AB5895 Sigma-AldrichAnti-P2X3 Receptor Antibody, pain

Experiments using P2X3 knock-out mice or more general P2X receptor antagonists suggest that P2X3 receptors contribute to visceral hypersensitivity. We aimed to investigate the effect of the selective P2X3 antagonist A-317491 on visceral sensitivity under physiological conditions, during acute colitis and in the post-inflammatory phase of colitis.Trinitrobenzene sulphonic-acid colitis was monitored by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Visceral sensitivity was assessed by quantifying visceromotor responses to colorectal distension in controls, rats with acute colitis and post-colitis rats. A-317491 was administered 30 min prior to visceral sensitivity testing. Expression of P2X3 receptors (RT-PCR and immunohistochemistry) and the intracellular signalling molecules cdk5, csk and CASK (RT-PCR) were quantified in colonic tissue and dorsal root ganglia. ATP release in response to colorectal distension was measured by luminiscence.Rats with acute TNBS-colitis displayed significant visceral hypersensitivity that was dose-dependently, but not fully, reversed by A-317491. Hypersenstivity was accompanied by an increased colonic release of ATP. Post-colitis rats also displayed visceral hypersensitivity that was dose-dependently reduced and fully normalized by A-317491 without increased release of ATP. A-317491 did not modify visceral sensitivity in controls. P2X3 mRNA and protein expression in the colon and dorsal root ganglia were similar in control, acute colitis and post-colitis groups, while colonic mRNA expression of cdk5, csk and CASK was increased in the post-colitis group only.These findings indicate that P2X3 receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X3 receptors as potential new targets in the treatment of abdominal pain syndromes.

A limited number of growth factors are capable of regulating numerous developmental processes, but how they accomplish this is unclear. The gustatory system is ideal for examining this issue because the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) have different developmental roles although both of them activate the same receptors, TrkB and p75. Here we first investigated whether the different roles of BDNF and NT4 are due to their differences in temporal and spatial expression patterns. Then, we asked whether or not these two neurotrophins exert their unique roles on the gustatory system by regulating different sets of downstream genes. By using Bdnf(Nt4/Nt4) mice, in which the coding region for BDNF is replaced with NT4, we examined whether the different functions of BDNF and NT4 are interchangeable during taste development. Our results demonstrated that NT4 could mediate most of the unique roles of BDNF during taste development. Specifically, caspase-3-mediated cell death, which was increased in the geniculate ganglion in Bdnf(-/-) mice, was rescued in Bdnf(Nt4/Nt4) mice. In BDNF knockout mice, tongue innervation was disrupted, and gustatory axons failed to reach their targets. However, disrupted innervation was rescued and target innervation is normal when NT4 replaced BDNF. Genome wide expression analyses revealed that BDNF and NT4 mutant mice exhibited different gene expression profiles in the gustatory (geniculate) ganglion. Compared to wild type, the expression of differentiation-, apoptosis- and axon guidance-related genes was changed in BDNF mutant mice, which is consistent with their different roles during taste development. However, replacement of BDNF by NT4 rescued these gene expression changes. These findings indicate that the functions of BDNF and NT4 in taste development are interchangeable. Spatial and temporal differences in BDNF and NT4 expression can regulate differential gene expression in vivo and determine their specific roles during development.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) regulate the survival of gustatory neurons, axon growth and branching, and innervation of taste buds during development. These actions are largely, but not completely, mediated through the tyrosine kinase receptor, TrkB. Here, we investigated the role of p75, the other major receptor for BDNF and NT4, in the development of the taste system.We found that p75-/-mice showed delayed axon outgrowth and reduced branching of gustatory axons at embryonic day (E)13.5. From E14.5 to E18.5, gustatory neurons innervated fewer papillae and completely failed to innervate the mid-region of the tongue in p75-/-mice. These early effects of the p75 mutation on gustatory axons preceded the loss of geniculate ganglion neurons starting at E14.5 and also contributed to a loss of taste buds at and after birth. Because knockouts for the TrkB receptor (TrkB-/-) do not lose as many taste buds as hybrid knockouts for its two ligands (BDNF and NT4), we asked if p75 maintains those additional taste buds in the absence of TrkB. It does not; hybrid TrkB-/-/p75-/-mice had more taste buds than TrkB-/-mice; these additional taste buds were not due to an increase in neurons or innervation.p75 regulates gustatory neuron axon branching and tongue innervation patterns during taste system development. This function is likely accomplished independently of BDNF, NT4, and TrkB. In addition, p75 does not support the remaining neurons or taste buds in TrkB-/-mice.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) are two neurotrophins that play distinct roles in geniculate (taste) neuron survival, target innervation, and taste bud formation. These two neurotrophins both activate the tropomyosin-related kinase B (TrkB) receptor and the pan-neurotrophin receptor p75. Although the roles of these neurotrophins have been well studied, the degree to which BDNF and NT-4 act via TrkB to regulate taste development in vivo remains unclear. In this study, we compared taste development in TrkB(-/-) and Bdnf(-/-)/Ntf4(-/-) mice to determine if these deficits were similar. If so, this would indicate that the functions of both BDNF and NT-4 can be accounted for by TrkB-signaling. We found that TrkB(-/-) and Bdnf(-/-)/Ntf4(-/-) mice lose a similar number of geniculate neurons by E13.5, which indicates that both BDNF and NT-4 act primarily via TrkB to regulate geniculate neuron survival. Surprisingly, the few geniculate neurons that remain in TrkB(-/-) mice are more successful at innervating the tongue and taste buds compared with those neurons that remain in Bdnf(-/-)/Ntf4(-/-) mice. The remaining neurons in TrkB(-/-) mice support a significant number of taste buds. In addition, these remaining neurons do not express the TrkB receptor, which indicates that either BDNF or NT-4 must act via additional receptors to influence tongue innervation and/or targeting.

Itch-specific neurons have been sought for decades. The existence of such neurons has been doubted recently as a result of the observation that itch-mediating neurons also respond to painful stimuli. We genetically labeled and manipulated MrgprA3(+) neurons in the dorsal root ganglion (DRG) and found that they exclusively innervated the epidermis of the skin and responded to multiple pruritogens. Ablation of MrgprA3(+) neurons led to substantial reductions in scratching evoked by multiple pruritogens and occurring spontaneously under chronic itch conditions, whereas pain sensitivity remained intact. Notably, mice in which TRPV1 was exclusively expressed in MrgprA3(+) neurons exhibited itch, but not pain, behavior in response to capsaicin. Although MrgprA3(+) neurons were sensitive to noxious heat, activation of TRPV1 in these neurons by noxious heat did not alter pain behavior. These data suggest that MrgprA3 defines a specific subpopulation of DRG neurons mediating itch. Our study opens new avenues for studying itch and developing anti-pruritic therapies.

Chronic pain associated with injury or disease can result from dysfunction of sensory afferents whereby the threshold for activation of pain-sensing neurons (nociceptors) is lowered. Neurotrophic factors control nociceptor development and survival, but also induce sensitization through activation of their cognate receptors, attributable, in part, to the modulation of ion channel function. Thermal pain is mediated by channels of the transient receptor potential (TRP) family, including the cold and menthol receptor TRPM8. Although it has been shown that TRPM8 is involved in cold hypersensitivity, the molecular mechanisms underlying this pain modality are unknown. Using microarray analyses to identify mouse genes enriched in TRPM8 neurons, we found that the glial cell line-derived neurotrophic factor (GDNF) family receptor GFRα3 is expressed in a subpopulation of TRPM8 sensory neurons that have the neurochemical profile of cold nociceptors. Moreover, we found that artemin, the specific GFRα3 ligand that evokes heat hyperalgesia, robustly sensitized cold responses in a TRPM8-dependent manner in mice. In contrast, GFRα1 and GFRα2 are not coexpressed with TRPM8 and their respective ligands GDNF and neurturin did not induce cold pain, whereas they did evoke heat hyperalgesia. Nerve growth factor induced mild cold sensitization, consistent with TrkA expression in TRPM8 neurons. However, bradykinin failed to alter cold sensitivity even though its receptor expresses in a subset of TRPM8 neurons. These results show for the first time that only select neurotrophic factors induce cold sensitization through TRPM8 in vivo, unlike the broad range of proalgesic agents capable of promoting heat hyperalgesia.

We recently developed an ex vivo lung slice model that allows for confocal live cell imaging (LCI) of neuroepithelial bodies (NEBs) in postnatal mouse lungs (postnatal days 1-21 and adult). NEBs are morphologically well-characterized, extensively innervated groups of neuroendocrine cells in the airway epithelium, which are shielded from the airway lumen by 'Clara-like' cells. The prominent presence of differentiated NEBs from early embryonic development onwards, strongly suggests that NEBs may exert important functions during late fetal and neonatal life. The main goal of the present study was to adapt the current postnatal LCI lung slice model to enable functional studies of fetal mouse lungs (gestational days 17-20).In vibratome lung slices of prenatal mice, NEBs could be unequivocally identified with the fluorescent stryryl pyridinium dye 4-Di-2-ASP. Changes in the intracellular free calcium concentration and in mitochondrial membrane potential could be monitored using appropriate functional fluorescent indicators (e.g. Fluo-4).It is clear that the described fetal mouse lung slice model is suited for LCI studies of Clara cells, ciliated cells, and the NEB microenvironment, and offers excellent possibilities to further unravel the significance of NEBs during the prenatal and perinatal period.

Adenosine 5'-triphosphate (ATP) plays an important role in nociceptive processing. We used a mouse model of skin cancer pain to investigate the role of ATP in cancer pain. Orthotopic inoculation of B16-BL6 melanoma cells into the hind paw produced spontaneous licking of the tumor-bearing paw. Intraperitoneal injection of the P2 purinoceptor antagonist suramin suppressed spontaneous licking dose-dependently. Two P2X purinoceptor antagonists also suppressed spontaneous licking. An intraplantar injection of ATP, which did not induce licking in the healthy paw, increased licking of the tumor-bearing paw. Spontaneous firing of the tibial nerve was significantly increased in tumor-bearing mice and was inhibited by suramin. Extracellular concentration of ATP was significantly increased in the tumor-bearing paw than in the normal paw. ATP is concentrated in the culture medium of melanoma, lung cancer and breast cancer cells, but not fibroblasts. The P2X(3) receptor was expressed in about 40% of peripherin-positive small and medium-sized neurons in the dorsal root ganglia. P2X(3)-positive neurons were significantly increased in melanoma-bearing mice. These results suggest that ATP and P2X, especially P2X(3), receptors are involved in skin cancer pain, due to the increased release of ATP and increased expression of P2X(3) receptors in the sensory neurons.

The brain receives sensory input from diverse peripheral tissues, including the skin, the body's largest sensory organ. Using genetically encoded axonal tracers expressed from the Mrgprd locus, we identify a subpopulation of nonpeptidergic, nociceptive neurons that project exclusively to the skin, and to no other peripheral tissue examined. Surprisingly, Mrgprd(+) innervation is restricted to the epidermis and absent from specialized sensory structures. Furthermore, Mrgprd(+) fibers terminate in a specific layer of the epidermis, the stratum granulosum. This termination zone is distinct from that innervated by most CGRP(+) neurons, revealing that peptidergic and nonpeptidergic epidermal innervation is spatially segregated. The central projections deriving from these distinct epidermal innervation zones terminate in adjacent laminae in the dorsal spinal cord. Thus, afferent input from different layers of the epidermis is conveyed by topographically segregated sensory circuits, suggesting that at least some aspects of sensory information processing may be organized along labeled lines.