Expression of the voltage-gated sodium channel NaV1.7 in sensory neurons is required for pain sensation. We examined the role of NaV1.7 in the dorsal horn of the spinal cord using an epitope-tagged NaV1.7 knock-in mouse. Immuno-electron microscopy showed the presence of NaV1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of NaV1.7 in the dorsal horn. Peripheral nervous system-specific NaV1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. NaV1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on NaV1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated NaV1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis.

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.

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.

Many studies support the pro-nociceptive role of brain-derived neurotrophin factor (BDNF) in pain processes in the peripheral and central nervous system. We have previously shown that nociceptor-derived BDNF is involved in inflammatory pain. Microglial-derived BDNF has also been shown to be involved in neuropathic pain. However, the distinct contribution of primary afferent-derived BNDF to chronic pain processing remains undetermined. In this study, we used Avil-CreERT2 mice to delete Bdnf from all adult peripheral sensory neurons. Conditional BDNF knockouts were healthy with no sensory neuron loss. Behavioural assays and in vivo electrophysiology indicated that spinal excitability was normal. Following formalin inflammation or neuropathy with a modified Chung model, we observed normal development of acute pain behaviour, but a deficit in second phase formalin-induced nocifensive responses and a reversal of neuropathy-induced mechanical hypersensitivity during the later chronic pain phase in conditional BDNF knockout mice. In contrast, we observed normal development of acute and chronic neuropathic pain in the Seltzer model, indicating differences in the contribution of BDNF to distinct models of neuropathy. We further used a model of hyperalgesic priming to examine the contribution of primary afferent-derived BDNF in the transition from acute to chronic pain, and found that primed BDNF knockout mice do not develop prolonged mechanical hypersensitivity to an inflammatory insult. Our data suggest that BDNF derived from sensory neurons plays a critical role in mediating the transition from acute to chronic pain.

Muscular dystrophies are severe genetic diseases for which no efficacious therapies exist. Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs). However, one of the limitations of this approach is the relatively low number of cells that engraft the diseased tissue, due, at least in part, to the sub-optimal efficiency of extravasation, whose mechanisms for MAB are unknown. Leukocytes emigrate into the inflamed tissues by crossing endothelial cell-to-cell junctions and junctional proteins direct and control leukocyte diapedesis. Here, we identify the endothelial junctional protein JAM-A as a key regulator of MAB extravasation. We show that JAM-A gene inactivation and JAM-A blocking antibodies strongly enhance MAB engraftment in dystrophic muscle. In the absence of JAM-A, the exchange factors EPAC-1 and 2 are down-regulated, which prevents the activation of the small GTPase Rap-1. As a consequence, junction tightening is reduced, allowing MAB diapedesis. Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.

Background: Functional deletion of the Scn9a (sodium voltage-gated channel alpha subunit 9) gene encoding sodium channel Nav1.7 makes humans and mice pain-free. Opioid signalling contributes to this analgesic state. We have used pharmacological and genetic approaches to identify the opioid receptors involved in this form of analgesia. We also examined the regulation of proenkephalin expression by the transcription factor Nfat5 that binds upstream of the Penk gene. Methods: We used specific µ-, δ- and κ-opioid receptor antagonists alone or in combination to examine which opioid receptors were necessary for Nav1.7 loss-associated analgesia in mouse behavioural assays of thermal pain. We also used µ- and δ-opioid receptor null mutant mice alone and in combination in behavioural assays to examine the role of these receptors in Nav1.7 knockouts pain free phenotype. Finally, we examined the levels of Penk mRNA in Nfat5-null mutant mice, as this transcription factor binds to consensus sequences upstream of the Penk gene. Results: The pharmacological block or deletion of both µ- and δ-opioid receptors was required to abolish Nav1.7-null opioid-related analgesia. κ-opioid receptor antagonists were without effect. Enkephalins encoded by the Penk gene are upregulated in Nav1.7 nulls. Deleting Nfat5, a transcription factor with binding motifs upstream of Penk, induces the same level of enkephalin mRNA expression as found in Nav1 .7 nulls, but without consequent analgesia. These data confirm that a combination of events linked to Scn9a gene loss is required for analgesia. Higher levels of endogenous enkephalins, potentiated opioid receptors, diminished electrical excitability and loss of neurotransmitter release together contribute to the analgesic phenotype found in Nav1.7-null mouse and human mutants. Conclusions: These observations help explain the failure of Nav1.7 channel blockers alone to produce analgesia and suggest new routes for analgesic drug development.

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. We generated an epitope-tagged NaV1.7 mouse that showed normal pain behaviours to identify channel-interacting proteins. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing-response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein interactors including membrane-trafficking protein synaptotagmin-2 (Syt2), L-type amino acid transporter 1 (Lat1) and transmembrane P24-trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co-immunoprecipitation (Co-IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein-regulated inducer of neurite outgrowth (Gprin1), an opioid receptor-binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope-tagged mouse should provide useful insights into the many functions now associated with the NaV1.7 channel.

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.