Gpr83 Tunes Nociceptor Function, Controlling Pain.

The function of peripheral nociceptors is frequently tuned by the action of G protein-coupled receptors (GPRs) that are expressed in them, which contribute to pain alteration. Expanding new information on such GPRs and predicting their potential outcomes can help to construct new analgesic strategies based on their modulations. In this context, we attempted to present a new GPR not yet acknowledged for its pain association. Gpr83 exhibits relatively high expressions in the peripheral nervous system compared to other tissues when we mined and reconstructed Gene Expression Omnibus (GEO) metadata, which we confirmed using immunohistochemistry on murine dorsal root ganglia (DRG). When Gpr83 expression was silenced in DRG, neuronal and behavioral nociception were all downregulated. Pathologic pain in hind paw inflammation and chemotherapy-induced peripheral neuropathy were also alleviated by this Gpr83 knockdown. Dependent on exposure time, the application of a known endogenous Gpr83 ligand PEN showed differential effects on nociceptor responses in vitro. Localized PEN administration mitigated pain in vivo, probably following Gq/11-involved GPR downregulation caused by the relatively constant exposure. Collectively, this study suggests that Gpr83 action contributes to the tuning of peripheral pain sensitivity and thus indicates that Gpr83 can be among the potential GPR targets for pain modulation.

In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment.

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca2+ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca2+ response because of degeneration of primary sensory nerve fibers in the skin.SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca2+ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca2+ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca2+ activity and postoperative pain.

Derivation of Peripheral Nociceptive, Mechanoreceptive, and Proprioceptive Sensory Neurons from the same Culture of Human Pluripotent Stem Cells.

The three peripheral sensory neuron (SN) subtypes, nociceptors, mechanoreceptors, and proprioceptors, localize to dorsal root ganglia and convey sensations such as pain, temperature, pressure, and limb movement/position. Despite previous reports, to date no protocol is available allowing the generation of all three SN subtypes at high efficiency and purity from human pluripotent stem cells (hPSCs). We describe a chemically defined differentiation protocol that generates all three SN subtypes from the same starting population, as well as methods to enrich for each individual subtype. The protocol yields high efficiency and purity cultures that are electrically active and respond to specific stimuli. We describe their molecular character and maturity stage and provide evidence for their use as an axotomy model; we show disease phenotypes in hPSCs derived from patients with familial dysautonomia. Our protocol will allow the modeling of human disorders affecting SNs, the search for treatments, and the study of human development.

Axonal extension from dorsal root ganglia on fibrillar and highly aligned poly(lactic acid)-polypyrrole substrates obtained by two different techniques: Electrospun nanofibres and extruded microfibres.

The biological behaviour of Schwann cells (SCs) and dorsal root ganglia (DRG) on fibrillar, highly aligned and electroconductive substrates obtained by two different techniques is studied. Mats formed by nanometer-sized fibres of poly(lactic acid) (PLA) are obtained by the electrospinning technique, while bundles formed by micrometer-sized extruded PLA fibres are obtained by grouping microfibres together. Both types of substrates are coated with the electrically conductive polymer polypyrrole (PPy) and their morphological, physical and electrical characterization is carried out. SCs on micrometer-sized substrates show a higher motility and cell-cell interaction, while a higher cell-material interaction with a lower cell motility is observed for nanometer-sized substrates. This higher motility and cell-cell interaction of SCs on the micrometer-sized substrates entails a higher axonal growth from DRG, since the migration of SCs from the DRG body is accelerated and, therefore, the SCs tapestry needed for the axonal growth is formed earlier on the substrate. A higher length and area of the axons is observed for these micrometer-sized substrates, as well as a higher level of axonal sprouting when compared with the nanometer-sized ones. These substrates offer the possibility of being electrically stimulated in different tissue engineering applications of the nervous system.

Accumulation of Neurofascin at Nodes of Ranvier Is Regulated by a Paranodal Switch.

The paranodal junctions flank mature nodes of Ranvier and provide a barrier between ion channels at the nodes and juxtaparanodes. These junctions also promote node assembly and maintenance by mechanisms that are poorly understood. Here, we examine their role in the accumulation of NF186, a key adhesion molecule of PNS and CNS nodes. We previously showed that NF186 is initially targeted/accumulates via its ectodomain to forming PNS (hemi)nodes by diffusion trapping, whereas it is later targeted to mature nodes by a transport-dependent mechanism mediated by its cytoplasmic segment. To address the role of the paranodes in this switch, we compared accumulation of NF186 ectodomain and cytoplasmic domain constructs in WT versus paranode defective (i.e., Caspr-null) mice. Both pathways are affected in the paranodal mutants. In the PNS of Caspr-null mice, diffusion trapping mediated by the NF186 ectodomain aberrantly persists into adulthood, whereas the cytoplasmic domain/transport-dependent targeting is impaired. In contrast, accumulation of NF186 at CNS nodes does not undergo a switch; it is predominantly targeted to both forming and mature CNS nodes via its cytoplasmic domain and requires intact paranodes. Fluorescence recovery after photobleaching analysis indicates that the paranodes provide a membrane diffusion barrier that normally precludes diffusion of NF186 to nodes. Linkage of paranodal proteins to the underlying cytoskeleton likely contributes to this diffusion barrier based on 4.1B and ßII spectrin expression in Caspr-null mice. Together, these results implicate the paranodes as membrane diffusion barriers that regulate targeting to nodes and highlight differences in the assembly of PNS and CNS nodes.SIGNIFICANCE STATEMENT Nodes of Ranvier are essential for effective saltatory conduction along myelinated axons. A major question is how the various axonal proteins that comprise the multimeric nodal complex accumulate at this site. Here we examine how targeting of NF186, a key nodal adhesion molecule, is regulated by the flanking paranodal junctions. We show that the transition from diffusion-trapping to transport-dependent accumulation of NF186 requires the paranodal junctions. We also demonstrate that these junctions are a barrier to diffusion of axonal proteins into the node and highlight differences in PNS and CNS node assembly. These results provide new insights into the mechanism of node assembly and the pathophysiology of neurologic disorders in which impaired paranodal function contributes to clinical disability.

Synaptic Dynamics of the Feed-forward Inhibitory Circuitry Gating Mechanical Allodynia in Mice.

BACKGROUND: The authors' previous studies have found that spinal protein kinase C γ expressing neurons are involved in the feed-forward inhibitory circuit gating mechanical allodynia in the superficial dorsal horn. The authors hypothesize that nerve injury enhances the excitability of spinal protein kinase C γ expressing interneurons due to disinhibition of the feed-forward inhibitory circuit, and enables Aß primary inputs to activate spinal protein kinase C γ expressing interneurons. METHODS: Prkcg-P2A-tdTomato mice were constructed using the clustered regularly interspaced short palindromic repeats and clustered regularly interspaced short palindromic repeats-associated nuclease 9 technology, and were used to analyze the electrophysiologic properties of spinal protein kinase C γ expressing neurons in both normal conditions and pathologic conditions induced by chronic constriction injury of the sciatic nerve. Patch-clamp whole cell recordings were used to identify the nature of the dynamic synaptic drive to protein kinase C γ expressing neurons. RESULTS: Aß fiber stimulation evoked a biphasic synaptic response in 42% (31 of 73) of protein kinase C γ expressing neurons. The inhibitory components of the biphasic synaptic response were blocked by both strychnine and bicuculline in 57% (16 of 28) of neurons. Toll-like receptor 5 immunoreactive fibers made close contact with protein kinase C γ expressing neurons. After nerve injury, the percentage of neurons double-labeled for c-fos and Prkcg-P2A-tdTomato in animals walking on a rotarod was significantly higher than that in the nerve injury animals (4.1% vs. 9.9%, 22 of 539 vs. 54 of 548,P < 0.001). Aß fiber stimulation evoked burst action potentials in 25.8% (8 of 31) of protein kinase C γ expressing neurons in control animals, while the proportion increased to 51.1% (23 of 45) in nerve injury animals (P = 0.027). CONCLUSIONS: The Prkcg-P2A-tdTomato mice the authors constructed provide a useful tool for further analysis on how the spinal allodynia gate works. The current study indicated that nerve injury enhanced the excitability of spinal protein kinase C γ expressing interneurons due to disinhibition of the feed-forward inhibitory circuit, and enabled Aß primary inputs to activate spinal protein kinase C γ expressing interneurons.

A selective and sensitive turn-on chemosensor for detection of Fe3+ in aqueous solution and its cell imaging in dorsal root ganglia neurons and MKN-45 cells.

A new turn-on fluorescent chemosensor (RBTM) for Fe3+ was designed based on Rhodamine B and a thiocarbonylimidazole moiety. The spectroscopic probe used for characterization of the synthesized system showed 300-fold fluorescence enhancement for the detection of Fe3+ with a 1:1 stoichiometry in EtOH/H2O solution (2:1, v/v, HEPES buffer, 1 mM, pH 7.30). Upon addition of Fe3+ in aqueous ethanol, the probe displayed a significant fluorescence enhancement and a distinct color change (colorless to pink) that can be detected by the naked eye. The binding constant between the probe and Fe3+ was determined to be 1.16 × 104 M-1 and the corresponding detection limit was calculated to be 0.256 µM. In addition, the energy gaps between the HOMO and LUMO in RBTM and RBTM-Fe3+ were calculated using DFT calculations to be 92.93 kcal/mol and 37.49 kcal/mol, respectively. The results indicate that binding of Fe3+ to RBTM lowered the HOMO-LUMO energy gap of the complex and stabilized the system. Fluorescence imaging experiments demonstrated that RBTM can be used as a fluorescent probe to detect Fe3+ in MKN-45 cells and dorsal root ganglia, thus revealing that RBTM could be used for biological applications.

Dynorphin and Enkephalin Opioid Peptides and Transcripts in Spinal Cord and Dorsal Root Ganglion During Peripheral Inflammatory Hyperalgesia and Allodynia.

Understanding molecular alterations associated with peripheral inflammation is a critical factor in selectively controlling acute and persistent pain. The present report employs in situ hybridization of the 2 opioid precursor mRNAs coupled with quantitative measurements of 2 peptides derived from the prodynorphin and proenkephalin precursor proteins: dynorphin A 1-8 and [Met5]-enkephalin-Arg6-Gly7-Leu8. In dorsal spinal cord ipsilateral to the inflammation, dynorphin A 1-8 was elevated after inflammation, and persisted as long as the inflammation was sustained. Qualitative identification by high performance liquid chromatography and gel permeation chromatography revealed the major immunoreactive species in control and inflamed extracts to be dynorphin A 1-8. In situ hybridization in spinal cord after administration of the inflammatory agent, carrageenan, showed increased expression of prodynorphin (Pdyn) mRNA somatotopically in medial superficial dorsal horn neurons. The fold increase in preproenkephalin mRNA (Penk) was comparatively lower, although the basal expression is substantially higher than Pdyn. While Pdyn is not expressed in the dorsal root ganglion (DRG) in basal conditions, it can be induced by nerve injury, but not by inflammation alone. A bioinformatic meta-analysis of multiple nerve injury datasets confirmed Pdyn upregulation in DRG across different nerve injury models. These data support the idea that activation of endogenous opioids, notably dynorphin, is a dynamic indicator of persistent pain states in spinal cord and of nerve injury in DRG. PERSPECTIVE: This is a systematic, quantitative assessment of dynorphin and enkephalin peptides and mRNA in dorsal spinal cord and DRG neurons in response to peripheral inflammation and axotomy. These studies form the foundational framework for understanding how endogenous spinal opioid peptides are involved in nociceptive circuit modulation.

Somatosensory corticospinal tract axons sprout within the cervical cord following a dorsal root/dorsal column spinal injury in the rat.

The corticospinal tract (CST) is the major descending pathway controlling voluntary hand function in primates, and though less dominant, it mediates voluntary paw movements in rats. As with primates, the CST in rats originates from multiple (albeit fewer) cortical sites, and functionally different motor and somatosensory subcomponents terminate in different regions of the spinal gray matter. We recently reported in monkeys that following a combined cervical dorsal root/dorsal column lesion (DRL/DCL), both motor and S1 CSTs sprout well beyond their normal terminal range. The S1 CST sprouting response is particularly dramatic, indicating an important, if poorly understood, somatosensory role in the recovery process. As rats are used extensively to model spinal cord injury, we asked if the S1 CST response is conserved in rodents. Rats were divided into sham controls, and two groups surviving post-lesion for ~6 and 10 weeks. A DRL/DCL was made to partially deafferent one paw. Behavioral testing showed a post-lesion deficit and recovery over several weeks. Three weeks prior to ending the experiment, S1 cortex was mapped electrophysiologically, for tracer injection placement to determine S1 CST termination patterns within the cord. Synaptogenesis was also assessed for labeled S1 CST terminals within the dorsal horn. Our findings show that the affected S1 CST sprouts well beyond its normal range in response to a DRL/DCL, much as it does in macaque monkeys. This, along with evidence for increased synaptogenesis post-lesion, indicates that CST terminal sprouting following a central sensory lesion, is a robust and conserved response.

The role of dorsal root ganglia PIM1 in peripheral nerve injury-induced neuropathic pain.

Neuropathic pain induced by peripheral nerve injury is a complex and chronic state that is accompanied by poor quality of life. However, whether PIM1 (proviral integration site 1) contributes to the development of nociceptive hypersensitivity induced by nerve injury remains unknown. The present study was designed to investigate the effects of PIM1 on spinal nerve ligation (SNL) induced pain hypersensitivity. Here, we found that PIM1 positive neurons in the dorsal root ganglion (DRG) were colocalized with nociceptive neuronal markers CGRP, IB4 and substance P and were upregulated after SNL surgery. Knockdown PIM1 in the DRG by AAV5-shPIM1 alleviated SNL-induced pain hypersensitivity. In neuroblastoma cells (neuro-2a), PIM1 regulated the expression of CXCR4 phosphorylated at ser339 (pCXCR4) as well as the CXCL12/CXCR4 pathway. In the DRG tissues, we found that PIM1 was co-expressed with CXCR4, and knockdown of PIM1 attenuated pCXCR4 (ser339) protein expression but had little effect on total CXCR4 protein expression after SNL surgery. These findings suggest that PIM1 contributes to nerve injury-induced nociceptive hypersensitivity. Based on these findings and the characteristics of PIM1, we speculate that PIM1 might be a viable therapeutic target for the treatment of neuropathic pain in the near future.

Ultrasound-Guided Pulsed Radiofrequency of C2 Dorsal Root Ganglion as Adjuvant Treatment for Chronic Headache Disorders: A Case Report.

Cluster headaches are characterized by unilateral sudden onset of intense, brief, sharp pain along the side of the face around the eye. Patients often can have symptoms that are resistant to medications, nerve blocks, and surgical treatments. There is increasing evidence of anatomical and functional connections between the trigeminal and occipital nerves. We present a patient with cluster headache presenting with chronic ipsilateral facial pain with nasal congestion and left eye pain who achieved sustained pain relief with an ultrasound-guided injection into the pterygopalatine fossa in conjunction with an ultrasound-guided pulsed radiofrequency ablation procedure involving the C2 dorsal root ganglion.

Microarray analyses of lncRNAs and mRNAs expression profiling associated with diabetic peripheral neuropathy in rats.

Diabetic peripheral neuropathy (DPN) is considered to be the most frequent neuropathic complication of diabetes, and severely affects the quality of life of patients. Long noncoding RNAs (lncRNAs) participate in various pathophysiological processes and associate with many diseases. However, the exact impact of lncRNAs on DPN remains obscure. To discover a potential connection, a microarray study was conducted to analyze the expression profiling of lncRNAs and messenger RNAs (mRNAs) in dorsal root ganglia (DRG) from streptozotocin-induced diabetic rats with DPN. As a result, 983 lncRNAs and 1357 mRNAs were aberrantly expressed compared with control samples. Using bioinformatics analyses, we identified 558 Gene Ontology terms and 94 Kyoto Encyclopedia of Genes and Genomes pathways to be significantly enriched. Additionally, the signal-net analysis indicated that integrin receptors, including Itgb3, Itgb1, Itgb8, and Itga6, might be important players in network regulation. Furthermore, the lncRNA-mRNA network analysis showed dynamic interactions between the dysregulated lncRNAs and mRNAs. This is the first study to present an overview of lncRNA and mRNA expressions in DRG tissues from DPN rats. Our results indicate that these differentially expressed lncRNAs may have crucial roles in pathological processes of DPN by regulating their coexpressed mRNAs. The data may provide novel targets for future studies, which should focus on validating their roles in the progression of DPN.

Role of GM-CSF in a mouse model of experimental autoimmune prostatitis.

The etiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is still unknown. Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to play an important role in the development of autoimmune and inflammatory diseases. Here, we investigated the expression and function of GM-CSF in patients with CP/CPPS and in a mouse model of experimental autoimmune prostatitis (EAP). GM-CSF mRNA levels were detected in expressed prostatic secretions samples from patients with CP/CPPS and in prostate tissue from a mouse model of EAP. The expression of GM-CSF receptor in mouse prostate and dorsal root ganglia were determined using PCR and immunohistochemistry. Behavioral testing and inflammation scoring were performed to evaluate the role of GM-CSF in disease development and symptom severity of EAP using GM-CSF knockout mice. mRNA levels of putative nociceptive and inflammatory markers were measured in the prostate after the induction of EAP. Elevated GM-CSF mRNA levels were observed in expressed prostatic secretions samples from patients with CP/CPPS compared with healthy volunteers. GM-CSF mRNA was also significantly increased in prostate tissue of the EAP mice model. The expression of GM-CSF receptors was confirmed in mouse prostate and dorsal root ganglia. GM-CSF knockout mice showed fewer Infiltrating leukocytes and pain symptoms after the induction of EAP. Deletion of GM-CSF significantly diminished EAP-induced increases of chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 3, and nerve growth factor mRNA expression. The results indicated that GM-CSF plays a functional role in the pathogenesis of EAP. GM-CSF may function as a signaling mediator for both inflammation and pain transduction in CP/CPPS.

Chronic pain induces nociceptive neurogenesis in dorsal root ganglia from Sox2-positive satellite cells.

Chronic pain is one of the most prevalent chronic diseases in the world. The plastic changes of sensory neurons in dorsal root ganglia (DRG) have been extensively studied as the underlying periphery mechanism. Recent studies revealed that satellite cells, the major glial cells in DRG, also played important roles in the development/modulation of chronic pain. Whether DRG satellite glial cells generate new neurons as their counterparts in enteric nerve ganglia and carotid body do under pathological conditions remains poorly investigated. Here, we report that chronic pain induces proliferation and upregulation of progenitor markers in the sex-determining region Y-box 2 (Sox2)- and platelet-derived growth factor receptor alpha (PDGFRα)-positive satellite glial cells. BrdU incorporation assay revealed the generation of IB4- and CGRP-positive neurons, but not NF200-positive neurons in DRG ipsilateral to injury. Genetic fate tracings showed that PDGFRα-positive cells did not generate neurons, whereas Sox2-positive cells produced both IB4- and CGRP-positive neurons. Interestingly, glial fibrillary acidic protein-positive cells, a subpopulation of Sox2-positive satellites, only gave birth to IB4-positive neurons. Local persistent delivery of tetrodotoxin to the sciatic nerve trunk significantly reduced the pain-induced neurogenesis. Furthermore, patch-clamp studies demonstrated that these glia-derived new neurons could fire action potentials and respond to capsaicin. Taken together, our data demonstrated a chronic pain-induced nociceptive neurogenesis in DRG from Sox2-positive satellite cells, indicating a possible contribution of DRG neurogenesis to the pathology of chronic pain.

Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

Quercetin Attenuates Neuropathic Pain in Rats with Spared Nerve Injury.

Quercetin is a flavonoid widely found in plants and marketed to the public as a supplement. Several studies have reported its effect on glial cells. This study aimed to examine the effect of quercetin on the development of neuropathic pain and the underlying mechanism in a spared nerve injury (SNI) rat model. Male Sprague-Dawley rats randomly assigned to the control or the quercetin group were subjected to SNI of the sciatic nerve. We measured pain behaviors on the hind paw and glial fibrillary acidic protein (GFAP) in the dorsal root ganglion (DRG) and spinal cord. Oral administration of 1% quercetin, begun before surgery, attenuated mechanical allodynia compared to the control group at days 7 and 10 after SNI. On the other hand, established pain was not attenuated in a post-dose group in which quercetin was begun 7 days after SNI. Quercetin inhibited GFAP in the satellite glial cells of the ipsilateral L5 DRG on day 7 compared to the control group. Quercetin suppressed the development of neuropathic pain through a mechanism partly involving the inhibition of satellite glial cells. As its safety is well established, quercetin has great potential for clinical use in pain treatment.

MBD1 Contributes to the Genesis of Acute Pain and Neuropathic Pain by Epigenetic Silencing of Oprm1 and Kcna2 Genes in Primary Sensory Neurons.

The transmission of normal sensory and/or acute noxious information requires intact expression of pain-associated genes within the pain pathways of nervous system. Expressional changes of these genes after peripheral nerve injury are also critical for neuropathic pain induction and maintenance. Methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, regulates gene transcriptional activity. We report here that MBD1 in the primary sensory neurons of DRG is critical for the genesis of acute pain and neuropathic pain as DRG MBD1-deficient mice exhibit the reduced responses to acute mechanical, heat, cold, and capsaicin stimuli and the blunted nerve injury-induced pain hypersensitivities. Furthermore, DRG overexpression of MBD1 leads to spontaneous pain and evoked pain hypersensitivities in the WT mice and restores acute pain sensitivities in the MBD1-deficient mice. Mechanistically, MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 is likely a key player under the conditions of acute pain and neuropathic pain.SIGNIFICANCE STATEMENT In the present study, we revealed that the mice with deficiency of methyl-CpG-binding domain protein 1 (MBD1), an epigenetic repressor, in the DRG displayed the reduced responses to acute noxious stimuli and the blunted neuropathic pain. We also showed that DRG overexpression of MBD1 produced the hypersensitivities to noxious stimuli in the WT mice and rescued acute pain sensitivities in the MBD1-deficient mice. We have also provided the evidence that MDB1 represses Oprm1 and Kcna2 gene expression by recruiting DNA methyltransferase DNMT3a into these two gene promoters in the DRG neurons. DRG MBD1 may participate in the genesis of acute pain and neuropathic pain likely through regulating DNMT3a-controlled Oprm1 and Kcna2 gene expression in the DRG neurons.

Optically Guided Single Cell Mass Spectrometry of Rat Dorsal Root Ganglia to Profile Lipids, Peptides and Proteins.

The mammalian dorsal root ganglia (DRG) are located on the dorsal roots of the spinal nerves and contain cell bodies of primary sensory neurons. DRG cells have been classified into subpopulations based on their size, morphology, intracellular markers, response to stimuli, and neuropeptides. To understand the connections between DRG chemical heterogeneity and cellular function, we performed optically guided, high-throughput single cell profiling using sequential matrix-assisted laser desorption/ionization mass spectrometry (MS) to detect lipids, peptides, and several proteins in individual DRG cells. Statistical analysis of the resulting mass spectra allows stratification of the DRG population according to cellular morphology and, presumably, major cell types. A subpopulation of small cells contained myelin proteins, which are abundant in Schwann cells, and mass spectra of several larger cells contained peaks matching neurofilament, vimentin, myelin basic protein S, and thymosin beta proteins. Of the over 1000 cells analyzed, approximately 78 % produced putative peptide-rich spectra, allowing the population to be classified into three distinct cell types. Two signals with m/z 4404 and 5487 were exclusively observed in a cell type, but could not be matched to results of our previous liquid chromatography-MS analyses.

Distribution and neurochemistry of porcine urinary bladder-projecting sensory neurons in subdomains of the dorsal root ganglia: A quantitative analysis.

The aim of the present study has been to verify the inter- and intraganglionic distribution pattern of porcine urinary bladder-projecting (UBP) neurons localized in the sacral dorsal root ganglia (DRGs). The morphology and chemical phenotype of these cells have also been investigated. These neurons were visualized using the fluorescent tracer Fast Blue (FB) which was injected bilaterally into the urinary bladder wall of five juvenile female pigs. The intraganglionic distribution showed that small- and medium-sized FB+ perikarya were mainly located in the central (S3-S4) and periphero-central (S2) region of the ganglia, while large cells were heterogeneously distributed. Immunohistochemistry revealed that the most frequently observed markers in small and medium-sized UBP perikarya were: neurofilament 200, lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) isolectin B4, substance P, calcitonin gene-related peptide, pituitary adenylate cyclase-activating polypeptide and transient receptor potential vanilloid 1. Moreover, UBP neurons containing these substances were also mainly observed in the central and periphero-central region of the ganglion. Differences in the percentage of traced cells and their neuropeptide content were observed between the S2, S3 and S4 DRGs. In conclusion, the present study, for the first time, describes the arrangement of UBP DRGs neurons within particular subdomains of sacral ganglia, taking into account their size and chemical phenotype.

Identification of Two Classes of Somatosensory Neurons That Display Resistance to Retrograde Infection by Rabies Virus.

Glycoprotein-deleted rabies virus-mediated monosynaptic tracing has become a standard method for neuronal circuit mapping, and is applied to virtually all parts of the rodent nervous system, including the spinal cord and primary sensory neurons. Here we identified two classes of unmyelinated sensory neurons (nonpeptidergic and C-fiber low-threshold mechanoreceptor neurons) resistant to direct and trans-synaptic infection from the spinal cord with rabies viruses that carry glycoproteins in their envelopes and that are routinely used for infection of CNS neurons (SAD-G and N2C-G). However, the same neurons were susceptible to infection with EnvA-pseudotyped rabies virus in tumor virus A receptor transgenic mice, indicating that resistance to retrograde infection was due to impaired virus adsorption rather than to deficits in subsequent steps of infection. These results demonstrate an important limitation of rabies virus-based retrograde tracing of sensory neurons in adult mice, and may help to better understand the molecular machinery required for rabies virus spread in the nervous system. In this study, mice of both sexes were used.SIGNIFICANCE STATEMENT To understand the neuronal bases of behavior, it is important to identify the underlying neural circuitry. Rabies virus-based monosynaptic tracing has been used to identify neuronal circuits in various parts of the nervous system. This has included connections between peripheral sensory neurons and their spinal targets. These connections form the first synapse in the somatosensory pathway. Here we demonstrate that two classes of unmyelinated sensory neurons, which account for >40% of dorsal root ganglia neurons, display resistance to rabies infection. Our results are therefore critical for interpreting monosynaptic rabies-based tracing in the sensory system. In addition, identification of rabies-resistant neurons might provide a means for future studies addressing rabies pathobiology.