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Paralleling the activation of dorsal horn microglia after peripheral nerve injury is a significant expansion and proliferation of macrophages around injured sensory neurons in dorsal root ganglia (DRG). Here we demonstrate a critical contribution of DRG macrophages, but not those at the nerve injury site, to both the initiation and maintenance of the mechanical hypersensitivity that characterizes the neuropathic pain phenotype. In contrast to the reported sexual dimorphism in the microglial contribution to neuropathic pain, depletion of DRG macrophages reduces nerve injury-induced mechanical hypersensitivity and expansion of DRG macrophages in both male and female mice. However, fewer macrophages are induced in the female mice and deletion of colony-stimulating factor 1 from sensory neurons, which prevents nerve injury-induced microglial activation and proliferation, only reduces macrophage expansion in male mice. Finally, we demonstrate molecular cross-talk between axotomized sensory neurons and macrophages, revealing potential peripheral DRG targets for neuropathic pain management. Interactions among spinal dorsal horn neurons and microglia contribute to the induction and maintenance of neuropathic pain after peripheral nerve injury. The authors show that depletion of macrophages in the dorsal root ganglia prevents and reverses ongoing nerve injury-induced hypersensitivity.

The σ 2 receptor has attracted intense interest in cancer imaging 1 , psychiatric disease 2 , neuropathic pain 3 – 5 and other areas of biology 6 , 7 . Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone 2 and the tool compound PB28 8 . These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ 1 receptor. Crystal structures of two ligands bound to the σ 2 receptor confirmed the docked poses. To investigate the contribution of the σ 2 receptor in pain, two potent σ 2 -selective ligands and one potent σ 1 /σ 2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model 9 , suggesting that the σ 2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology. Crystal structures of the σ 2 receptor are determined and used to perform a docking screen of nearly 500 million molecules, identifying σ 2 -selective ligands and providing insight into the role of σ 2 in neuropathic pain.

TRPV1, a capsaicin- and heat-activated ion channel, is expressed by peripheral nociceptors and has been implicated in various inflammatory and neuropathic pain conditions. Although pharmacological modulation of TRPV1 has attracted therapeutic interest, many TRPV1 agonists and antagonists produce thermomodulatory side effects in animal models and human clinical trials, limiting their utility. These on-target effects may result from the perturbation of TRPV1 receptors on nociceptors, which transduce signals to central thermoregulatory circuits and release proinflammatory factors from their peripheral terminals, most notably the potent vasodilative neuropeptide, calcitonin gene-related peptide (CGRP). Alternatively, these body temperature effects may originate from the modulation of TRPV1 on vascular smooth muscle cells (vSMCs), where channel activation promotes arteriole constriction. Here, we ask which of these pathways is most responsible for the body temperature perturbations elicited by TRPV1 drugs in vivo. We address this question by selectively eliminating TRPV1 expression in sensory neurons or vSMCs and show that only the former abrogates agonist-induced hypothermia and antagonist-induced hyperthermia. Furthermore, lesioning the central projections of TRPV1-positive sensory nerve fibers also abrogates drug-mediated thermomodulation, whereas eliminating CGRP has no effect. Thus, TRPV1 drugs alter core body temperature by modulating sensory input to the central nervous system, rather than through peripheral actions on the vasculature. These findings suggest how mechanistically distinct TRPV1 antagonists may diminish inflammatory pain without affecting core body temperature.

Voltage-gated calcium channels (VGCCs) are multimeric proteins composed of alpha 1, β and γ subunits, as well as one of four auxiliary α2δ subunits. Although there is considerable preclinical and clinical evidence for a contribution of VGCCs to nociceptive processing, notably the gabapentin-targeted α2δ-1 subunit, unclear is the extent to which other α2δ subunits contribute to baseline or injury-altered pain and itch processing. Here, we investigated the anatomical and behavioral consequences of deleting α2δ-2, α2δ-3 or α2δ-4 in the mouse and report that selectively ablating each α2δ subunit leads to different, and in some cases, opposite effects on behavioral indices of pain and itch. Specifically, deleting α2δ2 resulted in mechanical and heat hypersensitivity, and an increase in spinal cord microglial immunoreactivity, but reduced scratching (presumptive) itch in response to a pruritogen. In contrast, ablation of α2δ3 led to thermal hyposensitivity, but no change in mechanical responsiveness or indices of itch. Mice deficient for α2δ4 exhibited hyposensitivity across pain modalities and only minor itch deficits. Interestingly, these differential effects were limited to baseline nociceptive responses, therefore we conclude that the α2δ-2, α2δ-3 and α2δ-4 subunits of VGCCs differentially contribute to pain and itch processing. The mechanisms underlying these differences remain however to be determined.

Advances in optical imaging and fluorescent biosensors enable study of the spatiotemporal and long-term neural dynamics in the brain of awake animals. However, methodological difficulties and fibrosis limit similar advances in the spinal cord. Here, to overcome these obstacles, we combined in vivo application of fluoropolymer membranes that inhibit fibrosis, a redesigned implantable spinal imaging chamber and improved motion correction methods that together permit imaging of the spinal cord in awake behaving mice, for months to over a year. We demonstrated a robust ability to monitor axons, identified a spinal cord somatotopic map, performed months-long imaging in freely moving mice, conducted Ca 2+ imaging of neural dynamics in behaving mice responding to pain-provoking stimuli and observed persistent microglial changes after nerve injury. The ability to couple in vivo imaging and behavior at the spinal cord level will drive insights not previously possible at a key location for somatosensory transmission to the brain. Long-term imaging in the spinal cord is achieved by placing a fluoropolymer membrane on the spinal cord, which reduces fibrosis. This approach, combined with deep-learning-based motion correction, enables months-long imaging of the same neurons.

A remarkable molecular and functional heterogeneity of the primary sensory neurons and dorsal horn interneurons transmits pain- and or itch-relevant information, but the molecular signature of the projection neurons that convey the messages to the brain is unclear. Here, using retro-TRAP (translating ribosome affinity purification) and RNA sequencing, we reveal extensive molecular diversity of spino- and trigeminoparabrachial projection neurons. Among the many genes identified, we highlight distinct subsets of Cck⁺-, Nptx2⁺-, Nmb⁺-, and Crh⁺-expressing projection neurons. By combining in situ hybridization of retrogradely labeled neurons with Fos-based assays, we also demonstrate significant functional heterogeneity, including both convergence and segregation of pain- and itch-provoking inputs into molecularly diverse subsets of NK1R- and non–NK1R-expressing projection neurons.

Interneurons in the brain have been garnering increasing attention. Southwell et al. ( 10.1126/science.1240622 ) review the development of this unique class of neurons. The cells migrate long distances during brain development. Transplantation of interneurons derived from embryonic stem cells is yielding insight into disease processes and may have therapeutic potential. For example, Parkinson's disease, epilepsy, certain psychiatric disorders, and even some sorts of chronic pain either involve interneurons or may respond to transplanted interneurons. Many neurologic and psychiatric disorders are marked by imbalances between neural excitation and inhibition. In the cerebral cortex, inhibition is mediated largely by GABAergic (γ-aminobutyric acid–secreting) interneurons, a cell type that originates in the embryonic ventral telencephalon and populates the cortex through long-distance tangential migration. Remarkably, when transplanted from embryos or in vitro culture preparations, immature interneurons disperse and integrate into host brain circuits, both in the cerebral cortex and in other regions of the central nervous system. These features make interneuron transplantation a powerful tool for the study of neurodevelopmental processes such as cell specification, cell death, and cortical plasticity. Moreover, interneuron transplantation provides a novel strategy for modifying neural circuits in rodent models of epilepsy, Parkinson’s disease, mood disorders, and chronic pain.

Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neurons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the molecular and behavioral consequences of challenging Grhl3 PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in trigeminal ganglia (TG), before and after HDM. We found that neither Grhl3 PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3 PAR2/+ mice. Despite the absence of scratching in untreated Grhl3 PAR2/+ mice, several TG genes in thesemice were up-regulated compared to WT. HDM treatment of the Grhl3 PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was upregulated in HDM-treated Grhl3 PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcriptional changes in IL31Tg mice, demonstrating that a common genetic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.

Behavioral responses to painful stimuli require peripheral sensory neurons called nociceptors. Electrophysiological studies show that most C-fiber nociceptors are polymodal (i.e., respond to multiple noxious stimulus modalities, such as mechanical and thermal); nevertheless, these stimuli are perceived as distinct. Therefore, it is believed that discrimination among these modalities only occurs at spinal or supraspinal levels of processing. Here, we provide evidence to the contrary. Genetic ablation in adulthood of unmyelinated sensory neurons expressing the G protein-coupled receptor Mrgprd reduces behavioral sensitivity to noxious mechanical stimuli but not to heat or cold stimuli. Conversely, pharmacological ablation of the central branches of TRPV1⁺ nociceptors, which constitute a nonoverlapping population, selectively abolishes noxious heat pain sensitivity. Combined elimination of both populations yielded an additive phenotype with no additional behavioral deficits, ruling out a redundant contribution of these populations to heat and mechanical pain sensitivity. This double-dissociation suggests that the brain can distinguish different noxious stimulus modalities from the earliest stages of sensory processing.

Calcium binding proteins are expressed throughout the central and peripheral nervous system and disruption of their activity has major consequences in a wide array of cellular processes, including transmission of nociceptive signals that are processed at the level of the spinal cord. We previously reported that the calcium binding protein, hippocalcin-like 4 (Hpcal4), is heavily expressed in interneurons of the superficial dorsal horn, and that its expression is significantly downregulated in a TR4 mutant mouse model that exhibits major pain and itch deficits due to loss of a subpopulation of excitatory interneurons. That finding suggested that Hpcal4 may be a contributor to the behavioral phenotype of the TR4 mutant mouse. To address this question, here we investigated the behavioral consequences of global deletion of Hpcal4 in a battery of acute and persistent pain and itch tests. Unexpectedly, with the exception of a mild reduction in acute baseline thermal responses, Hpcal4-deficient mice exhibit no major deficits in pain or itch responses, under normal conditions or in the setting of tissue or nerve injury. Taken together, our results indicate that the neural calcium sensor Hpcal4 likely makes a limited contribution to pain and itch processing.