Currents In Dorsal Root Ganglion Neurons Research Articles

Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X 3 receptor

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X 3 receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X 3 receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X 3 agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X 3 mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X 3 agonist-activated currents and P2X 3 mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X 3 receptor during CCI.

Effect of tetramethylpyrazine on primary afferent transmission mediated by P2X 3 receptor in neuropathic pain states

Neuropathic pain is the most difficult type of pain to treat. The P2X 3 receptors play a crucial role in facilitating pain transmission at peripheral and spinal sites. The present research investigated the effects of tetramethylpyrazine (TMP) on the primary afferent transmission induced by P2X 3 receptor in neuropathic pain states. Chronic constriction injury (CCI) model was adopted. Sprague–Dawley male rats ( n = 30) had been randomly divided into normal saline (sham + NS) group (I), TMP group (II), sham group (III), CCI + TMP group (IV), and CCI group (V). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and P2X 3 immunoreactivity in L4/L5 dorsal root ganglion (DRG) and spinal cord was detected by immunohistochemistry. The mechanical withdrawal threshold and thermal withdrawal latency in group V were lower than those in groups I–III or IV ( p < 0.05), while P2X 3 receptor expression of L4/L5 DRG and spinal cord in group V was higher than those in groups I–III ( p < 0.01) or group IV ( p < 0.05). The mechanical withdrawal threshold, thermal withdrawal latency and P2X 3 immunoreactivity of L4/L5 DRG and spinal cord in group IV showed no significant difference compared with those in groups I, II or III ( p > 0.05). The amplitudes of the currents in group V (CCI) were much larger than those obtained in other groups after application of same concentration adenosine 5′-triphosphate disodium (ATP) ( p < 0.01). α,β-Methylene-ATP (α,β-meATP)-activated currents in DRG neurons of CCI rats were more obvious than those obtained in other group rats ( p < 0.01). The results showed that TMP may inhibit the primary afferent transmission of neuropathic pain induced by P2X 3 receptor.

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

A critical role of the cAMP sensor Epac in switching protein kinase signalling in prostaglandin E2‐induced potentiation of P2X3 receptor currents in inflamed rats

Sensitization of purinergic P2X receptors is one of the mechanisms responsible for exaggerated pain responses to inflammatory injuries. Prostaglandin E2 (PGE2), produced by inflamed tissues, is known to contribute to abnormal pain states. In a previous study, we showed that PGE2 increases fast inactivating ATP currents that are mediated by homomeric P2X3 receptors in dorsal root ganglion (DRG) neurons isolated from normal rats. Protein kinase A (PKA) is the signalling pathway used by PGE2. Little is known about the action of PGE2 on ATP currents after inflammation, although the information is crucial for understanding the mechanisms underlying inflammation-induced sensitization of P2X receptors. We therefore studied the effects of PGE2 on P2X3 receptor-mediated ATP currents in DRG neurons dissociated from complete Freund's adjuvant (CFA)-induced inflamed rats. We found that PGE2 produces a large increase in ATP currents. PKCepsilon, in addition to PKA, becomes involved in the modulatory action of PGE2. Thus, PGE2 signalling switches from a solely PKA-dependent pathway under normal conditions to both PKA- and PKC-dependent pathways after inflammation. Studying the mechanisms underlying the switch, we demonstrated that cAMP-responsive guanine nucleotide exchange factor 1 (Epac1) is up-regulated after inflammation. The Epac agonist CPT-OMe mimics the potentiating effect of PGE2 and occludes the PKC-mediated PGE2 action on ATP currents. These results suggest that Epac plays a critical role in P2X3 sensitization by activation of de novo PKC-dependent signalling of PGE2 after inflammation and would be a useful therapeutic target for pain therapies.

Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice.

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease-activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.

ω‐Conotoxin CVIB differentially inhibits native and recombinant N‐ and P/Q‐type calcium channels

Omega-conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist omega-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. Omega-conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 +/- 0.05 and 7.64 +/- 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (approximately 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (> or = 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (approximately 22% of total) was attributable to P/Q-type VGCCs. Omega-conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Ca(V)2.2) and P/Q- (Ca(V)2.1), but not R- (Ca(V)2.3) type VGCCs expressed in Xenopus oocytes. The alpha2delta1 auxiliary subunit co-expressed with Ca(V)2.2 and Ca(V)2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. Omega-conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.

Does prostaglandin upregulate the tetrodotoxin-resistant Na + current in DRG neurons?

One possible mechanism underlying the inflammation-induced sensitization of primary afferent neurons is the upregulation of tetrodotoxin-resistant (TTX-R) Na + current by inflammatory mediators such as prostaglandins. This notion is based on reports that showed an augmentation of TTX-R Na + current following an application of prostaglandin E 2 (PGE 2 ) in dorsal root ganglion (DRG) neurons. However, no information was available on the property of the novel type of TTX-R Na + channel, Na V 1.9, at the time when these reports were published. Hence, the contribution of Na V 1.9 to the PGE 2 -induced upregulation of TTX-R Na + current remains to be elucidated. To further examine the modulation of TTX-R Na + current by PGE 2 , two components of TTX-R Na + current have been recorded in isolation from small (<25 μm in diameter) DRG neurons using wild-type and Na V 1.8 knock-out mice. Unexpectedly, neither the component mediated by Na V 1.8 nor the persistent component mediated by Na V 1.9 was affected by PGE 2 (1 and 10 μM). These results raise a question regarding the well-known modulatory role of PGE 2 on TTX-R Na + current in inflammatory hyperalgesia. Here we will review these recent data in context with the current literature relating to this issue.

Prostaglandin E2 Receptor EP4 Contributes to Inflammatory Pain Hypersensitivity

Prostaglandin E(2) (PGE(2)) is both an inflammatory mediator released at the site of tissue inflammation and a neuromodulator that alters neuronal excitability and synaptic processing. The effects of PGE(2) are mediated by four G-protein-coupled EP receptors (EP1-EP4). Here we show that the EP4 receptor subtype is expressed by a subset of primary sensory dorsal root ganglion (DRG) neurons, and that its levels, but not that of the other EP1-3 subtypes, increase in the DRG after complete Freund' adjuvant-induced peripheral inflammation. Administration of both an EP4 antagonist [AH23848, (4Z)-7-[(rel-1S,2S,5R)-5-((1,1'-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid] and EP4 knockdown with intrathecally delivered short hairpin RNA attenuates inflammation-induced thermal and mechanical behavioral hypersensitivity, without changing basal pain sensitivity. AH23848 also reduces the PGE(2)-mediated sensitization of capsaicin-evoked currents in DRG neurons in vitro. These data suggest that EP4 is a potential target for the pharmacological treatment of inflammatory pain.

Modulation of dragon’s blood on tetrodotoxin-resistant sodium currents in dorsal root ganglion neurons and identification of its material basis for efficacy

To clarify the modulation of dragon's blood on the tetrodotoxin-resistant (TTX-R) sodium currents in dorsal root ganglion (DRG) neurons and explore its corresponding material basis for the efficacy, using whole-cell patch clamp technique, the effects of dragon's blood and the combined effects of three components (cochinchinenin A, cochinchinenin B, and loureirin B) extracted from dragon's blood on the TTX-R sodium currents in acute-isolated DRG neurons of rats were observed. According to the operational definition of material basis for the efficacy of TCM established, the material basis of the modulation on the TTX-R sodium currents in DRG neurons of dragon's blood was judged from the experimental results. The drug interaction equation of Greco et al. was used to assess the interaction of the three components extracted from dragon's blood. This investigation demonstrated that dragon's blood suppressed the peak TTX-R sodium currents in a dose-dependent way and affected the activations of TTX-R sodium currents. The effects of the combination of cochinchinenin A, cochinchinenin B, and loureirin B were in good agreement with those of dragon's blood. Although the three components used alone could modulate TTX-R sodium currents, the concentrations of the three components used alone were respectively higher than those used in combination when the inhibition rates on the TTX-R sodium currents of them used alone and in combination were the same. The combined effects of the three components were synergistic. These results suggested that the interference with pain messages caused by the modulation of dragon's blood on TTX-R sodium currents in DRG neurons may explain some of the analgesic effect of dragon's blood and the corresponding material basis for the efficacy is the combination of cochinchinenin A, cochinchinenin B, and loureirin B.

Mechanisms underlying enhanced P2X receptor-mediated responses in the neuropathic pain state

P2X3 and P2X2/3 receptors in dorsal root ganglia (DRG) appear to participate in producing nociceptive responses after nerve injury. However, the mechanisms underlying the receptor-mediated nociception in the neuropathic state remain unclear. Using spared nerve injury (SNI) rats, we found that allodynic and nocifensive (flinch) behavioral responses developed after injury can be reversed by P2X receptor antagonists, indicating an involvement of P2X receptors. Immunocytochemical studies revealed that P2X3 receptors are expressed in small and medium but rarely in large DRG neurons of both normal and SNI rats. Thus, contrary to the conventional view that only large Aβ cells mediate allodynia, small and medium cells are intimately involved in P2X3 receptor-mediated allodynia. Measuring ATP levels in the subcutaneous space of the rat paw, we showed that ATP release does not change after SNI. On the other hand, the P2X receptor agonist, αβ-methylene ATP produces 3.5-fold larger flinch responses at a 8.0-fold lower dose. Thus, sensitization of P2X3 receptors rather than a change in ATP release is responsible for the neuropathic pain behaviors. We further demonstrated that sensitization of P2X3 receptors arises from an increase in receptor function. ATP-induced P2X3 receptor-mediated currents in DRG neurons is 2.5-fold larger after SNI. The expression of P2X3 receptors on the cell membrane is significantly enhanced while the total expression of P2X3 receptors remained unchanged. Thus, the enhancement of trafficking of P2X3 receptors is likely an important mechanism contributing to the increase in receptor function after nerve injury.

Calcitonin gene-related peptide enhances TTX-resistant sodium currents in cultured dorsal root ganglion neurons from adult rats

The neuropeptide calcitonin gene-related peptide (CGRP) binds to a subpopulation of dorsal root ganglion (DRG) neurons, elevates intracellular calcium, and causes inward currents in about 30% of lumbar DRG neurons. Using whole-cell patch clamp recordings, we found in the present study that application of CGRP to isolated and cultured DRG neurons from the adult rat enhances voltage-gated TTX-resistant (TTX-R) Na + inward currents in about 30% of small- to medium-sized DRG neurons. During CGRP, peak densities of Na + currents increased significantly. CGRP shifted the membrane conductance of the CGRP-responsive cells towards hyperpolarization without changing the slope of the peak conductance curve. The effect of CGRP was blocked by coadministration of CGRP 8–37, an antagonist at the CGRP receptor. The effect of CGRP was also blocked after bath application of PKA 14–22, a membrane-permeant blocker of protein kinase A, and PKC 19–31, a PKC inhibitor, in the recording pipette. These data show pronounced facilitatory effects of CGRP on TTX-R Na + currents in DRG neurons which are mediated through CGRP receptors and intracellular pathways involving protein kinases A and C. Thus, in addition to prostaglandins, CGRP is another mediator that affects TTX-R Na + currents which are thought to occur mainly in nociceptive DRG neurons.

Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia.

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

Modulation of sodium currents in rat sensory neurons by nucleotides

The effects of various nucleotides on the fast tetrodotoxin-sensitive (f-TTX-S) and the slow tetrodotoxin-resistant (s-TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were investigated using the patch-clamp technique. Nucleoside triphosphates (NTPs; ATP, GTP, UTP and CTP) and nucleoside diphosphates (NDPs; ADP, GDP, UDP and CDP) decreased f-TTX-S current, whereas they increased s-TTX-R current, when currents were evoked by step depolarizations to 0 mV from a holding potential of −80 mV. NTPs and NDPs shifted both the conductance–voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction in both types of sodium currents. Most of them also increased the maximum conductance of s-TTX-R current. ITP, a derivative of ribonucleotide, and dTTP, a deoxyribonucleotide, modulated both types of sodium currents similarly to NTPs and NDPs. However, nucleoside monophosphates (NMPs; AMP, GMP, UMP and CMP) and adenosine had little or no effect on either type of sodium current. Therefore, it seems that nucleotides, regardless of the kind of base, should have two or more phosphates to be able to modulate sodium currents in DRG neurons. Extracellular nucleotides with di- or tri-phosphates would influence the perception by modulating sodium currents in sensory neurons. Particularly, the increase of the maximum conductance and the hyperpolarizing shift of the conductance–voltage relationship of s-TTX-R sodium current would result in an intensified nociception.

Heterologous expression and functional analysis of rat Na V1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7–23

The voltage-gated sodium channel Na V1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na + current. TTX-R/Na V1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant Na V1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells. Here, we show stable expression and functional analysis of rat Na V1.8 (rNa V1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7–23. Rat Na V1.8 Na + currents were recorded (789±89 pA, n =62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNa V1.8 (mean IC 50 value 12.5 μM) and in repeated gating paradigms (2–10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNa V1.8 ( r =0.72, P <0.001). RT-PCR analysis of wild type ND7–23 cells revealed endogenous expression of the β1 and β3 accessory Na + channel subunits—the possibility that the presence of these subunits assists and stabilises expression of rNa V1.8 is discussed. We conclude that the neuroblastoma ND7–23 cell line is a suitable heterologous expression system for rNa V1.8 Na + channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove useful in the search for pharmacological inhibitors of rNa V1.8 as novel analgesics.

Local anesthetic inhibition of voltage-activated potassium currents in rat dorsal root ganglion neurons.

Local anesthetic actions on the K+ channels of dorsal root ganglion (DRG) and dorsal horn neurons may modulate sensory blockade during neuraxial anesthesia. In dorsal horn neurons, local anesthetics are known to inhibit transient but not sustained K+ currents. The authors characterized the effects of local anesthetics on K+ currents of isolated DRG neurons. The effects of lidocaine, bupivacaine, and tetracaine on K+ currents in isolated rat DRG neurons were measured with use of a whole cell patch clamp method. The currents measured were fast-inactivating transient current (I(Af)), slow-inactivating transient current (I(As)), and noninactivating sustained current (I(Kn)). One group of cells (type 1) expressed I(Af) and I(Kn). The other group (type 2) expressed I(As) and I(Kn). The diameter of type 2 cells was smaller than that of type 1 cells. Lidocaine and bupivacaine inhibited all three K+ currents. Tetracaine inhibited I(As) and I(Kn) but not I(Af) For bupivacaine, the concentration for half-maximal inhibition (IC50) of I(Kn) in type 2 cells was lower than that for I(Kn) in type 1 cells (57 vs. 121 microM). Similar results were obtained for tetracaine (0.6 vs. 1.9 mM) and for lidocaine (2.2 vs. 5.1 mM). Local anesthetics inhibited both transient and sustained K+ currents in DRG neurons. Because K+ current inhibition is known to potentiate local anesthetic-induced impulse inhibition, the lower IC50 for I(Kn) of small type 2 cells may reflect preferential inhibition of impulses in nociceptive neurons. The overall modulatory actions of local anesthetics probably are determined by their differential effects on presynaptic (DRG) and postsynaptic (dorsal horn neurons) K+ currents.

Synaptogenesis in Co-Culture of Neurons of Dorsal Root Ganglia and Spinal Cord

In co-culture of spinal cord and dorsal root ganglion (DRG) neurons, we studied at different terms of culturing postsynaptic currents in DRG neurons evoked by direct electrical stimulation of single spinal neurons using a voltage-clamp technique in the whole-cell configuration. According to the reversal potential and sensitivity to bicuculline, these currents were classified as inhibitory postsynaptic currents (IPSC) carried by Cl- ions through GABAA receptors. During neuronal development in dissociated co-culture, the amplitude of evoked IPSC and their time to peak significantly increased. The time to peak of spontaneous IPSC (sIPSC) in DRG neurons remained unchanged, while the frequency of these currents increased with increasing culturing time. It is concluded that under culturing conditions spinal neurons establish inhibitory synaptic contacts with the somata of DRG neurons, and the number of such functional contacts increases in the course of culturing. Our findings show that in dissociated co-culture the process of formation of inhibitory synapses on the axon terminals of primary afferent neurons is akin to that realized in vivo, but with dissimilar topography of distribution of such synapses.

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.

CLINICAL CONCENTRATIONS OF ISOFLURANE SIGNIFICANTLY SUPPRESSES SODIUM CURRENTS IN RAT DORSAL ROOT GANGLIA

S519 Introduction: Over the last decade a number of studies have revealed that clinical concentrations of volatile anesthetics alter the discharge pattern and action potential properties of mammalian CNS and peripheral neurons [1]. In thin and unmyelinated neurons, which represent a substantial fraction of intracortical fibers and sensory afferents, anesthetics also have been found to alter signal propagation. [1] These neuronal functions all depend on the proper function of voltage-dependent sodium channels, making these integral membrane proteins potentially important molecular targets of general anesthetics. Although early studies examining anesthetic modification of sodium channel currents in non-mammalian preparations found them to be relatively insensitive targets compared with ligand-gated channels, a recent report has found that rat brain-Ila sodium channels expressed in a mammalian cell line are as sensitive as GABA-A receptors to clinical concentrations of volatile anesthetics [2]. It therefore has become essential to determine whether sodium channels in mammalian neurons have a similar anesthetic sensitivity as the expressed channels. To examine this question, we examined isoflurane modification of sodium currents in cultured rat Dorsal Root Ganglia (DRG). Methods: Freshly dissociated and primary cultured neurons of DRG from 5-30 day rats were obtained after enzymatic digestion with collagenase and/or trypsin. Sodium currents were measured using the whole cell patch clamp configuration; the extracellular Hanks' balanced salt solution additionally contained 5 mM HEPES and 0.1 mM CdCl2, pH 7.2, while the internal solution was 140 mM CsF, 10 mM NaCl, 5 mM HEPES, pH 7.4. Control measurements of the cells elicited normal action potentials and ionic currents. Sodium currents and anesthetic suppression were measured with depolarizing test potentials from a hyperpolarizing holding potential. Aqueous concentrations of isoflurane, which are relatively independent of experimental temperature [3], were measured by gas chromatography. All experiments were conducted at room temperature (22-25[degree sign]C). Results: Similar to results with the rat brain Ila sodium channels, isoflurane suppressed sodium currents through at least two mechanisms: a) a voltage-independent suppression of resting or open channels at hyperpolarized potentials, and b) a hyperpolarizing shift in the voltage-dependence of channel inactivation. Unlike the expressed channels, however, even high concentrations of isoflurane could not completely block sodium currents at potentials more negative than -80 mV. At -80 mV, isoflurane maximally suppressed 54 +/- 7.0% of sodium current (n=11, concentrations up to 5.4 mM isoflurane). The range of maximal suppression was 40-75%. Averaging data from the 11 cells and fitting to a rectangular hyperbola yielded a maximal suppression of 0.49 with an ED50=0.17 mM, slightly lower than aqueous MAC concentrations. The midpoint of sodium channel inactivation was about -50 mV. Similar to rat brain sodium channels, 0.54 mM isoflurane (twice MAC) shifted the inactivation midpoint by about -20 mV, resulting in significantly greater current suppression at these potentials (about 80% block at -60mV, compared with about 20% at -80 mV). Conclusions: Clinical concentrations of isoflurane caused a large depolarizing shift in steady-state channel inactivation, resulting in the block of a significant fraction (>50%) of sodium currents in DRG neurons at resting membrane potentials. These results support the hypothesis that some mammalian neuronal sodium channels are sensitive to clinical concentrations of volatile anesthetics in situ, and that their anesthetic modification may directly alter neuronal function.

Selective loss of slow and enhancement of fast Na+currents in cutaneous afferent dorsal root ganglion neurones following axotomy

Voltage-dependent Na+currents were recorded via patch-clamp from identified adult rat lumbar cutaneous dorsal root ganglion (DRG) neurones using whole-cell and bleb-patch-clamp configurations. Na+currents in DRG neurones studied 18 days after sciatic nerve ligation were compared with those in control neurones. Control neurones tended to have a singular kinetically slow Na+current or net Na+current suggestive of two kinetic varieties of channel in a single neurone. Three changes occurred following axotomy: (1) the peak Na+current increased significantly, (2) kinetically slow TTX-resistant Na+current, which predominated in controls, was significantly attenuated or lacking altogether, and (3) a singular, TTX-sensitive kinetically fast form of Na+current predominated. These findings suggest that as part of the response to axonal injury, DRG neurones increase Na+channel biosynthesis and/or modify preexisting channels such that their kinetics are accelerated. The results are consistent with the idea that axotomized DRG neurones become hyperexcitable due to the emergence of a high density of kinetically fast, pharmacologically distinguishable, Na+channels in the membrane.