Quantitative Proteomic Analysis of the Central Amygdala in Neuropathic Pain Model Rats.

Pain and emotional distress have a reciprocal relation. The amygdala has been implicated in emotional processing. The central nucleus of the amygdala (CeA) receives nociceptive information from the dorsal horn of spinal cord and is responsible for the central plasticity in chronic pain. Neuropathic pain is a type of severe chronic pain and can be strongly influenced by emotional components. Plastic changes in the CeA may play a key role in the development or maintenance or both of neuropathic pain. We studied the expression levels of proteins in the CeA of spinal nerve transection (SNT) model rats. Total tissue lysate proteins were separated by two-dimensional-gel electrophoresis (2D-PAGE). Gels from different time points were compared using Progenesis SameSpot software, and the spots with Fold Change greater than 2 were excised for protein identification by mass spectrometry. We identified more than 50 cytosolic proteins as significantly altered in their expression levels in the CeA of SNT rats, and most of these changes have been validated at mRNA levels by qRT-PCR. We also identified more than 40 membrane proteins as notably up- or down-regulated in the CeA of SNT model rats relative to a control using stable isotope dimethyl labeling nano-LC-MS/MS based proteomics and found that one such protein, doublecortin (DCX), a microtubule-associated protein expressed by neuronal precursor cells during development, is specifically localized in the membrane fraction without changes in total amount of the protein. Immunohistochemistry showed that doublecortin is expressed in processes in the CeA of rats 7 and 21 days after SNT surgery, suggesting that doublecortin is one of the proteins that may contribute to the plastic changes, namely, redevelopment or rewiring of neural networks, in the CeA in the neuropathic pain model. These dysregulated proteins may play roles in reciprocal relationships between pain and psychological distress in the amygdala and contribute to central sensitization. Data are available via ProteomeXchange with identifier PXD017473.

VGF: a biomarker and potential target for the treatment of neuropathic pain?

Neuropathic pain (NP) remains an area of considerable unmet medical need. A persistent challenge in the management of NP is to target the specific mechanisms leading to a change from normal to abnormal sensory perception while ensuring that the defensive pain perception remains intact. Targeting VGF-derived neuropeptides may offer this opportunity. VGF was first identified in 1985 and is highly expressed after nerve injury and inflammation in neurons of both the peripheral and central nervous system. Subsequent studies implicate the vgf gene and its products in pain pathways. This narrative review was supported by a systematic search to identify, select, and critically appraise all relevant research investigating the role of VGF-derived neuropeptides in pain pathways. It predominantly focuses on in vivo investigations of the role of VGF in the initiation and maintenance of NP. VGF expression levels are very low under normal physiological conditions and nerve injury results in rapid and robust upregulation, increasing mechanical and thermal hypersensitivity. The identification of the 2 complement receptors with which VGF neuropeptides interact suggests a novel interplay of neuronal and immune signalling mediators. The understanding of the molecular mechanisms and signalling events by which VGF-derived active neuropeptides exert their physiological actions is in its infancy. Future work should aim to improve understanding of the downstream consequences of VGF neuropeptides thereby providing novel insights into pain mechanisms potentially leading to the identification of novel therapeutic targets.

Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

The possible association of intracellular Ca2+ with metastasis in human cancer cells is poorly understood. We have studied Ca2+ signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca2+ was measured using a membrane-permeant fluorescent Ca2+-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca2+ oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca2+ level could be induced by applying a high-K+ solution. Various parameters of the oscillations depended on extracellular Ca2+ and voltage-gated Na+ channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na+ channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca2+ oscillations. It is concluded that the functional voltage-gated Na+ channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca2+ activity. Possible mechanisms and consequences of the Ca2+ oscillations are discussed.

Identification of a receptor for neuropeptide VGF and its role in neuropathic pain.

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca(2+) levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca(2+) levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.

A clinically relevant rodent model of the HIV antiretroviral drug stavudine induced painful peripheral neuropathy.

HIV-associated sensory neuropathy is the most frequent manifestation of HIV disease, afflicting 40-50% of patients whose HIV disease is otherwise controlled by antiretroviral therapy. It often presents with significant neuropathic pain and is consistently associated with previous exposure to nucleoside reverse transcriptase inhibitors including stavudine (d4T), which is widely used in resource-limited settings. Here we investigated complex pain-related behaviours associated with d4T treatment using ethologically relevant thigmotaxis and burrowing behaviours in adult rats. Detailed neuropathological response was also examined using neurochemistry, electron microscopy, and proteomics. After 2 intravenous injections of d4T (50 mg/kg, 4 days apart), rats developed hind paw mechanical hypersensitivity, which plateaued at 21 days after initial d4T injection, a time that these animals also had significant changes in thigmotaxis and burrowing behaviours when compared to the controls; reductions in hind paw intraepidermal nerve fibre density and CGRP/IB4 immunoreactivity in L5 spinal dorsal horn, suggesting injury to both the peripheral and central terminals of L5 dorsal root ganglion neurons; and increases in myelinated and unmyelinated axon diameters in the sural nerve, suggesting axonal swelling. However, no significant glial and inflammatory cell response to d4T treatment was observed. Sural nerve proteomics at 7 days after initial d4T injection revealed down-regulated proteins associated with mitochondrial function, highlighting distal axons vulnerability to d4T neurotoxicity. In summary, we have reported complex behavioural changes and a distinctive neuropathology in a clinically relevant rat model of d4T-induced sensory neuropathy that is suitable for further pathophysiological investigation and preclinical evaluation of novel analgesics.

Association between tetrodotoxin resistant channels and lipid rafts regulates sensory neuron excitability.

Voltage-gated sodium channels (VGSCs) play a key role in the initiation and propagation of action potentials in neurons. Na(V)1.8 is a tetrodotoxin (TTX) resistant VGSC expressed in nociceptors, peripheral small-diameter neurons able to detect noxious stimuli. Na(V)1.8 underlies the vast majority of sodium currents during action potentials. Many studies have highlighted a key role for Na(V)1.8 in inflammatory and chronic pain models. Lipid rafts are microdomains of the plasma membrane highly enriched in cholesterol and sphingolipids. Lipid rafts tune the spatial and temporal organisation of proteins and lipids on the plasma membrane. They are thought to act as platforms on the membrane where proteins and lipids can be trafficked, compartmentalised and functionally clustered. In the present study we investigated Na(V)1.8 sub-cellular localisation and explored the idea that it is associated with lipid rafts in nociceptors. We found that Na(V)1.8 is distributed in clusters along the axons of DRG neurons in vitro and ex vivo. We also demonstrated, by biochemical and imaging studies, that Na(V)1.8 is associated with lipid rafts along the sciatic nerve ex vivo and in DRG neurons in vitro. Moreover, treatments with methyl-ß-cyclodextrin (MßCD) and 7-ketocholesterol (7KC) led to the dissociation between rafts and Na(V)1.8. By calcium imaging we demonstrated that the lack of association between rafts and Na(V)1.8 correlated with impaired neuronal excitability, highlighted by a reduction in the number of neurons able to conduct mechanically- and chemically-evoked depolarisations. These findings reveal the sub-cellular localisation of Na(V)1.8 in nociceptors and highlight the importance of the association between Na(V)1.8 and lipid rafts in the control of nociceptor excitability.

Building excitable membranes: lipid rafts and multiple controls on trafficking of electrogenic molecules.

Multiple plasma membrane proteins such as ion transporters and ion channels are involved in electrogenesis by setting resting membrane potentials and triggering/propagating action potentials. Recent findings strongly suggest that some of these membrane proteins are selectively transported into membrane microdomains termed lipid rafts. There appear to be multiple mechanisms for the specific protein translocation to lipid rafts, and many of these proteins exhibit distinct properties when inserted into the raft microdomains. Here the authors review the plasma membrane ion channels specifically localized at membrane lipid rafts in neurons. The mechanisms to selectively translocate these molecules to the lipid rafts and the consequences of the trafficking are also discussed.

In vitro and intrathecal siRNA mediated K(V)1.1 knock-down in primary sensory neurons.

K(V)1.1 is a Shaker homologue K(+) channel that contributes to the juxta-paranodal membrane conductance in myelinated axons, and is blocked by fampridine (4-aminopyridine), used to treat the symptoms of multiple sclerosis. The present experiments investigate K(V)1.1 function in primary sensory neurons and A-fibres, and help define its characteristics as a drug-target using sequence specific small-interfering RNAs (siRNAs). siRNA (71nM) was used to knock-down functional expression of K(V)1.1 in sensory neurons (>25µm in apparent diameter) in culture, and was also delivered intrathecally in vivo (9.3µg). K(+) channel knock-down in sensory neurons was found to make the voltage-threshold for action potential generation significantly more negative than in control (p=0.02), led to the breakdown of accommodation and promoted spontaneous action potential firing. Exposure to dendrotoxin-K (DTX-K, 10-100nM) also selectively abolished K(+) currents at negative potentials and made voltage-threshold more negative, consistent with K(V)1.1 controlling excitability close to the nominal resting potential of the neuron cell body, near -60mV. Introduction of one working siRNA sequence into the intrathecal space in vivo was associated with a small increase in the amplitude of the depolarising after-potential in sacral spinal roots (p<0.02), suggesting a reduction in the number of working K(+) channels in internodal axon membrane. Our study provides evidence that K(V)1.1 contributes to the control of peripheral sensory nerve excitability, and suggests that its characteristics as a putative drug target can be assessed by siRNA transfection in primary sensory neurons in vitro and in vivo.

The trafficking of Na(V)1.8.

The α-subunit of tetrodotoxin-resistant voltage-gated sodium channel Na(V)1.8 is selectively expressed in sensory neurons. It has been reported that Na(V)1.8 is involved in the transmission of nociceptive information from sensory neurons to the central nervous system in nociceptive [1] and neuropathic [24] pain conditions. Thus Na(V)1.8 has been a promising target to treat chronic pain. Here we discuss the recent advances in the study of trafficking mechanism of Na(V)1.8. These pieces of information are particularly important as such trafficking machinery could be new targets for painkillers.

A multi PDZ-domain protein Pdzd2 contributes to functional expression of sensory neuron-specific sodium channel Na(V)1.8.

The voltage-gated sodium channel Na(V)1.8 is expressed exclusively in nociceptive sensory neurons and plays an important role in pain pathways. Na(V)1.8 cannot be functionally expressed in non-neuronal cells even in the presence of beta-subunits. We have previously identified Pdzd2, a multi PDZ-domain protein, as a potential interactor for Na(V)1.8. Here we report that Pdzd2 binds directly to the intracellular loops of Na(V)1.8 and Na(V)1.7. The endogenous Na(V)1.8 current in sensory neurons is inhibited by antisense- and siRNA-mediated downregulation of Pdzd2. However, no marked change in pain behaviours is observed in Pdzd2-decificent mice. This may be due to compensatory upregulation of p11, another regulatory factor for Na(V)1.8, in dorsal root ganglia of Pdzd2-deficient mice. These findings reveal that Pdzd2 and p11 play collaborative roles in regulation of Na(V)1.8 expression in sensory neurons.

Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression.

Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness.

Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIV-associated neuropathic pain.

To elucidate the mechanisms underlying peripheral neuropathic pain in the context of HIV infection and antiretroviral therapy, we measured gene expression in dorsal root ganglia (DRG) of rats subjected to systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) and concomitant delivery of HIV-gp120 to the rat sciatic nerve. L4 and L5 DRGs were collected at day 14 (time of peak behavioural change) and changes in gene expression were measured using Affymetrix whole genome rat arrays. Conventional analysis of this data set and Gene Set Enrichment Analysis (GSEA) was performed to discover biological processes altered in this model. Transcripts associated with G protein coupled receptor signalling and cell adhesion were enriched in the treated animals, while ribosomal proteins and proteasome pathways were associated with gene down-regulation. To identify genes that are directly relevant to neuropathic mechanical hypersensitivity, as opposed to epiphenomena associated with other aspects of the response to a sciatic nerve lesion, we compared the gp120+ddC-evoked gene expression with that observed in a model of traumatic neuropathic pain (L5 spinal nerve transection), where hypersensitivity to a static mechanical stimulus is also observed. We identified 39 genes/expressed sequence tags that are differentially expressed in the same direction in both models. Most of these have not previously been implicated in mechanical hypersensitivity and may represent novel targets for therapeutic intervention. As an external control, the RNA expression of three genes was examined by RT-PCR, while the protein levels of two were studied using western blot analysis.

Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves.

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha2delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S35methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.

Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue.

In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5'-exon ('neonatal') and the 3'-exon ('adult') forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.

Pain signalling pathways: from cytokines to ion channels.

Pain is the major reason patients seek medical care. The treatment of pain, particularly chronic pain associated with cancer and damage to the nervous system, is at present inadequate. Lack of effective analgesics is partly due to the fact that pain signalling mechanisms are still not fully understood. Over the recent years, many channels, receptors, and regulatory proteins involved in pain pathways have bee identified, and novel pain signalling mechanisms and pathways at peripheral and spinal levels have been discovered. It is anticipated that increased understanding of the molecular mechanisms of pain would provide a hope for the future development of effective pain killers. This review examines the currently available information on the molecular aspects of pain signalling pathways, and discusses novel and promising therapeutic targets for the treatment of pain in humans.

Deletion of annexin 2 light chain p11 in nociceptors causes deficits in somatosensory coding and pain behavior.

The S100 family protein p11 (S100A10, annexin 2 light chain) is involved in the trafficking of the voltage-gated sodium channel Na(V)1.8, TWIK-related acid-sensitive K+ channel (TASK-1), the ligand-gated ion channels acid-sensing ion channel 1a (ASIC1a) and transient receptor potential vanilloid 5/6 (TRPV5/V6), as well as 5-hydroxytryptamine receptor 1B (5-HT1B), a G-protein-coupled receptor. To evaluate the role of p11 in peripheral pain pathways, we generated a loxP-flanked (floxed) p11 mouse and used the Cre-loxP recombinase system to delete p11 exclusively from nociceptive primary sensory neurons in mice. p11-null neurons showed deficits in the expression of Na(V)1.8, but not of annexin 2. Damage-sensing primary neurons from these animals show a reduced tetrodotoxin-resistant sodium current density, consistent with a loss of membrane-associated Na(V)1.8. Noxious coding in wide-dynamic-range neurons in the dorsal horn was markedly compromised. Acute pain behavior was attenuated in certain models, but no deficits in inflammatory pain were observed. A significant deficit in neuropathic pain behavior was also apparent in the conditional-null mice. These results confirm an important role for p11 in nociceptor function.

Intense isolectin-B4 binding in rat dorsal root ganglion neurons distinguishes C-fiber nociceptors with broad action potentials and high Nav1.9 expression.

Binding to isolectin-B4 (IB4) and expression of tyrosine kinase A (trkA) (the high-affinity NGF receptor) have been used to define two different subgroups of nociceptive small dorsal root ganglion (DRG) neurons. We previously showed that only nociceptors have high trkA levels. However, information about sensory and electrophysiological properties in vivo of single identified IB4-binding neurons, and about their trkA expression levels, is lacking. IB4-positive (IB4+) and small dark neurons had similar size distributions. We examined IB4-binding levels in >120 dye-injected DRG neurons with sensory and electrophysiological properties recorded in vivo. Relative immunointensities for trkA and two TTX-resistant sodium channels (Nav1.8 and Nav1.9) were also measured in these neurons. IB4+ neurons were classified as strongly or weakly IB4+. All strongly IB4+ neurons were C-nociceptor type (C-fiber nociceptive or unresponsive). Of 32 C-nociceptor-type neurons examined, approximately 50% were strongly IB4+, approximately 20% were weakly IB4+ and approximately 30% were IB4-. Adelta low-threshold mechanoreceptive (LTM) neurons were weakly IB4+ or IB4-. All 33 A-fiber nociceptors and all 44 Aalpha/beta-LTM neurons examined were IB4-. IB4+ compared with IB4- C-nociceptor-type neurons had longer somatic action potential durations and rise times, slower conduction velocities, more negative membrane potentials, and greater immunointensities for Nav1.9 but not Nav1.8. Immunointensities of IB4 binding in C-neurons were positively correlated with those of Nav1.9 but not Nav1.8. Of 23 C-neurons tested for both trkA and IB4, approximately 35% were trkA+/IB4+ but with negatively correlated immunointensities; 26% were IB4+/trkA-, and 35% were IB4-/trkA+. We conclude that strongly IB4+ DRG neurons are exclusively C-nociceptor type and that high Nav1.9 expression may contribute to their distinct membrane properties.

Annexin II light chain p11 promotes functional expression of acid-sensing ion channel ASIC1a.

Acid-sensing ion channels (ASICs) have been implicated in a wide variety of physiological functions. We have used a rat dorsal root ganglion cDNA library in a yeast two-hybrid assay to identify sensory neuron proteins that interact with ASICs. We found that annexin II light chain p11 physically interacts with the N terminus of ASIC1a, but not other ASIC isoforms. Immunoprecipitation studies confirmed an interaction between p11 and ASIC1 in rat dorsal root ganglion neurons in vivo. Coexpression of p11 and ASIC1a in CHO-K1 cells led to a 2-fold increase in expression of the ion channel at the cell membrane as determined by membrane-associated immunoreactivity and cell-surface biotinylation. Consistent with these findings, peak ASIC1a currents in transfected CHO-K1 cells were up-regulated 2-fold in the presence of p11, whereas ASIC3-mediated currents were unaffected by p11 expression. Neither the pH dependence of activation nor the rates of desensitization were altered by p11, suggesting that its primary role in regulating ASIC1a activity is to enhance cell-surface expression of ASIC1a. These data demonstrate that p11, already known to traffic members of the voltage-gated sodium and potassium channel families as well as transient receptor potential and chloride channels, also plays a selective role in enhancing ASIC1a functional expression.

trkA is expressed in nociceptive neurons and influences electrophysiological properties via Nav1.8 expression in rapidly conducting nociceptors.

To test the hypothesis that trkA (the high-affinity NGF receptor) is selectively expressed in nociceptive dorsal root ganglion (DRG) neurons, we examined the intensity of trkA immunoreactivity in single dye-injected rat DRG neurons, the sensory receptor properties of which were identified in vivo with mechanical and thermal stimuli. We provide the first evidence in single identified neurons that strong trkA expression in DRGs is restricted to nociceptive neurons, probably accounting for the profound influence of NGF on these neurons. Furthermore, we demonstrate that trkA expression is as high in rapidly conducting (Aalpha/beta) as in more slowly conducting (Adelta and C) nociceptors. All Aalpha/beta low-threshold mechanoreceptors (LTMs) are trkA negative, although weak but detectable trkA is present in some C and Adelta LTMs. NGF can influence electrophysiological properties of DRG neurons, probably by binding to trkA. We found positive correlations for single identified Aalpha/beta (but not C or Adelta) nociceptors between trkA immunocytochemical intensity and electrophysiological properties typical of nociceptors, namely long action potential and afterhyperpolarization durations and large action potential amplitudes. Furthermore, for Aalpha/beta (notCorAdelta) nociceptors, trkA intensity is inversely correlated with conduction velocity. Similar relationships, again only in Aalpha/beta nociceptors, between electrophysiological properties and trkA expression exist for sodium channel Nav1.8 but not Nav1.9 immunoreactivities. These findings suggest that in Aalpha/beta nociceptors, influences of NGF on expression levels of Nav1.8 are related to, and perhaps limited by, expression levels of trkA. This view is supported by a positive correlation between immuno-intensities of trkA and Nav1.8 in A-fiber, but not C-fiber, nociceptors.

Ion channel activities implicated in pathological pain.

Altered expression of voltage-gated sodium, calcium and potassium channels has been associated with neuropathic pain conditions. In addition, roles for the ligand-gated P2X3 and NMDA receptors, as well as pacemaker HCN channels have also been invoked in the pathogenesis of neuropathic pain. In this chapter, evidence of an important role for post-translational regulation of Nav1.9 in setting pain thresholds is presented. Despite the importance of tactile allodynia and mechanical hyperalgesia in chronic pain, we remain ignorant of the molecular nature of mechanosensors present in sensory neurons. A number of candidate mechanosensor genes, identified because of their structural similarity with mechanosensors in Caenorbabditis elegans and Drosophila melanogaster have been identified. Acid-sensing ion channels (ASICs) are structurally related to putative mechanosensors in C. elegans, whilst transient receptor potential channels (TRPs) have been implicated in mechanosensation in the Drosophila acoustic system. Evidence against a role for ASICs as primary transducers of mechanosensation is provided here, and recent evidence implicating TRP channels is reviewed. Finally, the use of sensory neuron-specific gene deletion approaches to unravel the significance of individual ion channels in the regulation of sensory neuron excitability and the induction of pain will be described.