Mispositioned Neurokinin-1 Receptor-Expressing Neurons Underlie Heat Hyperalgesia in Disabled-1 Mutant Mice.

Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I-II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I-II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/- mice induced Fos in incorrectly positioned NK1R neurons in Laminae I-II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity

Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain.

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

Ablation of spinal cord estrogen receptor α-expressing interneurons reduces chemically induced modalities of pain and itch

Estrogens are presumed to underlie, at least in part, the greater pain sensitivity and chronic pain prevalence that women experience compared to men. Although previous studies revealed populations of estrogen receptor‐expressing neurons in primary afferents and in superficial dorsal horn neurons, there is little to no information as to the contribution of these neurons to the generation of acute and chronic pain. Here we molecularly characterized neurons in the mouse superficial spinal cord dorsal horn that express estrogen receptor α (ERα) and explored the behavioral consequences of their ablation. We found that spinal ERα‐positive neurons are largely excitatory interneurons and many coexpress substance P, a marker for a discrete subset of nociceptive, excitatory interneurons. After viral, caspase‐mediated ablation of spinal ERα‐expressing cells, we observed a significant decrease in the first phase of the formalin test, but in male mice only. ERα‐expressing neuron‐ablation also reduced pruritogen‐induced scratching in both male and female mice. There were no ablation‐related changes in mechanical or heat withdrawal thresholds or in capsaicin‐induced nocifensive behavior. In chronic pain models, we found no change in Complete Freund's adjuvant‐induced thermal or mechanical hypersensitivity, or in partial sciatic nerve injury‐induced mechanical allodynia. We conclude that ERα labels a subpopulation of excitatory interneurons that are specifically involved in chemically evoked persistent pain and pruritogen‐induced itch

Keratinocyte–TRPV1 sensory neuron interactions in a genetically controllable mouse model of chronic neuropathic itch

Our understanding of neural circuits that respond to skin dysfunction, triggering itch, and pathophysiological scratching remains incomplete. Here, we describe a profound chronic itch phenotype in transgenic mice expressing the tetracycline transactivator (tTA) gene within the Phox2a lineage. Phox2a; tTA mice exhibit intense, localized scratching and regional skin lesions, controllable by the tTA inhibitor, doxycycline. As gabapentin and the kappa opioid receptor agonist, nalfurafine, but not morphine, significantly reduce scratching, this phenotype has a pharmacological profile of neuropathic pruritus. Importantly, the Phox2a; tTA expression occurs in a spatially restricted population of skin keratinocytes that overlaps precisely with the skin area that is scratched. Localized Gi-DREADD-mediated inactivation of these Phox2a-keratinocytes completely reverses the skin lesions, while inducible tTA activation of keratinocytes initiates the condition. Notably, ablation of TRPV1-expressing primary afferent neurons also reduces scratching and skin lesions, but this occurs slowly, over a course of two months. In contrast denervation induced loss of all cutaneous input rapidly blocks scratching. These findings identify the cellular, molecular, and topographic basis of a robust and chronic sensory neuron-dependent and gabapentin-responsive neuropathic itch that is initiated by genetic factors within keratinocytes

Docking 14 Million Virtual Isoquinuclidines against the μ and κ Opioid Receptors Reveals Dual Antagonists–Inverse Agonists with Reduced Withdrawal Effects

Large library docking of tangible molecules has revealed potent ligands across many targets. While make-on-demand libraries now exceed 75 billion enumerated molecules, their synthetic routes are dominated by a few reaction types, reducing diversity and inevitably leaving many interesting bioactive-like chemotypes unexplored. Here, we investigate the large-scale enumeration and targeted docking of isoquinuclidines. These “natural-product-like” molecules are rare in current libraries and are functionally congested, making them interesting as receptor probes. Using a modular, four-component reaction scheme, we built and docked a virtual library of over 14.6 million isoquinuclidines against both the μ- and κ-opioid receptors (MOR and KOR, respectively). Synthesis and experimental testing of 18 prioritized compounds found nine ligands with low μM affinities. Structure-based optimization revealed low- and sub-nM antagonists and inverse agonists targeting both receptors. Cryo-electron microscopy structures illuminate the origins of activity on each target. In mouse behavioral studies, a potent joint MOR-antagonist and KOR-inverse-agonist reversed morphine-induced analgesia, phenocopying the MOR-selective antioverdose agent naloxone. Encouragingly, the isoquinuclidine induced less severe opioid-withdrawal symptoms versus naloxone and did not induce conditioned-place aversion, reflecting reduced dysphoria, consistent with its KOR-inverse agonism. The strengths and weaknesses of bespoke library docking and of docking for opioid receptor polypharmacology will be considered

Morphological and functional properties distinguish the substance P and gastrin-releasing peptide subsets of excitatory interneuron in the spinal cord dorsal horn

Excitatory interneurons account for the majority of neurons in the superficial dorsal horn, but despite their presumed contribution to pain and itch, there is still limited information about their organisation and function. We recently identified 2 populations of excitatory interneuron defined by expression of gastrin-releasing peptide (GRP) or substance P (SP). Here, we demonstrate that these cells show major differences in their morphological, electrophysiological, and pharmacological properties. Based on their somatodendritic morphology and firing patterns, we propose that the SP cells correspond to radial cells, which generally show delayed firing. By contrast, most GRP cells show transient or single-spike firing, and many are likely to correspond to the so-called transient central cells. Unlike the SP cells, few of the GRP cells had long propriospinal projections, suggesting that they are involved primarily in local processing. The 2 populations also differed in responses to neuromodulators, with most SP cells, but few GRP cells, responding to noradrenaline and 5-HT; the converse was true for responses to the μ-opioid agonist DAMGO. Although a recent study suggested that GRP cells are innervated by nociceptors and are strongly activated by noxious stimuli, we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signal–regulated kinases in response to noxious stimuli. These findings indicate that the SP and GRP cells differentially process somatosensory information