A central mechanism of analgesia in mice and humans lacking the sodium channel NaV1.7.

Deletion of SCN9A encoding the voltage-gated sodium channel NaV1.7 in humans leads to profound pain insensitivity and anosmia. Conditional deletion of NaV1.7 in sensory neurons of mice also abolishes pain, suggesting that the locus of analgesia is the nociceptor. Here we demonstrate, using in vivo calcium imaging and extracellular recording, that NaV1.7 knockout mice have essentially normal nociceptor activity. However, synaptic transmission from nociceptor central terminals in the spinal cord is greatly reduced by an opioid-dependent mechanism. Analgesia is also reversed substantially by central but not peripheral application of opioid antagonists. In contrast, the lack of neurotransmitter release from olfactory sensory neurons is opioid independent. Male and female humans with NaV1.7-null mutations show naloxone-reversible analgesia. Thus, inhibition of neurotransmitter release is the principal mechanism of anosmia and analgesia in mouse and human Nav1.7-null mutants.

Silent cold-sensing neurons contribute to cold allodynia in neuropathic pain.

Patients with neuropathic pain often experience innocuous cooling as excruciating pain. The cell and molecular basis of this cold allodynia is little understood. We used in vivo calcium imaging of sensory ganglia to investigate how the activity of peripheral cold-sensing neurons was altered in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, partial sciatic nerve ligation, and ciguatera poisoning. In control mice, cold-sensing neurons were few in number and small in size. In neuropathic animals with cold allodynia, a set of normally silent large diameter neurons became sensitive to cooling. Many of these silent cold-sensing neurons responded to noxious mechanical stimuli and expressed the nociceptor markers Nav1.8 and CGRPα. Ablating neurons expressing Nav1.8 resulted in diminished cold allodynia. The silent cold-sensing neurons could also be activated by cooling in control mice through blockade of Kv1 voltage-gated potassium channels. Thus, silent cold-sensing neurons are unmasked in diverse neuropathic pain states and cold allodynia results from peripheral sensitization caused by altered nociceptor excitability.

Molecular mechanisms of cold pain.

The sensation of cooling is essential for survival. Extreme cold is a noxious stimulus that drives protective behaviour and that we thus perceive as pain. However, chronic pain patients suffering from cold allodynia paradoxically experience innocuous cooling as excruciating pain. Peripheral sensory neurons that detect decreasing temperature express numerous cold-sensitive and voltage-gated ion channels that govern their response to cooling in health and disease. In this review, we discuss how these ion channels control the sense of cooling and cold pain under physiological conditions, before focusing on the molecular mechanisms by which ion channels can trigger pathological cold pain. With the ever-rising number of patients burdened by chronic pain, we end by highlighting the pressing need to define the cells and molecules involved in cold allodynia and so identify new, rational drug targets for the analgesic treatment of cold pain.

Somatosensation a la mode: plasticity and polymodality in sensory neurons.

Our understanding of how peripheral damage-sensing neurons (nociceptors) respond to noxious stimuli is fundamental to the development of effective analgesics. To date, numerous studies have presented diverging hypotheses on how nociceptors encode modality-specific stimuli, including labelled-line, intensity dependence or pattern theory. In this short review, we appraise data from electrophysiological, behavioural, imaging and molecular expression studies from the last 60 years, in order to obtain a coherent view of modality-specific sensing in peripheral sensory neurons. We propose a mechanistic explanation for the broad range of values obtained for the incidence of polymodal nociceptors that reconciles apparently contradictory data.

Distinct transcriptional responses of mouse sensory neurons in models of human chronic pain conditions.

Background: Sensory neurons play an essential role in almost all pain conditions, and have recently been classified into distinct subsets on the basis of their transcriptomes. Here we have analysed alterations in dorsal root ganglia (DRG) gene expression using microarrays in mouse models related to human chronic pain. Methods: Six different pain models were studied in male C57BL/6J mice: (1) bone cancer pain using cancer cell injection in the intramedullary space of the femur; (2) neuropathic pain using partial sciatic nerve ligation; (3) osteoarthritis pain using mechanical joint loading; (4) chemotherapy-induced pain with oxaliplatin; (5) chronic muscle pain using hyperalgesic priming; and (6) inflammatory pain using intraplantar complete Freund's adjuvant. Microarray analyses were performed using RNA isolated from dorsal root ganglia and compared to sham/vehicle treated controls. Results: Differentially expressed genes (DEGs) were identified. Known and previously unreported genes were found to be dysregulated in each pain model. The transcriptomic profiles for each model were compared and expression profiles of DEGs within subsets of DRG neuronal populations were analysed to determine whether specific neuronal subsets could be linked to each of the pain models.  Conclusions: Each pain model exhibits a unique set of altered transcripts implying distinct cellular responses to different painful stimuli. No simple direct link between genetically distinct sets of neurons and particular pain models could be discerned.

A novel human pain insensitivity disorder caused by a point mutation in ZFHX2.

Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A unique approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. Bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human pain-insensitive phenotype are therefore potential novel targets for the development of new analgesic drugs.awx326media15680039660001.

Synergistic regulation of serotonin and opioid signaling contributes to pain insensitivity in Nav1.7 knockout mice.

Genetic loss of the voltage-gated sodium channel Nav1.7 (Nav1.7-/-) results in lifelong insensitivity to pain in mice and humans. One underlying cause is an increase in the production of endogenous opioids in sensory neurons. We analyzed whether Nav1.7 deficiency altered nociceptive heterotrimeric guanine nucleotide-binding protein-coupled receptor (GPCR) signaling, such as initiated by GPCRs that respond to serotonin (pronociceptive) or opioids (antinociceptive), in sensory neurons. We found that the nociceptive neurons of Nav1.7 knockout (Nav1.7-/-) mice, but not those of Nav1.8 knockout (Nav1.8-/-) mice, exhibited decreased pronociceptive serotonergic signaling through the 5-HT4 receptors, which are Gαs-coupled GPCRs that stimulate the production of cyclic adenosine monophosphate resulting in protein kinase A (PKA) activity, as well as reduced abundance of the RIIß regulatory subunit of PKA. Simultaneously, the efficacy of antinociceptive opioid signaling mediated by the Gαi-coupled mu opioid receptors was increased. Consequently, opioids inhibited more efficiently tetrodotoxin-resistant sodium currents, which are important for pain-initiating neuronal activity in nociceptive neurons. Thus, Nav1.7 controls the efficacy and balance of GPCR-mediated pro- and antinociceptive intracellular signaling, such that without Nav1.7, the balance is shifted toward antinociception, resulting in lifelong endogenous analgesia.

In vivo characterization of distinct modality-specific subsets of somatosensory neurons using GCaMP.

Mechanistic insights into pain pathways are essential for a rational approach to treating this vast and increasing clinical problem. Sensory neurons that respond to tissue damage (nociceptors) may evoke pain sensations and are typically classified on the basis of action potential velocity. Electrophysiological studies have suggested that most of the C-fiber nociceptors are polymodal, responding to a variety of insults. In contrast, gene deletion studies in the sensory neurons of transgenic mice have frequently resulted in modality-specific deficits. We have used an in vivo imaging approach using the genetically encoded fluorescent calcium indicator GCaMP to study the activity of dorsal root ganglion sensory neurons in live animals challenged with painful stimuli. Using this approach, we can visualize spatially distinct neuronal responses and find that >85% of responsive dorsal root ganglion neurons are modality-specific, responding to either noxious mechanical, cold, or heat stimuli. These observations are mirrored in behavioral studies of transgenic mice. For example, deleting sodium channel Nav1.8 silences mechanical- but not heat-sensing sensory neurons, consistent with behavioral deficits. In contrast, primary cultures of axotomized sensory neurons show high levels of polymodality. After intraplantar treatment with prostaglandin E2, neurons in vivo respond more intensely to noxious thermal and mechanical stimuli, and additional neurons (silent nociceptors) are unmasked. Together, these studies define polymodality as an infrequent feature of nociceptive neurons in normal animals.

Nav1.7 and other voltage-gated sodium channels as drug targets for pain relief.

INTRODUCTION: Chronic pain is a massive clinical problem. We discuss the potential of subtype selective sodium channel blockers that may provide analgesia with limited side effects. AREAS COVERED: Sodium channel subtypes have been linked to human pain syndromes through genetic studies. Gain of function mutations in Nav1.7, 1.8 and 1.9 can cause pain, whilst loss of function Nav1.7 mutations lead to loss of pain in otherwise normal people. Intriguingly, both human and mouse Nav1.7 null mutants have increased opioid drive, because naloxone, an opioid antagonist, can reverse the analgesia associated with the loss of Nav1.7 expression. EXPERT OPINION: We believe there is a great future for sodium channel antagonists, particularly Nav1.7 antagonists in treating most pain syndromes. This review deals with recent attempts to develop specific sodium channel blockers, the mechanisms that underpin the Nav1.7 null pain-free phenotype and new routes to analgesia using, for example, gene therapy or combination therapy with subtype specific sodium channel blockers and opioids. The use of selective Nav1.7 antagonists together with either enkephalinase inhibitors or low dose opioids has the potential for side effect-free analgesia, as well as an important opioid sparing function that may be clinically very significant.

Novel SCN9A mutations underlying extreme pain phenotypes: unexpected electrophysiological and clinical phenotype correlations.

The importance of NaV1.7 (encoded by SCN9A) in the regulation of pain sensing is exemplified by the heterogeneity of clinical phenotypes associated with its mutation. Gain-of-function mutations are typically pain-causing and have been associated with inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). IEM is usually caused by enhanced NaV1.7 channel activation, whereas mutations that alter steady-state fast inactivation often lead to PEPD. In contrast, nonfunctional mutations in SCN9A are known to underlie congenital insensitivity to pain (CIP). Although well documented, the correlation between SCN9A genotypes and clinical phenotypes is still unclear. Here we report three families with novel SCN9A mutations. In a multiaffected dominant family with IEM, we found the heterozygous change L245 V. Electrophysiological characterization showed that this mutation did not affect channel activation but instead resulted in incomplete fast inactivation and a small hyperpolarizing shift in steady-state slow inactivation, characteristics more commonly associated with PEPD. In two compound heterozygous CIP patients, we found mutations that still retained functionality of the channels, with two C-terminal mutations (W1775R and L1831X) exhibiting a depolarizing shift in channel activation. Two mutations (A1236E and L1831X) resulted in a hyperpolarizing shift in steady-state fast inactivation. To our knowledge, these are the first descriptions of mutations with some retained channel function causing CIP. This study emphasizes the complex genotype-phenotype correlations that exist for SCN9A and highlights the C-terminal cytoplasmic region of NaV1.7 as a critical region for channel function, potentially facilitating analgesic drug development studies.

Stimulation of GLP-1 secretion downstream of the ligand-gated ion channel TRPA1.

Stimulus-coupled incretin secretion from enteroendocrine cells plays a fundamental role in glucose homeostasis and could be targeted for the treatment of type 2 diabetes. Here, we investigated the expression and function of transient receptor potential (TRP) ion channels in enteroendocrine L cells producing GLP-1. By microarray and quantitative PCR analysis, we identified trpa1 as an L cell-enriched transcript in the small intestine. Calcium imaging of primary L cells and the model cell line GLUTag revealed responses triggered by the TRPA1 agonists allyl-isothiocyanate (mustard oil), carvacrol, and polyunsaturated fatty acids, which were blocked by TRPA1 antagonists. Electrophysiology in GLUTag cells showed that carvacrol induced a current with characteristics typical of TRPA1 and triggered the firing of action potentials. TRPA1 activation caused an increase in GLP-1 secretion from primary murine intestinal cultures and GLUTag cells, an effect that was abolished in cultures from trpa1(-/-) mice or by pharmacological TRPA1 inhibition. These findings present TRPA1 as a novel sensory mechanism in enteroendocrine L cells, coupled to the facilitation of GLP-1 release, which may be exploitable as a target for treating diabetes.

Bacterial metabolite indole modulates incretin secretion from intestinal enteroendocrine L cells.

It has long been speculated that metabolites, produced by gut microbiota, influence host metabolism in health and diseases. Here, we reveal that indole, a metabolite produced from the dissimilation of tryptophan, is able to modulate the secretion of glucagon-like peptide-1 (GLP-1) from immortalized and primary mouse colonic L cells. Indole increased GLP-1 release during short exposures, but it reduced secretion over longer periods. These effects were attributed to the ability of indole to affect two key molecular mechanisms in L cells. On the one hand, indole inhibited voltage-gated K(+) channels, increased the temporal width of action potentials fired by L cells, and led to enhanced Ca(2+) entry, thereby acutely stimulating GLP-1 secretion. On the other hand, indole slowed ATP production by blocking NADH dehydrogenase, thus leading to a prolonged reduction of GLP-1 secretion. Our results identify indole as a signaling molecule by which gut microbiota communicate with L cells and influence host metabolism.

Inflammatory and neuropathic pain are rapidly suppressed by peripheral block of hyperpolarisation-activated cyclic nucleotide-gated ion channels.

Previous studies have shown that hyperpolarisation-activated cyclic nucleotide-gated (HCN)-2 ion channels regulate the firing frequency of nociceptive sensory neurons and thus play a central role in both inflammatory and neuropathic pain conditions. Here we use ivabradine, a clinically approved anti-anginal agent that blocks all HCN channel isoforms approximately equally, to investigate the effect on inflammatory and neuropathic pain of HCN ion channel block. We show that ivabradine does not have major off-target effects on a sample group of Na, Ca, and K ion channels, and that it is peripherally restricted because it is a substrate for the P-glycoprotein (PgP) multidrug transporter that is expressed in the blood-brain barrier. Its effects are therefore likely to be due to an action on HCN ion channels in peripheral sensory neurons. Using patch clamp electrophysiology, we found that ivabradine was a use-dependent blocker of native HCN channels expressed in small sensory neurons. Ivabradine suppressed the action potential firing that is induced in nociceptive neurons by elevation of intracellular cAMP. In the formalin model of inflammatory pain, ivabradine reduced pain behaviour only in the second (inflammatory) phase. In nerve injury and chemotherapy models of neuropathic pain, we observed rapid and effective analgesia as effective as that with gabapentin. We conclude that both inflammatory and neuropathic pain are rapidly inhibited by blocking HCN-dependent repetitive firing in peripheral nociceptive neurons.

Molecular mechanisms of incretin hormone secretion.

Incretin peptides (glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP)) are secreted from enteroendocrine cells in the intestinal epithelium, and help to coordinate metabolic responses to food ingestion. A number of molecular mechanisms have recently been defined that underlie carbohydrate, lipid and protein sensing in gut endocrine cells. Knockout mice lacking sodium glucose tranporter-1 (SGLT-1) or the short chain fatty acid sensing receptor FFAR2 (GPR43), for example, have highlighted the importance of these molecules in incretin secretion. This review outlines our current understanding of sensory pathways in incretin secreting cells and highlights the therapeutic potential of targeting them for the development of novel therapies for obesity and diabetes.

HCN2 ion channels: an emerging role as the pacemakers of pain.

Acute nociceptive pain is caused by the direct action of a noxious stimulus on pain-sensitive nerve endings, whereas inflammatory pain (both acute and chronic) arises from the actions of a wide range of inflammatory mediators released following tissue injury. Neuropathic pain, which is triggered by nerve damage, is often considered to be very different in its origins, and is particularly difficult to treat effectively. Here we review recent evidence showing that members of the hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channel family - better known for their role in the pacemaker potential of the heart - play important roles in both inflammatory and neuropathic pain. Deletion of the HCN2 isoform from nociceptive neurons abolishes heat-evoked inflammatory pain and all aspects of neuropathic pain, but acute pain sensation is unaffected. This work shows that inflammatory and neuropathic pain have much in common, and suggests that selective blockers of HCN2 may have value as analgesics in the treatment of pain.

HCN2 ion channels play a central role in inflammatory and neuropathic pain.

The rate of action potential firing in nociceptors is a major determinant of the intensity of pain. Possible modulators of action potential firing include the HCN ion channels, which generate an inward current, I(h), after hyperpolarization of the membrane. We found that genetic deletion of HCN2 removed the cyclic adenosine monophosphate (cAMP)-sensitive component of I(h) and abolished action potential firing caused by an elevation of cAMP in nociceptors. Mice in which HCN2 was specifically deleted in nociceptors expressing Na(V)1.8 had normal pain thresholds, but inflammation did not cause hyperalgesia to heat stimuli. After a nerve lesion, these mice showed no neuropathic pain in response to thermal or mechanical stimuli. Neuropathic pain is therefore initiated by HCN2-driven action potential firing in Na(V)1.8-expressing nociceptors.