Abstract
We have recently reported that systemic delivery of A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide], a selective Nav1.8 sodium channel blocker, reduces behavioral measures of chronic pain. In the current study, the effects of A-803467 on evoked and spontaneous firing of wide dynamic range (WDR) neurons were measured in uninjured and rats with spinal nerve ligations (SNLs). Administration of A-803467 (10–30 mg/kg i.v.) reduced mechanically evoked (10-g von Frey hair) and spontaneous WDR neuronal activity in SNL rats. In uninjured rats, A-803467 (20 mg/kg i.v.) transiently reduced evoked but not spontaneous firing of WDR neurons. The systemic effects of A-803467 in SNL rats were not altered by spinal transection or by systemic pretreatment with the transient receptor potential vanilloid type 1 (TRPV1) receptor agonist, resiniferatoxin, at doses that impair the function of TRPV1-expressing fibers. To determine sites of action, A-803467 was administered into spinal tissue, into the uninjured L4 dorsal root ganglion (DRG), or into the neuronal receptive field. Injections of A-803467 into the L4 DRG (30–100 nmol/1 μl) or into the hindpaw receptive field (300 nmol/50 μl) reduced evoked but not spontaneous WDR firing. In contrast, intraspinal (50–150 nmol/0.5 μl) injection of A-803467 decreased both evoked and spontaneous discharges of WDR neurons. Thus, Nav1.8 sodium channels on the cell bodies/axons within the L4 DRG as well as on peripheral and central terminals of primary afferent neurons regulate the inflow of low-intensity mechanical signals to spinal WDR neurons. However, Nav1.8 sodium channels on central terminals seem to be key to the modulation of spontaneous firing in SNL rats.
Voltage-gated sodium channels (VGSCs) are fundamental components to the induction and propagation of neuronal signals (Waxman et al., 1999). There are at least nine different VGSC subtypes in the nervous system, and each subtype can be functionally classified as either tetrodotoxin-sensitive or tetrodotoxin-resistant. The tissue distribution for the different VGSC subtypes is varied, for some subtypes widespread, and includes different populations of primary afferent sensory neurons (Waxman et al., 1994; Black et al., 1996; Renganathan et al., 2002). Expression on afferent neurons has made VGSCs attractive targets to regulate the flow of nociceptive signals to the spinal cord. Nonselective inhibitors of VGSCs, such as local anesthetics, have been used for the treatment of acute and pathological pain, but the therapeutic appeal of these agents is limited due to undesirable side effects (Petersen et al., 1986; Bach et al., 1990; Chaplan et al., 1995; Mao and Chen, 2000). Thus, there has been a growing interest in developing channel blockers that are selective for one VGSC subtype over all others for use in pain management and potentially limiting adverse events.
Nav1.8 is a tetrodotoxin-resistant sodium channel with a distribution restricted to primary afferent neurons (Akopian et al., 1996, 1999). The majority of Nav1.8-containing afferents transmit nociceptive signals to the spinal cord (Djouhri et al., 2003). Knockout of the Nav1.8 gene reduces the occurrence of spontaneous firing as well as the responses of spinal wide dynamic range (WDR) neurons to mechanical stimulation (Matthews et al., 2006). Following peripheral nerve damage, the functional expression of Nav1.8 channels is decreased in injured neurons but elevated in uninjured axons (Gold et al., 2003). Activity in the uninjured neurons is likely critical to the heightened sensory sensitivity observed in neuropathic pain states (Li et al., 2000; Jang et al., 2007), and the redistribution of Nav1.8 channels to the axons of these neurons is potentially a key component of this altered sensitivity (Gold et al., 2003). Further validation of the Nav1.8 role in neuropathic pain was generated from studies using antisense oligodeoxynucleotides, showing clear attenuation of hyperalgesia and allodynia in nerve-injured animals (Porreca et al., 1999; Lai et al., 2002; Joshi et al., 2006). Furthermore, systemic or spinal delivery of nonselective and preferential Nav1.8 channel blockers also reduced hyperalgesia and allodynia in animal models of pathological pain (Veneroni et al., 2003; Gaida et al., 2005; Akada et al., 2006; Brochu et al., 2006; Ekberg et al., 2006).
Our group has recently reported that systemic administration of A-803467 (Fig. 1), a novel sodium channel blocker that has high affinity and selectivity for Nav1.8 channels, is effective in several behavioral models of acute and chronic pain (Jarvis et al., 2007). In particular, A-803467 reduced acute mechanical nociception, inflammation-related thermal hyperalgesia, and mechanical allodynia in two models of neuropathic pain. A-803467 is 300 to 1000-fold more potent at blocking Nav1.8 channels than other VGSCs, shows no significant activity at other ion channels, receptors, or enzymes in a broad screening panel, and has good penetration into the central nervous system (Jarvis et al., 2007). To enhance our understanding of the contributions of Nav1.8 channels to the transmission of low-intensity mechanical stimulation in a neuropathic state, the present study evaluated the systemic and local effects of A-803467 on mechanically evoked and spontaneous firing of spinal WDR neurons in rats with spinal nerve ligations (SNLs).
Materials and Methods
All animal handling and experimental protocols were approved by Abbott's Institutional Animal Care and Use Committee and were conducted in accordance with the ethical principles for pain-related animal research of the American Pain Society. Male Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA; 350–400 g) were used for all experiments and were housed in a temperature controlled room with a 12-/12-h day/night cycle. Food and water were available ad libitum.
To induce neuropathy, a unilateral tight ligation of the L5 and L6 spinal nerves (SNL) was performed on anesthetized rats. Spinal neuronal recording occurred 14 days after surgery. On the day of recording, all SNL animals were confirmed allodynic by exhibiting robust responses to 6-g von Frey hair stimulation of the ipsilateral hindpaw. Animals that did not respond to the 6-g von Frey hair stimulation were excluded from electrophysiological experiments. Uninjured and SNL rats were then initially anesthetized with pentobarbital (50 mg/kg i.p.). A catheter was placed into the left and/or right external jugular vein(s), and a laminectomy was performed to remove vertebral segments T12 to L3. For SNL animals receiving an intradorsal root ganglion (DRG) injection of compound (McGaraughty et al., 2006), the L4, uninjured, DRG was exposed, and a small membrane incision was made distal to the DRG. PE-5 catheters (external PE-10; Marsil Enterprises, San Diego, CA) were inserted through the membrane opening up to the DRG. Animals were then secured in a stereotaxic apparatus (Kopf Instruments, Tujunga, CA) supported by clamps attached to the vertebral processes on either side of the exposure site. The exposed lumbar area was first enveloped by agar and then filled with mineral oil. A stable plane of anesthesia was maintained throughout the experiment by a continuous infusion of propofol at a rate of 8 to 12 mg/kg/h i.v. Body temperature was kept at approximately 37°C by placing the animals on a circulating water blanket.
Platinum-iridium microelectrodes (Frederick Haer, Brunswick, ME) were used to record extracellular activity of WDR neurons located in the dorsal horn of the spinal cord. To confirm that the neuron under study was a WDR neuron (responding to noxious and non-noxious stimuli but maximally to noxious stimuli), each neuron was characterized by their responses to both non-noxious and noxious stimuli applied to the ipsilateral hindpaw. In order, the neuronal receptive field was gently tapped, brushed, given a noxious pinch with forceps, and finally stimulated with a 10-g von Frey hair. Spike waveforms were monitored on an oscilloscope throughout the experiment, digitized, and then stored for off-line analysis (SciWorks; Datawave Technologies, Longmont, CO) to ensure that the unit under study was unambiguously discriminated throughout the experiment. Except for four experiments in which two easily distinguished neurons were simultaneously recorded on one electrode, only one neuron was studied per rat.
At the onset of each experiment, spontaneous neuronal firing was recorded for 5 min to determine baseline levels. Neurons were then characterized as described above. Before the administration of A-803467 or vehicle, three baseline evoked responses (separated by 5 min each) to von Frey hair stimulation (10 g) of the receptive field were recorded. The actions of A-803467 on spontaneous and evoked WDR neuronal activity in SNL rats were then examined after systemic (10–30 mg/kg i.v.), intra-DRG (30 and 100 nmol in 1 μl), intraspinal (50 and 150 nmol in 0.5 μl), or intrareceptive field (300 nmol in 50 μl) delivery. Von Frey-evoked (10 g) and spontaneous activity was measured 1 (for DRG injections only), 5, 15, 25, and 35 min after A-803467 or vehicle injection. For comparison, uninjured rats were administered 20 mg/kg (i.v.) A-803467 and followed the same testing protocol as the SNL rats.
To investigate possible contributions from supraspinal sites, A-803467 (20 mg/kg i.v.) was administered systemically to SNL rats with a complete spinal T9 transection and compared with results from intact SNL rats. Another group of animals received 0.3 mg/kg s.c. of the potent transient receptor potential vanilloid type 1 (TRPV1) receptor agonist, resiniferatoxin, 2 days prior to experimentation to impair TRPV1-containing fibers. We have shown previously that this dose of resiniferatoxin reduces the occurrence of nocifensive behaviors induced by administration of a TRPV1 receptor agonist as well as other irritants that do not directly act at TRPV1 receptors (Wismer et al., 2003). Furthermore, others have used similar doses of resiniferatoxin to observe disruption of TRPV1-related activity in addition to demonstrating that the TRPV1 fibers, not just the receptor itself, are impaired (Szallasi et al., 1989; Xu et al., 1997; Chen et al., 2007). RTX has been shown to degenerate TRPV1-containing fibers (Olah et al., 2001). The effects of A-803467 (20 mg/kg i.v.) on this group of animals were also compared with effects in intact SNL rats to determine whether TRPV1-containing fibers are necessary for the effects of A-803467 on WDR neurons.
Delivery and Preparation of Compounds. For systemic injection, the solution was infused over a 5- to 7-min period at a volume of 1 ml/kg. Intraplantar injections (50 μl) were made to both the ipsilateral and contralateral hindpaws in separate experiments. For direct DRG injections (McGaraughty et al., 2006), A-803467 or vehicle, in 1 μl, was injected over a period of 1 min onto the L4 DRG through the indwelling catheter attached to a 10-μl Hamilton syringe. If the intra-DRG injection of A-803467 or vehicle was without effect on neuronal activity, 5% lidocaine (1 μl) was then infused onto the L4 DRG. If evoked activity was unaffected by lidocaine, it was determined that the recorded spinal neuron did not receive direct/indirect input from the L4 DRG and was not used for data analysis. For intraspinal injections (McGaraughty et al., 2006), a glass infusion pipette (outer diameter 75–80 μm) with an angled beveled tip was attached to the recording electrode in such a way that the tips were separated by approximately 300 μm laterally and by 30 to 100 μm dorsoventrally. The electrode and pipette were simultaneously lowered into the spinal tissue. The infusion pipette was attached to a1-μl Hamilton syringe with a length of PE-50 tubing, and 0.5 μlof solution was delivered over a 6-min period. A-803467 was synthesized at Abbott Laboratories (Abbott Park, IL) and was dissolved in 5% (systemic), 10% (intra-DRG), or 20% (intraspinal) dimethylsulfoxide and polyethylene glycol. The vehicle for intrareceptive field delivery of A-803467 and for systemic injection of resiniferatoxin (Sigma-Aldrich, St. Louis, MO) was 10% dimethylsulfoxide and 34% 2-hydroxypropyl-β-cyclodextrin in saline.
Data Analysis. Baseline levels of spontaneous and evoked firing were compared using a Student's t test (p < 0.05). Postdrug spontaneous and mechanically evoked neuronal activity were calculated as a percentage of their respective baseline levels. All data are presented as mean ± S.E.M. For comparisons with baseline firing levels, statistical significance was established by using a Wilcoxon's matched pairs test. A Kruskal-Wallis analysis of variance followed by a Mann-Whitney U test was used for comparison across groups (p < 0.05).
Results
Baseline Activity. Discharge activity was recorded from 113 WDR neurons (95 from SNL rats and eight from uninjured rats) with a mean depth of 884.1 ± 83.6 μm from the surface of the spinal cord. The mean baseline (predrug) spontaneous and von Frey-evoked (10 g) firing across all WDR neurons recorded from uninjured rats was 1.2 ± 0.36 and 9.8 ± 2.6 spikes/s, respectively. Baseline spontaneous (2.6 ± 0.26 spikes/s), but not mechanically evoked (12.1 ± 0.8 spikes/s), firing of WDR neurons was significantly (p < 0.01) elevated in SNL rats compared with the uninjured rats.
Effects of Systemic A-803467 on WDR Neuronal Activity. Systemic i.v. administration of A-803467 (10–30 mg/kg) attenuated both spontaneous and von Frey-evoked (10 g) firing of WDR neurons in SNL rats (Fig. 2). WDR responses to 10-g von Frey stimulation of the hindpaw was significantly (p < 0.05) decreased from baseline levels 5 min after injection of all doses. Spontaneous activity of WDR neurons was also significantly (p < 0.05) decreased 5 min after injection of the 20 and 30 mg/kg doses of A-803467 and remained significantly decreased for the rest of the recording period. Maximal effects were observed 35 min after injection, when 30 mg/kg of A-803467 reduced spontaneous firing by 97.7 ± 1.3% and decreased the evoked responses of WDR neurons by 94.4 ± 2.6%. At the 35-min time point, spontaneous and evoked WDR neuronal firing was also significantly (p < 0.05) attenuated by 20 mg/kg (66.2 ± 13.1% and 53.2 ± 18.4%, respectively) but not by 10 mg/kg A-803467.
Administration of 20 mg/kg i.v. A-803467 to uninjured rats significantly decreased (p < 0.05) WDR neuronal responses to 10-g von Frey hair stimulation by 40.9 ± 11.5% from baseline levels 5 min after injection (Fig. 2A). This effect of A-803467 on evoked WDR activity in uninjured rats (significant against both baseline activity and the vehicle-treated group) was transient as it was not observed at any other time point after 5 min. Although systemic administration of A-803467 (20 mg/kg i.v.) decreased spontaneous WDR firing in uninjured rats between 15 and 35% across the different time points, the decreases in activity were not significant (Fig. 2B).
Site of A-803467 Action on WDR Activity. A-803467 was injected onto the L4 DRG region to affect both axons and cell bodies within this area. Within 1 min of injection onto the uninjured L4 DRG, A-803467 (30 and 100 nmol in 1 μl) significantly (p < 0.05) decreased the responses of WDR neurons to 10-g von Frey hair stimulation (Fig. 3, A and C). This effect lasted for the remainder of the recording period with a maximal observed reduction (68.8 ± 5.5%) occurring 35 min postadministration. Neither dose of A-803467 affected the spontaneous firing of WDR neurons after intra-DRG injection (Fig. 3C).
Needle insertion and administration of 50 μl of vehicle into the neuronal receptive field on the hindpaw significantly increased the von Frey-evoked (10 g) responses of WDR neurons for the first 5 min after injection but thereafter returned to baseline levels. Compared with the vehicle group, injection of A-803467 (300 nmol) into the neuronal receptive field significantly reduced (p < 0.05) evoked discharges of WDR neurons at each time point examined (5–35 min) with the largest effect (47.4 ± 12.7%) observed 35 min after intrareceptive field injection (Fig. 3B). Administration of A-803467 into the neuronal receptive field did not alter the spontaneous firing of WDR neurons (data not shown).
Unlike the peripheral injections, intraspinal delivery of A-803467 (50 and 150 nmol in 0.5 μl) reduced both the spontaneous and von Frey-evoked firing (10 g) of WDR neurons (Fig. 4), thus resembling the effects observed after systemic injection of A-803467. The onset of action after intraspinal delivery seems to occur earlier for evoked firing than for spontaneous firing. At the 5-min time point after intraspinal injection of 150 nmol A-803467, there was at least a 20% drop (20–79.9% range) in evoked firing for six of eight neurons, whereas only three of eight neurons showed at least a 20% drop (20.1–91.1% range) in spontaneous firing. Similar numbers were observed with the 50 nmol dose; seven of eight and one of eight WDR neurons showed at least a 20% decrease in evoked and spontaneous firing, respectively, 5 min after intraspinal injection. However, by 15 min postinjection, A-803467 significantly (p < 0.05) reduced both evoked (50 and 150 nmol) and spontaneous (150 nmol only) discharges of WDR neurons. It took 35 min for the 50 nmol dose of A-803467 to significantly decrease spontaneous firing of WDR neurons. The largest reduction in evoked (85.7 ± 4.3%) and spontaneous (86.9 ± 2.4%) activity of WDR neurons occurred 35 min after intraspinal injection of 150 nmol A-803467 (Fig. 4).
It is unlikely that supraspinal sites contributed to the systemic actions of A-803467 because spinal transection did not alter the effects of A-803467 (20 mg/kg i.v.) on either spontaneous or von Frey-evoked (10 g) firing of WDR neurons (Fig. 5). Furthermore, the observed influence of A-803467 on WDR neurons does not require TRPV1-containing fibers. Systemic administration of the TRPV1 receptor agonist, resiniferatoxin, 2 days prior to testing at a dose (0.3 mg/kg s.c.) that impairs TRPV1-expressing fibers (Szallasi et al., 1989; Xu et al., 1997; Wismer et al., 2003; Chen et al., 2007) did not alter the actions of A-803467 (20 mg/kg i.v.) on the spontaneous or evoked activity of WDR neurons (Fig. 6).
Discussion
The past decade has seen mounting evidence supporting a key role for Nav1.8 sodium channels in nociceptive transmission (Rogers et al., 2006). Localization studies of Nav1.8 sodium channels have shown a distribution that is restricted to primary sensory afferents (Akopian et al., 1996, 1999), thus limiting the functional contributions of this channel and avoiding undesired additional activity that typically accompanies a wider expression. As a result, direct targeting of the Nav1.8 sodium channel with genetic deletions and antisense treatment has confirmed this channel's contribution to normal and pathological nociception (Akopian et al., 1999; Porreca et al., 1999; Lai et al., 2002; Joshi et al., 2006; Matthews et al., 2006). The recent development of a small molecule selective blocker for Nav1.8 sodium channels, A-803467 (Jarvis et al., 2007), has added another tool to define the contributions of Nav1.8 sodium channels. Systemic delivery of A-803467 reduced behavioral responses to acute mechanical stimulation, thermal hyperalgesia, secondary mechanical hyperalgesia, and mechanical allodynia in models of neuropathic pain (Jarvis et al., 2007). The current data showing that systemic administration of A-803467 attenuated the responses of WDR neurons to low-intensity mechanical stimulation in SNL rats is consistent with the compound's antiallodynic action in behavioral tests and further delineates the influence of Nav1.8 sodium channels on the transmission of “painful” signals to the spinal cord.
WDR responses to peripheral mechanical stimulation are impaired in Nav1.8-null mice (Matthews et al., 2006). The impairment spanned a range of intensities and included reductions in response to both noxious and non-noxious mechanical stimuli. Likewise, blockade of Nav1.8 channels by injection of A-803467 attenuated the evoked responses of WDR neurons to application of a “non-noxious” von Frey hair in uninjured rats. These data are consistent with behavioral effects of A-803467 in uninjured animals (Jarvis et al., 2007) and indicate that Nav1.8 sodium channels contribute to normal mechanotransmission. However, the effects of A-803467 on evoked WDR neuronal activity in uninjured rats were relatively short-lived, compared with effects in the SNL animals, occurring only in the first 5 min after injection. Injection of the same dose (20 mg/kg i.v.) of A-803467 had a sustained effect on WDR neuronal activity in SNL rats. The plasma elimination half-life of A-803467 is 4.9 h and behavioral effects of A-803467 in the SNL model lasted at least 90 min after injection (Jarvis et al., 2007), indicating that the compound is not rapidly eliminated and should be effective for a substantial period. Although the exact mechanism mediating the increased effect of A-803467 in SNL rats has yet to be determined, the current data suggest that Nav1.8 sodium channels have an enhanced role in mechanotransmission after an injury to the peripheral nerves.
The afferent input to WDR neurons affected by A-803467 in the current study probably came via low-threshold mechanoreceptors (LTMs) given the low intensity of the stimulus. Nav1.8 sodium channels are found predominantly on C-fibers, both with and without TRPV1 receptors, larger diameter nociceptors, and are also on a small percentage of LTM fibers (Akopian et al., 1996; Amaya et al., 2000; Djouhri et al., 2003). Involvement of TRPV1-containing fibers was ruled out because pretreatment with systemic resiniferatoxin, at doses sufficient to impair the function of TRPV1-expressing neurons (Szallasi et al., 1989; Xu et al., 1997; Wismer et al., 2003; Chen et al., 2007), did not alter the effects of A-803467 on WDR neuronal firing. In addition, cutaneous thresholds of nociceptors have been reported to shift lower after an SNL injury (Shim et al., 2005), raising the possibility that A-803467 blocked mechanical input from non-TRPV1 nociceptors in addition to LTM fibers.
A-803467 acted at multiple sites along the primary afferent to interfere with mechanical-related input to WDR neurons. Intrareceptive field injection of A-803467 decreased WDR responses to von Frey hair stimulation, indicating an action at Nav1.8 channels on the peripheral terminals. Injection of A-803467 into the region of the uninjured L4 DRG also disrupted the flow of mechanical signals to WDR neurons. After an SNL injury, Nav1.8 sodium channels are functionally unaltered on the cell bodies of the uninjured L4 afferents (Gold et al., 2003). Furthermore, although expression of Nav1.8 sodium channels is decreased in the injured fibers and unchanged in the L4 cell bodies, there is an increase of channels in uninjured axons of SNL rats (Lai et al., 2002; Gold et al., 2003). Thus, an action of A-803467 on the cell bodies and axons in the region of the L4 DRG injection was not unexpected. Intraspinal application of A-803467 also attenuated WDR responses to low-intensity mechanical stimulation in SNL rats. This is consistent with the antiallodynia produced by i.t. injection of the preferential Nav1.8 sodium channel blocker, μO-conotoxin MrVIB, in an animal model of neuropathic pain (Ekberg et al., 2006). Immunostaining for Nav1.8 sodium channels is found in the superficial dorsal horn as well as in deeper laminae (Amaya et al., 2000). Thus, A-803467 may have blocked presynaptic Nav1.8 sodium channels at superficial and/or deeper laminae to prevent evoked signals from reaching WDR neurons. Because Nav1.8 sodium channels have not been detected in supraspinal tissue (Amaya et al., 2000), it was not surprising that spinal transection did not alter the systemic effects of A-803467.
In addition to effects on mechanical-related input, systemic injection of A-803467 also significantly decreased the spontaneous firing of WDR neurons in SNL rats. After an SNL injury, spontaneous WDR firing is elevated and is an indication of sensitization (Chapman et al., 1998; Chu et al., 2004; McGaraughty et al., 2006, 2007; Suzuki and Dickenson, 2006), possibly reflecting a certain degree of spontaneous or “nagging” pain in the animal. The decrease in WDR spontaneous firing after A-803467 administration to SNL rats suggests that Nav1.8 sodium channels contribute to the heightened state of sensory sensitivity. In Nav1.8-ablated mice, there was a lower occurrence of spontaneously active WDR neurons (Matthews et al., 2006). It seems that Nav1.8 sodium channels on peripheral terminals and on cell bodies/axons around the L4 DRG do not affect the ongoing activity of WDR neurons because direct injections of A-803467 to these peripheral sites did not reduce the spontaneous discharges of WDR neurons. Instead, direct delivery of A-803467 into the spinal cord reduced both evoked and spontaneous firing; therefore, this was the only site of action to mimic the effects of systemic A-803467. Previous studies have shown that heightened levels of WDR spontaneous firing can be modulated via spinal mechanisms in models of chronic pain (McGaraughty et al., 2006; Suzuki and Dickenson, 2006). Indeed, injection of lidocaine into spinal tissue almost eliminated the firing of hyperactive WDR neurons, but injection of lidocaine in the neuronal receptive field or onto a single DRG did not affect heightened ongoing activity (Sotgiu et al., 1992; McGaraughty et al., 2006). However, injection of lidocaine onto multiple DRGs (L4–L6) did partially reduce the firing of hyperactive WDR neurons (Sotgiu and Biella, 2000). Thus, blocking Nav1.8 sodium channels on central terminals in the spinal cord, possibly affecting input from multiple DRGs, decreases spontaneous firing of WDR neurons in SNL rats.
It is initially difficult to reconcile how A-803467 might affect the spontaneous firing of WDR neurons by acting only on central terminals and not the L4 DRG or peripheral terminals. One possibility is that intraspinal injections affect a broader range of inputs to WDR neurons than either of the peripheral injections. Intraspinal A-803467 will access afferent terminals in addition to those arising from the L4 DRG. In addition, orthodromic, ectopic input from the ligated nerves would have been minimally affected by intrareceptive field or L4 injection of A-803467 but could have been modulated by intraspinal injections. The delayed action of spinal A-803467 on spontaneous neuronal firing may reflect a necessary diffusion to affect additional central terminals or to indirectly trigger downstream mechanisms. Another possibility is that there are distinct functional consequences after blockade of either central presynaptic or peripheral Nav1.8 sodium channels. For example, in hippocampal mossy fibers, somatodendritic VGSCs are responsible for the propagation of action potentials, whereas presynaptic VGSCs amplify action potential amplitudes, leading to greater inflow of Ca2+ and transmitter release (Engel and Jonas, 2005). Thus, presynaptic VGSCs have a direct contribution to synaptic transmission. Finally, it cannot be excluded that the effects on spontaneous WDR firing were due to nonselective actions of intraspinally injected A-803467. This is a concern because high concentrations of the compound are used for this type of local injection. Nonetheless, spontaneous activity was also decreased by systemic A-803467 at plasma concentrations 1700-fold less than those in the intraspinal cannula. Although there are high concentrations of A-803467 in the cannula before injection, it is unlikely that this is the actual concentration interacting with channels after expulsion and diffusion of the very small volume through spinal tissue.
In summary, systemic injection of a selective Nav1.8 sodium channel blocker, A-803467, decreased both the mechanically evoked and spontaneous firing of spinal neurons in nerve-injured rats. This outcome is consistent with behavioral studies showing that A-803467 is effective against mechanical allodynia in animal models of neuropathic pain. The effect on evoked WDR firing seems to be mediated by Nav1.8 sodium channels located along the length of the primary afferent, including both central and peripheral terminals. The reduction in ongoing activity of WDR neurons seems to be through an action on central terminals and needs to be examined further to determine the exact mechanism. A-803467 represents an important tool to further understand the contribution of Nav1.8 sodium channels to normal and pathological nociception.
Footnotes
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.107.134148.
-
ABBREVIATIONS: VGSC, voltage-gated sodium channel; WDR, wide dynamic range; SNL, sciatic nerve ligation; DRG, dorsal root ganglion; TRPV1, transient receptor potential vanilloid type 1; LTM, low-threshold mechanoreceptor.
- Received November 9, 2007.
- Accepted December 17, 2007.
- The American Society for Pharmacology and Experimental Therapeutics