Regulation of Mitochondrial Function by Epac2 Contributes to Acute Inflammatory Hyperalgesia.

Gαs-coupled receptors signaling through cAMP provide a key mechanism for the sensitization of nociceptive sensory neurons, and the cAMP effector Epac has been implicated in the transition from acute to chronic pain. Epac exerts its effects through Rap1 and protein kinase C (PKC). To identify targets of Epac-PKC signaling in sensory neurons of the mouse dorsal root ganglion (DRG), we profiled PKC substrate proteins phosphorylated in response to the activation of Epac with the proinflammatory prostaglandin E2 (PGE2). A prominent Epac-dependent phospho-protein band induced by PGE2 was identified by mass spectrometry as the mitochondrial enzyme pyruvate dehydrogenase (Pdha1). In dissociated DRG from both males and females, the recruitment of Pdha1 to phospho-protein fractions was rapidly induced by PGE2 and prevented by selective inhibition of Epac2. Epac activation increased mitochondrial respiration, consistent with an increase in Pdha1 function mediated by Epac2. Hindpaw injection of PGE2 induced heat hyperalgesia in males and females, but Pdha1 phosphorylation occurred only in males. Hyperalgesia was attenuated in males but not in females by systemic inhibition of Epac2, and also by a mitochondrial membrane potential uncoupler, dinitrophenol, supporting a role for mitochondrial regulation in acute hyperalgesia. These findings identify a mechanism for the regulation of mitochondrial function by Epac2 that contributes to acute inflammatory hyperalgesia in male mice. Systemic administration of the cyclooxygenase 2 inhibitor celecoxib suppressed both PGE2-induced heat hyperalgesia and Pdha1 phosphorylation in DRG of males but not females, suggesting that prostaglandin synthesis within the DRG mediates the phosphorylation of Pdha1 in response to hindpaw insult.SIGNIFICANCE STATEMENT There has been extensive investigation of mitochondrial dysfunction as a causative factor in neuropathic pain disorders. In contrast, results reported here implicate enhanced mitochondrial function as a contributing factor in the development of acute inflammatory hyperalgesia. We describe a mechanism in which Epac2 activation by prostaglandin receptors leads to phosphorylation of pyruvate dehydrogenase and an increase in mitochondrial respiration in peripheral sensory neurons. Although Epac2 activation leads to Pdha1 (pyruvate dehydrogenase) phosphorylation in dissociated neurons from mice of both sexes, induction of this pathway in vivo by hindpaw insult is restricted to males and appears to require intraganglionic prostaglandin synthesis. These findings support a model in which Gs-coupled receptor modulation of mitochondrial function promotes acute nociceptive signaling and inflammatory hyperalgesia.

Phospho-substrate profiling of Epac-dependent protein kinase C activity.

Exchange protein directly activated by cAMP (Epac) and protein kinase A are effectors for cAMP with distinct actions and regulatory mechanisms. Epac is a Rap guanine nucleotide exchange factor that activates Rap1; protein kinase C (PKC) is a major downstream target of Epac-Rap1 signaling that has been implicated in a variety of pathophysiological processes, including cardiac hypertrophy, cancer, and nociceptor sensitization leading to chronic pain. Despite the implication of both Epac and PKC in these processes, few downstream targets of Epac-PKC signaling have been identified. This study characterized the regulation of PKC activity downstream of Epac activation. Using an antibody that recognizes phospho-serine residues within the consensus sequence phosphorylated by PKC, we analyzed the 1-dimensional banding profile of PKC substrate protein phosphorylation from the Neuro2A mouse neuroblastoma cell line. Activation of Epac either indirectly by prostaglandin PGE2, or directly by 8-pCPT-2-O-Me-cAMP-AM (8pCpt), produced distinct PKC phospho-substrate protein bands that were suppressed by co-administration of the Epac inhibitor ESI09. Different PKC isoforms contributed to the induction of individual phospho-substrate bands, as determined using isoform-selective PKC inhibitors. Moreover, the banding profile after Epac activation was altered by disruption of the cytoskeleton, suggesting that the orchestration of Epac-dependent PKC signaling is regulated in part by interactions with the cytoskeleton. The approach described here provides an effective means to characterize Epac-dependent PKC activity.

Neurturin overexpression in skin enhances expression of TRPM8 in cutaneous sensory neurons and leads to behavioral sensitivity to cool and menthol.

Neurturin (NRTN) is a member of the glial cell line-derived neurotrophic factor family of ligands that exerts its actions via Ret tyrosine kinase and GFRα2. Expression of the Ret-GFRα2 coreceptor complex is primarily restricted to the peripheral nervous system and is selectively expressed by sensory neurons that bind the isolectin B(4) (IB(4)). To determine how target-derived NRTN affects sensory neuron properties, transgenic mice that overexpress NRTN in keratinocytes (NRTN-OE mice) were analyzed. Overexpression of NRTN increased the density of PGP9.5-positive, but not calcitonin gene-related peptide-positive, free nerve endings in footpad epidermis. GFRα2-immunopositive somata were hypertrophied in NRTN-OE mice. Electron microscopic analysis further revealed hypertrophy of unmyelinated sensory axons and a subset of myelinated axons. Overexpression of NRTN increased the relative level of mRNAs encoding GFRα2 and Ret, the ATP receptor P2X(3) (found in IB(4)-positive, GFRα2-expressing sensory neurons), the acid-sensing ion channel 2a, and transient receptor potential cation channel subfamily member M8 (TRPM8) in sensory ganglia. Behavioral testing of NRTN-OE mice revealed an increased sensitivity to mechanical stimuli in glabrous skin of the hindpaw. NRTN-OE mice also displayed increased behavioral sensitivity to cool temperature (17°C-20°C) and oral sensitivity to menthol. The increase in cool and menthol sensitivity correlated with a significant increase in TRPM8 expression and the percentage of menthol-responsive cutaneous sensory neurons. These data indicate that the expression level of NRTN in the skin modulates gene expression in cutaneous sensory afferents and behavioral sensitivity to thermal, chemical, and mechanical stimuli.

Members of the CUGBP Elav-like family of RNA-binding proteins are expressed in distinct populations of primary sensory neurons.

Primary sensory dorsal root ganglia (DRG) neurons are diverse, with distinct populations that respond to specific stimuli. Previously, we observed that functionally distinct populations of DRG neurons express mRNA transcript variants with different 3' untranslated regions (3'UTRs). 3'UTRs harbor binding sites for interaction with RNA-binding proteins (RBPs) for transporting mRNAs to subcellular domains, modulating transcript stability, and regulating the rate of translation. In the current study, analysis of publicly available single-cell RNA-sequencing data generated from adult mice revealed that 17 3'UTR-binding RBPs were enriched in specific populations of DRG neurons. This included four members of the CUG triplet repeat (CUGBP) Elav-like family (CELF): CELF2 and CELF4 were enriched in peptidergic, CELF6 in both peptidergic and nonpeptidergic, and CELF3 in tyrosine hydroxylase-expressing neurons. Immunofluorescence studies confirmed that 60% of CELF4+ neurons are small-diameter C fibers and 33% medium-diameter myelinated (likely Aδ) fibers and showed that CELF4 is distributed to peripheral termini. Coexpression analyses using transcriptomic data and immunofluorescence revealed that CELF4 is enriched in nociceptive neurons that express GFRA3, CGRP, and the capsaicin receptor TRPV1. Reanalysis of published transcriptomic data from macaque DRG revealed a highly similar distribution of CELF members, and reanalysis of single-nucleus RNA-sequencing data derived from mouse and rat DRG after sciatic injury revealed differential expression of CELFs in specific populations of sensory neurons. We propose that CELF RBPs may regulate the fate of mRNAs in populations of nociceptors, and may play a role in pain and/or neuronal regeneration following nerve injury.

The P2Y2 receptor sensitizes mouse bladder sensory neurons and facilitates purinergic currents.

Sensitization of bladder afferents is an underlying contributor to the development and maintenance of painful bladder syndrome/interstitial cystitis. Extracellular purines and pyrimidines (e.g., ATP and UTP), released during bladder distension or from damaged cells after tissue insult, are thought to play an important role in bladder physiological and pathological states by actions at ionotropic P2X and metabotropic P2Y receptors. In the present study, we examined the ability of P2Y receptors to sensitize and modulate P2X-mediated responses in mouse bladder sensory neurons. UTP (a P2Y(2) and P2Y(4) agonist) increased excitability of bladder neurons by depolarizing resting membrane potential, increasing action potential firing, and facilitating responses to suprathreshold current injection as well as to P2X agonist application. These effects of UTP on bladder neuron excitability were blocked by the P2Y(2) receptor antagonist suramin. UTP also facilitated bladder neuron homomeric P2X(2) sustained currents and homomeric P2X(3) fast currents. The facilitatory effect of UTP on P2X(2) sustained currents was mediated by a G-protein-coupled P2Y(2) receptor/PKC pathway, whereas the effect of UTP on P2X(3) fast currents was G-protein independent. We also examined P2X and P2Y receptor expression in bladder neurons. P2Y(2) and P2Y(4) transcripts were detected in approximately 50 and approximately 20% of bladder neurons, respectively. Approximately 50% of P2X(2)- and P2X(3)-positive bladder neurons expressed P2Y(2) transcripts, whereas < or =25% of the same bladder neurons expressed P2Y(4) transcripts. These results support involvement of P2Y(2) receptors in bladder sensation, suggesting an important contribution to bladder neuron excitability and hypersensitivity.

The ADP receptor P2Y1 is necessary for normal thermal sensitivity in cutaneous polymodal nociceptors.

BACKGROUND: P2Y1 is a member of the P2Y family of G protein-coupled nucleotide receptors expressed in peripheral sensory neurons. Using ratiometric calcium imaging of isolated dorsal root ganglion neurons, we found that the majority of neurons responding to adenosine diphosphate, the preferred endogenous ligand, bound the lectin IB4 and expressed the ATP-gated ion channel P2X3. These neurons represent the majority of epidermal afferents in hairy skin, and are predominantly C-fiber polymodal nociceptors (CPMs), responding to mechanical stimulation, heat and in some cases cold. RESULTS: To characterize the function of P2Y1 in cutaneous afferents, intracellular recordings from sensory neuron somata were made using an ex vivo preparation in which the hindlimb skin, saphenous nerve, DRG and spinal cord were dissected in continuum, and cutaneous receptive fields characterized using digitally-controlled mechanical and thermal stimuli in male wild type mice. In P2Y1-/- mice, CPMs showed a striking increase in mean heat threshold and a decrease in mean peak firing rate during a thermal ramp from 31-52°C. A similar change in mean cold threshold was also observed. Interestingly, mechanical testing of CPMs revealed no significant differences between P2Y1-/- and WT mice. CONCLUSIONS: These results strongly suggest that P2Y1 is required for normal thermal signaling in cutaneous sensory afferents. Furthermore, they suggest that nucleotides released from peripheral tissues play a critical role in the transduction of thermal stimuli in some fiber types.

Gi- and Gq-coupled ADP (P2Y) receptors act in opposition to modulate nociceptive signaling and inflammatory pain behavior.

BACKGROUND: Investigations of nucleotide signaling in nociception to date have focused on actions of adenosine triphosphate (ATP). Both ATP-gated ion channels (P2X receptors) and G protein-coupled (P2Y) receptors contribute to nociceptive signaling in peripheral sensory neurons. In addition, several studies have implicated the Gq-coupled adenosine diphosphate (ADP) receptor P2Y1 in sensory transduction. In this study, we examined the expression and function of P2Y1 and the Gi-coupled receptors P2Y12, P2Y13 and P2Y14 in sensory neurons to determine their contribution to nociception. RESULTS: We detected mRNA and protein for ADP receptors P2Y12 and P2Y13 in mouse dorsal root ganglia (DRG). P2Y14, a homologous Gi-coupled nucleotide receptor, is also expressed in DRG. Immunohistochemical analysis of receptor distribution indicated that these receptors are widely expressed in nociceptive neurons. Using ratiometric calcium imaging, we found that ADP evokes increases in intracellular calcium in isolated DRG neurons and also produces a pertussis toxin-sensitive inhibition of depolarization-evoked calcium transients. The inhibitory effect of ADP was unaltered in the presence of the selective P2Y1 antagonist MRS2179 and in neurons isolated from P2Y1 knockout mice, whereas ADP-evoked calcium transients were greatly reduced. Analysis of behavioral responses to noxious heat before and after inflammatory injury (injection of complete Freund's adjuvant into the hindpaw) revealed that P2Y1 is required for the full expression of inflammatory hyperalgesia, whereas local injection of agonists for Gi-coupled P2Y receptors reduced hyperalgesia. CONCLUSIONS: We report that Gi-coupled P2Y receptors are widely expressed in peripheral sensory neurons. Agonists for these receptors inhibit nociceptive signaling in isolated neurons and reduce behavioral hyperalgesia in vivo. Anti-nociceptive actions of these receptors appear to be antagonized by the Gq-coupled ADP receptor, P2Y1, which is required for the full expression of inflammatory hyperalgesia. We propose that nociceptor sensitivity is modulated by the integration of nucleotide signaling through Gq- and Gi-coupled P2Y receptors, and this balance is altered in response to inflammatory injury. Taken together, our data suggest that Gi-coupled P2Y receptors are broadly expressed in nociceptors, inhibit nociceptive signaling in vivo, and represent potential targets for the development of novel analgesic drugs.

A Novel Mechanism for Zika Virus Host-Cell Binding.

Zika virus (ZIKV) recently emerged in the Western Hemisphere with previously unrecognized or unreported clinical presentations. Here, we identify two putative binding mechanisms of ancestral and emergent ZIKV strains featuring the envelope (E) protein residue asparagine 154 (ASN154) and viral phosphatidylserine (PS). Synthetic peptides representing the region containing ASN154 from strains PRVABC59 (Puerto Rico 2015) and MR_766 (Uganda 1947) were exposed to neuronal cells and fibroblasts to model ZIKV E protein/cell interactions and bound MDCK or Vero cells and primary neurons significantly. Peptides significantly inhibited Vero cell infectivity by ZIKV strains MR_766 and PRVABC59, indicating that this region represents a putative binding mechanism of ancestral African ZIKV strains and emergent Western Hemisphere strains. Pretreatment of ZIKV strains MR_766 and PRVABC59 with the PS-binding protein annexin V significantly inhibited replication of PRVABC59 but not MR_766, suggesting that Western hemisphere strains may additionally be capable of utilizing PS-mediated entry to infect host cells. These data indicate that the region surrounding E protein ASN154 is capable of binding fibroblasts and primary neuronal cells and that PS-mediated entry may be a secondary mechanism for infectivity utilized by Western Hemisphere strains.

Glial cell line-derived neurotrophic factor family members sensitize nociceptors in vitro and produce thermal hyperalgesia in vivo.

Nerve growth factor (NGF) has been implicated as an effector of inflammatory pain because it sensitizes primary afferents to noxious thermal, mechanical, and chemical [e.g., capsaicin, a transient receptor potential vanilloid receptor 1 (TRPV1) agonist] stimuli and because NGF levels increase during inflammation. Here, we report the ability of glial cell line-derived neurotrophic factor (GDNF) family members artemin, neurturin and GDNF to potentiate TRPV1 signaling and to induce behavioral hyperalgesia. Analysis of capsaicin-evoked Ca2+ transients in dissociated mouse dorsal root ganglion (DRG) neurons revealed that a 7 min exposure to GDNF, neurturin, or artemin potentiated TRPV1 function at doses 10-100 times lower than NGF. Moreover, GDNF family members induced capsaicin responses in a subset of neurons that were previously insensitive to capsaicin. Using reverse transcriptase-PCR, we found that artemin mRNA was profoundly upregulated in response to inflammation induced by hindpaw injection of complete Freund's adjuvant (CFA): artemin expression increased 10-fold 1 d after CFA injection, whereas NGF expression doubled by day 7. No increase was seen in neurturin or GDNF. A corresponding increase in mRNA for the artemin coreceptor GFRalpha3 (for GDNF family receptor alpha) was seen in DRG, and GFRalpha3 immunoreactivity was widely colocalized with TRPV1 in epidermal afferents. Finally, hindpaw injection of artemin, neurturin, GDNF, or NGF produced acute thermal hyperalgesia that lasted up to 4 h; combined injection of artemin and NGF produced hyperalgesia that lasted for 6 d. These results indicate that GDNF family members regulate the sensitivity of thermal nociceptors and implicate artemin in particular as an important effector in inflammatory hyperalgesia.

Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.

Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRalpha3 and the receptor tyrosine kinase Ret. Expression of the GFRalpha3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRalpha3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRalpha3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.

Deletion of the murine ATP/UTP receptor P2Y2 alters mechanical and thermal response properties in polymodal cutaneous afferents.

P2Y2 is a member of the P2Y family of G protein-coupled nucleotide receptors that is widely co-expressed with TRPV1 in peripheral sensory neurons of the dorsal root ganglia. To characterize P2Y2 function in cutaneous afferents, intracellular recordings from mouse sensory neurons were made using an ex vivo preparation in which hindlimb skin, saphenous nerve, dorsal root ganglia and spinal cord are dissected intact. The peripheral response properties of individual cutaneous C-fibers were analyzed using digitally controlled mechanical and thermal stimuli in male P2Y2(+/+) and P2Y2(-/-) mice. Selected sensory neurons were labeled with Neurobiotin and further characterized by immunohistochemistry. In wildtype preparations, C-fibers responding to both mechanical and thermal stimuli (CMH or CMHC) preferentially bound the lectin marker IB4 and were always immunonegative for TRPV1. Conversely, cells that fired robustly to noxious heat, but were insensitive to mechanical stimuli, were TRPV1-positive and IB4-negative. P2Y2 gene deletion resulted in reduced firing by TRPV1-negative CMH fibers to a range of heat stimuli. However, we also identified an atypical population of IB4-negative, TRPV1-positive CMH fibers. Compared to wildtype CMH fibers, these TRPV1-positive neurons exhibited lower firing rates in response to mechanical stimulation, but had increased firing to noxious heat (43-51°C). Collectively, these results demonstrate that P2Y2 contributes to response properties of cutaneous afferents, as P2Y2 deletion reduces responsiveness of conventional unmyelinated polymodal afferents to heat and appears to result in the acquisition of mechanical responsiveness in a subset of TRPV1-expressing afferents.

ASIC3, an acid-sensing ion channel, is expressed in metaboreceptive sensory neurons.

BACKGROUND: ASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1) ASIC3 might trigger ischemic pain in heart and muscle; 2) it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses. RESULTS: Less than half (40%) of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small (<25 microm) sensory neurons that innervate muscle are more likely to express ASIC3 than those that innervate skin (51% of small muscle afferents vs. 28% of small skin afferents). Over 80% of ASIC3+ muscle afferents co-express CGRP (a vasodilatory peptide). Remarkably few (9%) ASIC3+ cells express P2X3 receptors (an ATP-gated ion channel), whereas 31% express TRPV1 (the noxious heat and capsaicin-activated ion channel also known as VR1). ASIC3+/CGRP+ sensory nerve endings were observed on muscle arterioles, the blood vessels that control vascular resistance; like the cell bodies, the endings are P2X3- and can be TRPV1+. The TrkC+/ASIC3+ cell bodies are uniformly large, possibly consistent with non-nociceptive mechanosensation. They are not proprioceptors because they fail two other tests: ASIC3+ cells do not express parvalbumin and they are absent from the mesencephalic trigeminal nucleus. CONCLUSION: Our data indicates that: 1) ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2) co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen.

Overexpression of NGF or GDNF alters transcriptional plasticity evoked by inflammation.

Transcriptional changes evoked in nociceptive sensory neurons by inflammatory injury play a substantial role in the generation of and recovery from painful hypersensitivity. Transgenic mice overexpressing nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) in the skin possess a greatly increased number of nociceptors. Surprisingly, NGF-overexpressers display reduced hypersensitivity and recovered more rapidly in response to inflammation, suggesting a compensatory suppression of nociceptive transmission in these mice. To determine whether these transgenic mice show changes in inflammation-evoked transcriptional plasticity, we examined the expression of a panel of genes implicated in nociceptive signaling in response to injection of complete Freund's adjuvant into the hindpaw. Relative mRNA levels were quantified 1, 4 and 15 days after injection using real-time PCR. In wild type mice CFA injection elicited a reproducible pattern of altered gene expression that returned to baseline over a 2-week period. In mice overexpressing NGF or GDNF the expression patterns for several genes were substantially altered; these changes in injury-evoked patterns of gene expression suggest the existence of endogenous regulatory mechanisms that can compensate for increased nociceptive input by modulating the expression of a limited subset of genes.

The P2Y agonist UTP activates cutaneous afferent fibers.

The majority of adenosine triphosphate (ATP)-induced nociceptive transduction and pain has been attributed to ionotropic P2X3 receptors. Metabotropic P2Y receptors, some of which bind pyrimidines as well as purines, have received little attention. Here we have examined the ability of P2Y receptor signaling to evoke action potential firing in functionally identified afferent fibers using the skin nerve preparation from adult mouse. The P2Y2/P2Y4 ligand UTP activated sustained action potential firing in 54% of C fibers in a concentration-dependent manner. The effect was specific for P2Y2/P2Y4 receptors, as the P2Y6 ligand UDP never activated C fibers. In comparison to C fibers, few thinly myelinated A-mechanoreceptors (AM) (12%) were activated by UTP. The majority (70-80%) of the UTP-sensitive C and Adelta fibers responded to the algogen capsaicin with a barrage of action potentials, whereas the UTP-insensitive fibers were largely unresponsive to capsaicin. Furthermore, 86% of the UTP-sensitive C fibers and 100% of the UTP-sensitive AM fibers also responded to the P2X agonist alpha,beta-methylene ATP, indicating that P2Y and P2X receptors are widely co-expressed. Surprisingly, a significant proportion (20-40%) of low threshold slowly and rapidly adapting Abeta fibers were also activated by UTP and alpha,beta-methylene ATP. These data indicate that P2Y receptors on the terminals of capsaicin-sensitive cutaneous sensory neurons effectively evoke nociceptive transmission, and support the hypothesis that UTP may be an endogenous nociceptive messenger. Furthermore, P2Y signaling may contribute to mechanotransduction in low threshold Abeta fibers under normal or pathological conditions.

Transgenic mice possessing increased numbers of nociceptors do not exhibit increased behavioral sensitivity in models of inflammatory and neuropathic pain.

At least two classes of neciceptors can be distinguished based on their growth factor requirements: glial cell-line derived neurotrophic factor (GDNF)- and nerve growth factor (NGF)-dependent primary afferent neurons. Based on numerous anatomical and biochemical differences, GDNF- and NGF-dependent neurons have been proposed to be involved in the development of different types of persistent pain. To examine this hypothesis we used two lines of transgenic mice that contained a supernormal number of either NGF- or GDNF-dependent neurons (referred to as NGF-OE and GDNF-OE mice, respectively). These mice were tested in a model of inflammatory pain (induced by injection of complete Freund's adjuvant) and neuropathic pain (using a spinal nerve ligation protocol). Contrary to expectations, neither line of transgenic mice became more hyperalgesic following induction of persistent pain. In fact, NGF-OE mice recovered more rapidly and became hypoalgesic despite extensive paw swelling in the inflammatory pain model. In the neuropathic pain model, only wildtype mice became hyperalgesic. Real-time PCR analysis showed that the NGF-OE and GDNF-OE mice exhibited changes in neuronal-specific mRNAs in the dorsal root ganglia but not the spinal cord dorsal horn. These results indicate that increasing the number of nociceptors results in potent compensatory mechanisms that may begin with changes in the sensory neurons themselves.

Purinergic receptor P2Y1 regulates polymodal C-fiber thermal thresholds and sensory neuron phenotypic switching during peripheral inflammation.

We have recently found that, following complete Freund's adjuvant (CFA)-induced inflammation, cutaneous polymodal nociceptors (CPM) lacking the transient receptor potential vanilloid 1 (TRPV1) are sensitized to heat stimuli. In order to determine possible mechanisms playing a role in this change, we examined gene expression in the L2/L3 sensory ganglia following CFA injection into the hairy hind paw skin and found that G-protein-coupled purinoreceptor P2Y1 expression was increased. This receptor is of particular interest, as most CPMs innervating mouse hairy skin bind isolectin B4, which co-localizes with P2Y1. Additionally, our recent findings have shown that cutaneous CPMs in P2Y1-/- mice displayed significantly reduced thermal sensitivity. Together, these findings suggested a possible role for P2Y1 in inflammation-induced heat sensitization in these fibers. To test this hypothesis, we utilized our in vivo small interfering RNA technique to knock down the inflammation-induced increase in P2Y1 expression and then examined the functional effects using ex vivo recording. We found that the normal reduction of heat thresholds in CPM fibers induced by CFA was completely blocked by inhibition of P2Y1. Surprisingly, inhibition of P2Y1 during inflammation also significantly increased the number of CPM neurons expressing TRPV1 without a change in the total number of TRPV1-positive cells in the L2 and L3 dorsal root ganglia. These results show that the inflammation-induced enhanced expression of P2Y1 is required for normal heat sensitization of cutaneous CPM fibers. They also suggest that P2Y1 plays a role in the maintenance of phenotype in cutaneous afferent fibers containing TRPV1.

Phenotypic switching of nonpeptidergic cutaneous sensory neurons following peripheral nerve injury.

In adult mammals, the phenotype of half of all pain-sensing (nociceptive) sensory neurons is tonically modulated by growth factors in the glial cell line-derived neurotrophic factor (GDNF) family that includes GDNF, artemin (ARTN) and neurturin (NRTN). Each family member binds a distinct GFRα family co-receptor, such that GDNF, NRTN and ARTN bind GFRα1, -α2, and -α3, respectively. Previous studies revealed transcriptional regulation of all three receptors in following axotomy, possibly in response to changes in growth factor availability. Here, we examined changes in the expression of GFRα1-3 in response to injury in vivo and in vitro. We found that after dissociation of adult sensory ganglia, up to 27% of neurons die within 4 days (d) in culture and this can be prevented by nerve growth factor (NGF), GDNF and ARTN, but not NRTN. Moreover, up-regulation of ATF3 (a marker of neuronal injury) in vitro could be prevented by NGF and ARTN, but not by GDNF or NRTN. The lack of NRTN efficacy was correlated with rapid and near-complete loss of GFRα2 immunoreactivity. By retrogradely-labeling cutaneous afferents in vivo prior to nerve cut, we demonstrated that GFRα2-positive neurons switch phenotype following injury and begin to express GFRα3 as well as the capsaicin receptor, transient receptor potential vanilloid 1(TRPV1), an important transducer of noxious stimuli. This switch was correlated with down-regulation of Runt-related transcription factor 1 (Runx1), a transcription factor that controls expression of GFRα2 and TRPV1 during development. These studies show that NRTN-responsive neurons are unique with respect to their plasticity and response to injury, and suggest that Runx1 plays an ongoing modulatory role in the adult.

In search of analgesia: emerging roles of GPCRs in pain.

Of all clinically marketed drugs, greater than thirty percent are modulators of G protein-coupled receptors (GPCRs). Nearly 400 GPCRs (i.e., excluding odorant and light receptors) are encoded within the human genome, but only a small fraction of these seven-transmembrane proteins have been identified as drug targets. Chronic pain affects more than one-third of the population, representing a substantial societal burden in use of health care resources and lost productivity. Furthermore, currently available treatments are often inadequate, underscoring the significant need for better therapeutic strategies. The expansion of the identified human GPCR repertoire, coupled with recent insights into the function and structure of GPCRs, offers new opportunities for the development of novel analgesic therapeutics.

Thermal nociception and TRPV1 function are attenuated in mice lacking the nucleotide receptor P2Y2.

Recent studies indicate that ATP and UTP act at G protein-coupled (P2Y) nucleotide receptors to excite nociceptive sensory neurons; nucleotides also potentiate signaling through the pro-nociceptive capsaicin receptor, TRPV1. We demonstrate here that P2Y(2) is the principal UTP receptor in somatosensory neurons: P2Y(2) is highly expressed in dorsal root ganglia and P2Y(2)-/- mice showed profound deficits in UTP-evoked calcium transients and potentiation of capsaicin responses. P2Y(2)-/- mice were also deficient in the detection of painful heat: baseline thermal response latencies were increased and mutant mice failed to develop thermal hypersensitivity in response to inflammatory injury (injection of complete Freund's adjuvant into the hindpaw). P2Y(2) was the only Gq-coupled P2Y receptor examined that showed an increase in DRG mRNA levels in response to inflammation. Surprisingly, TRPV1 function was also attenuated in P2Y(2)-/- mice, as measured by the frequency and magnitude of capsaicin responses in vitro and behavioral responses to capsaicin administration in vivo. However, TRPV1 mRNA levels and immunoreactivity were not reduced, and behavioral sensitivity to capsaicin could be largely restored in P2Y(2)-/- mice by pretreatment with bradykinin, suggesting that normal function of TRPV1 requires ongoing modulation by G protein-coupled receptors. These results indicate that nucleotide signaling through P2Y(2) plays a key role in thermal nociception.

Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity.

Dissociated primary sensory neurons are commonly used to study growth factor-dependent cell survival, axon outgrowth, differentiation and basic mechanisms of sensory physiology and pain. Spinal or trigeminal sensory neurons can be collected from embryos, neonates or adults, treated with enzymes that degrade the extracellular matrix, triturated and grown in defined media with or without growth factors and additional animal sera. Production of cultures can take as little as 2.5 h. Cells can be used almost immediately or maintained for as long as 1 month. Ease of production and the ability to control growth conditions make sensory neuron culture a powerful model system for studying basic neurobiology of central and peripheral nervous systems.