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AP traces and algesic response profile for sub-classes of CGRP-cre + DRG neurons. For each CGRP-cre + sensory neuronal group recorded from mouse DRG, the AP, response to ATP (30μM), capsaicin (100nM; CAP), 5-HT (30μM) and mustard oil (10μM; MO) are presented from left to right. The AP time scale (horizontal bar) is 5 msec for each panel. I ATP time scale is 1 sec for each panel. I ATP magnitude (vertical bar) scales are indicated for each panel. Name of the groups are also indicated when two I ATP traces are presented on a panel. I CAP time scale is 10 sec for each panel. I CAP magnitude scale is indicated. I 5-HT was recorded from S3, but not S2 neurons. I 5-HT time and magnitude scales are 1 sec and 50 pA, respectively, for each panel. An exception is the M1/M1a group that has a large I 5-HT current. MO responses were measured by Ca 2+ imaging. CGRP-cre + sensory neuronal groups are shown for each row. Drug application times are illustrated by horizontal bar above traces. More complete information on subgroups is presented in Table 2.
Source publication
Peptidergic sensory neurons play a critical role in nociceptive pathways. To precisely define the function and plasticity of sensory neurons in detail, new tools such as transgenic mouse models are needed. We employed electrophysiology and immunohistochemistry to characterize in detail dorsal root ganglion (DRG) neurons expressing an inducible CGRP...
Citations
... In our previous report, we characterized and validated ChAT-Cre/Ai9, PV-Cre/Ai9, and Npy2r-Cre/Ai9 mice for the study of motoneurons, proprioceptors, and cutaneous mechanoreceptors, respectively (Bolívar and Udina, 2022). Here, we added TRPV1-Cre/Ai9 mice for the study of nociceptors, as described previously (Patil et al., 2018). We corroborated that the labeled neurons in the DRG were mostly peptidergic (42.8 ± 1.2% co-labeling with CGRP) and nonpeptidergic (30.1 ± 1.4% IB4-reactive) small-diameter neurons. ...
Peripheral neurons are heterogeneous and functionally diverse, but all share the capability to switch to a pro-regenerative state after nerve injury. Despite the assumption that the injury response is similar among neuronal subtypes, functional recovery may differ. Understanding the distinct intrinsic regenerative properties between neurons may help to improve the quality of regeneration, prioritizing the growth of axon subpopulations to their targets. Here, we present a comparative analysis of regeneration across four key peripheral neuron populations: motoneurons, proprioceptors, cutaneous mechanoreceptors, and nociceptors. Using Cre/Ai9 mice that allow fluorescent labeling of neuronal subtypes, we found that nociceptors showed the greater regeneration after a sciatic crush, followed by motoneurons, mechanoreceptors, and, finally, proprioceptors. By breeding these Cre mice with Ribotag mice, we isolated specific translatomes and defined the regenerative response of these neuronal subtypes after axotomy. Only 20% of the regulated genes were common, revealing a diverse response to injury among neurons, which was also supported by the differential influence of neurotrophins among neuron subtypes. Among differentially regulated genes, we proposed MED12 as a specific regulator of the regeneration of proprioceptors. Altogether, we demonstrate that the intrinsic regenerative capacity differs between peripheral neuron subtypes, opening the door to selectively modulate these responses.
... However, the SNS-CKO is based on Na v 1.8-promoter driven Cre recombinase-induced deletion of miR-183C 76,78,82 . Although it is expressed in nearly all small-diameter nociceptors, Na v 1.8-Cre is expressed in less than a third of the largediameter mechanoreceptive and proprioceptive neurons 78,[114][115][116] . Therefore, miR-183C is possibly intact in two thirds of the mechanoreceptors in the SNS-CKO mice, resulting in only modest decrease of corneal sensitivity to mechanical stimuli measured by the Cochet and Bonnet aesthesiometer in this study. ...
The conserved miR-183/96/182 cluster (miR-183C) is expressed in both corneal resident myeloid cells (CRMCs) and sensory nerves (CSN) and modulates corneal immune/inflammatory responses. To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contributions to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult naïve mice; 3′ RNA sequencing (Seq) and proteomics in the trigeminal ganglion (TG), cornea and CRMCs. Our results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. The number of CRMCs was increased in male vs female MS-CKO mice, suggesting sex-dependent regulation of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation.
... 18 HTMRs of nociceptive afferent fibers have also been tagged genetically using Cre mouse lines, including Mrgprd cre , CGRP cre , TRPV1 cre , Nav1.8 cre , and others. 8,19 Nav1.8 are voltagegated Na + channels expressed mainly in small-sized C-fiber nociceptors involved in mechanical and thermal nociception. [20][21][22] A recent study characterized electrophysiological properties and immunochemical profiles of subpopulations of nociceptive dorsal root ganglion (DRG) neurons using Nav1.8 cre , CGRP cre , and TRPV1 cre mouse lines. ...
... [20][21][22] A recent study characterized electrophysiological properties and immunochemical profiles of subpopulations of nociceptive dorsal root ganglion (DRG) neurons using Nav1.8 cre , CGRP cre , and TRPV1 cre mouse lines. 19 The study shows that the Nav1.8 cre mouse line labels almost all C-fibers, many Aδ-fibers, and some Aβ-fibers. 19 Transgenic Cre mice have also been used to express channel rhodopsin 2 (ChR2) in distinct subpopulations of afferents, allowing for opto-tagged electrophysiological studies on the properties of different mechanoreceptors. ...
... 19 The study shows that the Nav1.8 cre mouse line labels almost all C-fibers, many Aδ-fibers, and some Aβ-fibers. 19 Transgenic Cre mice have also been used to express channel rhodopsin 2 (ChR2) in distinct subpopulations of afferents, allowing for opto-tagged electrophysiological studies on the properties of different mechanoreceptors. 12 We recently generated Nav1.8 cre/ChR2+ (Nav1.8-ChR2) ...
We recently used Nav1.8ChR2 mice in which Nav1.8-expressing afferents were optogenetically tagged to classify mechanosensitive afferents into Nav1.8ChR2-postive and Nav1.8ChR2-negative mechanoreceptors. We found that the former were mainly high threshold mechanoreceptors (HTMRs), while the latter were low threshold mechanoreceptors (LTMRs). In the present study, we further investigated whether the properties of these mechanoreceptors were altered following tissue inflammation. Nav1.8ChR2 mice received a subcutaneous injection of saline or Complete Freund's Adjuvant (CFA) in the hindpaws. Using the hindpaw glabrous skin-tibial nerve preparation and the pressure-clamped single-fiber recordings, we found that CFA-induced hindpaw inflammation lowered the mechanical threshold of many Nav1.8ChR2-postive Aβ-fiber mechanoreceptors but heightened the mechanical threshold of many Nav1.8ChR2-negative Aβ-fiber mechanoreceptors. Spontaneous action potential impulses were not observed in Nav1.8ChR2-postive Aβ-fiber mechanoreceptors, but occurred in Nav1.8ChR2-negative Aβ-fiber mechanoreceptors with a lower mechanical threshold in the saline group and a higher mechanical threshold in the CFA group. No significant change was observed in the mechanical sensitivity of Nav1.8ChR2-postive and Nav1.8ChR2-negative Aδ-fiber mechanoreceptors as well as Nav1.8ChR2-postive C-fiber mechanoreceptors following hindpaw inflammation. Collectively, inflammation significantly altered the functional properties of both Nav1.8ChR2-postive and Nav1.8ChR2-negative Aβ-fiber mechanoreceptors, which may contribute to mechanical allodynia during inflammation.
... The diameter of neurons was calculated using an automated analysis pipeline and validated using DRG tissue from TRPV1-lineage td-tomato reporter mice, which identifies TRPV1-lineage neurons as putative nociceptors (S6 Fig in S1 File). TRPV1 is a broad embryonic marker for many small diameter nociceptors including transient receptor potential cation channel subfamily V member (TRPV1), isolectin-B4 (IB4), and a subset of Aδ neurons [37], so all of these nociceptors will express td-tomato protein even though some subpopulations may lose TRPV1 expression after development (i.e., IB4 and Aδ) [38]. Although MHCII protein was detected in large diameter neurons (>30μM [39]) in naïve female DRG tissue, the majority of MHCII + neurons were small diameter (<25μM: 63.63% ± 3.056) (Fig 5A). ...
Chemotherapy is often a life-saving treatment, but the development of intractable pain caused by chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting toxicity that restricts cancer survival rates. Recent reports demonstrate that paclitaxel (PTX) robustly increases anti-inflammatory CD4 ⁺ T cells in the dorsal root ganglion (DRG), and that T cells and anti-inflammatory cytokines are protective against CIPN. However, the mechanism by which CD4 ⁺ T cells are activated, and the extent cytokines released by CD4 ⁺ T cells target DRG neurons are unknown. Here, we are the first to detect major histocompatibility complex II (MHCII) protein in mouse DRG neurons and to find CD4 ⁺ T cells breaching the satellite glial cell barrier to be in close proximity to neurons, together suggesting CD4 ⁺ T cell activation and targeted cytokine release. MHCII protein is primarily expressed in small nociceptive neurons in male and female mouse DRG but increased after PTX in small nociceptive neurons in only female DRG. Reducing one copy of MHCII in small nociceptive neurons decreased anti-inflammatory IL-10 and IL-4 producing CD4 ⁺ T cells in naïve male DRG and increased their hypersensitivity to cold. Administration of PTX to male and female mice that lacked one copy of MHCII in nociceptive neurons decreased anti-inflammatory CD4 ⁺ T cells in the DRG and increased the severity of PTX-induced cold hypersensitivity. Collectively, our results demonstrate expression of MHCII protein in mouse DRG neurons, which modulates cytokine producing CD4 ⁺ T cells in the DRG and attenuates cold hypersensitivity during homeostasis and after PTX treatment.
... A standard marker for peptidergic nerves and neurons is CGRP 43,53 . We used validated anti-CGRP antibodies, which exhibited strong labeling in a subset of TG neurons in adult male marmosets (Suppl Fig. 2). ...
... Data from Figs. 1, 2, 3, 4, 5, 6 imply that masticatory muscles have relatively smaller subset of C-fibers compared to skin and dura mater and are predominantly innervated by A-fibers 52,54,55 . Here, we have evaluated expressions of trpV1, a marker for a subset of C-fiber sensory neurons 43 , and MrgprD, a marker for non-peptidergic C-fiber sensory neurons 43,53 , in the masticatory muscles. TrpV1 and MrgprD antibodies were validated on TG sections, and produced strong labeling in subsets of adult male marmoset TG neurons (Suppl Overall, we found that MM, TM and LPM did not show immunoreactivity for trpV1 or MrgprD, a marker for non-peptidergic sensory fibers. ...
... However, PEP2 and PEP3 represent A-fibers (A-HTMR groups) and are similar to mouse CGRP-eta 44,59 . Considering that primate masticatory muscles have NFH + peptidergic fibers, which could belong to the A-HTMR group 53,59 and be similar to reported PEP2 and PEP3 transcriptomic clusters in primates (Fig. 12). Nevertheless, precise transcriptomic identity of C-fibers and A-HTMR fibers in primate masticatory muscles are not clear. ...
Myogenous temporomandibular disorders is associated with an increased responsiveness of nerves innervating the masseter (MM), temporal (TM), and lateral pterygoid muscles (LPM). This study aimed to examine sensory nerve types innervating MM, TM and LPM of adult non-human primate—common marmosets. Sensory nerves were localized in specific regions of these muscles. Pgp9.5, marker for all nerves, and NFH, a marker for A-fibers, showed that masticatory muscles were primarily innervated with A-fibers. The proportion of C- to A-fibers was highest in LPM, and lowest in MM. All C-fibers (pgp9.5⁺/NFH⁻) observed in masticatory muscles were peptidergic (CGRP⁺) and lacked mrgprD and CHRNA3, a silent nociceptive marker. TrpV1 was register in 17% of LPM nerves. All fibers in masticatory muscles were labeled with GFAP⁺, a myelin sheath marker. There were substantially more peptidergic A-fibers (CGRP⁺/NFH⁺) in TM and LPM compared to MM. MM, TM and LPM NFH⁺ fibers contained different percentages of trkC⁺ and parvalbumin⁺, but not trkB⁺ fibers. Tyrosine hydroxylase antibodies, which did not label TG, highlighted sympathetic fibers around blood vessels of the masticatory muscles. Overall, masticatory muscle types of marmosets have similarities and differences in innervation patterns.
... Functional and transcript studies have shown that TRPA1 expression in the DRG correlates with TRPV1 expression. In addition to the expected tdT 1 TRPV1 1 population, our data also suggest evidence of a TRPA1 1 TRPV1subset in DRGs, and this is consistent with some reports (Malin et al., 2011;Usoskin et al., 2015;Patil et al., 2018) whereas others argue that TRPA1 is expressed exclusively in TRPV1 1 neurons (Story et al., 2003;Mishra et al., 2011;Pogorzala et al., 2013;Wilson et al., 2013;Weng et al., 2015). The tdT 1 TRPV1 -DRG neurons observed here were larger than tdT 1 TRPV1 1 DRG neurons but were much smaller than tdT -TRPV1neurons, which are likely to be non-nociceptive mechanoreceptors. ...
Transient receptor potential ankyrin 1 (TRPA1) is a polymodal cation channel that is activated by electrophilic irritants, oxidative stress, cold temperature, and GPCR signaling. TRPA1 expression has been primarily identified in subsets of nociceptive sensory afferents and is considered a target for future analgesics. Nevertheless, TRPA1 has been implicated in other cell types including keratinocytes, epithelium, enterochromaffin cells, endothelium, astrocytes, and CNS neurons. Here, we developed a knock-in mouse that expresses the recombinase Flp O in TRPA1-expressing cells. We crossed the TRPA1 Flp mouse with the R26 ai65f mouse that expresses tdTomato in a Flp-sensitive manner. We found tdTomato expression correlated well with TRPA1 mRNA expression and sensitivity to TRPA1 agonists in subsets of TRPV1 (transient receptor potential vanilloid receptor type 1)-expressing neurons in the vagal ganglia and dorsal root ganglia (DRGs), although tdTomato expression efficiency was limited in DRG. We observed tdTomato-expressing afferent fibers centrally (in the medulla and spinal cord) and peripherally in the esophagus, gut, airways, bladder, and skin. Furthermore, chemogenetic activation of TRPA1-expressing nerves in the paw evoked flinching behavior. tdTomato expression was very limited in other cell types. We found tdTomato in subepithelial cells in the gut mucosa but not in enterochromaffin cells. tdTomato was also observed in supporting cells within the cochlea, but not in hair cells. Lastly, tdTomato was occasionally observed in neurons in the somatomotor cortex and the piriform area, but not in astrocytes or vascular endothelium. Thus, this novel mouse strain may be useful for mapping and manipulating TRPA1-expressing cells and deciphering the role of TRPA1 in physiological and pathophysiological processes.
... In our previous report, we characterized and validated ChAT-Cre/Ai9, PV-Cre/Ai9, and Npy2r-Cre/Ai9 mice for the study of motoneurons, proprioceptors, and cutaneous mechanoreceptors, respectively (Bolívar and Udina, 2022 ). Here, we added TRPV1-Cre/Ai9 mice for the study of nociceptors, as described previously (Patil et al., 2018 ). We corroborated that the labeled neurons in the DRG were mostly peptidergic (42.8 ± 1.2% colabeling with CGRP) and non-peptidergic (30.1 ± 1.4% IB4-reactive) small-diameter neurons. ...
Peripheral neurons are heterogeneous and functionally diverse, but all share the capability to switch to a pro-regenerative state after nerve injury. Despite the assumption that the injury response is similar among neuronal subtypes, functional recovery may differ. Understanding the distinct intrinsic regenerative properties between neurons may help to improve the quality of regeneration, prioritizing the growth of axon subpopulations to their targets. Here, we present a comparative analysis of regeneration across four key peripheral neuron populations: motoneurons, proprioceptors, cutaneous mechanoreceptors, and nociceptors. Using Cre/Ai9 mice that allow fluorescent labelling of neuronal subtypes, we found that nociceptors showed the greater regeeneration after a sciatic crush, followed by motoneurons, mechanoreceptors and, finally, proprioceptors. By breeding these Cre mice with Ribotag mice, we isolated specific translatomes and defined the regenerative response of these neuronal subtypes after axotomy. Only 20% of the regulated genes were common, revealing a diverse response to injury among neurons, which was also supported by the differential influence of neurotrophins among neuron subtypes. Among differentially regulated genes, we proposed MED12 as a specific regulator of the regeneration of proprioceptors. Altogether, we demonstrate that the intrinsic regenerative capacity differs between peripheral neuron subtypes, opening the door to selectively modulate these responses.
... However, the SNS-CKO is based on Na v 1.8-promoter driven Cre recombinase-induced deletion of miR-183C 76,78,82 . Although it is expressed in nearly all small-diameter nociceptors, Na v 1.8-Cre is expressed in less than a third of the largediameter mechanoreceptive and proprioceptive neurons 78,[114][115][116] . Therefore, miR-183C is possibly intact in two thirds of the mechanoreceptors in the SNS-CKO mice, resulting in only modest decrease of corneal sensitivity to mechanical stimuli measured by the Cochet and Bonnet aesthesiometer in this study. ...
Purpose:
The conserved miR-183/96/182 cluster (miR-183C) regulates both corneal sensory innervation and corneal resident immune cells (CRICs). This study is to uncover its role in CRICs and in shaping the corneal cellular landscape at a single-cell (sc) level.
Methods:
Corneas of naïve, young adult [2 and 6 months old (mo)], female miR-183C knockout (KO) mice and wild-type (WT) littermates were harvested and dissociated into single cells. Dead cells were removed using a Dead Cell Removal kit. CD45+ CRICs were enriched by Magnetic Activated Cell Sorting (MACS). scRNA libraries were constructed and sequenced followed by comprehensive bioinformatic analyses.
Results:
The composition of major cell types of the cornea stays relatively stable in WT mice from 2 to 6 mo, however the compositions of subtypes of corneal cells shift with age. Inactivation of miR-183C disrupts the stability of the major cell-type composition and age-related transcriptomic shifts of subtypes of corneal cells. The diversity of CRICs is enhanced with age. Naïve mouse cornea contains previously-unrecognized resident fibrocytes and neutrophils. Resident macrophages (ResMϕ) adopt cornea-specific function by expressing abundant extracellular matrix (ECM) and ECM organization-related genes. Naïve cornea is endowed with partially-differentiated proliferative ResMϕ and contains microglia-like Mϕ. Resident lymphocytes, including innate lymphoid cells (ILCs), NKT and γδT cells, are the major source of innate IL-17a. miR-183C limits the diversity and polarity of ResMϕ.
Conclusion:
miR-183C serves as a checkpoint for CRICs and imposes a global regulation of the cellular landscape of the cornea.
... Here, we added TRPV1-Cre/Ai9 mice for the study of nociceptors, as described previously (Patil et al., 2018). We corroborated that the labeled neurons in the DRG were mostly peptidergic (42.8 ± 1.2% colabeling with CGRP) and non-peptidergic (30.1 ± 1.4% IB4-reactive) smalldiameter neurons. ...
Peripheral neurons are heterogeneous and functionally diverse, but all share the capability to switch to a pro-regenerative state after nerve injury. Despite the assumption that the injury response is similar among neuronal subtypes, functional recovery may differ. Understanding the distinct intrinsic regenerative properties between neurons may help to improve the quality of regeneration, prioritizing the growth of axon subpopulations to their targets. Here, we present a comparative analysis of regeneration across four key peripheral neuron populations: motoneurons, proprioceptors, cutaneous mechanoreceptors, and nociceptors. Using Cre/Ai9 mice that allow fluorescent labelling of neuronal subtypes, we found that nociceptors showed the greater regeeneration after a sciatic crush, followed by motoneurons, mechanoreceptors and, finally, proprioceptors. By breeding these Cre mice with Ribotag mice, we isolated specific translatomes and defined the regenerative response of these neuronal subtypes after axotomy. Only 20% of the regulated genes were common, revealing a diverse response to injury among neurons, which was also supported by the differential influence of neurotrophins among neuron subtypes. Among differentially regulated genes, we proposed MED12 as a specific regulator of the regeneration of proprioceptors. Altogether, we demonstrate that the intrinsic regenerative capacity differs between peripheral neuron subtypes, opening the door to selectively modulate these responses.
... RET RunX [7,9,13,24,26,28,29,31,33,34,36,41,[44][45][46][47][48] LTMR <0.5 m/s VGluT 3 TH TRPM8 Nav1.8 Nav1 .9 TrkB I, II, III GfrA2 PIEZO2 RET [14,28,29,34,36,48] There are protein markers or transcription factors that allow the differentiation of C fibers. ...
... RET RunX [7,9,13,24,26,28,29,31,33,34,36,41,[44][45][46][47][48] LTMR <0.5 m/s VGluT 3 TH TRPM8 Nav1.8 Nav1 .9 TrkB I, II, III GfrA2 PIEZO2 RET [14,28,29,34,36,48] There are protein markers or transcription factors that allow the differentiation of C fibers. One important protein family is the transient receptor potential (TRP) channel, a cationic channel that senses the injurious information. ...
... The sodium channels play an important role in the generation of excitability for the transmission of pain towards the dorsal horn of the spinal cord and are distributed within the C fibers. There are nine isoforms of this protein (Nav1.1 to Nav1.9), of which Nav1.8 is found in peptidergic and nonpeptidergic fibers, while the Nav1.7 channel is expressed in peptidergic C fibers, and Nav1.9 in IB4+ neurons [28]. ...
Pain is a complex experience that involves physical, emotional, and cognitive aspects. This review focuses specifically on the physiological processes underlying pain perception, with a particular emphasis on the various types of sensory neurons involved in transmitting pain signals to the central nervous system. Recent advances in techniques like optogenetics and chemogenetics have allowed researchers to selectively activate or inactivate specific neuronal circuits, offering a promising avenue for developing more effective pain management strategies. The article delves into the molecular targets of different types of sensory fibers such as channels, for example, TRPV1 in C-peptidergic fiber, TRPA1 in C-non-peptidergic receptors expressed differentially as MOR and DOR, and transcription factors, and their colocalization with the vesicular transporter of glutamate, which enable researchers to identify specific subtypes of neurons within the pain pathway and allows for selective transfection and expression of opsins to modulate their activity.
