Localization of ATP-gated P2X2 and P2X3 receptor immunoreactive nerves in rat taste buds.

P2X receptors have been suggested to play a role in the transduction of sensory signals such as pain and sound. In the present study, polyclonal antibodies against P2X1 to P2X6 receptors were used to localize P2X receptors in circumvallate and fungiform papillae of rats. Nerve fibres innervating the taste buds stained intensely with P2X3 receptor antibodies. P2X3 receptor-positive nerves were observed in the intra- and subgemmal regions. The nerve fibres were also stained with P2X2 receptor antibodies, but the intensity was much lower. The distribution of P2X2 receptor immunoreactivity overlaps with that of P2X3. These results suggest that ATP might be a neurotransmitter in taste reception cells in the taste buds, where it transducts the taste signals to the afferent taste nerves by activating P2X receptors at the synapses. This is the first experiment indicating such a role for ATP, although supplementary functional studies are required.     

On the distribution and origins of substance P in the papillae of the rat tongue: an experimental and immunohistochemical study

The distribution and the orgins of substance P (SP)-positive fibers in the papillae of the rat tongue were investigated by means of the indirect immunofluorescent method. Three types of papilla contain SP-positive fibers, though the number ofthese fibers varies from papilla to papilla. The circumvallate papilla contains the greatest number of SP-positive fibers, followed by the foliate papillae and the fungiform papillae; the filiform papillae lack SP-positive fibers. The papillar SP-positive fibers form dense bands in the lamina propria just beneath the epithelium. Some of the fibers enter the epithelium and the taste buds. It should be stressed that not every taste bud is provided with SP-positive fibers: Only 95% of the taste buds in the foliate papillae, 70% of the taste buds in the fungiform papillae, and 40% of the taste buds in the circumvallate papillae contain detectable SP-positive fibers. Unilateral section of the glossopharyngeal nerve resulted in a complete disappearance of SP-positive fibers in the foliate papillae on the operated side, and a slight decrease in the circumvallate papillae on both sides. Bilateral section of the glossopharyngeal nerve resulted in a complete disappearance of SP-positive fibers in the foliate and circumvallate papillae. Following unilateral section of the chorda tympani, SP-positive fibers in the tastebuds of the fungiform papillae disappeared completely. In addition, unilateral neurotomy of the mandibular nerve resulted in a complete disappearance of SP-positive fibers in the epithelium of the fungiform papillae. These facts strongly indicate that SP in the foliate and circumvallate papillae is supplied by the glossopharyngeal nerve, SP in the taste buds of the fungiform papillae by the chorda tympani, and SP in the epithelium of the fungiform papillae by the third division of the trigeminal nerve.     

Immunohistochemical,electrophysiological, and electron microscopical study of rat fungiform taste buds after regeneration of chorda tympani through the non-gustatory lingual nerve

The sensory innervation of fungiform papillae on the rat dorsal tongue is derived from branches of two cranial nerves: the lingual branch of the trigeminal nerve which provides somatosensory innervation and the chorda tympani (CT) branch of the facial nerve, which provides innervation to the taste buds. Removal of the CT results in degeneration of the taste buds. Removal of both nerves results in reduction in size of fungiform papillae and an altered pattern of keratinization in its epithelium. Regeneration of nerves to the epithelium restores the pre-operative condition. Thus, in addition to their sensory functions, both the CT and lingual seem to exert trophic effects on the phenotypic expression of epithelial cells in the fungiform papillae. We severed both the CT and lingual nerves in rats and sutured the proximal stump of the CT to the distal stump of the lingual to promote regeneration of the CT along the lingual nerve pathway. At the same time, we prevented the proximal stump of the lingual from regenerating into the tongue. Our purpose was to determine whether and how the innervation pattern of the regenerated taste bud might be different from normal under these experimental conditions. We found that reinnervation by the CT through the lingual nerve occurs, that this restores the anatomical and functional integrity of the fungiform taste buds and papillae, and that some papillae, but not all, were richly innervated with subgemmal, extragemmal, and perigemmal neuron-specific enolase, calcitonin gene-related peptide, substance P, and neurokinin A-positive fibers. Moreover, responses to taste stimuli were recorded electrophysiologically from the CT. © 1996 Wiley-Liss, Inc.     

Development of fungiform papillae,taste buds,and their innervation in the hamster

Fungiform taste buds in mature hamsters are less subject to neurotrophic influences than those of other species. This study evaluates taste-bud neurotrophism during development in hamsters by examining the relation between growing nerves and differentiating fungiform papillae. Chorda tympani (CT) or lingual (trigeminal) nerve (LN) fibers were labelled with Lucifer Yellow as they grew into (CT fibers) or around (LN fibers) developing taste buds. Developing fungiform papillae and taste pores were counted with the aid of a topical tongue stain. The tongue forms on embryonic days (E) 10.5–11 and contains deeply placed CT and LN fibers but no papillae. By E12, the tongue epithelium develops scattered elevations. These “eminences” selectively become innervated by LN fibers that grow to the epithelium earlier and in larger numbers than CT fibers. Definitive fungiform papillae form rapidly during E13–14 and become heavily innervated by LN fibers. Intraepithelial CT fibers, rare at E13, invariably innervate fungiform papillae containing nascent taste buds at E14. During E14–15 (birth = E15–16), most papillae contain taste buds with pores, extensive perigemmal LN innervation, and extensive intragemmal CT innervation. At birth, numbers of fungiform papillae and taste pores are adultlike. The results show that fungiform eminences begin forming in the absence of innervation. The subsequent differentiation of definitive fungiform papillae and their innervation by LN fibers occur synchronously, prior to the differentiation of taste buds and their CT innervation. The hamster is precocious (e.g., compared to rat) in terms of LN development and the structural maturity of the anterior tongue at birth. © Wiley-Liss, Inc.     

P2X7 receptors in cerebral ischemia

Cerebral ischemia is one of the most common diseases resulting in death and disability in aged people. It leads immediately to rapid energy failure, ATP depletion, and ionic imbalance, which increase extracellular ATP levels and accordingly activate P2X₇ receptors. These receptors are ATP-gated cation channels and widely distributed in nerve cells, especially in the immunocompetent cells of the brain. Currently, interest in the roles of P2X₇ receptors in ischemic brain injury is growing. In this review, we discuss recent research progress on the actions of P2X₇ receptors, their possible mechanisms in cerebral ischemia, and the potential therapeutic value of P2X₇ receptor antagonists which may provide a new target both for clinical and for research purposes.     

Brain-derived neurotrophic factor-, neurotrophin-3-, and tyrosine kinase receptor-like immunoreactivity in lingual taste bud fields of mature hamster

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), as well as their respective tyrosine kinase (Trk) receptors, TrkB and TrkC, influence peripheral target cell innervation, survival, and proliferation. In the mature taste system the role of neurotrophins and their receptors is not known. The mature hamster is an intriguing model because anterior lingual fungiform, unlike posterior lingual foliate and circumvallate, taste buds survive denervation. In light of this difference, we examined whether the degree of neurotrophin- or neurotrophin receptor-like immunoreactivity (IR) normally differs among lingual gemmal fields. In single- and double-labeled immunofluorescent experiments, 3,209 taste bud sections (profiles) from 13 hamsters were examined for immunopositive gemmal cells or nerve fibers using antibodies to BDNF and NT-3, their respective receptors TrkB and TrkC, and the neural marker ubiquitin c-terminal hydrolase L-1 [protein gene product (PGP) 9.5]. In each gemmal field, more than 75% of taste bud profiles showed immunopositivity to BDNF, NT-3, and TrkB. Across bud fields, BDNF-, TrkB-, and BDNF/TrkB-like IR, as well as PGP 9.5 and PGP 9.5/BDNF-like IR in centrally located, fungiform bud cells was greater (P < 0.0001 to P < 0.002) than in circumvallate or foliate buds. Within bud fields, the number of BDNF-like, labeled bud cells/bud profile was greater than that for NT-3-like IR in fungiform (P < 0.0002) and foliate (P < 0.0001) buds. TrkC was immunonegative in gemmal cells. The average density of TrkB- and TrkC-like fiber IR was more pronounced in fungiform than posterior gemmal-bearing papillae. Thus, fungiform papillae, whose taste buds are least affected by denervation, exhibit specific neurotrophin and receptor enrichment.     

TRPM8 protein localization in trigeminal ganglion and taste papillae

TRPM8 is a TRP family cation channel which can be activated by cold stimuli or l-menthol. However, TRPM8 protein localization of nerve terminals in sensory organs remains unknown. Here we generated an antibody against TRPM8 and analyzed TRPM8 protein localization in trigeminal ganglia (TG) and in sensory nerve fibers in the tongue. TRPM8 immunoreactivity was detected in a subset of neurons with a small diameter in TG and in nerve fibers in the tongue. TRPM8-immunoreactive nerve fibers were rich in fungiform papillae, but sparse in foliate and circumvallate papillae. The TRPM8-immunoreactive nerve fibers reached the outer epithelial layer in each papilla, while no TRPM8-immunoreactive nerve fibers penetrated into taste buds. Double labeling analysis revealed that TRPM8 immunoreactivity was co-expressed with a part of TRPV1 or CGRP-immunoreactive neurons in TG. However, TRPM8 immunoreactivity was not observed in TRPV1- or CGRP-positive nerve fibers in fungiform, foliate, and circumvallate papillae. These results suggest that TRPM8 protein is present in sensory lingual nerve fibers mainly projected from TG and might work as cold and l-menthol receptors on tongue.     

Involvement of peripheral adenosine 5′‐triphosphate and P2X purinoceptor in pain‐related behavior produced by orthotopic melanoma inoculation in mice

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 P2X₃ receptor was expressed in about 40% of peripherin‐positive small and medium‐sized neurons in the dorsal root ganglia. P2X₃‐positive neurons were significantly increased in melanoma‐bearing mice. These results suggest that ATP and P2X, especially P2X₃, receptors are involved in skin cancer pain, due to the increased release of ATP and increased expression of P2X₃ receptors in the sensory neurons.     

Localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamus

Previous pharmacological studies have indicated that ATP receptors may be involved in the regulation of physiological functions in hypothalamus. In the present study, the distribution of P2X₂ receptor in the rat hypothalamus was studied with immunohistochemistry. It was shown that P2X₂ immunoreactivity-positive neurons and nerve fibres were localized in many hypothalamic nuclei. Intense labelling of both neuronal cell bodies and nerve fibres was observed in the paraventricular nucleus, arcuate nucleus, retrochiasmatic area, periventricular nucleus, and the ventral part of tuber cinereum area. In supraoptic, circular, and ventral tuberomammillary nuclei the neuronal cell bodies were strongly positive, but few nerve fibres were positive. Axons with strong P2X₂ immunoreactivity were found in the organum vasculosum of the lamina terminalis and median eminence. Some scattered positive neurons and nerve fibres were found in many hypothalamic nuclei including preoptic nucleus. The results of the present study demonstrated the existence of P2X receptors in hypothalamus, as a basis for detailed studies of the roles of P2X receptors in the regulation of hypothalamic functions.     

Expressions of P2X2 and P2X3 receptors in rat nodose neurons after myocardial ischemia injury

The role of ATP is as a functional neurotransmitter and local intercellular signaling molecule. The nodose neurons express both P2X₂ and P2X₃ subunits in their plasma membrane. This study wants to observe the expression of P2X₂ receptor and the expression relationship between P2X₂ and P2X₃ in nodose neurons after myocardial ischemic injury. The expressions of P2X₃ immunoreactivity, mRNA and protein were analyzed by immunohistochemistry, in situ hybridization and western blotting. P2X₂ and P2X₃ immunoreactivity, mRNA expression had been increased after myocardial ischemia in nodose neurons. Myocardial ischemia enhanced P2X₂ and P2X₃ protein level in nodose ganglia after myocardial ischemia. P2X₂ receptor in nodose neurons participated in the transmission of cardiac pain. The changes of P2X₂ and P2X₃ immunoreactivities, mRNA and protein that occurred following myocardial ischemic injury in the nodose ganglia showed that a correlation existed between P2X₂ and P2X₃ expression. It suggests that P2X₂ receptor subtype in company with P2X₃ receptor subtype plays the important role in cardiac vagal sensory nociceptors with a sensitivity to ATP.     

P2X7R‐mediated Ca2+‐independent d‐serine release via pannexin‐1 of the P2X7R‐pannexin‐1 complex in astrocytes

d ‐serine is a coagonist of N‐methyl‐d ‐aspartate (NMDA) subtype of glutamate receptor and plays a role in regulating activity‐dependent synaptic plasticity. In this study, we examined the mechanism by which extracellular ATP triggers the release of d ‐serine from astrocytes and discovered a novel Ca²⁺‐independent release mechanism mediated by P2X₇ receptors (P2X₇R). Using [³H] d ‐serine, which was loaded into astrocytes via the neutral amino acid transporter 2 (ASCT2), we observed that ATP and a potent P2X₇R agonist, 2′(3′)‐O‐(4‐benzoylbenzoyl)adenosine‐5′‐triphosphate (BzATP), stimulated [³H]D‐serine release and that were abolished by P2X₇R selective antagonists and by shRNAs, whereas enhanced by removal of intracellular or extracellular Ca²⁺. The P2X₇R‐mediated d ‐serine release was inhibited by pannexin‐1 antagonists, such as carbenoxolone (CBX), probenecid (PBN), and ¹⁰Panx‐1 peptide, and shRNAs, and stimulation of P2X₇R induced P2X₇R‐pannexin‐1 complex formation. Simply incubating astrocytes in Ca²⁺/Mg²⁺‐free buffer also induced the complex formation, and that enhanced basal d ‐serine release through pannexin‐1. The P2X₇R‐mediated d ‐serine release assayed in Ca²⁺/Mg²⁺‐free buffer was enhanced as well, and that was inhibited by CBX. Treating astrocytes with general protein kinase C (PKC) inhibitors, such as chelerythrine, GF109203X, and staurosporine, but not Ca²⁺‐dependent PKC inhibitor, Gö6976, inhibited the P2X₇R‐mediated d ‐serine release. Thus, we conclude that in astrocytes, P2X₇R‐pannexin‐1 complex formation is crucial for P2X₇R‐mediated d ‐serine release through pannexin‐1 hemichannel. The release is Ca²⁺‐independent and regulates by a Ca²⁺‐independent PKC. The activated P2X₇R per se is also functioned as a permeation channel to release d ‐serine in part. This P2X₇R‐mediated d ‐serine release represents an important mechanism for activity‐dependent neuron‐glia interaction. GLIA 2015;63:877–893     

The role of substance P and calcitonin gene-related peptide containing nerve fibers in maintaining fungiform taste buds in the rat after a chronic chorda tympani nerve injury.

Taste buds in the anterior part of the tongue of adult rats were denervated by unilateral resection of the chorda tympani nerve in the middle ear. Three months later one group of animals was perfused and their tongues were processed for demonstration of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity. Fungiform taste buds found on the denervated side showed increased numbers of intragemmal SP- and CGRP-immunoreactive (IR) fibers compared to the normal side. Compared to the normal side, the number of taste buds appeared to be fewer on the denervated side. Moreover, taste buds on this side seemed to be only partially restored. Another group of animals was given the neurotoxin capsaicin which causes a depletion of SP and CGRP from sensory axons. The animals were perfused 2 or 3 weeks after the capsaicin treatment, and their tongues prepared for SP and CGRP immunohistochemistry or for histological examination of taste buds. Very few SP- and CGRP-IR fibers were present in capsaicin-treated animals. In these animals almost all fungiform taste buds and papillae on the chorda tympani-injured side disappeared. In contrast, normal numbers of taste buds were still present on the contralateral side where the chorda tympani innervation remained intact. It is conceivable that taste buds on the chorda tympani-innervated part of the tongue, deprived of the normal chorda tympani-innervation, can regenerate and become reinnervated by SP- and CGRP-containing fibers, and that these are essential for partially restoring and maintaining the structure of the denervated taste buds and the fungiform papillae.     

Purinergic signaling mediated by P2X7 receptors controls myelination in sciatic nerves

Adenosine‐5′‐triphosphate, the physiological ligand of P2X receptors, is an important factor in peripheral nerve development. P2X₇ receptor is expressed in Schwann cells (SCs), but the specific effects of P2X₇ purinergic signaling on peripheral nerve development, myelination, and function are largely unknown. In this study, sciatic nerves from P2X₇ knockout mice were analyzed for altered expression of myelin‐associated proteins and for alterations in nerve morphology. Immunohistochemical analyses revealed that, in the wild‐type peripheral nerves, the P2X₇ receptor was localized mainly in myelinating SCs, with only a few immunopositive nonmyelinating SCs. Complete absence of P2X₇ receptor protein was confirmed in the sciatic nerves of the knockout mice by Western blot and immunohistochemistry. Western blot analysis revealed that expression levels of the myelin proteins protein zero and myelin‐associated glycoprotein are reduced in P2X₇ knockout nerves. In accordance with the molecular results, transmission electron microscopy analyses revealed that P2X₇ knockout nerves possess significantly more unmyelinated axons, contained in a higher number of Remak bundles. The myelinating/nonmyelinating SC ratio was also decreased in knockout mice, and we found a significantly increased number of irregular fibers compared with control nerves. Nevertheless, the myelin thickness in the knockout was unaltered, suggesting a stronger role for P2X₇ in determining SC maturation than in myelin formation. In conclusion, we present morphological and molecular evidence of the importance of P2X₇ signaling in peripheral nerve maturation and in determining SC commitment to a myelinating phenotype. © 2014 Wiley Periodicals, Inc.     

Spinal P2X(7) receptor mediates microglia activation-induced neuropathic pain in the sciatic nerve injury rat model

P2X₇ receptor is an important member of ATP-sensitive ionotropic P2X receptors family, which includes seven receptor subtypes (P2X₁-P2X₇). Recent evidence indicates that P2X₇R participates in the onset and persistence of neuropathic pain. In tetanic stimulation of the sciatic nerve model, P2X₇R was involved in the activation of microglia, but whether this happens in other neuropathic pain models remains unclear. In this study we used immunohistochemistry and Western blot to explore the relationship of P2X₇R expression with microglia activation, and with mechanical allodynia and thermal hypersensitivity in the chronic constriction of the sciatic nerve (CCI) rat model. The results show that following nerve ligature, mechanical allodynia and thermal hypersensitivity were developed within 3 days (d), peaked at 14 d and persisted for 21 d on the injured side. P2X₇R levels in the ipsilateral L4-6 spinal cord were increased markedly after injury and the highest levels were observed on day 14, significant difference was observed at I-IV layers of the dorsal horn. The change in P2X₇R levels in the spinal cord was consistent with the development of mechanical allodynia and thermal hypersensitivity. Intrathecal administration of the P2X₇R antagonist Brilliant Blue G (BBG) reversed CCI-induced mechanical allodynia and thermal hypersensitivity. Double-labeled immunofluorescence showed that P2X₇R expression were restricted to microglia, spinal microglia were activated after nerve injury, which was inhibited by BBG. These results indicated that spinal P2X₇R mediate microglia activation, this process may play an important role in development of mechanical allodynia and thermal hypersensitivity in CCI model.     

Dystonin deficiency reduces taste buds and fungiform papillae in the anterior part of the tongue

The anterior part of the tongue was examined in wild type and dystonia musculorum mice to assess the effect of dystonin loss on fungiform papillae. In the mutant mouse, the density of fungiform papillae and their taste buds was severely decreased when compared to wild type littermates (papilla, 67% reduction; taste bud, 77% reduction). The mutation also reduced the size of these papillae (17% reduction) and taste buds (29% reduction). In addition, immunohistochemical analysis demonstrated that the dystonin mutation reduced the number of PGP 9.5 and calbindin D28k-containing nerve fibers in fungiform papillae. These data together suggest that dystonin is required for the innervation and development of fungiform papillae and taste buds.     

Epithelial Na+ channel subunits in rat taste cells: localization and regulation by aldosterone

Amiloride-sensitive Na+ channels play an important role in transducing Na+ salt taste. Previous studies revealed that in rodent taste cells, the channel shares electrophysiological and pharmacological properties with the epithelial Na+ channel, ENaC. Using subunit-specific antibodies directed against alpha, beta, and gamma subunits of rat ENaC (rENaC), we observed cytoplasmic immunoreactivity for all three subunits in nearly all taste cells of fungiform papillae, and in about half of the taste cells in foliate and vallate papillae. The intensity of labeling in cells of vallate papillae was significantly lower than that of fungiform papillae, especially for beta and gamma subunits. Dual localization experiments showed that immunoreactivity for the taste cell-specific G protein, gustducin, occurs in a subset ofrENaC positive taste cells. Aldosterone is known to increase the amiloride sensitivity of the NaCl taste response. In our study, increases in blood aldosterone levels enhanced the intensity of apical immunoreactivity for beta and gamma rENaC in taste cells of all papillae. In addition, whole cell recordings from isolated taste cells showed that in fungiform papillae, aldosterone increased the number of amiloride-sensitive taste cells and enhanced the current amplitude. In vallate taste cells, which are normally unresponsive to amiloride, aldosterone treatment induced an amiloride sensitive current in about half of the cells. Immunoreactivity for rENaC subunits also was present in nonsensory epithelial cells, especially in the anterior portion of the tongue. In addition, immunoreactivity for all subunits, but especially beta and gamma, was associated with some nerve fibers innervating taste papillae. These extragustatory sites of rENaC expression may indicate a role for this channel in paracellular transduction of sodium ions.     

Microglia‐derived purines modulate mossy fibre synaptic transmission and plasticity through P2X4 and A1 receptors

Recent data have provided evidence that microglia, the brain‐resident macrophage‐like cells, modulate neuronal activity in both physiological and pathophysiological conditions, and microglia are therefore now recognized as synaptic partners. Among different neuromodulators, purines, which are produced and released by microglia, have emerged as promising candidates to mediate interactions between microglia and synapses. The cellular effects of purines are mediated through a large family of receptors for adenosine and for ATP (P2 receptors). These receptors are present at brain synapses, but it is unknown whether they can respond to microglia‐derived purines to modulate synaptic transmission and plasticity. Here, we used a simple model of adding immune‐challenged microglia to mouse hippocampal slices to investigate their impact on synaptic transmission and plasticity at hippocampal mossy fibre (MF) synapses onto CA3 pyramidal neurons. MF–CA3 synapses show prominent forms of presynaptic plasticity that are involved in the encoding and retrieval of memory. We demonstrate that microglia‐derived ATP differentially modulates synaptic transmission and short‐term plasticity at MF–CA3 synapses by acting, respectively, on presynaptic P2X₄ receptors and on adenosine A₁ receptors after conversion of extracellular ATP to adenosine. We also report that P2X₄ receptors are densely located in the mossy fibre tract in the dentate gyrus–CA3 circuitry. In conclusion, this study reveals an interplay between microglia‐derived purines and MF–CA3 synapses, and highlights microglia as potent modulators of presynaptic plasticity.     

The reduction of intraepidermal P2X3 nerve fiber density correlates with behavioral hyperalgesia in a rat model of nerve injury‐induced pain

Skin biopsies from patients with neuropathic pain often show changes in epidermal innervation, although it remains to be elucidated to what extent such changes can be linked to a particular subgroup of nerve fibers and how these changes are correlated with pain intensity. Here, we investigated to what extent behavioral signs of hyperalgesia are correlated with immunohistochemical changes of peptidergic and non‐peptidergic epidermal nerve fibers in a rat model of nerve injury‐induced pain. Rats subjected to unilateral partial ligation of the sciatic nerve developed significant mechanical and thermal hyperalgesia as tested by the withdrawal responses of the ipsilateral footpad to von Frey hairs and hotplate stimulation. At day 14, epidermal nerve fiber density and total epidermal nerve fiber length/mm² were significantly and consistently reduced compared to the contralateral side, following testing and re‐testing by two blinded observers. The expression of calcitonin gene‐related peptide, a marker for peptidergic nerve fibers, was not significantly changed on the ipsilateral side. In contrast, the expression of the P2X₃ receptor, a marker for non‐peptidergic nerve fibers, was not only significantly reduced but could also be correlated with behavioral hyperalgesia. When labeling both peptidergic and non‐peptidergic nerve fibers with the pan‐neuronal marker PGP9.5, the expression was significantly reduced, albeit without a significant correlation with behavioral hyperalgesia. In conjunction, our data suggest that the pathology of the P2X₃ epidermal nerve fibers can be selectively linked to neuropathy, highlighting the possibility that it is the degeneration of these fibers that drives hyperalgesia.