Nonpeptidergic neurons suppress mast cells via glutamate to maintain skin homeostasis.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.

The functional organization of cutaneous low-threshold mechanosensory neurons.

Innocuous touch of the skin is detected by distinct populations of neurons, the low-threshold mechanoreceptors (LTMRs), which are classified as Aß-, Aδ-, and C-LTMRs. Here, we report genetic labeling of LTMR subtypes and visualization of their relative patterns of axonal endings in hairy skin and the spinal cord. We found that each of the three major hair follicle types of trunk hairy skin (guard, awl/auchene, and zigzag hairs) is innervated by a unique and invariant combination of LTMRs; thus, each hair follicle type is a functionally distinct mechanosensory end organ. Moreover, the central projections of Aß-, Aδ-, and C-LTMRs that innervate the same or adjacent hair follicles form narrow LTMR columns in the dorsal horn. These findings support a model of mechanosensation in which the activities of Aß-, Aδ-, and C-LTMRs are integrated within dorsal horn LTMR columns and processed into outputs that underlie the perception of myriad touch sensations.

Parallel ascending spinal pathways for affective touch and pain.

The anterolateral pathway consists of ascending spinal tracts that convey pain, temperature and touch information from the spinal cord to the brain1-4. Projection neurons of the anterolateral pathway are attractive therapeutic targets for pain treatment because nociceptive signals emanating from the periphery are channelled through these spinal projection neurons en route to the brain. However, the organizational logic of the anterolateral pathway remains poorly understood. Here we show that two populations of projection neurons that express the structurally related G-protein-coupled receptors (GPCRs) TACR1 and GPR83 form parallel ascending circuit modules that cooperate to convey thermal, tactile and noxious cutaneous signals from the spinal cord to the lateral parabrachial nucleus of the pons. Within this nucleus, axons of spinoparabrachial (SPB) neurons that express Tacr1 or Gpr83 innervate distinct sets of subnuclei, and strong optogenetic stimulation of the axon terminals induces distinct escape behaviours and autonomic responses. Moreover, SPB neurons that  express Gpr83 are highly sensitive to cutaneous mechanical stimuli and receive strong synaptic inputs from both high- and low-threshold primary mechanosensory neurons. Notably, the valence associated with activation of SPB neurons that express Gpr83 can be either positive or negative, depending on stimulus intensity. These findings reveal anatomically, physiologically and functionally distinct subdivisions of the SPB tract that underlie affective aspects of touch and pain.

Neuronally expressed PDL1, not PD1, suppresses acute nociception.

PDL1 is a protein that induces immunosuppression by binding to PD1 expressed on immune cells. In line with historical studies, we found that membrane-bound PD1 expression was largely restricted to immune cells; PD1 was not detectable at either the mRNA or protein level in peripheral neurons using single neuron qPCR, immunolabeling and flow cytometry. However, we observed widespread expression of PDL1 in both sensory and sympathetic neurons that could have important implications for patients receiving immunotherapies targeting this pathway that include unexpected autonomic and sensory related effects. While signaling pathways downstream of PD1 are well established, little to no information is available regarding the intracellular signaling downstream of membrane-bound PDL1 (also known as reverse signaling). Here, we administered soluble PD1 to engage neuronally expressed PDL1 and found that PD1 significantly reduced nocifensive behaviors evoked by algogenic capsaicin. We used calcium imaging to examine the underlying neural mechanism of this reduction and found that exogenous PD1 diminished TRPV1-dependent calcium transients in dissociated sensory neurons. Furthermore, we observed a reduction in membrane expression of TRPV1 following administration of PD1. Exogenous PD1 had no effect on pain-related behaviors in sensory neuron specific PDL1 knockout mice. These data indicate that neuronal PDL1 activation is sufficient to modulate sensitivity to noxious stimuli and as such, may be an important homeostatic mechanism for regulating acute nociception.

Unique Molecular Characteristics of Visceral Afferents Arising from Different Levels of the Neuraxis: Location of Afferent Somata Predicts Function and Stimulus Detection Modalities.

Viscera receive innervation from sensory ganglia located adjacent to multiple levels of the brainstem and spinal cord. Here we examined whether molecular profiling could be used to identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and nodose ganglia (NG) in male and female mice. Profiling of TL and LS bladder afferents was also performed. Visceral afferents were back-labeled using retrograde tracers injected into proximal and distal regions of colon or bladder, followed by single-cell qRT-PCR and analysis via an automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for colon) based on their established role in stimulus detection, regulation of sensitivity/function, or neuroimmune interaction. A total of 132 colon afferents (from NG, TL, and LS ganglia) and 128 bladder afferents (from TL and LS ganglia) were analyzed. Retrograde labeling from the colon showed that NG and TL afferents innervate proximal and distal regions of the colon, whereas 98% of LS afferents only project to distal regions. There were clusters of colon and bladder afferents, defined by mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was found. In addition, transcriptionally, NG colon afferents were almost completely segregated from colon TL and LS neurons. Furthermore, colon and bladder afferents expressed genes at similar levels, although different gene combinations defined the clusters. These results indicate that genes implicated in both homeostatic regulation and conscious sensations are found at all anatomic levels, suggesting that afferents from different portions of the neuraxis have overlapping functions.SIGNIFICANCE STATEMENT Visceral organs are innervated by sensory neurons whose cell bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, where sensation and pain are mediated by spinal afferents and fear and anxiety (the affective aspects of visceral pain) are the domain of nodose afferents. The transcriptomic analysis performed here reveals that genes implicated in both homeostatic regulation and pain are found in afferents across all ganglia types, suggesting that conscious sensation and homeostatic regulation are the result of convergence, and not segregation, of sensory input.

Genetic identification of C fibres that detect massage-like stroking of hairy skin in vivo.

Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons in mice that express the G-protein-coupled receptor MRGPRB4 (refs 5, 6). These neurons exclusively innervate hairy skin with large terminal arborizations that resemble the receptive fields of C-tactile (CT) afferents in humans. Unlike other molecularly defined mechanosensory C-fibre subtypes, MRGPRB4(+) neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in the spinal projections of these neurons during stimulation of the periphery in intact mice. Here we show that MRGPRB4(+) neurons are activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C fibres expressing MRGPRD was activated by pinching but not by stroking, consistent with previous physiological and behavioural data. Pharmacogenetic activation of Mrgprb4-expressing neurons in freely behaving mice promoted conditioned place preference, indicating that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MRGPRB4 neurons during natural behaviours, and provide a general approach to the functional characterization of genetically identified subsets of somatosensory neurons in vivo.

Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes.

The extent to which the skin instructs peripheral somatosensory neuron maturation is unknown. We studied this question in Merkel cell-neurite complexes, where slowly adapting type I (SAI) neurons innervate skin-derived Merkel cells. Transgenic mice lacking Merkel cells had normal dorsal root ganglion (DRG) neuron numbers, but fewer DRG neurons expressed the SAI markers TrkB, TrkC, and Ret. Merkel cell ablation also decreased downstream TrkB signaling in DRGs, and altered the expression of genes associated with SAI development and function. Skin- and Merkel cell-specific deletion of Bdnf during embryogenesis, but not postnatal Bdnf deletion or Ntf3 deletion, reproduced these results. Furthermore, prototypical SAI electrophysiological signatures were absent from skin regions where Bdnf was deleted in embryonic Merkel cells. We conclude that BDNF produced by Merkel cells during a precise embryonic period guides SAI neuron development, providing the first direct evidence that the skin instructs sensory neuron molecular and functional maturation. SIGNIFICANCE STATEMENT: Peripheral sensory neurons show incredible phenotypic and functional diversity that is initiated early by cell-autonomous and local environmental factors found within the DRG. However, the contribution of target tissues to subsequent sensory neuron development remains unknown. We show that Merkel cells are required for the molecular and functional maturation of the SAI neurons that innervate them. We also show that this process is controlled by BDNF signaling. These findings provide new insights into the regulation of somatosensory neuron development and reveal a novel way in which Merkel cells participate in mechanosensation.

Cutaneous neurturin overexpression alters mechanical, thermal, and cold responsiveness in physiologically identified primary afferents.

Neurotrophic factors play an important role in the regulation of functional properties of sensory neurons under normal and pathological conditions. The GDNF family member neurturin is one such factor that has been linked to modulating responsiveness to peripheral stimuli. Neurturin binds to the GFRα2 receptor, a receptor found primarily in isolectin B4-expressing polymodal cutaneous nociceptors. Previous work has shown that knockout of GFRα2 alters heat, but not mechanical, responses in dissociated sensory neurons and reduces pain-related behaviors during the second phase of the formalin test. Research has also shown that overexpression of neurturin in basal keratinocytes increases behavioral responsiveness to mechanical stimulation and innocuous cooling of the skin without affecting noxious heat responses. Here we directly examined the impact of neurturin overexpression on cutaneous afferent function. We compared physiological responses of individual sensory neurons to mechanical and thermal stimulation of the skin, using an ex vivo skin-nerve-dorsal root ganglion-spinal cord preparation produced from neurturin-overexpressing (NRTN/OE) mice and wild-type littermate controls. We found that neurturin overexpression increases responsiveness to innocuous mechanical stimuli in A-fiber nociceptors, alters thermal responses in the polymodal subpopulation of C-fiber sensory neurons, and changes the relative numbers of mechanically sensitive but thermally insensitive C-fiber afferents. These results demonstrate the potential roles of different functional groups of sensory neurons in the behavioral changes observed in mice overexpressing cutaneous neurturin and highlight the importance of neurturin in regulating cutaneous afferent response properties.NEW & NOTEWORTHY GDNF family neurotrophic factors regulate the development and function of primary sensory neurons. Of these, neurturin has been shown to modulate mechanical and cooling sensitivity behaviorally. Here we show that overexpression of neurturin in basal keratinocytes regulates mechanical responsiveness in A-fiber primary sensory neurons while increasing the overall numbers of cold-sensing units. Results demonstrate a crucial role for cutaneous neurturin in modulating responsiveness to peripheral stimuli at the level of the primary afferent.

Dynamic changes in heat transducing channel TRPV1 expression regulate mechanically insensitive, heat sensitive C-fiber recruitment after axotomy and regeneration.

Peripheral injury leads to a significant increase in the prevalence of mechanically insensitive, heat-sensitive C-fibers (CH) that contain the heat transducing TRPV1 (transient receptor potential vanilloid type I) channel in mice. We have recently shown that this recruitment of CH fibers is associated with increased expression of the receptor for GDNF (glial cell line-derived neurotrophic factor) family neurotrophic factor artemin (GFRα3), and that in vivo inhibition of GFRα3 prevented the increase in TRPV1 expression normally observed following axotomy. Here we have directly tested the hypothesis that the recruitment of functional CH fibers following nerve regeneration requires enhanced TRPV1 levels. We used in vivo siRNA-mediated knockdown to inhibit the injury-induced expression of TRPV1 coupled with ex vivo recording to examine response characteristics and neurochemical phenotypes of different functionally defined cutaneous sensory neurons after regeneration. We confirmed that inhibition of TRPV1 did not affect the axotomy-induced decrease in polymodal C-fiber (CPM) heat threshold, but transiently prevented the recruitment of CH neurons. Moreover, a recovery of TRPV1 protein was observed following resolution of siRNA-mediated inhibition that was correlated with a concomitant rebound in CH neuron recruitment. Thus dynamic changes in TRPV1 expression, not absolute levels, may underlie the functional alterations observed in CH neurons and may contribute to the development of heat hyperalgesia after nerve injury.

Comprehensive phenotyping of group III and IV muscle afferents in mouse.

While much is known about the functional properties of cutaneous nociceptors, relatively little is known about the comprehensive functional properties of group III and IV muscle afferents. We have developed a mouse ex vivo forepaw muscle, median and ulnar nerve, dorsal root ganglion (DRG), spinal cord recording preparation to examine the functional response properties, neurochemical phenotypes, and spinal projections of individual muscle afferents. We found that the majority of group III and IV muscle afferents were chemosensitive (52%) while only 34% responded to mechanical stimulation and fewer (32%) responded to thermal stimuli. The chemosensitive afferents could be grouped into those that responded to a "low"-metabolite mixture containing amounts of lactate and ATP at pH 7.0 simulating levels observed in muscle during exercise (metaboreceptors) and a "high"-metabolite mixture containing lactic acid concentrations and ATP at pH 6.6 mimicking levels observed during ischemic contractions (metabo-nociceptors). While the majority of the metabo-nociceptive fibers responding to the higher concentration levels were found to contain acid-sensing ion channel 3 (ASIC3) and/or transient receptor potential vanilloid type 1 (TRPV1), metaboreceptors responding to the lower concentration levels lacked these receptors. Anatomically, group III muscle afferents were found to have projections into laminae I and IIo, and deeper laminae in the spinal cord, while all functional types of group IV muscle afferents projected primarily into both laminae I and II. These results provide novel information about the variety of sensory afferents innervating the muscle and provide insight into the types of fibers that may exhibit plasticity after injuries.

Identification of a convergent spinal neuron population that encodes itch.

Itch is a protective sensation that drives scratching. Although specific cell types have been proposed to underlie itch, the neural circuit basis for itch remains unclear. Here, we used two-photon Ca2+ imaging of the dorsal horn to visualize the neuronal populations that are activated by itch-inducing agents. We identify a convergent population of spinal neurons that is defined by the expression of GRPR. Moreover, we discover that itch is conveyed to the brain via GRPR-expressing spinal output neurons that target the lateral parabrachial nucleus. Further, we show that nalfurafine, a clinically effective kappa opioid receptor agonist, relieves itch by inhibiting GRPR spinoparabrachial neurons. Finally, we demonstrate that a subset of GRPR spinal neurons show persistent, cell-intrinsic Ca2+ oscillations. These experiments provide the first population-level view of the spinal neurons that respond to pruritic stimuli, pinpoint the output neurons that convey itch to the brain, and identify the cellular target of kappa opioid receptor agonists for the inhibition of itch.

Cell type-specific calcium imaging of central sensitization in mouse dorsal horn.

Allodynia is a state in which pain is elicited by innocuous stimuli. Capsaicin applied to the skin results in an allodynia that extends to a broad region beyond the application site. This sensitization is thought to be mediated by spinal networks; however, we do not have a clear picture of which spinal neurons mediate this phenomenon. To address this gap, we used two-photon calcium imaging of excitatory interneurons and spinal projection neurons in the mouse spinal dorsal horn. To distinguish among neuronal subtypes, we developed CICADA, a cell profiling approach to identify cell types during calcium imaging. We then identified capsaicin-responsive and capsaicin-sensitized neuronal populations. Capsaicin-sensitized neurons showed emergent responses to innocuous input and increased receptive field sizes consistent with psychophysical reports. Finally, we identified spinal output neurons that showed enhanced responses from innocuous input. These experiments provide a population-level view of central sensitization and a framework with which to model somatosensory integration in the dorsal horn.

Enhanced artemin/GFRα3 levels regulate mechanically insensitive, heat-sensitive C-fiber recruitment after axotomy and regeneration.

We have shown recently that following saphenous nerve transection and successful regeneration, cutaneous polymodal nociceptors (CPMs) lacking transient receptor potential vanilloid 1 (TRPV1) are sensitized to heat stimuli and that mechanically insensitive, heat-sensitive C-fibers (CHs) that contain TRPV1 increase in prevalence. Target-derived neurotrophic factor levels were also enhanced after axotomy and regeneration. In particular, the glial-cell line-derived neurotrophic factor (GDNF) family member artemin was found to be significantly enhanced in the hairy hindpaw skin and its receptor GDNF family receptor α3 (GFRα3) was increased in the L2/L3 dorsal root ganglia (DRGs) following nerve injury. In this study, we assessed the role of enhanced artemin/GFRα3 levels on the changes in mouse cutaneous CH neurons following saphenous nerve regeneration. We used a newly developed siRNA-mediated in vivo knockdown strategy to specifically inhibit the injury-induced expression of GFRα3 and coupled this with an ex vivo recording preparation to examine response characteristics and neurochemical phenotype of different types of functionally defined neurons after injury. We found that inhibition of GFRα3 did not affect the axotomy-induced decrease in CPM threshold, but transiently prevented the recruitment of CH neurons. Western blot and real-time PCR analysis of hairy hindpaw skin and L2/L3 DRGs after saphenous nerve regeneration suggested that inhibition of the potential initial injury-induced increase in enhanced target-derived artemin signaling resulted in dynamic changes in TRPV1 expression after regeneration. These changes in TRPV1 expression may underlie the functional alterations observed in CH neurons after nerve regeneration.

The ADP receptor P2Y1 is necessary for normal thermal sensitivity in cutaneous polymodal nociceptors.

BACKGROUND: P2Y1 is a member of the P2Y family of G protein-coupled nucleotide receptors expressed in peripheral sensory neurons. Using ratiometric calcium imaging of isolated dorsal root ganglion neurons, we found that the majority of neurons responding to adenosine diphosphate, the preferred endogenous ligand, bound the lectin IB4 and expressed the ATP-gated ion channel P2X3. These neurons represent the majority of epidermal afferents in hairy skin, and are predominantly C-fiber polymodal nociceptors (CPMs), responding to mechanical stimulation, heat and in some cases cold. RESULTS: To characterize the function of P2Y1 in cutaneous afferents, intracellular recordings from sensory neuron somata were made using an ex vivo preparation in which the hindlimb skin, saphenous nerve, DRG and spinal cord were dissected in continuum, and cutaneous receptive fields characterized using digitally-controlled mechanical and thermal stimuli in male wild type mice. In P2Y1-/- mice, CPMs showed a striking increase in mean heat threshold and a decrease in mean peak firing rate during a thermal ramp from 31-52°C. A similar change in mean cold threshold was also observed. Interestingly, mechanical testing of CPMs revealed no significant differences between P2Y1-/- and WT mice. CONCLUSIONS: These results strongly suggest that P2Y1 is required for normal thermal signaling in cutaneous sensory afferents. Furthermore, they suggest that nucleotides released from peripheral tissues play a critical role in the transduction of thermal stimuli in some fiber types.

MrgprdCre lineage neurons mediate optogenetic allodynia through an emergent polysynaptic circuit.

ABSTRACT: Most cutaneous C fibers, including both peptidergic and nonpeptidergic subtypes, are presumed to be nociceptors and respond to noxious input in a graded manner. However, mechanically sensitive, nonpeptidergic C fibers also respond to mechanical input in the innocuous range, so the degree to which they contribute to nociception remains unclear. To address this gap, we investigated the function of nonpeptidergic afferents using the MrgprdCre allele. In real-time place aversion studies, we found that low-frequency optogenetic activation of MrgrpdCre lineage neurons was not aversive in naive mice but became aversive after spared nerve injury (SNI). To address the underlying mechanisms of this allodynia, we recorded responses from lamina I spinoparabrachial (SPB) neurons using the semi-intact ex vivo preparation. After SNI, innocuous brushing of the skin gave rise to abnormal activity in lamina I SPB neurons, consisting of an increase in the proportion of recorded neurons that responded with excitatory postsynaptic potentials or action potentials. This increase was likely due, at least in part, to an increase in the proportion of lamina I SPB neurons that received input on optogenetic activation of MrgprdCre lineage neurons. Intriguingly, in SPB neurons, there was a significant increase in the excitatory postsynaptic current latency from MrgprdCre lineage input after SNI, consistent with the possibility that the greater activation post-SNI could be due to the recruitment of a new polysynaptic circuit. Together, our findings suggest that MrgprdCre lineage neurons can provide mechanical input to the dorsal horn that is nonnoxious before injury but becomes noxious afterwards because of the engagement of a previously silent polysynaptic circuit in the dorsal horn.

Sensitization of cutaneous nociceptors after nerve transection and regeneration: possible role of target-derived neurotrophic factor signaling.

Damage to peripheral nerves is known to contribute to chronic pain states, including mechanical and thermal hyperalgesia and allodynia. It is unknown whether the establishment of these states is attributable to peripheral changes, central modifications, or both. In this study, we used several different approaches to assess the changes in myelinated (A) and unmyelinated (C) cutaneous nociceptors after transection and regeneration of the saphenous nerve. An ex vivo recording preparation was used to examine response characteristics and neurochemical phenotype of different types of functionally defined neurons. We found that myelinated nociceptors had significantly lower mechanical and thermal thresholds after regeneration, whereas C-polymodal nociceptors (CPMs) had lower heat thresholds. There was a significant increase in the percentage of mechanically insensitive C-fibers that responded to heat (CHs) after regeneration. Immunocytochemical analysis of identified afferents revealed that most CPMs were isolectin B4 (IB4) positive and transient receptor potential vanilloid 1 (TRPV1) negative, whereas CHs were always TRPV1 positive and IB4 negative in naive animals (Lawson et al., 2008). However, after regeneration, some identified CPMs and CHs stained positively for both markers, which was apparently attributable to an increase in the total number of IB4-positive neurons. Real-time PCR analysis of L2/L3 DRGs and hairy hindpaw skin at various times after saphenous nerve axotomy suggested multiple changes in neurotrophic factor signaling that correlated with either denervation or reinnervation of the cutaneous target. These changes may underlie the functional alterations observed after nerve regeneration and may explain how nerve damage leads to chronic pain conditions.

Mrgprd enhances excitability in specific populations of cutaneous murine polymodal nociceptors.

The Mas-related G protein-coupled receptor D (Mrgprd) is selectively expressed in nonpeptidergic nociceptors that innervate the outer layers of mammalian skin. The function of Mrgprd in nociceptive neurons and the physiologically relevant somatosensory stimuli that activate Mrgprd-expressing (Mrgprd(+)) neurons are currently unknown. To address these issues, we studied three Mrgprd knock-in mouse lines using an ex vivo somatosensory preparation to examine the role of the Mrgprd receptor and Mrgprd(+) afferents in cutaneous somatosensation. In mouse hairy skin, Mrgprd, as marked by expression of green fluorescent protein reporters, was expressed predominantly in the population of nonpeptidergic, TRPV1-negative, C-polymodal nociceptors. In mice lacking Mrgprd, this population of nociceptors exhibited decreased sensitivity to cold, heat, and mechanical stimuli. Additionally, in vitro patch-clamp studies were performed on cultured dorsal root ganglion neurons from Mrgprd(-/-) and Mrgprd(+/-) mice. These studies revealed a higher rheobase in neurons from Mrgprd(-/-) mice than from Mrgprd(+/-) mice. Furthermore, the application of the Mrgprd ligand beta-alanine significantly reduced the rheobase and increased the firing rate in neurons from Mrgprd(+/-) mice but was without effect in neurons from Mrgprd(-/-) mice. Our results demonstrate that Mrgprd influences the excitability of polymodal nonpeptidergic nociceptors to mechanical and thermal stimuli.

Cutaneous C-polymodal fibers lacking TRPV1 are sensitized to heat following inflammation, but fail to drive heat hyperalgesia in the absence of TPV1 containing C-heat fibers.

BACKGROUND: Previous studies have shown that the TRPV1 ion channel plays a critical role in the development of heat hyperalgesia after inflammation, as inflamed TRPV1-/- mice develop mechanical allodynia but fail to develop thermal hyperalgesia. In order to further investigate the role of TRPV1, we have used an ex vivo skin/nerve/DRG preparation to examine the effects of CFA-induced-inflammation on the response properties of TRPV1-positive and TRPV1-negative cutaneous nociceptors. RESULTS: In wildtype mice we found that polymodal C-fibers (CPMs) lacking TRPV1 were sensitized to heat within a day after CFA injection. This sensitization included both a drop in average heat threshold and an increase in firing rate to a heat ramp applied to the skin. No changes were observed in the mechanical response properties of these cells. Conversely, TRPV1-positive mechanically insensitive, heat sensitive fibers (CHs) were not sensitized following inflammation. However, results suggested that some of these fibers may have gained mechanical sensitivity and that some previous silent fibers gained heat sensitivity. In mice lacking TRPV1, inflammation only decreased heat threshold of CPMs but did not sensitize their responses to the heat ramp. No CH-fibers could be identified in naïve nor inflamed TRPV1-/- mice. CONCLUSIONS: Results obtained here suggest that increased heat sensitivity in TRPV1-negative CPM fibers alone following inflammation is insufficient for the induction of heat hyperalgesia. On the other hand, TRPV1-positive CH fibers appear to play an essential role in this process that may include both afferent and efferent functions.

Selective-cold output through a distinct subset of lamina I spinoparabrachial neurons.

Spinal projection neurons are a major pathway through which somatic stimuli are conveyed to the brain. However, the manner in which this information is coded is poorly understood. Here, we report the identification of a modality-selective spinoparabrachial (SPB) neuron subtype with unique properties. Specifically, we find that cold-selective SPB neurons are differentiated by selective afferent input, reduced sensitivity to substance P, distinct physiological properties, small soma size, and low basal drive. In addition, optogenetic experiments reveal that cold-selective SPB neurons do not receive input from Nos1 inhibitory interneurons and, compared with other SPB neurons, show significantly smaller inhibitory postsynaptic currents upon activation of Pdyn inhibitory interneurons. Together, these data suggest that cold output from the spinal cord to the parabrachial nucleus is mediated by a specific cell type with distinct properties.