Oxidative DNA Damage and Cisplatin Neurotoxicity Is Exacerbated by Inhibition of OGG1 Glycosylase Activity and APE1 Endonuclease Activity in Sensory Neurons.

Cisplatin can induce peripheral neuropathy, which is a common complication of anti-cancer treatment and negatively impacts cancer survivors during and after completion of treatment; therefore, the mechanisms by which cisplatin alters sensory neuronal function to elicit neuropathy are the subject of much investigation. Our previous work suggests that the DNA repair activity of APE1/Ref-1, the rate-limiting enzyme of the base excision repair (BER) pathway, is critical for neuroprotection against cisplatin. A specific role for 8-oxoguanine DNA glycosylase-1 (OGG1), the glycosylase that removes the most common oxidative DNA lesion, and putative coordination of OGG1 with APE1/Ref-1 in sensory neurons, has not been investigated. We investigated whether inhibiting OGG1 glycosylase activity with the small molecule inhibitor, TH5487, and/or APE1/Ref-1 endonuclease activity with APE Repair Inhibitor III would alter the neurotoxic effects of cisplatin in sensory neuronal cultures. Sensory neuron function was assessed by calcitonin gene-related peptide (CGRP) release, as a marker of sensitivity and by neurite outgrowth. Cisplatin altered neuropeptide release in an inverse U-shaped fashion, with low concentrations enhancing and higher concentrations diminishing CGRP release. Pretreatment with BER inhibitors exacerbated the functional effects of cisplatin and enhanced 8oxo-dG and adduct lesions in the presence of cisplatin. Our studies demonstrate that inhibition of OGG1 and APE1 endonuclease activity enhances oxidative DNA damage and exacerbates neurotoxicity, thus limiting oxidative DNA damage in sensory neurons that might alleviate cisplatin-induced neuropathy.

Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds.

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

The impact of SBF2 on taxane-induced peripheral neuropathy.

Taxane-induced peripheral neuropathy (TIPN) is a devastating survivorship issue for many cancer patients. In addition to its impact on quality of life, this toxicity may lead to dose reductions or treatment discontinuation, adversely impacting survival outcomes and leading to health disparities in African Americans (AA). Our lab has previously identified deleterious mutations in SET-Binding Factor 2 (SBF2) that significantly associated with severe TIPN in AA patients. Here, we demonstrate the impact of SBF2 on taxane-induced neuronal damage using an ex vivo model of SBF2 knockdown of induced pluripotent stem cell-derived sensory neurons. Knockdown of SBF2 exacerbated paclitaxel changes to cell viability and neurite outgrowth while attenuating paclitaxel-induced sodium current inhibition. Our studies identified paclitaxel-induced expression changes specific to mature sensory neurons and revealed candidate genes involved in the exacerbation of paclitaxel-induced phenotypes accompanying SBF2 knockdown. Overall, these findings provide ex vivo support for the impact of SBF2 on the development of TIPN and shed light on the potential pathways involved.

Mechanical stimulus-evoked signal transduction between keratinocytes and sensory neurons via extracellular ATP.

The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca2+ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca2+ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca2+ and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca2+ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca2+ response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 µM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis.

Profiling sensory neuron microenvironment after peripheral and central axon injury reveals key pathways for neural repair.

Sensory neurons with cell bodies in dorsal root ganglia (DRG) represent a useful model to study axon regeneration. Whereas regeneration and functional recovery occurs after peripheral nerve injury, spinal cord injury or dorsal root injury is not followed by regenerative outcomes. Regeneration of sensory axons in peripheral nerves is not entirely cell autonomous. Whether the DRG microenvironment influences the different regenerative capacities after injury to peripheral or central axons remains largely unknown. To answer this question, we performed a single-cell transcriptional profiling of mouse DRG in response to peripheral (sciatic nerve crush) and central axon injuries (dorsal root crush and spinal cord injury). Each cell type responded differently to the three types of injuries. All injuries increased the proportion of a cell type that shares features of both immune cells and glial cells. A distinct subset of satellite glial cells (SGC) appeared specifically in response to peripheral nerve injury. Activation of the PPARα signaling pathway in SGC, which promotes axon regeneration after peripheral nerve injury, failed to occur after central axon injuries. Treatment with the FDA-approved PPARα agonist fenofibrate increased axon regeneration after dorsal root injury. This study provides a map of the distinct DRG microenvironment responses to peripheral and central injuries at the single-cell level and highlights that manipulating non-neuronal cells could lead to avenues to promote functional recovery after CNS injuries or disease.

Quantitative description of the interactions among kinase cascades underlying long-term plasticity of Aplysia sensory neurons.

Kinases play critical roles in synaptic and neuronal changes involved in the formation of memory. However, significant gaps exist in the understanding of how interactions among kinase pathways contribute to the mechanistically distinct temporal domains of memory ranging from short-term memory to long-term memory (LTM). Activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) pathways are critical for long-term enhancement of neuronal excitability (LTEE) and long-term synaptic facilitation (LTF), essential processes in memory formation. This study provides new insights into how these pathways contribute to the temporal domains of memory, using empirical and computational approaches. Empirical studies of Aplysia sensory neurons identified a positive feedforward loop in which the PKA and ERK pathways converge to regulate RSK, and a negative feedback loop in which p38 MAPK inhibits the activation of ERK and RSK. A computational model incorporated these findings to simulate the dynamics of kinase activity produced by different stimulus protocols and predict the critical roles of kinase interactions in the dynamics of these pathways. These findings may provide insights into the mechanisms underlying aberrant synaptic plasticity observed in genetic disorders such as RASopathies and Coffin-Lowry syndrome.

Mini-Review: Mitochondrial dysfunction and chemotherapy-induced neuropathic pain.

Chemotherapy-induced peripheral neuropathy (CIPN) is a somatosensory axonopathy in cancer patients receiving any of a variety of widely-use antitumor agents. CIPN can lead to long-lasting neuropathic pain that limits the dose or length of otherwise life-saving cancer therapy. Accumulating evidence over the last two decades indicates that many chemotherapeutic agents cause mitochondrial injury in the peripheral sensory nerves by disrupting mitochondrial structure and bioenergetics, increasing nitro-oxidative stress and altering mitochondrial transport, fission, fusion and mitophagy. The accumulation of abnormal and dysfunctional mitochondria in sensory neurons are linked to axonal growth defects resulting in the loss of intraepidermal nerve fibers in the hands and feet, increased spontaneous discharge and the sensitization of peripheral sensory neurons that provoke and promote changes in the central nervous system that establish a chronic neuropathic pain state. This has led to the propose mitotoxicity theory of CIPN. Strategies that improve mitochondrial function have shown success in preventing and reversing CIPN in pre-clinical animal models and have begun to show some progress toward translation to the clinic. In this review, we will review the evidence for, the causes and effects of and current strategies to target mitochondrial dysfunction in CIPN.

Distinct subtypes of proprioceptive dorsal root ganglion neurons regulate adaptive proprioception in mice.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice.

Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Airway Sensory Nerve Density Is Increased in Chronic Cough.

Rationale: Chronic cough is characterized by frequent urges to cough and a heightened sensitivity to inhaled irritants. Airway sensory nerves trigger cough. We hypothesized that sensory nerve density is increased in chronic cough, which may contribute to excessive and persistent coughing.Objectives: To measure airway nerve density (axonal length) and complexity (nerve branching, neuropeptide expression) in humans with and without chronic cough.Methods: Bronchoscopic human airway biopsies were immunolabeled for nerves and the sensory neuropeptide substance P. Eosinophil peroxidase was also quantified given previous reports showing associations between eosinophils and nerve density. Three-dimensional image z-stacks of epithelium and subepithelium were generated using confocal microscopy, and from these z-stacks, total nerve length, the number of nerve branch points, substance P expression, and eosinophil peroxidase were quantified within each airway compartment.Measurements and Main Results: Nerve length and the number of branch points were significantly increased in epithelium, but not subepithelium, in chronic cough compared with healthy airways. Substance P expression was scarce and was similar in chronic cough and healthy airways. Nerve length and branching were not associated with eosinophil peroxidase nor with demographics such as age and sex in either group.Conclusions: Airway epithelial sensory nerve density is increased in chronic cough, suggesting sensory neuroplasticity contributes to cough hypersensitivity.

Human Induced Pluripotent Stem Cell Derived Sensory Neurons are Sensitive to the Neurotoxic Effects of Paclitaxel.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse event associated with treatment with paclitaxel and other chemotherapeutic agents. The prevention and treatment of CIPN are limited by a lack of understanding of the molecular mechanisms underlying this toxicity. In the current study, a human induced pluripotent stem cell-derived sensory neuron (iPSC-SN) model was developed for the study of chemotherapy-induced neurotoxicity. The iPSC-SNs express proteins characteristic of nociceptor, mechanoreceptor, and proprioceptor sensory neurons and show Ca2+ influx in response to capsaicin, α,ß-meATP, and glutamate. The iPSC-SNs are relatively resistant to the cytotoxic effects of paclitaxel, with half-maximal inhibitory concentration (IC50 ) values of 38.1 µM (95% confidence interval (CI) 22.9-70.9 µM) for 48-hour exposure and 9.3 µM (95% CI 5.7-16.5 µM) for 72-hour treatment. Paclitaxel causes dose-dependent and time-dependent changes in neurite network complexity detected by ßIII-tubulin staining and high content imaging. The IC50 for paclitaxel reduction of neurite area was 1.4 µM (95% CI 0.3-16.9 µM) for 48-hour exposure and 0.6 µM (95% CI 0.09-9.9 µM) for 72-hour exposure. Decreased mitochondrial membrane potential, slower movement of mitochondria down the neurites, and changes in glutamate-induced neuronal excitability were also observed with paclitaxel exposure. The iPSC-SNs were also sensitive to docetaxel, vincristine, and bortezomib. Collectively, these data support the use of iPSC-SNs for detailed mechanistic investigations of genes and pathways implicated in chemotherapy-induced neurotoxicity and the identification of novel therapeutic approaches for its prevention and treatment.

Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling.

Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nervous system biology, particularly dendrite arborization. We sought to test if EC-tagging, a biosynthetic RNA tagging and purification method that avoids the caveats of physical isolation, would enable discovery of novel regulators of md neuron dendrite arborization. Our aims were twofold: discover novel md neuron transcripts and test the sensitivity of EC-tagging. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Only CD:UPRT-expressing cells are competent to convert EC into 5-ethynyluridine-monophosphate which is subsequently incorporated into nascent RNA transcripts. Tagged RNAs were purified and used for RNA-sequencing. Reference RNA was prepared in a similar manner using 5-ethynyluridine (EUd) to tag RNA in all cells and negative control RNA-seq was performed on "mock tagged" samples to identify non-specifically purified transcripts. Differential expression analysis identified md neuron enriched and depleted transcripts. Three candidate genes encoding RNA-binding proteins (RBPs) were tested for a role in md neuron dendrite arborization. Loss-of-function for the m6A-binding factor Ythdc1 did not cause any dendrite arborization defects while RNAi of the other two candidates, the poly(A) polymerase Hiiragi and the translation regulator Hephaestus, caused significant defects in dendrite arborization. This work provides an expanded view of transcription in md neurons and a technical framework for combining EC-tagging with RNA-seq to profile transcription in cells that may not be amenable to physical isolation.

Single cell RNA sequencing identifies early diversity of sensory neurons forming via bi-potential intermediates.

Somatic sensation is defined by the existence of a diversity of primary sensory neurons with unique biological features and response profiles to external and internal stimuli. However, there is no coherent picture about how this diversity of cell states is transcriptionally generated. Here, we use deep single cell analysis to resolve fate splits and molecular biasing processes during sensory neurogenesis in mice. Our results identify a complex series of successive and specific transcriptional changes in post-mitotic neurons that delineate hierarchical regulatory states leading to the generation of the main sensory neuron classes. In addition, our analysis identifies previously undetected early gene modules expressed long before fate determination although being clearly associated with defined sensory subtypes. Overall, the early diversity of sensory neurons is generated through successive bi-potential intermediates in which synchronization of relevant gene modules and concurrent repression of competing fate programs precede cell fate stabilization and final commitment.

Sensory neurons in the human jugular ganglion.

The jugular ganglion (JG) contains sensory neurons of the vagus nerve which innervate somatic and visceral structures in cranial and cervical regions. In this study, the number of sensory neurons in the human JG was investigated. And, the morphology of sensory neurons in the human JG and nodose ganglion (NG) was compared. The estimated number of JG neurons was 2721.8-9301.1 (average number of sensory neurons ±â€¯S.D. = 7975.1 ±â€¯3312.8). There was no significant difference in sizes of the neuronal cell body and nucleus within the JG (cell body, 1128.8 ±â€¯99.7 µâ€¯m2; nucleus, 127.7 ±â€¯20.8 µâ€¯m2) and NG (cell body, 963.8 ±â€¯225.7 µâ€¯m2; nucleus, 123.2 ±â€¯32.3 µâ€¯m2). These findings indicate that most of sensory neurons show the similar morphology in the JG and NG. Our immunohistochemical method also demonstrated the distribution of ion channels, neurotransmitter agents and calcium-binding proteins in the human JG. Numerous JG neurons were immunoreactive for transient receptor potential cation channel subfamily V member 1 (TRPV1, mean ±â€¯SD = 19.9 ±â€¯11.5 %) and calcitonin gene-related peptide (CGRP, 28.4 ±â€¯6.7 %). A moderate number of JG neurons contained TRPV2 (12.0 ±â€¯4.7 %), substance P (SP, 15.7 ±â€¯6.9 %) and secreted protein, acidic and rich in cysteine-like 1 (SPARCL1, 14.6 ±â€¯7.4 %). A few JG neurons had vesicular glutamate transporter 2 (VGLUT2, 5.6 ±â€¯2.9 %) and parvalbumin (PV, 2.3 ±â€¯1.4 %). SP- and TRPV2-containing JG neurons had mainly small and medium-sized cell bodies, respectively. TRPV1- and VGLUT2- containing JG neurons were small to medium-sized. CGRP- and SPARCL1-containing JG neurons were of various cell body sizes. Sensory neurons in the human JG were mostly free of vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH) and neuropeptide Y (NPY). In the external auditory canal skin, subepithelial nerve fibers contained TRPV1, TRPV2, SP, CGRP and VGLUT2. Perivascular nerve fibers also had TRPV1, TRPV2, SP, CGRP, VIP, NPY and TH. However, PV- and SPARCL1-containing nerve endings could not be seen in the external auditory canal. It is likely that sensory neurons in the human JG can transduce nociceptive and mechanoreceptive information from the external auditory canal. Theses neurons may be also associated with neurogenic inflammation in the external auditory canal and ear-cough reflex through the vagus nerve.

Dendritic cells in tumor microenvironment promoted the neuropathic pain via paracrine inflammatory and growth factors.

Neuropathic pain associated with cancers was caused by tumor itself or tumor therapy, which was aggravated by sensitizing nociceptor sensory neurons. The tumor microenvironment contributed to tumorigenesis, tumor progress, tumor metastasis, tumor immune resistance, tumor chemotherapy, and tumor immunotherapy. In the current study, we explored the contributions of the infiltrated dendritic cells insulted by Wnt1 in tumor microenvironment to neuropathic pain associated with cancers. The different transcriptome of infiltrated dendritic cells from lung adenocarcinoma and from juxtatumor indicated that thousands of genes were up-regulated by the tumor microenvironment, some of which were enriched in pain pathway. The paracrine factors such as TNF, WNT10A, PDGFA, and NRG1 were also elevated in tumor-infiltrating dendritic cells. The receptors of paracrine factors were highly expressed on dorsal root ganglia (DRG), and not altered in pain conditions. Single-cell RNA-seq data unveiled that TNFSF1 was expressed in neurons, microglial cells, and endothelial cells. PDGFRA was only expressed in microglial cells. ERBB3 was only expressed in neurons. FZD1 and 3 were extensively expressed in various cells. The components composed of signaling pathways associated with the above paracrine factors participated in pain networks. The transcription factors activated by paracrine factor signaling regulated the expression of genes associated with pain. TNF, WNT10A, and PDGFA were extensively expressed in multiple cancers, but their expression in patients did not distribute normally. These data indicated that infiltrated dendritic cells in tumor microenvironment promoted neuropathic pain by sensitizing nociceptor sensory neurons via paracrine factors. Blockage of paracrine factor signaling might alleviate cancer pain.

Regulation of axonal morphogenesis by the mitochondrial protein Efhd1.

During development, neurons adjust their energy balance to meet the high demands of robust axonal growth and branching. The mechanisms that regulate this tuning are largely unknown. Here, we show that sensory neurons lacking liver kinase B1 (Lkb1), a master regulator of energy homeostasis, exhibit impaired axonal growth and branching. Biochemical analysis of these neurons revealed reduction in axonal ATP levels, whereas transcriptome analysis uncovered down-regulation of Efhd1 (EF-hand domain family member D1), a mitochondrial Ca2+-binding protein. Genetic ablation of Efhd1 in mice resulted in reduced axonal morphogenesis as well as enhanced neuronal death. Strikingly, this ablation causes mitochondrial dysfunction and a decrease in axonal ATP levels. Moreover, Efhd1 KO sensory neurons display shortened mitochondria at the axonal growth cones, activation of the AMP-activated protein kinase (AMPK)-Ulk (Unc-51-like autophagy-activating kinase 1) pathway and an increase in autophagic flux. Overall, this work uncovers a new mitochondrial regulator that is required for axonal morphogenesis.

Differentiation of Intrafusal Fibers from Human Induced Pluripotent Stem Cells.

Human-based "body-on-a-chip" technology provides powerful platforms in developing models for drug evaluation and disease evaluations in phenotypic models. Induced pluripotent stem cells (iPSCs) are ideal cell sources for generating different cell types for these in vitro functional systems and recapitulation of the neuromuscular reflex arc would allow for the study of patient specific neuromuscular diseases. Regarding relevant afferent (intrafusal fibers, sensory neurons) and efferent (extrafusal fibers, motoneurons) cells, in vitro differentiation of intrafusal fiber from human iPSCs has not been established. This work demonstrates a protocol for inducing an enrichment of intrafusal bag fibers from iPSCs using morphological analysis and immunocytochemistry. Phosphorylation of the ErbB2 receptors and S46 staining indicated a 3-fold increase of total intrafusal fibers further confirming the efficiency of the protocol. Integration of induced intrafusal fibers would enable more accurate reflex arc models and application of this protocol on patient iPSCs would allow for patient-specific disease modeling.

Expression Analysis of Cnidarian-Specific Neuropeptides in a Sea Anemone Unveils an Apical-Organ-Associated Nerve Net That Disintegrates at Metamorphosis.

Neuropeptides are ancient neuronal signaling molecules that have diversified across Cnidaria (e.g., jellyfish, corals, and sea anemones) and its sister group Bilateria (e.g., vertebrates, insects, and worms). Over the course of neuropeptide evolution emerged lineage-specific neuropeptides, but their roles in the evolution and diversification of nervous system function remain enigmatic. As a step toward filling in this knowledge gap, we investigated the expression pattern of a cnidarian-specific neuropeptide-RPamide-during the development of the starlet sea anemone Nematostella vectensis, using in situ hybridization and immunohistochemistry. We show that RPamide precursor transcripts first occur during gastrulation in scattered epithelial cells of the aboral ectoderm. These RPamide-positive epithelial cells exhibit a spindle-shaped, sensory-cell-like morphology, and extend basal neuronal processes that form a nerve net in the aboral ectoderm of the free-swimming planula larva. At the aboral end, RPamide-positive sensory cells become integrated into the developing apical organ that forms a bundle of long cilia referred to as the apical tuft. Later during planula development, RPamide expression becomes evident in sensory cells in the oral ectoderm of the body column and pharynx, and in the developing endodermal nervous system. At metamorphosis into a polyp, the RPamide-positive sensory nerve net in the aboral ectoderm degenerates by apoptosis, and RPamide expression begins in ectodermal sensory cells of growing oral tentacles. In addition, we find that the expression pattern of RPamide in planulae differs from that of conserved neuropeptides that are shared across Cnidaria and Bilateria, indicative of distinct functions. Our results not only provide the anatomical framework necessary to analyze the function of the cnidarian-specific neuropeptides in future studies, but also reveal previously unrecognized features of the sea anemone nervous system-the apical organ neurons of the planula larva, and metamorphosis-associated reorganization of the ectodermal nervous system.

Identifying spinal afferent (sensory) nerve endings that innervate the marrow cavity and periosteum using anterograde tracing.

While sensory and sympathetic neurons are known to innervate bone, previous studies have found it difficult to unequivocally identify and characterize only those that are of sensory origin. In this study, we have utilized an in vivo anterograde tracing technique to selectively label spinal afferent (sensory) nerve endings that innervate the periosteum and marrow cavity of murine long bones. Unilateral injections of dextran-biotin (anterograde tracer; 20% in saline, 50-100 nl) were made into L3-L5 dorsal root ganglia. After a 10-day recovery period to allow sufficient time for selective anterograde transport of the tracer to nerve terminal endings in bone, the periosteum (whole-mount) and underlying bone were collected, processed to reveal anterograde labeling, and immuno-labeled with antibodies directed against protein gene product (pan-neuronal marker; PGP9.5), tyrosine hydroxylase (sympathetic neuron marker; TH), calcitonin gene-related protein (peptidergic nociceptor marker; CGRP), and/or neurofilament 200 (myelinated axon marker; NF200). Anterograde-labeled nerve endings were dispersed throughout the periosteum and marrow cavity and could be identified in close apposition to blood vessels and at sites distant from them. The periosteum and the marrow cavity were each innervated by myelinated (NF200+) sensory neurons, and unmyelinated (NF200-) sensory neurons that were either peptidergic (CGRP+) or nonpeptidergic (CGRP-). Spinal afferent nerve endings did not express TH, and lacked the cylindrical morphology around blood vessels characteristic of sympathetic innervation. This approach to selective labeling of sensory nerve terminal endings will help to better identify how different sub-populations of sensory neurons, and their peripheral nerve terminal endings, interact with bone.

Histologic Analysis of Sensory and Motor Axons in Branches of the Human Brachial Plexus.

BACKGROUND: The topographic distribution through histologic analysis of motor and sensory axons within peripheral nerves at the brachial plexus level is not clearly defined, as there has previously been little need to appreciate this microanatomy. A desire to better understand the topography of fascicle groups developed with the introduction of targeted muscle reinnervation. METHODS: Fourteen bilateral brachial plexus specimens from seven fresh human cadavers were harvested at the time of organ donation, and immunofluorescent staining of motor and sensory nerves with choline acetyltransferase and Neurofilament 200 was performed to determine whether a consistent somatotopic orientation exists at the brachial plexus level. RESULTS: There was significant variability in the number of fascicles at the level of the brachial plexus. Qualitative analysis of choline acetyltransferase staining demonstrated that although motor axons tended to be grouped in clusters, there were high degrees of variability in somatotopic orientation across specimens. The radial nerve demonstrated the highest number of total myelinated axons, whereas the median nerve exhibited the greatest number of motor axons. The ulnar nerve contained only 13 percent motor axons, which was significantly lower than the median, radial, and musculocutaneous nerves. CONCLUSIONS: There was no consistent somatotopic organization of motor and sensory axons of the mixed major nerves of the arm just distal to the brachial plexus, but clustering of motor axons may facilitate the splitting of nerves into primarily "motor" and "sensory" fascicles.