Nitric oxide is an autocrine regulator of Na(+) currents in axotomized C-type DRG neurons

In this study, we examined whether nitric oxide synthase (NOS) is upregulated in small dorsal root ganglion (DRG) neurons after axotomy and, if so, whether the upregulation of NOS modulates Na(+) currents in these cells. We identified axotomized C-type DRG neurons using a fluorescent label, hydroxystilbamine methanesulfonate and found that sciatic nerve transection upregulates NOS activity in 60% of these neurons. Fast-inactivating tetrodotoxin-sensitive (TTX-S) Na(+) ("fast") current and slowly inactivating tetrodotoxin-resistant (TTX-R) Na(+) ("slow") current were present in control noninjured neurons with current densities of 1.08 +/- 0. 09 nA/pF and 1.03 +/- 0.10 nA/pF, respectively (means +/- SE). In some control neurons, a persistent TTX-R Na(+) current was observed with current amplitude as much as approximately 50% of the TTX-S Na(+) current amplitude and 100% of the TTX-R Na(+) current amplitude. Seven to 10 days after axotomy, current density of the fast and slow Na(+) currents was reduced to 0.58 +/- 0.05 nA/pF (P < 0.01) and 0.2 +/- 0.05 nA/pF (P < 0.001), respectively. Persistent TTX-R Na(+) current was not observed in axotomized neurons. Nitric oxide (NO) produced by the upregulation of NOS can block Na(+) currents. To examine the role of NOS upregulation on the reduction of the three types of Na(+) currents in axotomized neurons, axotomized DRG neurons were incubated with 1 mM N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. The current density of fast and slow Na(+) channels in these neurons increased to 0.82 +/- 0.08 nA/pF (P < 0.01) and 0.34 +/- 0.04 nA/pF (P < 0.05), respectively. However, we did not observe any persistent TTX-R current in axotomized neurons incubated with L-NAME. These results demonstrate that endogenous NO/NO-related species block both fast and slow Na(+) current in DRG neurons and suggest that NO functions as an autocrine regulator of Na(+) currents in injured DRG neurons.     

In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons

Recent evidence suggests that changes in sodium channel expression and localization may be involved in some pathological pain syndromes. SNS, a tetrodotoxin-resistant (TTX-R) sodium channel, is preferentially expressed in small dorsal root ganglion (DRG) neurons, many of which are nociceptive. TTX-R sodium currents and SNS mRNA expression have been shown to be modulated by nerve growth factor (NGF) in vitro and in vivo. To determine whether SNS expression and TTX-R currents in DRG neurons are affected by reduced levels of systemic NGF, we immunized adult rats with NGF, which causes thermal hypoalgesia in rats with high antibody titers to NGF. DRG neurons cultured from rats with high antibody titers to NGF, which do not bind the isolectin IB4 (IB4(-)) but do express TrkA, were studied with whole cell patch-clamp and in situ hybridization. Mean TTX-R sodium current density was decreased from 504 +/- 77 pA/pF to 307 +/- 61 pA/pF in control versus NGF-deprived neurons, respectively. In comparison, the mean TTX-sensitive sodium current density was not significantly different between control and NGF-deprived neurons. Quantification of SNS mRNA hybridization signal showed a significant decrease in the signal in NGF-deprived neurons compared with the control neurons. The data suggest that NGF has a major role in the maintenance of steady-state levels of TTX-R sodium currents and SNS mRNA in IB4(-) DRG neurons in adult rats in vivo.     

Inhibitory action of protein kinase Cbeta inhibitor on tetrodotoxin-resistant Na+ current in small dorsal root ganglion neurons in diabetic rats

Experimental evidence has been presented to suggest that protein kinase Cbeta isoform-selective inhibitor LY333531 is effective at alleviating diabetic hyperalgesia. In the present study, we isolated small (< or =25 microm in soma diameter) dorsal root ganglion (DRG) neurons from control and streptozocin (STZ)-induced diabetic rats, and examined the acute action of LY333531 (1-1000 nM) on the tetrodotoxin-resistant Na(+) current (TTX-R I(Na)), which plays an essential role in transmitting nociceptive impulses, using the whole-cell patch-clamp method. TTX-R I(Na) in diabetic DRG neurons was enhanced in amplitude (71.5+/-3.6pA/pF, n=10 versus 41.2+/-3.3pA/pF, n=8) and was activated at more negative potentials (V(1/2), -15.1+/-1.3 mV versus -9.6+/-1.4 mV), compared with that in control neurons. Bath application of LY333531 acutely inhibited TTX-R I(Na) in both control and diabetic DRG neurons, and the degree of inhibition by the drug at concentrations of 1, 10 and 100 nM was significantly greater in diabetic DRG neurons than in control DRG neurons. Thus, TTX-R I(Na), which is upregulated in the diabetic state, is likely to be more potently inhibited by submicromolar concentrations of LY333531. These results suggest that an acute inhibition of TTX-R I(Na) by LY333531 attenuates the exaggerated excitability of DRG neurons in the diabetic state, which appears to be related at least partly to anti-hyperalgesic actions of the drug in diabetic neuropathy.     

Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons

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 Na(v)1.8 (+/+) and (-/-) small DRG neurons maintained for 2-8 h in vitro to examine the role of sodium channel Na(v)1.8 (alpha-SNS) in action potential electrogenesis. Although there was no significant difference in resting membrane potential, input resistance, current threshold, or voltage threshold in Na(v)1.8 (+/+) and (-/-) DRG neurons, there were significant differences in action potential electrogenesis. Most Na(v)1.8 (+/+) neurons generate all-or-none action potentials, whereas most of Na(v)1.8 (-/-) neurons produce smaller graded responses. The peak of the response was significantly reduced in Na(v)1.8 (-/-) neurons [31.5 +/- 2.2 (SE) mV] compared with Na(v)1.8 (+/+) neurons (55.0 +/- 4.3 mV). The maximum rise slope was 84.7 +/- 11.2 mV/ms in Na(v)1.8 (+/+) neurons, significantly faster than in Na(v)1.8 (-/-) neurons where it was 47.2 +/- 1.3 mV/ms. Calculations based on the action potential overshoot in Na(v)1.8 (+/+) and (-/-) neurons, following blockade of Ca(2+) currents, indicate that Na(v)1.8 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 Na(v)1.8 (-/-) neurons but, presumably as a result of steady-state inactivation of these channels, electrogenesis in Na(v)1.8 (-/-) neurons is more sensitive to membrane depolarization than in Na(v)1.8 (+/+) neurons, and, in the absence of Na(v)1.8, is attenuated with even modest depolarization. These observations indicate that Na(v)1.8 contributes substantially to action potential electrogenesis in C-type DRG neurons.     

Multiple types of Na+ currents mediate action potential electrogenesis in small neurons of mouse dorsal root ganglia

Small (<25 μm in diameter) neurons of the dorsal root ganglion (DRG) express multiple voltage-gated Na⁺ channel subtypes, two of which being resistant to tetrodotoxin (TTX). Each subtype mediates Na⁺ current with distinct kinetic property. However, it is not known how each type of Na⁺ channel contributes to the generation of action potentials in small DRG neurons. Therefore, we investigated the correlation between Na⁺ currents in voltage-clamp recordings and corresponding action potentials in current-clamp recordings, using wild-type (WT) and Na_V1.8 knock-out (KO) mice, to clarify the action potential electrogenesis in small DRG neurons. We classified Na⁺ currents in small DRG neurons into three categories on the basis of TTX sensitivity and kinetic properties, i.e., TTX-sensitive (TTX-S)/fast Na⁺ current, TTX-resistant (TTX-R)/slow Na⁺ current, and TTX-R/persistent Na⁺ current. Our concurrent voltage- and current-clamp recordings from the same neuron revealed that the action potentials in WT small DRG neurons were mainly dependent on TTX-R/slow Na⁺ current mediated by Na_V1.8. It was surprising that a large portion of TTX-S/fast Na⁺ current was switched off in WT small DRG neurons due to a hyperpolarizing shift of the steady-state inactivation (h _∞), whereas in KO small DRG neurons which are devoid of TTX-R/slow Na⁺ current, the action potentials were generated by TTX-S/fast Na⁺ current possibly through a compensatory shift of h _∞ in the positive direction. We also confirmed that TTX-R/persistent Na⁺ current mediated by Na_V1.9 actually regulates subthreshold excitability in small DRG neurons. In addition, we demon strated that TTX-R/persistent Na⁺ current can carry an action potential when the amplitude of this current was abnormally increased. Thus, our results indicate that the action potentials in small DRG neurons are generated and regulated with a combination of multiple mechanisms that may give rise to unique functional properties of small DRG neurons.     

Characterization and developmental changes of Na+ currents of petrosal neurons with projections to the carotid body

Carotid body chemoreceptors transduce a decrease in arterial oxygen tension into an increase in spiking activity on the sinus nerve, and this response increases with postnatal age over the first week or two of life. Previous work from our laboratory has suggested a major role of axonal Na(+) channels in the initiation of afferent spiking activity. Using RT-PCR of the petrosal ganglia we identified Na(+) channel TTX-S isoforms Na(v)1.1, Na(v)1.6, and Na(v)1.7 and the TTX-resistant (TTX-R) isoforms Na(v)1.8 and Na(v)1.9 at high levels. Electrophysiologic recordings (at 3 ages: 3 days, 9 days, 18-20 days) of neurons that project to the carotid body exhibited predominantly fast-inactivating sodium currents, with a bimodal recovery from inactivation at -80 mV (fast component approximately 8 ms; slow component approximately 90 ms). Developmental age had little effect with no change in peak current density (approximately 1.4 nA/pF) and was associated with a slight, but significant increase in the speed of recovery from inactivation at -140 and -120 mV but not at other potentials. Assuming that the same Na(+) channel complement is present at the nerve terminal as at the soma, the association of a sensory modality (chemoreception) with a relatively uniform Na(+) channel profile suggests that the rapid kinetics of TTX-S channels may be essential for some aspects of chemoreceptor function beyond mediating simple axonal conduction.     

Characteristics of sodium currents in rat geniculate ganglion neurons

Geniculate ganglion (GG) cell bodies of chorda tympani (CT), greater superficial petrosal (GSP), and posterior auricular (PA) nerves transmit orofacial sensory information to the rostral nucleus of the solitary tract. We have used whole cell recording to investigate the characteristics of the Na(+) channels in isolated Fluorogold-labeled GG neurons that innervate different peripheral receptive fields. GG neurons expressed two classes of Na(+) channels, TTX sensitive (TTX-S) and TTX resistant (TTX-R). The majority of GG neurons expressed TTX-R currents of different amplitudes. TTX-R currents were relatively small in 60% of the neurons but were large in 12% of the sampled population. In a further 28% of the neurons, TTX completely abolished all Na(+) currents. Application of TTX completely inhibited action potential generation in all CT and PA neurons but had little effect on the generation of action potentials in 40% of GSP neurons. Most CT, GSP, and PA neurons stained positively with IB(4), and 27% of the GSP neurons were capsaicin sensitive. The majority of IB(4)-positive GSP neurons with large TTX-R Na(+) currents responded to capsaicin, whereas IB(4)-positive GSP neurons with small TTX-R Na(+) currents were capsaicin insensitive. These data demonstrate the heterogeneity of GG neurons and indicate the existence of a subset of GSP neurons sensitive to capsaicin, usually associated with nociceptors. Since there are no reports of nociceptors in the GSP receptive field, the role of these capsaicin-sensitive neurons is not clear.     

Role of TTX-sensitive and TTX-resistant sodium channels in Adelta- and C-fiber conduction and synaptic transmission

Thin afferent axons conduct nociceptive signals from the periphery to the spinal cord. Their somata express two classes of Na+ channels, TTX-sensitive (TTX-S) and TTX-resistant (TTX-R), but their relative contribution to axonal conduction and synaptic transmission is not well understood. We studied this contribution by comparing effects of nanomolar TTX concentrations on currents associated with compound action potentials in the peripheral and central branches of Adelta- and C-fiber axons as well as on the Adelta- and C-fiber-mediated excitatory postsynaptic currents (EPSCs) in spinal dorsal horn neurons of rat. At room temperature, TTX completely blocked Adelta-fibers (IC50, 5-7 nM) in dorsal roots (central branch) and spinal, sciatic, and sural nerves (peripheral branch). The C-fiber responses were blocked by 85-89% in the peripheral branch and by 65-66% in dorsal roots (IC50, 14-33 nM) with simultaneous threefold reduction in their conduction velocity. At physiological temperature, the degree of TTX block in dorsal roots increased to 93%. The Adelta- and C-fiber-mediated EPSCs in dorsal horn neurons were also sensitive to TTX. At room temperature, 30 nM blocked completely Adelta-input and 84% of the C-fiber input, which was completely suppressed at 300 nM TTX. We conclude that in mammals, the TTX-S Na+ channels dominate conduction in all thin primary afferents. It is the only type of functional Na+ channel in Adelta-fibers. In C-fibers, the TTX-S Na+ channels determine the physiological conduction velocity and control synaptic transmission. TTX-R Na+ channels could not provide propagation of full-amplitude spikes able to trigger synaptic release in the spinal cord.     

Reduction in potassium currents in identified cutaneous afferent dorsal root ganglion neurons after axotomy.

Potassium currents have an important role in modulating neuronal excitability. We have investigated the effects of axotomy on three voltage-activated K(+) currents, one sustained and two transient, in cutaneous afferent dorsal root ganglion (DRG) neurons. Fourteen to 21 days after axotomy, L(4) and L(5) DRG neurons were acutely dissociated and were studied 2-8 h after plating. Whole cell patch-clamp recordings were obtained from identified cutaneous afferent neurons (46-50 microm diam); K(+) currents were isolated by blocking Na(+) and Ca(2+) currents with appropriate ion replacement and channel blockers. Separation of the current components was achieved on the basis of sensitivity to dendrotoxin or 4-aminopyridine and by the response to variation in conditioning voltage. Both control and injured neurons displayed qualitatively similar complex K(+) currents composed of distinct kinetic and pharmacological components. Three distinct K(+) current components, a sustained (I(K)) and two transient (I(A) and I(D)), were identified in variable proportions. However, total peak current was reduced by 52% in the axotomized cells when compared with control cells. Two current components were reduced after ligation, I(A) by 60%, I(K) by over 65%, compared with control cells. I(D) appeared unaffected after acute ligation. These results indicate a large reduction in overall K(+) current, resulting from reductions in I(K) and I(A), on large cutaneous afferent neurons after nerve ligation and have implications for excitability changes of injured primary afferent neurons.     

The modulation effects of BmK I, an alpha-like scorpion neurotoxin, on voltage-gated Na(+) currents in rat dorsal root ganglion neurons

The present study investigated the effects of BmK I, a Na(+) channel receptor site 3 modulator purified from the Buthus martensi Karsch (BmK) venom, on the voltage-gated sodium currents in dorsal root ganglion (DRG) neurons. Whole-cell patch-clamping was used to record the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) components of voltage-gated Na(+) currents in small DRG neurons. It was found that the inhibitory effect of BmK I on open-state inactivation of TTX-S Na(+) currents was stronger than that of TTX-R Na(+) currents. In addition, BmK I exhibited a selective enhancing effect on voltage-dependent activation of TTX-S currents, and an opposite effect on time-dependent activation of TTX-S and TTX-R Na(+) currents. The results suggested that the inhibitory effect of BmK I on open-state inactivation might contribute to the increase of peak TTX-S and TTX-R currents, and the enhancing effect of BmK I on time-dependent activation might also contribute to the increase of peak TTX-S currents. It was further suggested that a combined effect of BmK I including inhibiting the inactivation of TTX-S and TTX-R channels, accelerating activation and decreasing the activation threshold of TTX-S channels, might produce a hyperexcitability of small DRG neurons, and thus contribute to the BmK I-induced hyperalgesia.     

Voltage-gated calcium currents in axotomized adult rat cutaneous afferent neurons

The effect of sciatic nerve injury on the somatic expression of voltage-gated calcium currents in adult rat cutaneous afferent dorsal root ganglion (DRG) neurons identified via retrograde Fluoro-gold labeling was studied using whole cell patch-clamp techniques. Two weeks after a unilateral ligation and transection of the sciatic nerve, the L(4)-L(5) DRG were dissociated and barium currents were recorded from cells 3-10 h later. Cutaneous afferents (35-50 microm diam) were classified as type 1 (possessing only high-voltage-activated currents; HVA) or type 2 (having both high- and low-voltage-activated currents). Axotomy did not change the percentage of neurons exhibiting a type 2 phenotype or the properties of low-threshold T-type current found in type 2 neurons. However, in type 1 neurons the peak density of HVA current available at a holding potential of -60 mV was reduced in axotomized neurons (83.9 +/- 5.6 pA/pF, n = 53) as compared with control cells (108.7 +/- 6.9 pA/pF, n = 58, P < 0.01, unpaired t-test). A similar reduction was observed at more negative holding potentials, suggesting differences in steady-state inactivation are not responsible for the effect. Separation of the type 1 cells into different size classes indicates that the reduction in voltage-gated barium current occurs selectively in the larger (capacitance >80 pF) cutaneous afferents (control: 112.4 +/- 10.6 pA/pF, n = 30; ligated: 72.6 +/- 5.0 pA/pF, n = 36; P < 0.001); no change was observed in cells with capacitances of 45-80 pF. Isolation of the N- and P?Q-type components of the HVA current in the large neurons using omega-conotoxin GVIA and omega-agatoxin TK suggests a selective reduction in N-type barium current after nerve injury, as the density of omega-CgTx GVIA-sensitive current decreased from 56.9 +/- 6.6 pA/pF in control cells (n = 13) to 31.3 +/- 4.6 pA/pF in the ligated group (n = 12; P < 0.005). The HVA barium current of large cutaneous afferents also demonstrates a depolarizing shift in the voltage dependence of inactivation after axotomy. Injured type 1 cells exhibited faster inactivation kinetics than control neurons, although the rate of recovery from inactivation was similar in the two groups. The present results indicate that nerve injury leads to a reorganization of the HVA calcium current properties in a subset of cutaneous afferent neurons.     

Voltage-dependent currents in isolated vestibular afferent calyx terminals

Na(+) currents were studied by whole cell patch clamp of chalice-shaped afferent terminals attached to type I hair cells isolated from the gerbil semicircular canal and utricle. Outward K(+) currents were blocked with intracellular Cs(+) or with extracellularly applied 20 microM linopirdine and 2.5 mM 4-aminopyridine (4-AP). With K(+) currents blocked, inward currents activated and inactivated rapidly, had a maximum mean peak amplitude of 0.92 +/- 0.60 (SD) nA (n = 24), and activated positive to -60 mV from holding potentials of -70 mV and more negative. The transient inward currents were blocked almost completely by 100 nM TTX, confirming their identity as Na(+) currents. Half-inactivation of Na(+) currents occurred at -82.6 +/- 0.9 mV, with a slope factor of 9.2 +/- 0.8 (n = 7) at room temperature. In current clamp, large overshooting action potential-like events were observed only after prior hyperpolarizing current injections. However, spontaneous currents consistent with quantal release from the hair cell were observed at holding potentials close to the zero-current potential. This is the first report of ionic conductances in calyx terminals postsynaptic to type I hair cells in the mammalian vestibular system.     

Voltage-dependent K+ currents in rat cardiac dorsal root ganglion neurons

We have assessed the expression and kinetics of voltage-gated K(+) currents in cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of a fluorescent tracer into the pericardial sack. Ninety-nine neurons were labelled: 24% small (diameter<30 microm), 66% medium-sized (diameter 30 microm>.48 microm) and 10% large (>48 microm) neurons. Current recordings were performed in small and medium-sized neurons. The kinetic and pharmacological properties of K(+) currents recorded in these two groups of neurons were identical and the results obtained from these neurons were pooled. Three types of K(+) currents were identified:a) I(As), slowly activating and slowly time-dependently inactivating current, with V(1/2) of activation -18 mV and current density at +30 mV equal to 164 pA/pF, V(1/2) of inactivation at -84 mV. b) I(Af) current, fast activating and fast time-dependently inactivating current, with V(1/2) of activation at two mV and current density at +30 mV equal to 180 pA/pF, V(1/2) of inactivation at -26 mV. At resting membrane potential I(As) was inactivated, whilst I(Af), available for activation. The I(As) currents recovered faster from inactivation than I(Af) current. 4-Aminopiridyne (4-AP) (10 mM) and tetraethylammonium (TEA) (100 mM) produced 98% and 92% reductions of I(Af) current, respectively and 27% and 66% of I(As) current, respectively. c) The I(K) current that did not inactivate over time. Its V(1/2) of activation was -11 mV and its current density equaled 67 pA/pF. This current was inhibited by 95% (100 mM) TEA, whilst 4-AP (10 mM) produced its 23% reduction. All three K(+) current components (I(As), I(Af) and I(K)) were present in every small and medium-sized cardiac DRG neuron.We suggest that at hyperpolarized membrane potentials the fast reactivating I(As) current limits the action potential firing rate of cardiac DRG neurons. At depolarised membrane potentials the I(Af) K(+) current, the reactivation of which is very slow, does not oppose the firing rate of cardiac DRG neurons.     

Prostaglandin E(2) modulates TTX-R I(Na) in rat colonic sensory neurons

This study was performed to determine the impact of the inflammatory mediator prostaglandin E(2) (PGE(2)) on the biophysical properties of tetrodotoxin resistant voltage-gated Na(+) currents (TTX-R I(Na)) in colonic dorsal root ganglion (DRG) neurons. TTX-R I(Na) was studied in DRG neurons from thoracolumbar (TL: T(13)-L(2)) and lumbosacral (LS: L(6)-S(2)) DRG retrogradely labeled following the injection of DiIC(18) (DiI) into the wall of the descending colon of adult male rats. TTX-R I(Na) in colonic DRG neurons had a high threshold for activation [V(0.5) of conductance-voltage (G-V) curve = -3.1 +/- 1.0 (SE) mV] and steady-state availability (V(0.5) for H-infinity curve = -18.4 +/- 1.4 mV), was slowly inactivating (10.6 +/- 1.4 ms at 0 mV), and recovered rapidly from inactivation (83.5 +/- 5.0% of the current recovered with a time constant of 1.3 +/- 0.1 ms at -80 mV). TTX-R I(Na) was present in every colonic DRG neuron studied (n = 62). PGE(2) induced a rapid (<15 s) increase in TTX-R I(Na) that was associated with a hyperpolarizing shift in the G-V curve (3.4 +/- 0.7 mV), an increase in the rate of inactivation (4.21 +/- 0.7 ms at 0 mV), and no change in steady-state availability. There was no statistically significant difference (P > 0.05) between TL and LS colonic DRG neurons with respect to the biophysical properties of TTX-R I(Na), the current density or the magnitude of PGE(2)-induced changes in the current. However, both the proportion of TL and LS neurons in which TTX-R I(Na) was modulated by PGE(2) (16 of 16 TL neurons and 12 of 14 LS neurons) as well as the magnitude of PGE(2)-induced changes in the current were significantly larger in colonic DRG neurons than in the total population of DRG neurons. These results suggest that changes in nociceptive processing associated with inflammation of the colon does not reflect differences between TL and LS neurons with respect to the properties of TTX-R I(Na), distribution of current, or magnitude of inflammatory mediator-induced changes in the current. However, these results do suggest modulation of TTX-R I(Na) in colonic afferents is an underlying mechanism of hyperalgesia and pain associated with inflammation of the colon and that this current constitutes a novel target for therapeutic relief of visceral inflammatory pain.     

TNF-alpha differentially modulates ion channels of nociceptive neurons

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain conditions. The mechanisms by which TNF-alpha elicits pain behavior are still incompletely understood. Numerous studies suggest that TNF-alpha sensitizes primary afferent neurons. Most recently, it was shown that TNF-alpha induced an enhancement of TTX-R Na(+) current in dorsal root ganglion (DRG) cells. In the present study, we have tested the effect of acute application of TNF-alpha on voltage-gated potassium, calcium and sodium channel currents as well as its influence on membrane conductance in isolated rat DRG neurons. We report that voltage-gated potassium channel currents of nociceptive DRG neurons are not influenced by TNF-alpha (100 ng/ml), while voltage-gated calcium channel currents were decreased voltage-dependently by -7.73+/-6.01% (S.D.), and voltage-activated sodium channels currents were increased by +5.62+/-4.27%, by TNF-alpha. In addition, TNF-alpha induced a significant increase in IV ramps at a potential of +20 mV, which did not exist when the experiments were conducted in a potassium-free solution, indicating that this effect is mainly the result of a change in potassium conductance. These different actions of TNF-alpha might help to explain how it sensitizes primary afferent neurons after nerve injury and thus facilitates pain.     

Effects of a chronic compression of the dorsal root ganglion on voltage-gated Na+ and K+ currents in cutaneous afferent neurons

A chronic compression of the dorsal root ganglion (CCD) produces ipsilateral cutaneous hyperalgesia that is associated with an increased excitability of neuronal somata in the compressed ganglion, as evidenced by spontaneous activity and a lower rheobase. We searched for differences in the properties of voltage-gated Na+ and K+ currents between somata of CCD- and control (unoperated) rats. CCD was produced in adult rats by inserting two rods through the intervertebral foramina, one compressing the L4, and the other, the ipsilateral, L5 dorsal root ganglion (DRG). After 5-9 days, DRG somata were dissociated and placed in culture for 16-26 h. Cutaneous neurons of medium size (35-45 microm), Fluorogold-labeled from the hindpaw, were selected for whole cell patch-clamp recording of action potentials and ion currents. In comparison with control neurons, CCD neurons had steady-state activation curves for TTX-sensitive (TTX-S) Na+ currents that were shifted in the hyperpolarizing direction, and CCD neurons had enhanced TTX-resistant (TTX-R) current. CCD neurons also had smaller, fast-inactivating K+ currents (Ka) at voltages from -30 to 50 mV. The reduction in Ka, the hyperpolarizing shift in TTX-S Na+ current activation, and the enhanced TTX-R Na+ current may all contribute to the enhanced neuronal excitability and thus to the pain and hyperalgesia associated with CCD.     

Neurotrophin regulation of sodium and calcium channels in human neuroblastoma cells

Neurotrophins, acting through tyrosine kinase family genes, are essential for neuronal differentiation. The expression of tyrosine kinase family genes is prognostic in neuroblastoma, and neurotrophins reduce proliferation and induce differentiation, indicating that neuroblastomas are regulated by neurotrophins. We tested the effects of nerve growth factor and brain-derived neurotrophic factor on Na(+) and Ca(2+) currents, using the whole-cell patch-clamp technique, in human neuroblastoma NB69 cells. Control cells exhibited a slow tetrodotoxin-resistant (IC(50)=98 nM) Na(+) current and a high-voltage-activated Ca(2+) current. Exposure to nerve growth factor (50 ng/ml) and/or brain-derived neurotrophic factor (5 ng/ml) produced the expression of a fast tetrodotoxin-sensitive (IC(50)=10 nM) Na(+) current after day 3, and suppressed the slow tetrodotoxin-resistant variety. The same type of high-voltage-activated Ca(2+) current was expressed in control and treated cells. The treatment increased the surface density of both Na(+) and Ca(2+) currents with time after plating, from 17 pA/pF at days 3-5 and 1-5 to 34 and 30 pA/pF after days 6-10, respectively. Therefore, both nerve growth factor and brain-derived neurotrophic factor, acting through different receptors of the tyrosine kinase family and also possibly the tumor necrosis factor receptor-II, were able to regulate differentiation and the expression of Na(+) and Ca(2+) channels, partially reproducing the modifications induced by diffusible astroglial factors.We show that neurotrophins induced differentiation to a neuronal phenotype and modified the expression of Na(+) and Ca(2+) currents, partially reproducing the effects of diffusible astroglial factors.     

Analysis of the variation in use-dependent inactivation of high-threshold tetrodotoxin-resistant sodium currents recorded from rat sensory neurons

This study addressed variation in the use-dependent inactivation (UDI) of high-threshold tetrodotoxin-resistant Na+ currents (TTX-R currents) and action potential firing behavior among acutely isolated rat dorsal root ganglion (DRG) cells. UDI was quantified as the percent decrease in current amplitude caused by increasing the current activation rate from 0.1-1.0 Hz for 20 s. TTX-R current UDI varied from 6% to 66% among 122 DRG cells examined, suggesting the existence of two or more levels of UDI. The voltage-dependency of the TTX-R currents was consistent with Na(V)1.8, regardless of UDI. However, TTX-R currents with more UDI had a more negative voltage-dependency of inactivation, a greater tendency to enter slow inactivation, and a slower recovery rate from slow inactivation, compared with those with less UDI. TTX-R currents with more UDI ran down faster than those with less UDI. However, UDI itself changed little over time, regardless of the initial UDI level observed in a particular DRG cell. Together, these two observations suggest that individual DRG cells did not express mixtures of TTX-R channels that varied regarding UDI. TTX-R current UDI was correlated with expression of a low-threshold A-current and whole-cell capacitance, suggesting that it varied among different nociceptor types. Whole-cell inward currents (WCI-currents), recorded without channel blockers, also exhibited UDI. WCI-current UDI varied similarly to TTX-R current UDI in magnitude, and relative to whole-cell capacitance and A-current expression, suggesting that the WCI-currents were carried predominantly by TTX-R channels. DRG cells with more WCI-current UDI exhibited a greater decrease in action potential amplitude and number, and a greater increase in action potential threshold over seven ramp depolarizations, compared with DRG cells with less WCI-current UDI. Variation in UDI of Na(V)1.8 channels expressed by different nociceptor types could contribute to shaping their individual firing patterns in response to noxious stimuli.     

Membrane properties and electrogenesis in the distal axons of small dorsal root ganglion neurons in vitro

Although it is generally thought that sensory transduction occurs at or close to peripheral nerve endings, with action potentials subsequently propagating along the axons of dorsal root ganglia (DRG) neurons toward the central nervous system, the small diameter of nociceptive axons and their endings have made it difficult to estimate their membrane properties and electrogenic characteristics. Even the resting potentials of nociceptive axons are unknown. In this study, we developed the capability to record directly with patch-clamp electrodes from the small-diameter distal axons of DRG neurons in vitro. We showed using current-clamp recordings that 1) these sensory axons have a resting potential of -60.2 ± 1 mV; 2) both tetrodotoxin (TTX)-sensitive (TTX-S) and TTX-resistant (TTX-R) Na(+) channels are present and available for activation at resting potential, at densities that can support action potential electrogenesis in these axons; 3) TTX-sensitive channels contribute to the amplification of small depolarizations that are subthreshold with respect to the action potential in these axons; 4) TTX-R channels can support the production of action potentials in these axons; and 5) these TTX-R channels can produce repetitive firing, even at depolarized membrane potentials where TTX-S channels are inactivated. Finally, using voltage-clamp recordings with an action potential as the command, we confirmed the presence of both TTX-S and TTX-R channels, which are activated sequentially during action potential in these axons. These results provide direct evidence for the presence of TTX-S and TTX-R Na(+) channels that are functionally available at resting potential and contribute to electrogenesis in small-diameter afferent axons.