Cutaneous Dorsal Root Ganglion Neurons Research Articles

CXCR3 chemokine receptor signaling mediates itch in experimental allergic contact dermatitis.

Persistent itch is a common symptom of allergic contact dermatitis (ACD) and represents a significant health burden. The chemokine CXCL10 is predominantly produced by epithelial cells during ACD. Although the chemokine CXCL10 and its receptor CXCR3 are implicated in the pathophysiology of ACD, it is largely unexplored for itch and pain accompanying this disorder. Here, we showed that CXCL10 and CXCR3 mRNA, protein, and signaling activity were upregulated in the dorsal root ganglion after contact hypersensitivity (CHS), a murine model of ACD, induced by squaric acid dibutylester. CXCL10 directly activated a subset of cutaneous dorsal root ganglion neurons innervating the area of CHS through neuronal CXCR3. In behavioral tests, a CXCR3 antagonist attenuated spontaneous itch- but not pain-like behaviors directed to the site of CHS. Injection of CXCL10 into the site of CHS elicited site-directed itch- but not pain-like behaviors, but neither type of CXCL10-evoked behaviors was observed in control mice. These results suggest that CXCL10/CXCR3 signaling mediates allergic itch but not inflammatory pain in the context of skin inflammation. Thus, upregulation of CXCL10/CXCR3 signaling in sensory neurons may contribute to itch associated with ACD. Targeting the CXCL10/CXCR3 signaling might be beneficial for the treatment of allergic itch.

Inflammation-induced increase in nicotinic acetylcholine receptor current in cutaneous nociceptive DRG neurons from the adult rat

The goals of the present study were to determine (1) the properties of the nicotinic acetylcholine receptor (nAChR) currents in rat cutaneous dorsal root ganglion (DRG) neurons; (2) the impact of nAChR activation on the excitability of cutaneous DRG neurons; and (3) the impact of inflammation on the density and distribution of nAChR currents among cutaneous DRG neurons. Whole-cell patch-clamp techniques were used to study retrogradely labeled DRG neurons from naïve and complete Freund’s adjuvant inflamed rats. Nicotine-evoked currents were detectable in ∼70% of the cutaneous DRG neurons, where only one of two current types, fast or slow currents based on rates of activation and inactivation, was present in each neuron. The biophysical and pharmacological properties of the fast current were consistent with nAChRs containing an α7 subunit while those of the slow current were consistent with nAChRs containing α3/β4 subunits. The majority of small diameter neurons with fast current were IB4− while the majority of small diameter neurons with slow current were IB4+. Preincubation with nicotine (1μM) produced a transient (1min) depolarization and increase in the excitability of neurons with fast current and a decrease in the amplitude of capsaicin-evoked current in neurons with slow current. Inflammation increased the current density of both slow and fast currents in small diameter neurons and increased the percentage of neurons with the fast current. With the relatively selective distribution of nAChR currents in putative nociceptive cutaneous DRG neurons, our results suggest that the role of these receptors in inflammatory hyperalgesia is likely to be complex and dependent on the concentration and timing of acetylcholine release in the periphery.

Activity-dependent hyperpolarization of EGABA is absent in cutaneous DRG neurons from inflamed rats

A shift in GABAA signaling from inhibition to excitation in primary afferent neurons appears to contribute to the inflammation-induced increase in afferent input to the CNS. An activity-dependent depolarization of the GABAA current equilibrium potential (EGABA) has been described in CNS neurons which drives a shift in GABAA signaling from inhibition to excitation. The purpose of the present study was to determine if such an activity-dependent depolarization of EGABA occurs in primary afferents and whether the depolarization is amplified with persistent inflammation. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion (DRG) neurons from naïve and inflamed rats were studied with gramicidin perforated patch recording. Rather than a depolarization, 200 action potentials delivered at 2Hz resulted in a ∼10mV hyperpolarization of EGABA in cutaneous neurons from naïve rats. No such hyperpolarization was observed in neurons from inflamed rats. The shift in EGABA was not blocked by 10μM bumetanide. Furthermore, because activity-dependent hyperpolarization of EGABA was fully manifest in the absence of HCO3− in the bath solution, this shift was not dependent on a change in HCO3−–Cl− exchanger activity, despite evidence of HCO3−–Cl− exchangers in DRG neurons that may contribute to the establishment of EGABA in the presence of HCO3−. While the mechanism underlying the activity-dependent hyperpolarization of EGABA has yet to be identified, because this mechanism appears to function as a form of feedback inhibition, facilitating GABA-mediated inhibition of afferent activity, it may serve as a novel target for the treatment of inflammatory pain.

Contribution of endoplasmic reticulum Ca2+ regulatory mechanisms to the inflammation-induced increase in the evoked Ca2+ transient in rat cutaneous dorsal root ganglion neurons

Persistent inflammation results in an increase in the magnitude and duration of high K+-evoked Ca2+ transients in putative nociceptive cutaneous dorsal root ganglion (DRG) neurons. The purpose of the present study was to determine whether recruitment of Ca2+-induced Ca2+ release (CICR) contributes to these inflammation-induced changes. Acutely dissociated, retrogradely labeled cutaneous DRG neurons from naïve and complete Freund's adjuvant inflamed adult male Sprague–Dawley rats were studied with ratiometric microfluorimetry. Ryanodine only attenuated the duration but not magnitude of the high K+-evoked Ca2+ transient in neurons from inflamed rats. However, there was no significant impact of inflammation on the potency or efficacy of ryanodine-induced block of the caffeine-evoked Ca2+ transient, or the impact of sarco-endoplasmic reticulum ATPase (SERCA) inhibition on the high K+-evoked Ca2+ transient. Furthermore, while there was no change in the magnitude, an inflammation-induced increase in the duration of the caffeine-evoked Ca2+ transient was only observed with a prolonged caffeine application. In contrast to the high K+-evoked Ca2+ transient, there was no evidence of direct mitochondrial involvement or that of the Ca2+ extrusion mechanism, the Na+/Ca2+ exchanger, on the caffeine-evoked Ca2+ transient, and block of SERCA only increased the duration of this transient. These results indicate the presence of Ca2+ regulatory domains in cutaneous nociceptive DRG neurons within which cytosolic Ca2+ increased via influx and release are highly segregated. Furthermore, our results suggest that changes in neither CICR machinery nor the coupling between Ca2+ influx and CICR are primarily responsible for the inflammation-induced changes in the evoked Ca2+ transient.

Persistent inflammation increases GABA-induced depolarization of rat cutaneous dorsal root ganglion neurons in vitro

Persistent inflammation is associated with a shift in spinal GABAA signaling from inhibition to excitation such that GABAA-receptor activation contributes to inflammatory hyperalgesia. We tested the hypothesis that the primary afferent is the site of the persistent inflammation-induced shift in GABAA signaling which is due to a Na+–K+–Cl−-co-transporter (NKCC1)-dependent depolarization of the GABAA current equilibrium potential (EGABA). Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion (DRG) neurons from naïve and inflamed (3days after a subcutaneous injection of complete Freund’s adjuvant) adult male rats were studied with Ca2+ imaging, western blot and gramicidin-perforated patch recording. GABA evoked a Ca2+ transient in a subpopulation of small- to medium-diameter capsaicin-sensitive cutaneous neurons. Inflammation was associated with a significant increase in the magnitude of GABA-induced depolarization as well as the percentage of neurons in which GABA evoked a Ca2+ transient. There was no detectable change in NKCC1 protein or phosphoprotein at the whole ganglia level. Furthermore, the increase in excitatory response was comparable in both HEPES- and HCO3--buffered solutions, but was only associated with a depolarization of EGABA in HCO3--based solution. In contrast, under both recording conditions, the excitatory response was associated with an increase in GABAA current density, a decrease in low threshold K+ current density, and resting membrane potential depolarization. Our results suggest that increasing K+ conductance in afferents innervating a site of persistent inflammation may have greater efficacy in the inhibition of inflammatory hyperalgesia than attempting to drive a hyperpolarizing shift in EGABA.

Inflammation-Induced Changes in BKCa Currents in Cutaneous Dorsal Root Ganglion Neurons from the Adult Rat

BackgroundInflammation-induced sensitization of primary afferents is associated with a decrease in K+ current. However, the type of K+ current and basis for the decrease varies as a function of target of innervation. Because glabrous skin of the rat hindpaw is used often to assess changes in nociception in models of persistent pain, the purpose of the present study was to determine the type and extent to which K+ currents contribute to the inflammation-induced sensitization of cutaneous afferents. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion neurons from naïve and inflamed (3 days post complete Freund’s adjuvant injection) rats were studied with whole cell and perforated patch techniques.ResultsInflammation-induced sensitization of small diameter cutaneous neurons was associated with an increase in action potential duration and rate of decay of the afterhyperpolarization. However, no changes in voltage-gated K+ currents were detected. In contrast, Ca2+ modulated iberiotoxin sensitive and paxilline sensitive K+ (BKCa) currents were significantly smaller in small diameter IB4+ neurons. This decrease in current was not associated with a detectable change in total protein levels of the BKCa channel α or β subunits. Single cell PCR analysis revealed a significant change in the pattern of expression of α subunit splice variants and β subunits that were consistent, at least in part, with inflammation-induced changes in the biophysical properties of BKCa currents in cutaneous neurons.ConclusionsResults of this study provide additional support for the conclusion that it may be possible, if not necessary to selectively treat pain arising from specific body regions. Because a decrease in BKCa current appears to contribute to the inflammation-induced sensitization of cutaneous afferents, BKCa channel openers may be effective for the treatment of inflammatory pain.

ASIC3, a sensor of acidic and primary inflammatory pain

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular acidosis that are expressed in both central and peripheral nervous systems. Although peripheral ASICs seem to be natural sensors of acidic pain (e.g., in inflammation, ischaemia, lesions or tumours), a direct demonstration is still lacking. We show that approximately 60% of rat cutaneous sensory neurons express ASIC3-like currents. Native as well as recombinant ASIC3 respond synergistically to three different inflammatory signals that are slight acidifications (approximately pH 7.0), hypertonicity and arachidonic acid (AA). Moderate pH, alone or in combination with hypertonicity and AA, increases nociceptors excitability and produces pain suppressed by the toxin APETx2, a specific blocker of ASIC3. Both APETx2 and the in vivo knockdown of ASIC3 with a specific siRNA also have potent analgesic effects against primary inflammation-induced hyperalgesia in rat. Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Survival and regeneration of cutaneous and muscular afferent neurons after peripheral nerve injury in adult rats

Peripheral nerve injury induces the retrograde degeneration of dorsal root ganglion (DRG) cells, which affects predominantly the small-diameter cutaneous afferent neurons. This study compares the time-course of retrograde cell death in cutaneous and muscular DRG cells after peripheral nerve transection as well as neuronal survival and axonal regeneration after primary repair or nerve grafting. For comparison, spinal motoneurons were also included in the study. Sural and medial gastrocnemius DRG neurons were retrogradely labeled with the fluorescent tracers Fast Blue (FB) or Fluoro-Gold (FG) from the homonymous transected nerves. Survival of labeled sural and gastrocnemius DRG cells was assessed at 3 days and 1-24 weeks after axotomy. To evaluate axonal regeneration, the sciatic nerve was transected proximally at 1 week after FB-labeling of the sural and medial gastrocnemius nerves and immediately reconstructed using primary repair or autologous nerve grafting. Twelve weeks later, the fluorescent tracer Fluoro-Ruby (FR) was applied 10 mm distal to the sciatic lesion in order to double-label sural and gastrocnemius neurons that had regenerated across the repair site. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. In contrast, sural axotomy induced a delayed loss of sural DRG cells, which amounted to 22% at 4 weeks and 43-48% at 8-24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after injury to the sural or gastrocnemius nerves neither further increased retrograde DRG degeneration, nor did it affect survival of sural or gastrocnemius motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53-60% of the spinal motoneurons and 47-49% of the muscular DRG neurons at 13 weeks postoperatively. In the cutaneous DRG neurons, primary repair or peripheral nerve grafting increased survival by 19-30% and promoted regeneration of 46-66% of the cells. The present results suggest that cutaneous DRG neurons are more sensitive to peripheral nerve injury than muscular DRG cells, but that their regenerative capacity does not differ from that of the latter cells. However, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit the recovery of cutaneous sensory functions.

Alpha-SNS produces the slow TTX-resistant sodium current in large cutaneous afferent DRG neurons.

In this study, we used sensory neuron specific (SNS) sodium channel gene knockout (-/-) mice to ask whether SNS sodium channel produces the slow Na(+) current ("slow") in large (>40 microm diam) cutaneous afferent dorsal root ganglion (DRG) neurons. SNS wild-type (+/+) mice were used as controls. Retrograde Fluoro-Gold labeling permitted the definitive identification of cutaneous afferent neurons. Prepulse inactivation was used to separate the fast and slow Na(+) currents. Fifty-two percent of the large cutaneous afferent neurons isolated from SNS (+/+) mice expressed only fast-inactivating Na(+) currents ("fast"), and 48% expressed both fast and slow Na(+) currents. The fast and slow current densities were 0.90 +/- 0.12 and 0.39 +/- 0.16 nA/pF, respectively. Fast Na(+) currents were blocked completely by 300 nM tetrodotoxin (TTX), while slow Na(+) currents were resistant to 300 nM TTX, confirming that the slow Na(+) currents observed in large cutaneous DRG neurons are TTX-resistant (TTX-R). Slow Na(+) currents could not be detected in large cutaneous afferent neurons from SNS (-/-) mice; these cells expressed only fast Na(+) current, and it was blocked by 300 nM TTX. The fast Na(+) current density in SNS (-/-) neurons was 1.47 +/- 0. 14 nA/pF, approximately 60% higher than the current density observed in SNS (+/+) mice (P < 0.02). A low-voltage-activated TTX-R Na(+) current ("persistent") observed in small C-type neurons is not present in large cutaneous afferent neurons from either SNS (+/+) or SNS (-/-) mice. These results show that the slow TTX-R Na(+) current in large cutaneous afferent DRG is produced by the SNS sodium channel.

Calbindin D28k-containing splanchnic and cutaneous dorsal root ganglion neurons of the rat

Calbindin D28k (CaBP)-containing splanchnic and cutaneous sensory neurons in the rat dorsal root ganglia (DRGs) were investigated immunocytochemically in combination with a fluorescent dye tracer (Fluoro gold). About 15% of the DRG neurons at Th 9–10 levels showed CaBP-like immunoreactivity. Eighty-four% of the splanchnic sensory neurons were immunoreactive to CaBP, while only 3% of the cutaneous sensory neurons were. The diameters of the splanchnic and cutaneous sensory neurons containing CaBP were 23.4 ± 6.3 μm and 38.4 ± 8.8 μm, respectively. Splanchnic sensory neurons containing CaBP were sensitive to capsaicin while cutaneous ones were not. These findings suggest that CaBP-containing splanchnic and cutaneous sensory neurons constitute different subgroups among the DRG neurons at the lower thoracic level.