Fluor 594 (3 Ag/ ml, Molecular Probes, Eugene, OR) in Tyrode solution for 10 min and then rinsed for at least 3 min. In some control experiments, recordings were performed on neurons before they were labeled with IB4–Alexa Fluor 594. All neurons selected for recordings had overshooting action potentials and membrane potentials more negative than 45 mV. To selectively record sodium currents and minimize the contribution from calcium and potassium currents [31], the extracellular solution contained (in mM): 134 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 20 tetraethylammonium Cl, 1 4-aminopyridine, 0.1 CdCl2 (pH 7.4 adjusted with NaOH, osmolarity 320 mosM). Electrodes were filled with solution containing (in mM): 124 CsCl, 20 TEA, 2 MgCl2, 3 EGTA, 10 HEPES, 4 Mg-ATP and 0.3 Na-GTP (pH 7.2 adjusted with CsOH, osmolarity 300 mosM). Action potentials in DRG neurons were elicited in Tyrode solution by injection of a series of depolarizing currents (from 0 to 250 pA in 10 pA increments, 400 ms duration). The intracellular solution contained (in mM): 124 KCl, 2 MgCl2, 1 EGTA, 10 HEPES, 4 Mg-ATP, 0.3 NaGTP (pH 7.2 adjusted with KOH, osmolarity 300 mosM).

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Analysis program (Synaptosoft, Leonia, NJ). The action potential threshold was defined as the lowest current injected that elicited an action potential with an overshoot. The frequency was calculated as the number of action potentials elicited at the activation threshold. The action potential amplitude was measured from the peak of overshoot to the bottom of afterhyperpolarization, and the action potential overshoot was determined from 0 mV to the peak of an action potential. The duration of the action potential was measured at 50% of the peak amplitude from the resting potential because the 50% amplitude was close to the base of the action potential. Statistical data are presented as meansFS.E.M. All comparisons between means were tested for significance using Student’s unpaired t-test unless otherwise indicated. Pb0.05 was considered to be statistically significant.

3. Results The diameter of most IB4-positive rat DRG neurons was in the range of 15–30 Am. Thus, the similar size of IB4negative cells was selected for recordings as the control. The diameter of IB4-positive (22.3F0.4 Am, n=48) and IB4negative neurons (21.9F1.2 Am, n=42) was not significantly different. 3.1. Voltage-activated Na+ currents in IB4-positive and IB4negative DRG neurons

2.3. Drug application Drugs were dissolved in distilled water at 1000 times the final concentration and kept frozen in aliquots. The stock solutions were diluted in extracellular solution just before use and held in a series of independent syringes connected to corresponding fused silica columns (i.d. 200 Am). The end of the parallel columns was connected to a common silica column. The distance from the column mouth to the cell examined was about 100 Am. Cells in the recording chamber were continuously bathed in Tyrode solution. Each drug was delivered to the recording chamber by gravity, and rapid solution exchange (about 200 ms) was achieved by controlling the corresponding valve switch (World Precision Instruments). Drugs and chemicals were purchased from Sigma except TTX (Alomone labs, Jerusalem, Israel). 2.4. Data analysis Data were analyzed using PulseFit software program (HEKA). Whole-cell current–voltage curves for individual neurons were generated by calculating the mean peak inward current at each testing potential and normalized for cell capacitance. TTX-S Na+ currents were obtained by subtracting the TTX-R Na+ current from the total Na+ currents. The action potential was analyzed with Mini-

The cells were depolarized by a series of command potentials (from 70 to 60 mV with 10 mV increments for 50 ms) from a holding potential of 90 mV. In 20 IB4negative cells, the Na+ currents were activated at 40 mV and reached the maximum at 20 or 10 mV (Fig. 2). However, in 19 IB4-positive cells, the Na+ currents were elicited at 30 mV and the maximal Na+ currents were evoked at 0 mV (Fig. 2). The Na+ currents had a faster activation and inactivation kinetics in most IB4-negative than IB4-positive cells (Fig. 3). The time to peak (0.7F0.05 vs. 1.08F0.11 ms, Pb0.05) and the decay time constant (0.86F0.16 vs. 3.05F0.26 ms, Pb0.05) of the Na+ currents were significantly faster in IB4-negative than IB4-positive cells at the test potential of 0 mV. Furthermore, the peak whole-cell Na+ current density was significantly greater in IB4-negative than IB4-positive cells at the test potentials ranging from 40 to 20 mV (Fig. 2). At 20, 10, and 0 mV, the Na+ current density was 413.1F49.8, 419.0F47.2, and 398.1F40.2 pA/pF, respectively, in 20 IB4-negative cells. Whereas in 19 IB4-positive neurons, the Na+ current density was 134.5F35.4, 207.9F32.5, and 241.3F30.3 pA/ pF when the cell was depolarized to 20, 10, and 0 mV, respectively. We then examined the composition of TTX-S and TTXR Na+ currents in 19 IB4-positive and 20 IB4-negative neurons before and after TTX (1 AM) treatment. The TTX-S

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398.9F48.9 and 106.5F34.7 pA/pF ( Pb0.05) in IB4negative and IB4-positive neurons, respectively, at the testing potential of 20 mV. When the effect of TTX on individual neurons was examined, TTX completely abolished the Na+ currents in 36% (7/20) IB4-negative cells. In another 13 (13/20, 65%) IB4-negative cells, TTX caused a large reduction of Na+ currents (Fig. 2). Following TTX treatment, the Na+ current density in these 13 cells was 80.2F27.0, 105.7F31.8, and 99.0F32.4 pA/pF at 10, 0, and 10 mV, respectively. Among these 13 IB4-negative cells, the TTX-R Na+ currents in 9 cells were 165.9F33.3 pA/pF at the testing potential of 0 mV. In the remaining 4 neurons, the TTX-R Na + currents were very small (27.7F4.2 pA/pF at the testing potential of 0 mV, Fig. 3). By contrast, most IB4-positive neurons (16/19, 84.2%) displayed predominately TTX-R Na+ currents and a small component of TTX-S Na+ currents (Fig. 3). A large component of TTX-S Na+ current was only present in 3 of 19 (15.8%) IB4-positive neurons (Fig. 3).

Fig. 2. Mean whole-cell sodium current density at different test potentials in 19 IB4-positive and 20 IB4-negative DRG neurons before (A) and after (B, TTX-R) treatment with 1 AM TTX. Neurons were depolarized from 70 to 60 mV for 50 ms with 10 mV increments from a holding potential of 90 mV. (C) TTX-S sodium current density was obtained by subtracting the TTX-R sodium current from the total sodium current. *Pb0.05, compared to IB4-positive neurons at the same test potential.

Na+ currents obtained by subtracting the TTX-R currents from the total Na+ currents were predominantly present in IB4-negative neurons. The TTX-S Na+ currents all reached their peak at 20 mV in 19 IB4-positive and 20 IB4negative neurons. The TTX-S Na+ currents in IB4-negative neurons were significantly larger than those in IB4-positive neurons (Fig. 2). The TTX-S Na+ current density was

Fig. 3. Representative whole-cell recordings showing the distribution of TTX-resistant (TTX-R) and TTX-sensitive (TTX-S) sodium currents in IB4-positive and IB4-negative DRG neurons. TTX-S sodium currents were obtained by subtracting the TTX-R sodium current from the sodium current before TTX (1 AM) treatment. Neurons were depolarized from 70 to 60 mV for 50 ms with 10 mV increments from a holding potential of 90 mV.

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We also performed control experiments to determine if the high density of TTX-R Na+ currents in IB4-positive neurons was due to IB4 binding to the membrane of DRG neurons. In separate seven IB4-positive DRG neurons, the I–V curve of Na+ currents was repeated in the presence of 1 AM TTX before and after perfusion of IB4–Alexa Fluor 594 solution to label the neurons. There was no significant difference in the TTX-R Na+ currents before and after IB4 labeling (Fig. 4). 3.2. Action potentials in IB4-positive and IB4-negative DRG neurons Because of the observed difference in TTX-S and TTX-R Na+ currents between IB4-positive and IB4-negative cells, we further determine the difference of the electrophysiological properties of action potentials between these two groups of DRG neurons. The action potential elicited by injection of depolarizing currents had a higher activation threshold (78.1F11.6 vs. 47.9F8.0 pA, Pb0.05) and a longer duration (3.4F0.4 vs. 2.1F0.2 ms, Pb0.05) in 20 IB4-positive than 19 IB4-negative cells. However, there

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Table 1 Action potential profiles of 22 IB4-positive and 19 IB4-negative rat DRG neurons

IB4 IB+4

Threshold (pA)

AP50 (ms)

Amplitude (mV)

Overshoot (mV)

#AP

47.9F8.0 78.1F11.6*

2.1F0.2 3.4F0.4*

115.7F3.7 113.1F2.9

42.0F3.1 43.08F2.6

3.1F0.6 2.7F0.5

AP50: action potential duration at 50% of the peak amplitude. #AP: number of action potentials elicited at the activation threshold. * Pb0.05, compared to respective values in IB4-negative neurons.

were no significant differences in the amplitude, overshoot, and number of action potentials between IB4-positive and IB4-negative neurons (Table 1). Application of 1 AM TTX completely inhibited the action potential generation in 47.4% (9/19, Fig. 5) IB4negative cells. TTX had no significant effect on the evoked action potential in the remaining (10/19, 52.6%) IB4negative neurons. In all 22 IB4-positive cells tested, TTX had no significant effect on the generation of action potentials (Fig. 5).

4. Discussion

Fig. 4. (A) Representative current traces showing lack of effect of IB4 labeling on whole-cell sodium currents at different test potentials in an IB4positive neuron. (B) Comparison of the mean TTX-R sodium current density before and after labeling with IB4–Alexa Fluor 594 in seven IB4positive DRG neurons.

In this study, we determined the difference in TTX-S and TTX-R Na+ channel currents between IB4-positive and IB4negative small-diameter rat DRG neurons. We found that there was a significantly higher density of whole-cell Na+ currents in IB4-negative than IB4-positive neurons. While IB4-negative neurons had a higher density of TTX-S Na+ currents, a significant proportion of the IB4-positive neurons exhibited a higher density of TTX-R Na+ currents. Furthermore, the action potential had a higher activation threshold and a longer duration in IB4-positive than IB4-negative cells. While TTX inhibited the action potential generation in about 50% IB4-negative neurons, it had no effect on the action potential elicited in IB4-positive cells. Thus, this study provides complementary functional evidence that a greater density of TTX-R Na+ channels is expressed in IB4-binding DRG neurons in rats. This quantitative difference of voltageactivated Na+ channels between IB4-positive and IB4negative neurons may explain the different membrane properties of these two populations of putative nociceptors. IB4-positive and IB4-negative DRG neurons in rats exhibited a clear difference in voltage-gated Na+ currents. We found that the dominant TTX-R Na+ currents are uniformly present in all IB4-positive rat DRG neurons. This finding is consistent with a previous study using mouse DRG neurons [31]. However, unlike DRG neurons in mice, we found that the total voltage-gated Na+ current density was significantly higher in IB4-negative than IB4-positive DRG neurons in rats. Furthermore, the TTX-S Na+ currents were more predominant in all IB4-negative rat DRG neurons. In adult DRG neurons, voltage-activated Na+ currents include at least four types: TTX-S fast-activating, TTX-R fast-

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Fig. 5. Representative recordings showing action potentials of IB4-negative and IB4-positive neurons before and after treatment with 1 AM TTX. Action potentials were elicited by injection of depolarizing currents from 0 to 250 pA for 400 ms with 10 pA increments and 3 s intervals.

inactivating, TTX-R slowly inactivating, and TTX-R persistent currents [3]. TTX-R fast-inactivating currents are only detectable in 3% of small DRG neurons of adult rats [26]. Multiple subtypes of voltage-gated TTX-S Na+ channels have been identified in primary sensory neurons. Small DRG neurons express high levels of NaV1.7 and NaV1.6 but a low level of NaV1.3 [3,6,12,36]. TTX-R slowly inactivating and persistent currents are carried through NaV1.8 and NaV1.9 channels, respectively [10,12,33]. The threshold for activation of NaV1.9 is close to the resting potential ( 70 mV), but NaV1.8 prefers relatively more positive potentials [10,33]. In our study, the threshold for activation and the kinetics of TTX-R Na+ currents were similar to the current properties reported for the NaV1.8 [1] but not NaV1.9 channels [10]. This is likely due to that the expression level of NaV1.8 channels is much more abundant than that of NaV1.9 in adult DRG neurons [4,12,25]. A recent study has shown that nociceptive neurons identified in vivo express a high level of NaV1.8 [12]. The different composition of TTX-R Na+ and TTX-S Na+ channels in IB4-positive and IB4-negative DRG neurons may be caused by the different neurotrophic factors that regulate them in the adulthood. For example, it has been demonstrated that treatment with GDNF causes a twofold increase in the peak amplitude of TTX-R Na+ currents in DRG neurons in rats and mice [11,15]. Since TTX-R Na+ channels are more dominant and uniformly expressed in IB4-

positive neurons, the IB4-conjugated fluorescence dye is a valuable tool to identify DRG cells with a high density of TTX-R Na+ currents in the in vitro electrophysiology study. We also found that the action potential had a higher activation threshold and a longer duration in IB4-positive than IB4-negative DRG neurons in rats. In small DRG neurons, both TTX-R and TTX-S Na+ currents are important for generation of action potentials. While TTX-S and TTX-R Na+ currents contribute significantly to the upstroke of the action potential, the falling phase of action potential is shaped mainly by TTX-R Na+ currents [7,25]. Therefore, a higher density of total Na+ and TTX-S Na+ channels may be responsible for a lower activation threshold of action potentials in IB4-negative than IB4-positive cells. On the other hand, a higher density of TTX-R Na+ currents may be the basis for a higher activation threshold and longer duration of action potentials in IB4-positive than IB4-negative neurons. The role of IB4-positive DRG neurons as nociceptors has been suggested because selective depletion of IB4binding sensory neurons results in an elevated thermal and mechanical nociceptive threshold in rats [34]. However, IB4negative (expressing TrkA receptors for NGF) small DRG neurons also play an important role in nociception. In this regard, rats and mice deprived of NGF during embryonic development by antibodies or gene targeting are unable to respond to painful stimuli [9,18,29]. Furthermore, recent

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studies have documented that humans with mutations of TrkA receptors or NGF beta gene are unable to detect pain [14,19]. We recently have shown that IB4-negative cells have a higher density of A opioid receptors and high voltage-gated Ca2+ channels in this group of DRG cells are more sensitive to inhibition by A opioids [38]. It should be noted that TTX-S and TTX-R Na+ channels are differentially altered in different pain states. It has been shown that the total Na+ and TTX-S Na+ currents are increased while the TTX-R Na+ currents are attenuated in DRG neurons following nerve injury [23,36]. By contrast, in the carageenan-induced inflammatory pain model, both the expression of NaV1.8 and the TTX-R Na+ currents are significantly increased in small rat DRG neurons [32]. Further studies are warranted to define the functional role s of TTX-S and TTX-R Na+ channels in IB4-positive and IB4-negative DRG neurons in various acute and chronic pain conditions. In summary, this study demonstrates a clear difference in TTX-S and TTX-R Na+ channel currents between IB4negative and IB4-positive rat DRG neurons. This complementary new information is important for a better understanding of the membrane properties of these two populations of putative nociceptors [31,37,38]. IB4-positive and IB4-negative neurons may relay different aspects of nociceptive information, and it is possible that the nociceptive function of IB4-positive and IB4-negative DRG neurons may be complementary and overlapping in certain degree. It has been shown that IB4 binds primarily nociceptive neurons and afferent nerves innervating the skin [24]. Since IB4binding DRG neurons are responsive to capsaicin and have a high threshold in response to mechanical stimulation [13], these neurons may be primarily involved in cutaneous thermal nociception. The longer duration of action potentials in IB4-positive neurons can allow more calcium influx in small DRG neurons, resulting in enhanced neurotransmitters release from afferent terminals in the spinal cord [27]. On the other hand, IB4-negative neurons may be involved in mechano-nociception due to the presence of a high density of total Na+ currents and a lower activation threshold for generation of action potentials. Since certain ion channels, especially the TTX-S and TTX-R Na+ channels, are altered differently in acute and chronic pain states, these channel subtypes could become important targets for development of new drugs to alleviate pain syndromes. Acknowledgments This study was supported by grants GM64830 and NS45602 from the National Institutes of Health.

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