The Input-Output Relation of Primary Nociceptive Neurons is Determined by the Morphology of the Peripheral Nociceptive Terminals.

The output from the peripheral terminals of primary nociceptive neurons, which detect and encode the information regarding noxious stimuli, is crucial in determining pain sensation. The nociceptive terminal endings are morphologically complex structures assembled from multiple branches of different geometry, which converge in a variety of forms to create the terminal tree. The output of a single terminal is defined by the properties of the transducer channels producing the generation potentials and voltage-gated channels, translating the generation potentials into action potential (AP) firing. However, in the majority of cases, noxious stimuli activate multiple terminals; thus, the output of the nociceptive neuron is defined by the integration and computation of the inputs of the individual terminals. Here, we used a computational model of nociceptive terminal tree to study how the architecture of the terminal tree affects the input-output relation of the primary nociceptive neurons. We show that the input-output properties of the nociceptive neurons depend on the length, the axial resistance (Ra), and location of individual terminals. Moreover, we show that activation of multiple terminals by a capsaicin-like current allows summation of the responses from individual terminals, thus leading to increased nociceptive output. Stimulation of the terminals in simulated models of inflammatory or neuropathic hyperexcitability led to a change in the temporal pattern of AP firing, emphasizing the role of temporal code in conveying key information about changes in nociceptive output in pathologic conditions, leading to pain hypersensitivity.SIGNIFICANCE STATEMENT Noxious stimuli are detected by terminal endings of primary nociceptive neurons, which are organized into morphologically complex terminal trees. The information from multiple terminals is integrated along the terminal tree, computing the neuronal output, which propagates toward the CNS, thus shaping the pain sensation. Here, we revealed that the structure of the nociceptive terminal tree determines the output of nociceptive neurons. We show that the integration of noxious information depends on the morphology of the terminal trees and how this integration and, consequently, the neuronal output change under pathologic conditions. Our findings help to predict how nociceptive neurons encode noxious stimuli and how this encoding changes in pathologic conditions, leading to pain.

Neurochemical and Ultrastructural Characterization of Unmyelinated Non-peptidergic C-Nociceptors and C-Low Threshold Mechanoreceptors Projecting to Lamina II of the Mouse Spinal Cord.

C-nociceptors (C-Ncs) and non-nociceptive C-low threshold mechanoreceptors (C-LTMRs) are two subpopulations of small unmyelinated non-peptidergic C-type neurons of the dorsal root ganglia (DRGs) with central projections displaying a specific pattern of termination in the spinal cord dorsal horn. Although these two subpopulations exist in several animals, remarkable neurochemical differences occur between mammals, particularly rat/humans from one side and mouse from the other. Mouse is widely investigated by transcriptomics. Therefore, we here studied the immunocytochemistry of murine C-type DRG neurons and their central terminals in spinal lamina II at light and electron microscopic levels. We used a panel of markers for peptidergic (CGRP), non-peptidergic (IB4), nociceptive (TRPV1), non-nociceptive (VGLUT3) C-type neurons and two strains of transgenic mice: the TAFA4Venus knock-in mouse to localize the TAFA4+ C-LTMRs, and a genetically engineered ginip mouse that allows an inducible and tissue-specific ablation of the DRG neurons expressing GINIP, a key modulator of GABABR-mediated analgesia. We confirmed that IB4 and TAFA4 did not coexist in small non-peptidergic C-type DRG neurons and separately tagged the C-Ncs and the C-LTMRs. We then showed that TRPV1 was expressed in only about 7% of the IB4+ non-peptidergic C-Ncs and their type Ia glomerular terminals within lamina II. Notably, the selective ablation of GINIP did not affect these neurons, whereas it reduced IB4 labeling in the medial part of lamina II and the density of C-LTMRs glomerular terminals to about one half throughout the entire lamina. We discuss the significance of these findings for interspecies differences and functional relevance.

Bursty spike trains of antennal thermo- and bimodal hygro-thermoreceptor neurons encode noxious heat in elaterid beetles.

The main purpose of this study was to explain the internal fine structure of potential antennal thermo- and hygroreceptive sensilla, their innervation specifics, and responses of the sensory neurons to thermal and humidity stimuli in an elaterid beetle using focused ion beam scanning electron microscopy and electrophysiology, respectively. Several essential, high temperature induced turning points in the locomotion were determined using automated video tracking. Our results showed that the sensilla under study, morphologically, are identical to the dome-shaped sensilla (DSS) of carabids. A cold-hot neuron and two bimodal hygro-thermoreceptor neurons, the moist-hot and dry-hot neuron, innervate them. Above 25-30 °C, all the three neurons, at different threshold temperatures, switch from regular spiking to temperature dependent spike bursting. The percentage of bursty DSS neurons on the antenna increases with temperature increase suggesting that this parameter of the neurons may encode noxious heat in a graded manner. Thus, we show that besides carabid beetles, elaterids are another large group of insects with this ability. The threshold temperature of the beetles for onset of elevated locomotor activity (OELA) was lower by 11.9 °C compared to that of critical thermal maximum (39.4 °C). Total paralysis occurred at 41.8 °C. The threshold temperatures for spike bursting of the sensory neurons in DSS and OELA of the beetles coincide suggesting that probably the spike bursts are responsible for encoding noxious heat when confronted. In behavioural thermoregulation, spike bursting DSS neurons serve as a fast and firm three-fold early warning system for the beetles to avoid overheating and death.

Cellular and subcellular localization of CXCL12 and CXCR4 in rat nociceptive structures: physiological relevance.

Initial studies implicated the chemokine CXC motif ligand 12 (CXCL12) and its cognate CXC motif receptor 4 (CXCR4) in pain modulation. However, there has been no description of the distribution, transport and axonal sorting of CXCL12 and CXCR4 in rat nociceptive structures, and their direct participation in nociception modulation has not been demonstrated. Here, we report that acute intrathecal administration of CXCL12 induced mechanical hypersensitivity in naive rats. This effect was prevented by a CXCR4-neutralizing antibody. To determine the morphological basis of this behavioural response, we used light and electron microscopic immunohistochemistry to map CXCL12- and CXCR4-immunoreactive elements in dorsal root ganglia, lumbar spinal cord, sciatic nerve and skin. Light microscopy analysis revealed CXCL12 and CXCR4 immunoreactivity in calcitonin gene related peptide-containing peptidergic primary sensory neurons, which were both conveyed to central and peripheral sensory nerve terminals. Electron microscopy clearly demonstrated CXCL12 and CXCR4 immunoreactivity in primary sensory nerve terminals in the dorsal horn; both were sorted into small clear vesicles and large dense-core vesicles. This suggests that CXCL12 and CXCR4 are trafficked from nerve cell bodies to the dorsal horn. Double immunogold labelling for CXCL12 and calcitonin gene related peptide revealed partial vesicular colocalization in axonal terminals. We report, for the first time, that CXCR4 receptors are mainly located on the neuronal plasma membrane, where they are present at pre-synaptic and post-synaptic sites of central terminals. Receptor inactivation experiments, behavioural studies and morphological analyses provide strong evidence that the CXCL12/CXCR4 system is involved in modulation of nociceptive signalling.

Translocation of GluR1-containing AMPA receptors to a spinal nociceptive synapse during acute noxious stimulation.

Potentiation of spinal nociceptive transmission by synaptic delivery of AMPA receptors, via an NMDA receptor- and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent pathway, has been proposed to underlie certain forms of hyperalgesia, the enhanced pain sensitivity that may accompany inflammation or tissue injury. However, the specific synaptic populations that may be subject to such plasticity have not been identified. Using neuronal tracing and postembedding immunogold labeling, we show that a model of acute inflammatory hyperalgesia is associated with an elevated density of GluR1-containing AMPA receptors, as well as an increased synaptic ratio of GluR1 to GluR2/3 subunits, at synapses established by C-fibers that lack the neuropeptide substance P. A more subtle increase in GluR1 immunolabeling was noted at synapses formed by substance P-containing nociceptors. No changes in either GluR1 or GluR2/3 contents were observed at synapses formed by low-threshold mechanosensitive primary afferent fibers. These results contrast with our previous observations in the same pain model of increased and decreased levels of activated CaMKII at synapses formed by peptidergic and nonpeptidergic nociceptive fibers, respectively, suggesting that the observed redistribution of AMPA receptor subunits does not depend on postsynaptic CaMKII activity. The present ultrastructural evidence of topographically specific, activity-dependent insertion of GluR1-containing AMPA receptors at a central synapse suggests that potentiation of nonpeptidergic C-fiber synapses by this mechanism contributes to inflammatory pain.

Molecular architecture of endocannabinoid signaling at nociceptive synapses mediating analgesia.

Cannabinoid administration suppresses pain by acting at spinal, supraspinal and peripheral levels. Intrinsic analgesic pathways also exploit endocannabinoids; however, the underlying neurobiological substrates of endocannabinoid-mediated analgesia have remained largely unknown. Compelling evidence shows that, upon exposure to a painful environmental stressor, an endocannabinoid molecule called 2-arachidonoylglycerol (2-AG) is mobilized in the lumbar spinal cord in temporal correlation with stress-induced antinociception. We therefore characterized the precise molecular architecture of 2-AG signaling and its involvement in nociception in the rodent spinal cord. Nonradioactive in situ hybridization revealed that dorsal horn neurons widely expressed the mRNA of diacylglycerol lipase-alpha (DGL-alpha), the synthesizing enzyme of 2-AG. Peroxidase-based immunocytochemistry demonstrated high levels of DGL-alpha protein and CB(1) cannabinoid receptor, a receptor for 2-AG, in the superficial dorsal horn, at the first site of modulation of the ascending pain pathway. High-resolution electron microscopy uncovered postsynaptic localization of DGL-alpha at nociceptive synapses formed by primary afferents, and revealed presynaptic positioning of CB(1) on excitatory axon terminals. Furthermore, DGL-alpha in postsynaptic elements receiving nociceptive input was colocalized with metabotropic glutamate receptor 5 (mGluR(5)), whose activation induces 2-AG biosynthesis. Finally, intrathecal activation of mGluR(5) at the lumbar level evoked endocannabinoid-mediated stress-induced analgesia through the DGL-2-AG-CB(1) pathway. Taken together, these findings suggest a key role for 2-AG-mediated retrograde suppression of nociceptive transmission at the spinal level. The striking positioning of the molecular players of 2-AG synthesis and action at nociceptive excitatory synapses suggests that pharmacological manipulation of spinal 2-AG levels may be an efficacious way to regulate pain sensation.

Nonselective innervation of lamina I projection neurons by cocaine- and amphetamine-regulated transcript peptide (CART)-immunoreactive fibres in the rat spinal dorsal horn.

Cocaine- and amphetamine-regulated transcript (CART) peptides have been implicated in spinal pain transmission. A dense plexus of CART-immunoreactive fibres has been described in the superficial laminae of the spinal cord, which are key areas in sensory information and pain processing. We demonstrated previously that the majority of these fibres originate from nociceptive primary afferents. Using tract tracing, multiple immunofluorescent labelling and electronmicroscopy we determined the proportion of peptidergic primary afferents expressing CART, looked for evidence for coexistence of CART with galanin in these afferents in lamina I and examined their targets. Almost all (97.9%) randomly selected calcitonin gene-related peptide (CGRP)-immunoreactive terminals were substance P (SP)-positive (+) and CART was detected in approximately half (48.6%) of them. Most (81.4%) of the CGRP/SPergic boutons were galanin+ and approximately half (49.0%) of these contained CART. Many (72.9%) of the CARTergic boutons which expressed CGRP were also immunoreactive for galanin, while only 8.6% of the CARTergic terminals were galanin+ without CGRP. Electron microscopy showed that most of the CART terminals formed asymmetrical synapses, mainly with dendrites. All different morphological and neurochemical subtypes of spinoparabrachial projection neurons in the lamina I received contacts from CART-immunoreactive nociceptive afferents. The innervation density from these boutons did not differ significantly between either the different neurochemical or the morphological subclasses of these cells. This suggests a nonselective innervation of lamina I projection neurons from a subpopulation of CGRP/SP afferents containing CART peptide. These results provide anatomical evidence for involvement of CART peptide in spinal pain transmission.

[Morpho-functional changes in the frog urinary bladder receptors under the influence of barbiturates].

The effect of hexenal and nembutal on the tissue bushy receptors was studied the living isolated frog urinary bladder using methylene blue staining. These drugs were shown to induce the changes in the receptor pulse activity which included three phases: an initial sharp increase, an abrupt decline and a low protracted plateau. Reactions to hexenal and nembutal, while possessing some common features, had their own peculiarities. Synchronously, the dynamics of methylene blue staining of the receptor elements was registered for the control of the intensity of oxidation-reduction processes in the receptor neuroplasm, that is for redox-system dynamics. It was found that the phases of this dynamics coincided in many respects with the phases of the receptor electric activity changes. No ultrastructural changes associated with the putative damaging effect of barbiturates on the receptors were recorded (during the exposure of 1-30 min). The most significant characteristic was an accumulation glycogen granules in the neuroplasm of the receptor elements, suggesting the prevalence of energy substrate deposition over its expenditure. Depression of the receptor pulse activity supports the assumption that barbiturates, besides their soporific and narcotic actions, apparently possess some anesthetic property.

Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion.

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca(2+) release from IP(3)-sensitive stores and Ca(2+) entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y(1) receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca(2+)-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.

Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat.

Trigeminal primary afferents expressing P2X(3) receptor are involved in the transmission of orofacial nociceptive information. However, little is known about their central projection pattern and ultrastructural features within the trigeminal brainstem sensory nuclei (TBSN). Here we use multiple immunofluorescence and electron microscopy to characterize the P2X(3)-immunopositive (+) neurons in the trigeminal ganglion and describe the distribution and synaptic organization of their central terminals within the rat TBSN, including nuclei principalis (Vp), oralis (Vo), interpolaris (Vi), and caudalis (Vc). In the trigeminal ganglion, P2X(3) immunoreactivity was mainly in small and medium-sized somata, but also frequently in large somata. Although most P2X(3) (+) somata costained for the nonpeptidergic marker IB4, few costained for the peptidergic marker substance P. Most P2X(3) (+) fibers in the sensory root of trigeminal ganglion (92.9%) were unmyelinated, whereas the rest were small myelinated. In the TBSN, P2X(3) immunoreactivity was dispersed in the rostral TBSN but was dense in the superficial laminae of Vc, especially in the inner lamina II. The P2X(3) (+) terminals contained numerous clear, round vesicles and sparse large, dense-core vesicles. Typically, they were presynaptic to one or two dendritic shafts and also frequently postsynaptic to axonal endings, containing pleomorphic vesicles. Such P2X(3) (+) terminals, showing glomerular shape and complex synaptic relationships, and those exhibiting axoaxonic contacts, were more frequently seen in Vp than in any other TBSN. These results suggest that orofacial nociceptive information may be transmitted via P2X(3) (+) afferents to all TBSN and that it may be processed differently in different TBSN.

Formation of neuromuscular junctions and synthesis of sensory neuropeptides in the co-cultures of dorsal root ganglion and cardiac myocytes.

AIM: The interactions between primary sensory neurons and cardiac myocytes are still unclear. In the present study, the co-culture model of dorsal root ganglion (DRG) explant and cardiac myocytes was used to characterize the morphological relationship between primary sensory nerve endings and cardiac myocytes and to investigate whether cardiac myocytes could induce substance P (SP) and calcitonin gene-related peptide (CGRP) synthesis in DRG neurons and release from DRG neurons in the neuromuscular co-cultures. METHODS: The formation of neuromuscular junctions was observed with scanning electron microscopy (SEM). SP and CGRP expression were detected by immunocytochemistry. Basal SP and CGRP release and capsaicin-evoked SP and CGRP release were analyzed by radioimmunoassay (RIA). RESULTS: In this study, neuromuscular junctions were observed in the co-cultures of DRG explant and cardiac myocytes. SP-immunoreactive (IR) and CGRP-IR neurons were detected in both neuromuscular co-cultures and DRG explant cultures, but the number of SP-IR and CGRP-IR neurons migrating from DRG explant was significantly increased in neuromuscular co-cultures. Capsaicin-evoked SP and CGRP release but not basal SP and CGRP release in neuromuscular co-cultures increased significantly as compared with that in the cultures of DRG explant alone. CONCLUSIONS: The results implicated that the morphological relationship between sensory nerve terminal and cardiac myocyte is much more close in vitro than it is in vivo. Cardiac myocytes may induce sensory neuropeptide synthesis and capsaicin-evoked neuropeptide release in neuromuscular co-cultures. Further experiment needs to be performed about the significance of neuropeptide synthesis and capsaicin-evoked neuropeptide release induced by target cardiac myocytes.

Sympathetic nerve fibers sprout into rat odontoblast layer, but not into dentinal tubules, in response to cavity preparation.

This study was designed to determine if sympathetic nerve fibers exist in dentinal tubules in rat normal dental pulp, and if they sprout into the dentinal tubules in response to artificial cavity preparation in dentin. Sympathetic nerve fibers in rat molar dental pulp were labeled using an anterograde axonal transport technique involving injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion (SCG). They were then observed using light and electron microscopes. In normal dental pulp (control), scattered WGA-HRP reaction products were observed in unmyelinated nerve endings in the odontoblast layer and subodontoblastic region. In injured pulp 3 weeks after cavity preparation, reaction products were about 1.8-times more plentiful in the above areas (versus control pulp). However, no labeled nerve fibers were observed in the dentinal tubules in either control or injured dental pulp. These results indicate that although sympathetic nerve fibers do indeed sprout in rat dental pulp in response to cavity preparation, they do not penetrate into the dentinal tubules in which postganglionic nerve endings derived from the SCG were not originally present.

The somatosensory system, with emphasis on structures important for pain.

Santiago Ramón y Cajal described a number of somatosensory structures, including several associated with pain, in his major work on the Histology of the Nervous System of Man and Vertebrates. Our knowledge of such structures has been considerably expanded since Cajal because of the introduction of a number of experimental approaches that were not available in his time. For example, Cajal made several drawings of peripheral mechanoreceptors, as well as of bare nerve endings, but later work by others described additional somatosensory receptors and investigated the ultrastructure of bare nerve endings. Furthermore, the transducer molecules responsible for responses to nociceptive, thermal or chemical stimuli are now becoming known, including a series of TRP (transient receptor potential) receptor molecules, such as TRPV1 (the capsaicin receptor). Cajal described the development of dorsal root and other sensory ganglion cells and related the disposition of their somata and neurites to his theory of the functional polarity of neurons. He described the entry of both large and small afferent fibers into the spinal cord, including the projections of their collaterals into different parts of the gray matter and into different white matter tracts. He described a number of types of neurons in the gray matter, including ones in the marginal zone, substantia gelatinosa and head and neck of the dorsal horn. He found neurons in the deep dorsal horn whose dendrites extend dorsally into the superficial dorsal horn. Some of these neurons have since been shown by retrograde labeling to be spinothalamic tract cells. Cajal clearly described the dorsal column/medial lemniscus pathway, but the presence and course of the spinothalamic tract was unknown at the time.

Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors.

Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8EGFPf/+) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain.

Ionotropic glutamate receptors are expressed in GABAergic terminals in the rat superficial dorsal horn.

Ionotropic glutamate receptors (IGR), including NMDA, AMPA, and kainate receptors, are expressed in terminals with varied morphology in the superficial laminae (I-III) of the dorsal horn of the spinal cord. Some of these terminals can be identified as endings of primary afferents, whereas others establish symmetric synapses, suggesting that they may be gamma-aminobutyric acid (GABA)-ergic. In the present study, we used confocal and electron microscopy of double immunostaining for GAD65, a marker for GABAergic terminals, and for subunits of IGRs to test directly whether IGRs are expressed in GABAergic terminals in laminae I-III of the dorsal horn. Although colocalization is hard to detect with confocal microscopy, electron microscopy reveals a substantial number of terminals immunoreactive for GAD65 also stained for IGRs. Among all GAD65-immunoreactive terminals counted, 37% express the NMDA receptor subunit NR1; 28% are immunopositive using an antibody for the GluR2/4 subunits of the AMPA receptor; and 20-35% are immunopositive using antibodies for the kainate receptor subunits GluR5, GluR6/7, KA1, or KA2. Terminals immunoreactive for IGR subunits and GAD65 establish symmetric synapses onto dendrites and perikarya and can be presynaptic to primary afferent terminals within both type 1 and type 2 synaptic glomeruli. Activation of presynaptic IGR may reduce neurotransmitter release. As autoreceptors in terminals of Adelta and C afferent fibers in laminae I-III, presynaptic IGRs may play a role in inhibiting nociception. As heteroreceptors in GABAergic terminals in the same laminae, on the other hand, presynaptic IGRs may have an opposite role and even contribute to central sensitization and hyperalgesia.

Morphological evidence for GABA/glycine-cocontaining terminals in synaptic contact with neurokinin-1 receptor-expressing neurons in the sacral dorsal commissural nucleus of the rat.

Previous studies have shown that neurons in the sacral dorsal commissural nucleus (SDCN) express neurokinin-1 receptor (NK1R) and can be modulated by the co-release of GABA and glycine (Gly) from single presynaptic terminal. These results raise the possibility that GABA/Gly-cocontaining terminals might make synaptic contacts with NK1R-expressing neurons in the SDCN. In order to provide morphological evidence for this hypothesis, the triple-immunohistochemical studies were performed in the SDCN. Triple-immunofluorescence histochemical study showed that some axon terminals in close association with NK1R-immunopositive (NK1R-ip) neurons in the SDCN were immunopositive for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). In electron microscopic dual- and triple-immunohistochemistry for GAD/GlyT2, GAD/NK1R, GlyT2/NK1R, or GAD/GlyT2/NK1R also revealed dually labeled (GAD/GlyT2-ip) synaptic terminals upon SDCN neurons, as well as GAD- and/or GlyT2-ip axon terminals in synaptic contact with NK1R-ip SDCN neurons. These results suggested that some synaptic terminals upon NK1R-expressing SDCN neurons co-released both GABA and Gly.

Structural and functional assessment of skin nerve fibres in small-fibre pathology.

Damage to nociceptor nerve fibres may give rise to peripheral neuropathies, some of which are pain free and some are painful. A hallmark of many peripheral neuropathies is the loss of small nerve fibres in the epidermis, a condition called small-fibre neuropathy (SFN) when it is predominantly the small nerve fibres that are damaged. Historically, SFN has been very difficult to diagnose as clinical examination and nerve conduction studies mainly detect large nerve fibres, and quantitative sensory testing is not sensitive enough to detect small changes in small nerve fibres. However, taking a 3-mm punch skin biopsy from the distal leg and quantification of the nerve fibre density has proven to be a useful method to diagnose SFN. However, the correlation between the nerve fibre loss and other test results varies greatly. Recent studies have shown that it is possible not only to extract information about the nerve fibre density from the biopsies but also to get an estimation of the nerve fibre length density using stereology, quantify sweat gland innervation and detect morphological changes such as axonal swelling, all of which may be additional parameters indicating diseased small fibres relating to symptoms reported by the patients. In this review, we focus on available tests to assess structure and function of the small nerve fibres, and summarize recent advances that have provided new possibilities to more specifically relate structural findings with symptoms and function in patients with SFN.

Selective conversion of fibroblasts into peripheral sensory neurons.

Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.

Perineural capsaicin induces the uptake and transganglionic transport of choleratoxin B subunit by nociceptive C-fiber primary afferent neurons.

The distribution of spinal primary afferent terminals labeled transganglionically with the choleratoxin B subunit (CTB) or its conjugates changes profoundly after perineural treatment with capsaicin. Injection of CTB conjugated with horseradish peroxidase (HRP) into an intact nerve labels somatotopically related areas in the ipsilateral dorsal horn with the exceptions of the marginal zone and the substantia gelatinosa, whereas injection of this tracer into a capsaicin-pretreated nerve also results in massive labeling of these most superficial layers of the dorsal horn. The present study was initiated to clarify the role of C-fiber primary afferent neurons in this phenomenon. In L5 dorsal root ganglia, analysis of the size frequency distribution of neurons labeled after injection of CTB-HRP into the ipsilateral sciatic nerve treated previously with capsaicin or resiniferatoxin revealed a significant increase in the proportion of small neurons. In the spinal dorsal horn, capsaicin or resiniferatoxin pretreatment resulted in intense CTB-HRP labeling of the marginal zone and the substantia gelatinosa. Electron microscopic histochemistry disclosed a dramatic, ∼10-fold increase in the proportion of CTB-HRP-labeled unmyelinated dorsal root axons following perineural capsaicin or resiniferatoxin. The present results indicate that CTB-HRP labeling of C-fiber dorsal root ganglion neurons and their central terminals after perineural treatment with vanilloid compounds may be explained by their phenotypic switch rather than a sprouting response of thick myelinated spinal afferents which, in an intact nerve, can be labeled selectively with CTB-HRP. The findings also suggest a role for GM1 ganglioside in the modulation of nociceptor function and pain.

C-fiber structure varies with location in peripheral nerve.

Recent advances in regeneration and pain research have revealed gaps in the understanding of normal C-fiber anatomy. In the rat PNS, C-fiber axons assemble into Remak bundles, but beyond this, features of C-fiber organization are not defined. Systematic sampling and quantitation reveals that Remak bundles exiting from the L5 dorsal root ganglion (DRG) contain large numbers of axons, for example, 56% of unmyelinated axons were in bundles of >20 axons. This is different from distal nerve segments such as the hindpaw plantar nerve where the median number of axons per bundle is 3. The cross-sectional area of unmyelinated axons in dorsal root was homogeneous near the DRG but variability in axonal area increased near the spinal cord (p = 0.00001) and the mean axonal area was unchanged. Unmyelinated axons in peripheral nerve were almost always isolated from one another by Schwann cell processes; however, in dorsal root 7% to 9% of unmyelinated axons were immediately adjacent within pockets containing 2 or more axons. Remak bundles in the distal peripheral nerve clustered with other Remak bundles. We observe that multiple unmyelinated axons are juxtaposed within the C-fiber/Remak bundle and that the close association of afferent axons may have important functional implications.