Gut-innervating nociceptors regulate the intestinal microbiota to promote tissue protection.

Nociceptive pain is a hallmark of many chronic inflammatory conditions including inflammatory bowel diseases (IBDs); however, whether pain-sensing neurons influence intestinal inflammation remains poorly defined. Employing chemogenetic silencing, adenoviral-mediated colon-specific silencing, and pharmacological ablation of TRPV1+ nociceptors, we observed more severe inflammation and defective tissue-protective reparative processes in a murine model of intestinal damage and inflammation. Disrupted nociception led to significant alterations in the intestinal microbiota and a transmissible dysbiosis, while mono-colonization of germ-free mice with Gram+Clostridium spp. promoted intestinal tissue protection through a nociceptor-dependent pathway. Mechanistically, disruption of nociception resulted in decreased levels of substance P, and therapeutic delivery of substance P promoted tissue-protective effects exerted by TRPV1+ nociceptors in a microbiota-dependent manner. Finally, dysregulated nociceptor gene expression was observed in intestinal biopsies from IBD patients. Collectively, these findings indicate an evolutionarily conserved functional link between nociception, the intestinal microbiota, and the restoration of intestinal homeostasis.

Neuronal substance P drives metastasis through an extracellular RNA-TLR7 axis.

Tumour innervation is associated with worse patient outcomes in multiple cancers1,2, which suggests that it may regulate metastasis. Here we observed that highly metastatic mouse mammary tumours acquired more innervation than did less-metastatic tumours. This enhanced innervation was driven by expression of the axon-guidance molecule SLIT2 in tumour vasculature. Breast cancer cells induced spontaneous calcium activity in sensory neurons and elicited release of the neuropeptide substance P (SP). Using three-dimensional co-cultures and in vivo models, we found that neuronal SP promoted breast tumour growth, invasion and metastasis. Moreover, patient tumours with elevated SP exhibited enhanced lymph node metastatic spread. SP acted on tumoral tachykinin receptors (TACR1) to drive death of a small population of TACR1high cancer cells. Single-stranded RNAs (ssRNAs) released from dying cells acted on neighbouring tumoural Toll-like receptor 7 (TLR7) to non-canonically activate a prometastatic gene expression program. This SP- and ssRNA-induced Tlr7 gene expression signature was associated with reduced breast cancer survival outcomes. Therapeutic targeting of this neuro-cancer axis with the TACR1 antagonist aprepitant, an approved anti-nausea drug, suppressed breast cancer growth and metastasis in multiple models. Our findings reveal that tumour-induced hyperactivation of sensory neurons regulates multiple aspects of metastatic progression in breast cancer through a therapeutically targetable neuropeptide/extracellular ssRNA sensing axis.

Allergens TRP a swITCH to Initiate Type 2 Immunity.

Allergens induce type-2 immunity, but unresolved questions remain about initiation of this response. In this issue, Perner et al. propose that cutaneous activation of TRPV1+ sensory neurons by protease allergens stimulates release of substance P to induce migration of Th2-skewing CD301b+ DC to draining lymph nodes.

Substance P Release by Sensory Neurons Triggers Dendritic Cell Migration and Initiates the Type-2 Immune Response to Allergens.

Dendritic cells (DCs) of the cDC2 lineage initiate allergic immunity and in the dermis are marked by their expression of CD301b. CD301b+ dermal DCs respond to allergens encountered in vivo, but not in vitro. This suggests that another cell in the dermis may sense allergens and relay that information to activate and induce the migration of CD301b+ DCs to the draining lymph node (dLN). Using a model of cutaneous allergen exposure, we show that allergens directly activated TRPV1+ sensory neurons leading to itch and pain behaviors. Allergen-activated sensory neurons released the neuropeptide Substance P, which stimulated proximally located CD301b+ DCs through the Mas-related G-protein coupled receptor member A1 (MRGPRA1). Substance P induced CD301b+ DC migration to the dLN where they initiated T helper-2 cell differentiation. Thus, sensory neurons act as primary sensors of allergens, linking exposure to activation of allergic-skewing DCs and the initiation of an allergic immune response.

C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Sensory nerves unlock the TOLL-7 gate for cancer spread.

Cancers hijack the nervous system for growth and spread. Thus, disrupting neuron-cancer crosstalk holds promise for blocking metastasis. Recently, Padmanaban et al. reported new therapeutic targets and showed that breast cancer cells activate sensory neurons to secrete the neuropeptide substance P (SP), leading to single-strand (ss)RNA release and noncanonical Toll-like receptor (TLR)7 signaling that drives metastasis.

Parvalbumin interneuron activity drives fast inhibition-induced vasoconstriction followed by slow substance P-mediated vasodilation.

The role of parvalbumin (PV) interneurons in vascular control is poorly understood. Here, we investigated the hemodynamic responses elicited by optogenetic stimulation of PV interneurons using electrophysiology, functional magnetic resonance imaging (fMRI), wide-field optical imaging (OIS), and pharmacological applications. As a control, forepaw stimulation was used. Stimulation of PV interneurons in the somatosensory cortex evoked a biphasic fMRI response in the photostimulation site and negative fMRI signals in projection regions. Activation of PV neurons engaged two separable neurovascular mechanisms in the stimulation site. First, an early vasoconstrictive response caused by the PV-driven inhibition is sensitive to the brain state affected by anesthesia or wakefulness. Second, a later ultraslow vasodilation lasting a minute is closely dependent on the sum of interneuron multiunit activities, but is not due to increased metabolism, neural or vascular rebound, or increased glial activity. The ultraslow response is mediated by neuropeptide substance P (SP) released from PV neurons under anesthesia, but disappears during wakefulness, suggesting that SP signaling is important for vascular regulation during sleep. Our findings provide a comprehensive perspective about the role of PV neurons in controlling the vascular response.

Therapeutic antagonism of the neurokinin 1 receptor in endosomes provides sustained pain relief.

The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK1R), Gαq/i, and ßarrestin-2. Whereas the FDA-approved NK1R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK1R+ve neurons in knockin mice expressing human NK1R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK1R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK1R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK1R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of diverse diseases.

Substance P Increases STAT6-Mediated Transcription Activation of Lymphocyte Cytosolic Protein 2 to Sustain M2 Macrophage Predominance in Pediatric Asthma.

Substance P (SP) is a neuropeptide released by neurons and participates in various biological processes, including inflammation. M2 macrophages are major immune cells associated with type 2 inflammation in asthma. This study investigated the effect of SP on macrophage phenotype in pediatric asthma and the underpinning factors. Asthmatic children exhibited an increased level of SP, along with a higher proportion of M2 macrophages in their bronchoalveolar lavage fluid. Flow cytometry revealed that SP treatment enhanced the M2 polarization of 12-O-tetradecanoylphorbol 13-acetate-treated THP-1 cells (macrophages) in vitro. By contrast, the administration of a neutralizing antibody of SP reduced the M2 macrophage population, mitigated inflammatory cell infiltration in mouse lung tissues, and decreased the population of immune cells in the mouse bronchoalveolar lavage fluid. SP up-regulated the expression of STAT6, which, in turn, activated the transcription of lymphocyte cytosolic protein 2 (LCP2). The population of macrophages and allergic inflammatory responses in mice were reduced by STAT6 inhibition but restored by LCP2 overexpression. Collectively, the present study demonstrated that SP sustains M2 macrophage predominance and allergic inflammation in pediatric asthma by enhancing STAT6-dependent transcription activation of LCP2.

Synaptotagmin 9 Modulates Spontaneous Neurotransmitter Release in Striatal Neurons by Regulating Substance P Secretion.

Synaptotagmin 9 (SYT9) is a tandem C2 domain Ca2+ sensor for exocytosis in neuroendocrine cells; its function in neurons remains unclear. Here, we show that, in mixed-sex cultures, SYT9 does not trigger rapid synaptic vesicle exocytosis in mouse cortical, hippocampal, or striatal neurons, unless it is massively overexpressed. In striatal neurons, loss of SYT9 reduced the frequency of spontaneous neurotransmitter release events (minis). We delved into the underlying mechanism and discovered that SYT9 was localized to dense-core vesicles that contain substance P (SP). Loss of SYT9 impaired SP release, causing the observed decrease in mini frequency. This model is further supported by loss of function mutants. Namely, Ca2+ binding to the C2A domain of SYT9 triggered membrane fusion in vitro, and mutations that disrupted this activity abolished the ability of SYT9 to regulate both SP release and mini frequency. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.SIGNIFICANCE STATEMENT Synaptotagmin 9 (SYT9) has been described as a Ca2+ sensor for dense-core vesicle (DCV) exocytosis in neuroendocrine cells, but its role in neurons remains unclear, despite widespread expression in the brain. This article examines the role of SYT9 in synaptic transmission across cultured cortical, hippocampal, and striatal neuronal preparations. We found that SYT9 regulates spontaneous neurotransmitter release in striatal neurons by serving as a Ca2+ sensor for the release of the neuromodulator substance P from DCVs. This demonstrates a novel role for SYT9 in neurons and uncovers a new field of study into neuromodulation by SYT9, a protein that is widely expressed in the brain.

Endogenous Inflammatory Mediators Produced by Injury Activate TRPV1 and TRPA1 Nociceptors to Induce Sexually Dimorphic Cold Pain That Is Dependent on TRPM8 and GFRα3.

The detection of environmental temperatures is critical for survival, yet inappropriate responses to thermal stimuli can have a negative impact on overall health. The physiological effect of cold is distinct among somatosensory modalities in that it is soothing and analgesic, but also agonizing in the context of tissue damage. Inflammatory mediators produced during injury activate nociceptors to release neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P, inducing neurogenic inflammation, which further exasperates pain. Many inflammatory mediators induce sensitization to heat and mechanical stimuli but, conversely, inhibit cold responsiveness, and the identity of molecules inducing cold pain peripherally is enigmatic, as are the cellular and molecular mechanisms altering cold sensitivity. Here, we asked whether inflammatory mediators that induce neurogenic inflammation via the nociceptive ion channels TRPV1 (vanilloid subfamily of transient receptor potential channel) and TRPA1 (transient receptor potential ankyrin 1) lead to cold pain in mice. Specifically, we tested cold sensitivity in mice after intraplantar injection of lysophosphatidic acid or 4-hydroxy-2-nonenal, finding that each induces cold pain that is dependent on the cold-gated channel transient receptor potential melastatin 8 (TRPM8). Inhibition of CGRP, substance P, or toll-like receptor 4 (TLR4) signaling attenuates this phenotype, and each neuropeptide produces TRPM8-dependent cold pain directly. Further, the inhibition of CGRP or TLR4 signaling alleviates cold allodynia differentially by sex. Last, cold pain induced by both inflammatory mediators and neuropeptides requires TRPM8, as well as the neurotrophin artemin and its receptor GDNF receptor α3 (GFRα3). These results are consistent with artemin-induced cold allodynia requiring TRPM8, demonstrating that neurogenic inflammation alters cold sensitivity via localized artemin release that induces cold pain via GFRα3 and TRPM8.SIGNIFICANCE STATEMENT The cellular and molecular mechanisms that generate pain are complex with a diverse array of pain-producing molecules generated during injury that act to sensitize peripheral sensory neurons, thereby inducing pain. Here we identify a specific neuroinflammatory pathway involving the ion channel TRPM8 (transient receptor potential cation channel subfamily M member 8) and the neurotrophin receptor GFRα3 (GDNF receptor α3) that leads to cold pain, providing select targets for potential therapies for this pain modality.

Cellular metabolism of substance P produces neurokinin-1 receptor peptide agonists with diminished cyclic AMP signaling.

Substance P (SP) is released from sensory nerves in the arteries and heart. It activates neurokinin-1 receptors (NK1Rs) causing vasodilation, immune modulation, and adverse cardiac remodeling. The hypothesis was tested: SP and SP metabolites activate different second messenger signaling pathways. Macrophages, endothelial cells, and fibroblasts metabolized SP to N- and C-terminal metabolites to varying extents. SP 5-11 was the most abundant metabolite followed by SP 1-4, SP 7-11, SP 6-11, SP 3-11, and SP 8-11. In NK1R-expressing human embryonic kidney 293 (HEK293) cells, SP and some C-terminal SP metabolites stimulate the NK1R, promoting the dissociation of several Gα proteins, including Gαs and Gαq from their ßγ subunits. SP increases intracellular calcium concentrations ([Ca]i) and cyclic 3',5'-adenosine monophosphate (cAMP) accumulation with similar -log EC50 values of 8.5 ± 0.3 and 7.8 ± 0.1 M, respectively. N-terminal metabolism of SP by up to five amino acids and C-terminal deamidation of SP produce peptides that retain activity to increase [Ca]i but not to increase cAMP. C-terminal metabolism results in the loss of both activities. Thus, [Ca]i and cAMP signaling are differentially affected by SP metabolism. To assess the role of N-terminal metabolism, SP and SP 6-11 were compared with cAMP-mediated activities in NK1R-expressing 3T3 fibroblasts. SP inhibits nuclear factor κB (NF-κB) activity, cell proliferation, and wound healing and stimulates collagen production. SP 6-11 had little or no activity. Cyclooxygenase-2 (COX-2) expression is increased by SP but not by SP 6-11. Thus, metabolism may select the cellular response to SP by inhibiting or redirecting the second messenger signaling pathway activated by the NK1R.NEW & NOTEWORTHY Endothelial cells, macrophages, and fibroblasts metabolize substance P (SP) to N- and C-terminal metabolites with SP 5-11 as the most abundant metabolite. SP activates neurokinin-1 receptors to increase intracellular calcium and cyclic AMP. In contrast, SP metabolites of N-terminal metabolism and C-terminal deamidation retain the ability to increase calcium but lose the ability to increase cyclic AMP. These new insights indicate that the metabolism of SP directs cellular functions by regulating specific signaling pathways.

Substance P promotes transforming growth factor-ß-induced collagen synthesis in human corneal fibroblasts.

Corneal fibroblasts maintain homeostasis of the corneal stroma by mediating the synthesis and degradation of extracellular collagen, and these actions are promoted by transforming growth factor-ß (TGF-ß) and interleukin-1ß (IL-1ß), respectively. The cornea is densely innervated with sensory nerve fibers that are not only responsible for sensation but also required for physiological processes such as tear secretion and wound healing. Loss or dysfunction of corneal nerves thus impairs corneal epithelial wound healing and can lead to neurotrophic keratopathy. The sensory neurotransmitter substance P (SP) promotes corneal epithelial wound healing by enhancing the stimulatory effects of growth factors and fibronectin. We have now investigated the role of SP in collagen metabolism mediated by human corneal fibroblasts in culture. Although SP alone had no effect on collagen synthesis or degradation by these cells, it promoted the stimulatory effect of TGF-ß on collagen type I synthesis without affecting that of IL-1ß on the expression of matrix metalloproteinase-1. This effect of SP on TGF-ß-induced collagen synthesis was accompanied by activation of p38 mitogen-activated protein kinase (MAPK) signaling and was attenuated by pharmacological inhibition of p38 or of the neurokinin-1 receptor. Our results thus implicate SP as a modulator of TGF-ß-induced collagen type I synthesis by human corneal fibroblasts, and they suggest that loss of this function may contribute to the development of neurotrophic keratopathy.NEW & NOTEWORTHY This study investigates the role of substance P (SP) in collagen metabolism mediated by human corneal fibroblasts in culture. We found that, although SP alone had no effect on collagen synthesis or degradation by corneal fibroblasts, it promoted the stimulatory effect of transforming growth factor-ß on collagen type I synthesis without affecting that of interleukin-1ß on the expression of matrix metalloproteinase-1.

Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Recent advances in the molecular signaling pathways of Substance P in Alzheimer's disease: Link to neuroinflammation associated with toll-like receptors.

A significant quantity of substance P (SP) and its receptor, the neurokinin 1 (NK1) receptors are found in the brain. SP is a neuropeptide distributed in the central nervous system and functions as a neurotransmitter, neuromodulator, and neurotrophic factor. The concentrations of SP in the brain and cerebrospinal fluid fluctuate in individuals with Alzheimer's disease (AD). SP is an endogenous ligand for NK1 receptor, enhancing the expression of toll-like receptors (TLR) and vice versa. So, both pathways are interconnected, where activation of one pathway activates the second pathway. Researchers have observed the interaction of TLR with SP in the pathophysiology of AD. Thus, this review discusses various TLRs involved in regulating amyloid processing and its interaction with SP in AD. Further, in AD pathology, SP can regulate the non-amyloidogenic pathway. Recent studies have also demonstrated the capacity of SP in regulating voltage-gated potassium channel currents, emphasizing SP's neuroprotective ability. Therefore, we corroborate the findings linking the SP, NK1R, and TLRs in AD.

The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep†.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon.

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm2 in the AC, 166/mm2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19-22% and 13-17%, respectively, P < 0.001-0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62-85%), whereas 18-38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33-36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions.

Melanopsin in the human and chicken choroid.

The choroid embedded in between retina and sclera is essential for retinal photoreceptor nourishment, but is also a source of growth factors in the process of emmetropization that converts retinal visual signals into scleral growth signals. Still, the exact control mechanisms behind those functions are enigmatic while circadian rhythms are involved. These rhythms are attributed to daylight influences that are melanopsin (OPN4) driven. Recently, OPN4-mRNA has been detected in the choroid, and while its origin is unknown we here seek to identify the underlying structures using morphological methods. Human and chicken choroids were prepared for single- and double-immunohistochemistry of OPN4, vasoactive intestinal peptide (VIP), substance P (SP), CD68, and α-smooth muscle actin (ASMA). For documentation, light-, fluorescence-, and confocal laser scanning microscopy was applied. Retinal controls proved the reliability of the OPN4 antibody in both species. In humans, OPN4 immunoreactivity (OPN4-IR) was detected in nerve fibers of the choroid and adjacent ciliary nerve fibers. OPN4+ choroidal nerve fibers lacked VIP, but were co-localized with SP. OPN4-immunoreactivity was further detected in VIP+/SP + intrinsic choroidal neurons, in a hitherto unclassified CD68-negative choroidal cell population thus not representing macrophages, as well as in a subset of choroidal melanocytes. In chicken, choroidal nerve fibers were OPN4+, and further OPN4-IR was detected in clustered suprachoroidal structures that were not co-localized with ASMA and therefore do not represent non-vascular smooth-muscle cells. In the choroidal stroma, numerous cells displayed OPN4-IR, the majority of which was VIP-, while a few of those co-localized with VIP and were therefore classified as avian intrinsic choroidal neurons. OPN4-immunoreactivity was absent in choroidal blood vessels of both species. In summary, OPN4-IR was detected in both species in nerve fibers and cells, some of which could be identified (ICN, melanocytes in human), while others could not be classified yet. Nevertheless, the OPN4+ structures described here might be involved in developmental, light-, thermally-driven or nociceptive mechanisms, as known from other systems, but with respect to choroidal control this needs to be proven in upcoming studies.

Yingxiang Acupoint Embedding Improves Mucosal Barrier Function in Rats with Local Allergic Rhinitis.

INTRODUCTION: Epithelial barrier disruption is the initial cause of various diseases. We previously reported that acupoint catgut embedding (AE) improves tight junction proteins (TJs) in rats with allergic rhinitis. However, whether AE improves the epithelial barrier in local allergic rhinitis (LAR) remains unknown. METHODS: A total of 36 Sprague Dawley (SD) male rats aged 5-7 weeks were divided into 6 groups with 6 rats each: control group, LAR model group, false acupoint embedding + LAR group, acupoint embedding + LAR group, capsaicin + LAR group, and tunicamycin + acupoint embedding + LAR group. Behavioral observation, ELISA to detect inflammatory factors in nasal lavage fluid and serum IgE, nasal mucosal permeability test, hematoxylin-eosin staining, PCR to detect Substance P (SP), Western blot, and immunofluorescence to detect endoplasmic reticulum stress (ERS) index and TJs were used to investigate the mechanism of AE in LAR. RESULTS: AE improved the symptoms and pathological features of nasal mucosa of LAR rats, reduced the inflammatory factors (IL4, IL5, IL13) of nasal lavage fluid, and showed no significant change in serum IgE levels in all groups. In addition, AE decreased the expression of SP in nasal mucosa of LAR rats, inhibited ERS, increased the expression of tight junction protein, reduced the permeability of nasal mucosa, and improved the function of nasal mucosal barrier. CONCLUSION: This study confirms that AE can improve the nasal mucosal barrier function of LAR by reducing the expression of SP, inhibiting ERS and increasing the expression of TJs, thus enhancing the nasal mucosal barrier function.

Dissociation of the opioid receptor mechanisms that control mechanical and heat pain.

Delta and mu opioid receptors (DORs and MORs) are inhibitory G protein-coupled receptors that reportedly cooperatively regulate the transmission of pain messages by substance P and TRPV1-expressing pain fibers. Using a DOReGFP reporter mouse we now show that the DOR and MOR are, in fact, expressed by different subsets of primary afferents. The MOR is expressed in peptidergic pain fibers, the DOR in myelinated and nonpeptidergic afferents. Contrary to the prevailing view, we demonstrate that the DOR is trafficked to the cell surface under resting conditions, independently of substance P, and internalized following activation by DOR agonists. Finally, we show that the segregated DOR and MOR distribution is paralleled by a remarkably selective functional contribution of the two receptors to the control of mechanical and heat pain, respectively. These results demonstrate that behaviorally relevant pain modalities can be selectively regulated through the targeting of distinct subsets of primary afferent pain fibers.