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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 TAFA4 Venus 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 GABA B R-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.

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., (Copyright © 2020 the authors.)

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 (TRPM8 EGFPf/+ ) 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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

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., (© 2014 European Pain Federation - EFIC®)

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.

Fine myelinated (Aδ) nociceptors are responsible for fast, well-localised pain, but relatively little is known about their postsynaptic targets in the spinal cord, and therefore about their roles in the neuronal circuits that process nociceptive information. Here we show that transganglionically transported cholera toxin B subunit (CTb) labels a distinct set of afferents in lamina I that are likely to correspond to Aδ nociceptors, and that most of these lack neuropeptides. The vast majority of lamina I projection neurons can be retrogradely labelled from the lateral parabrachial area, and these can be divided into 2 major groups based on expression of the neurokinin 1 receptor (NK1r). We show that CTb-labelled afferents form contacts on 43% of the spinoparabrachial lamina I neurons that lack the NK1r, but on a significantly smaller proportion (26%) of those that express the receptor. We also confirm with electron microscopy that these contacts are associated with synapses. Among the spinoparabrachial neurons that received contacts from CTb-labelled axons, contact density was considerably higher on NK1r-lacking cells than on those with the NK1r. By comparing the density of CTb contacts with those from other types of glutamatergic bouton, we estimate that nonpeptidergic Aδ nociceptors may provide over half of the excitatory synapses on some NK1r-lacking spinoparabrachial cells. These results provide further evidence that synaptic inputs to dorsal horn projection neurons are organised in a specific way. Taken together with previous studies, they suggest that both NK1r(+) and NK1r-lacking lamina I projection neurons are directly innervated by Aδ nociceptive afferents., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (IIi ) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina IIi PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A- and C-fibers low threshold mechanoreceptors (LTMRs) terminate in lamina IIi raise the general issue of synaptic inputs to lamina IIi PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ-immunoreactivity is mostly restricted to interneurons in lamina IIi of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ-immunoreactive interneurons are asymmetric (likely excitatory). PKCγ-immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3-immunoreactive bouton, specific to C-LTMRs, on PKCγ-immunoreactive interneurons. PKCγ-immunoreactive interneurons contain GABAA ergic and glycinergic receptors. At the subcellular level, PKCγ-immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ-immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia., (Copyright © 2013 Wiley Periodicals, Inc.)

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., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Introduction: Vesicular glutamate transporters (VGLUTs) are involved in the transport of transmitter glutamate into synaptic vesicles and are used as markers for glutamatergic neurons., Methods: To assess which types of VGLUTs are involved in the glutamate signaling in pulpal axons and to investigate their distribution, we performed light microscopic immunohistochemistry by using antibodies against VGLUT1, VGLUT2, calcitonin gene-related peptide, and Western blot analysis in human dental pulp., Results: VGLUT1 was expressed in a large number of pulpal axons, especially in the peripheral pulp where the axons branch extensively. The VGLUT1 immunopositive axons showed bead-like appearance, and the majority of these also expressed calcitonin gene-related peptide. VGLUT2 was expressed in few axons throughout the pulp., Conclusions: Our findings suggest that VGLUT1 is involved mainly in the glutamate-mediated signaling of pain, primarily at the level of the peripheral pulp., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Sensory dendrites fall into many different morphological and functional classes. Polymodal nociceptors are one subclass of sensory neurons, which are of particular note owing to their elaborate dendritic arbors. Complex developmental programs are required to form these arbors and there is striking conservation of morphology, function and molecular determinants between vertebrate and invertebrate polymodal nociceptors. Based on these studies, we argue that arbor morphology plays an important role in the function of polymodal nociceptors. Similar associations between form and function might explain the plethora of dendrite morphologies seen among all sensory neurons., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The study of projections and synaptic connectivity of trigeminal sensory and proprioceptive afferents in the 1(st) relay nucleus of the brainstem, helps us to understand where and how the specific craniofacial neural information is transmitted and processed in the CNS. This paper reviews recent findings on the synaptic connectivity of specific craniofacial sensory and proprioceptive afferents in the brainstem. It also deals with neurotransmitters and receptors involved in the presynaptic modulation of the trigeminal primary afferents. Here, we will also review recent findings on the projection and synaptic connectivity of the axons, and terminals in the trigeminal sensory nuclei that express nociceptive markers such as theromosensitive TRP channels TRPV1 and TRPA1, and the purinergic receptor P2X3. The dental pulp is a good model for the study of peripheral pain because it is densely innervated by nociceptive afferents. Finally, we describe the axons innervating the dental pulp and the morphological changes that the myelinated axons undergo during their intradental course., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The development of neuronal dendritic trees involves positive and negative control of growth and branching, as well as modulation of the spacing and orientation of branches. A new study reveals the importance of a membrane fusogen in the dendrite arborization of a pair of highly-branched worm sensory neurons., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is a recently identified, endogenous ligand of the alpha7 subunit of nicotinic acetylcholine receptors. SLURP-1 is also the causative gene for an autosomal recessive palmoplantar keratoderma, Mal de Meleda. Although the function of SLURP-1 in keratinocyte development and differentiation has been extensively studied, little is known about its role in the nervous system. In the present study, we analyzed SLURP-1 expression in the spinal cord of rats, as a number of studies suggest spinal nicotinic acetylcholine receptors are important modulators of pain transmission. We detected intense SLURP-1 immunoreactivity in the dorsal horn of the spinal cord, especially in lamina I and outer II. In dorsal root ganglia, SLURP-1 immunoreactivity was detected in small- to medium-sized neurons, where in situ hybridization also revealed the presence of SLURP-1 mRNA. Fluorescent labeling of SLURP-1 partially overlapped that of calcitonin-gene related peptide (CGRP) or substance P (SP) in both the spinal cord dorsal horn and glabrous skin, and electron microscopic analysis revealed colocalization of SLURP-1 with SP or CGRP, in large synaptic vesicles in terminals within the superficial layer of the spinal cord. Finally, sciatic nerve axotomy reduced levels of SLURP-1 immunoreactivity in parallel with that of SP and CGRP in the ipsilateral superficial dorsal horn. These findings suggest that SLURP-1 is expressed in a subset of primary peptidergic sensory neurons.

Several lines of evidence indicate that peripheral 5-HT2A receptors are involved in the development of inflammatory and neuropathic pain. However, their localization in sensory cell bodies is not accurately known. We therefore studied 5-HT2A receptor distribution in rat lumbar dorsal root ganglia using immunocytochemistry. Forty percent of L3 lumbar dorsal root ganglion cells were immunoreactive for 5-HT2A receptor. Most were small- to medium-sized cell bodies. Double-labeled experiments revealed that they expressed various chemical phenotypes. The smaller 5-HT2AR cell bodies often bind the isolectin B4 although some 5-HT2AR cell bodies also express substance P (SP). Many 5-HT2A-positive small dorsal root ganglion cells expressed the capsaicin receptor transient receptor potential vanilloid type 1 receptor (TRPV1), confirming their nociceptive nature. In addition, a few large cell bodies were labeled for 5-HT2A, and they also expressed NF200 suggesting that they were at the origin of Adelta or Abeta fibers. A total absence of double labeling with parvalbumin showed that they were not proprioceptors. 5-HT2A immunoreactivity in dorsal root ganglia cells was found in the cytoplasm and along the plasma membrane at the interface between sensory cell and the adjacent satellite cells; this distribution was confirmed under the electron microscope, and suggested a functional role for the 5-HT2A receptor at these sites. We therefore investigated the presence of 5-HT and 5-HIAA in lumbar dorsal root ganglia by high performance liquid chromatography. There were 5.75+/-0.80 ng 5-HT and 3.19+/-0.37 ng 5-hydroxyindoleacetic acid (5-HIAA) per mg of protein with a ratio 5-HIAA/5-HT of 0.67+/-0.10, similar to values typically observed in brain tissues. These findings suggest that 5-HT, via the 5-HT2AR, may be involved in the peripheral control of sensory afferents, mainly unmyelinated nociceptors and to a lesser extent neurons with Adelta or Abeta fibers, and in the control of cellular excitability of some dorsal root cell bodies through a paracrine mechanism of action.

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.

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.

Agonists acting at alpha(2)-adrenergic and opioid receptors (alpha(2)ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although the existence of alpha(2)AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of heterooligomers has been proposed as a molecular basis for interactions between neuronal G-protein-coupled receptors. The relevance of heterooligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between alpha(2)ARs and ORs in the rat spinal cord to determine whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between alpha(2A)-adrenergic and delta-opioid receptors (DOP) on substance P (SP)-immunoreactive (-ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. alpha(2A)AR- and DOP-ir elements were colocalized in subcellular structures of 0.5 mum or less in diameter in isolated nerve terminals. Furthermore, coincubation of isolated synaptosomes with alpha(2)AR and DOP agonists resulted in a greater-than-additive increase in the inhibition of K(+)-stimulated neuropeptide release. These findings suggest that coexpression of the synergistic receptor pair alpha(2A)AR-DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein-protein interactions such as heterooligomerization., ((c) 2009 Wiley-Liss, Inc.)

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.

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.

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.

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.

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., ((c) 2007 Wiley-Liss, Inc.)

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.

Agmatine (decarboxylated arginine) was originally identified in the CNS as an imidazoline receptor ligand. Further studies demonstrated that agmatine antagonizes NMDA receptors and inhibits nitric oxide synthase. Intrathecally administered agmatine inhibits opioid tolerance and hyperalgesia evoked by inflammation, nerve injury, and intrathecally administered NMDA. These actions suggest an anti-glutamatergic role for agmatine in the spinal cord. We have previously reported that radiolabeled agmatine is transported into spinal synaptosomes in an energy- and temperature-dependent manner. In the present study, we demonstrate that agmatine is releasable from purified spinal nerve terminals upon depolarization. When exposed to either elevated potassium or capsaicin, tritiated agmatine (but not its precursor L-arginine or its metabolite putrescine) is released in a calcium-dependent manner. Control experiments confirmed that the observed release was specific to depolarization and not due to permeabilization of or degradation of synaptosomes. That capsaicin-evoked stimulation results in agmatine release implicates the participation of primary afferent nerve terminals. Radiolabeled agmatine also accumulates in purified spinal synaptosomal vesicles in a temperature-dependent manner, suggesting that the source of releasable agmatine may be vesicular in origin. These results support the proposal that agmatine may serve as a spinal neuromodulator involved in pain processing.

Recent studies show that excitatory glutamatergic transmission is potentiated by BDNF in superficial dorsal horn, both at the pre- and the postsynaptic site. The role of BDNF in modulating GABA and glycine-mediated inhibitory transmission has not been fully investigated. To determine whether the neurotrophin is effective in regulating the spontaneous release of the two neurotransmitters, we have recorded miniature inhibitory postsynaptic currents (mIPSCs) in lamina II of post-natal rats. We show that application of BDNF enhanced the spontaneous release of GABA and glycine, in presence of tetrodotoxin. The effect was blocked by the trk-receptor inhibitor k-252a. Amplitude and kinetics of mIPSCs were not altered. Evoked GABA and glycine IPSCs (eIPSCs) were depressed by BDNF and the coefficient of variation of eIPSC amplitude was significantly increased. By recording glycine eIPSCs with the paired-pulse protocol, an increase of paired-pulse ratio during BDNF application was observed. We performed parallel ultrastructural studies to unveil the circuitry involved in the effects of BDNF. These studies show that synaptic interactions between full length functional trkB receptors and GABA-containing profiles only occur at non peptidergic synaptic glomeruli of types I and II. Expression of trkB in presynaptic vesicle-containing dendrites originating from GABAergic islet cells, indicates these profiles as key structures in the modulation of inhibitory neurotransmission by the neurotrophin. Our results thus describe a yet uncharacterized effect of BDNF in lamina II, giving further strength to the notion that the neurotrophin plays an important role in pain neurotransmission.

Eimer's organ is a small, densely innervated sensory structure found on the glabrous rhinarium of most talpid moles. This structure consists of an epidermal papilla containing a central circular column of cells associated with intraepidermal free nerve endings, Merkel cell neurite complexes, and lamellated corpuscles. The free nerve endings within the central cell column form a ring invested in the margins of the column, surrounding 1-2 fibers that pass through the center of the column. A group of small-diameter nociceptive free nerve endings that are immunoreactive for substance P surrounds this central ring of larger-diameter free nerve endings. Transmission electron microscopy revealed a high concentration of tonofibrils in the epidermal cells of the central column, suggesting they are more rigid than the surrounding keratinocytes and may play a mechanical role in transducing stimuli to the different receptor terminals. The intraepidermal free nerve endings within the central column begin to degrade 15 microm from the base of the stratum corneum and do not appear to be active within the keratinized outer layer. The peripheral free nerve endings are structurally distinct from their counterparts in the central column and immunocytochemical double labeling with myelin basic protein and substance P indicates these afferents are unmyelinated. Merkel cell-neurite complexes and lamellated corpuscles are similar in morphology to those found in a range of other mammalian skin., ((c) 2007 Wiley-Liss, Inc.)

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.

Opioid and tachykinin systems are involved in modulation of pain transmission in the spinal cord. Regulation of surface opioid receptors on nociceptive afferents is critical for opioid analgesia. Plasma-membrane insertion of delta-opioid receptors (DORs) is induced by stimulus-triggered exocytosis of DOR-containing large dense-core vesicles (LDCVs), but how DORs become sorted into the regulated secretory pathway is unknown. Here we report that direct interaction between protachykinin and DOR is responsible for sorting of DORs into LDCVs, allowing stimulus-induced surface insertion of DORs and DOR-mediated spinal analgesia. This interaction is mediated by the substance P domain of protachykinin and the third luminal domain of DOR. Furthermore, deletion of the preprotachykinin A gene reduced stimulus-induced surface insertion of DORs and abolished DOR-mediated spinal analgesia and morphine tolerance. Thus, protachykinin is essential for modulation of the sensitivity of nociceptive afferents to opioids, and the opioid and tachykinin systems are directly linked by protachykinin/DOR interaction.

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., (Copyright 2005 Wiley-Liss, Inc.)

Postsynaptic autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr286/287 is crucial for the induction of long-term potentiation at many glutamatergic synapses, and has also been implicated in the persistence of synaptic potentiation. However, the availability of CaMKII phosphorylated at Thr286/287 at individual glutamatergic synapses in vivo is unclear. We used post-embedding immunogold labelling to quantitatively analyse the ultrastructural localization of CaMKII phosphorylated at Thr286/287 (pCaMKII) at synapses formed by presumed nociceptive and low-threshold mechanosensitive primary afferent nerve endings in laminae I-IV of rat spinal cord. Immunogold labelling was enriched in the postsynaptic densities of such synapses, consistent with observations in pre-embedding immunoperoxidase-stained dorsal horn. Presynaptic axoplasm also exhibited sparse immunogold labelling, in peptidergic terminals partly associated with dense core vesicles. Analysis of single or serial pCaMKII-immunolabelled sections indicated that the large majority of synapses formed either by presumed peptidergic or non-peptidergic nociceptive primary afferent terminals in laminae I-II of the spinal cord, or by presumed low-threshold mechanosensitive primary afferent terminals in laminae IIi-IV, contained pCaMKII in their postsynaptic density. However, the postsynaptic levels of pCaMKII immunolabelling at low-threshold primary afferent synapses were only approximately 50% of those at nociceptive synapses. These results suggest that constitutively autophosphorylated CaMKII in the postsynaptic density is a common characteristic of glutamatergic synapses, thus potentially contributing to maintenance of synaptic efficacy. Furthermore, pCaMKII appears to be differentially regulated between high- and low-threshold primary afferent synapses, possibly reflecting different susceptibility to synaptic plasticity between these afferent pathways.

A variety of studies indicate that spinal nicotinic acetylcholine receptors modulate the behavioral and autonomic responses elicited by afferent stimuli. To examine the location of and role played by particular subtypes of nicotinic receptors in mediating cardiovascular and nociceptive responses, we treated neonatal and adult rats with capsaicin to destroy C-fibers in primary afferent terminals. Reduction of C-fiber terminals was ascertained by the loss of isolectin B4, CGRP and vanilloid receptors as monitored by immunofluorescence. Receptor autoradiography shows a reduction in number of epibatidine binding sites following capsaicin treatment. The reduction is particularly marked in the dorsal horn and primarily affects the class of high affinity epibatidine binding sites thought to modulate nociceptive responses. Accompanying the loss of terminals and nicotinic binding sites were significant reductions in the expression of alpha 3, alpha 4, alpha 5, beta 2 and beta 4 nicotinic receptor subunits in the superficial layers of the spinal cord as determined by antibody staining and confocal microscopy. The loss of nicotinic receptors that follows capsaicin treatment results in attenuation of the nociceptive responses to both spinal cytisine and epibatidine. Capsaicin treatment also diminishes the capacity of cytisine to desensitize nicotinic receptors mediating nociception, but it shows little effect on intrathecal nicotinic agonist elicited pressor and heart rate responses. Hence, our data suggest that alpha 3, alpha 4, alpha 5, beta 2 and beta 4 subunits of nicotinic receptors are localized in the spinal cord on primary afferent terminals that mediate nociceptive input. A variety of convergent data based on functional studies and subunit expression suggest that alpha 3 and alpha 4, in combination with beta 2 and alpha 5 subunits, form the majority of functional nicotinic receptors on C-fiber primary afferent terminals. Conversely, spinal nicotinic receptors not located on C-fibers play a primary role in the spinal pathways evoking spinally coordinated autonomic cardiovascular responses.

The 5-HT5A receptor is a seven-transmembrane receptor negatively coupled to adenylate cyclase, whose activation opens K+ channels. The 5-HT5A receptor may thus exert an inhibitory effect on neuronal activity. However, the function of this receptor is still largely unknown, in particular at the spinal level, and this is partly due to lack of specific ligands. Immunocytochemistry using specific anti-5-HT5A antibodies reveals a particularly dense labeling in the two superficial layers of the dorsal horn, suggesting that the 5-HT5A receptor may be involved in the spinal modulation of pain. In addition, a very intense staining in the lumbar dorsolateral nucleus (Onuf nucleus) in both males and females suggests that the 5-HT5A receptor is also involved in micturition through the control of urethral sphincter muscles. Colchicine pretreatment allows the staining of numerous cell bodies in lamina II. Fewer labeled cell bodies are seen in laminae I and III-VI, in the lateral spinal nucleus, and in lamina X. Electron microscope examination of 5-HT5A receptor immunoreactivity in spinal cords from untreated animals confirmed the postsynaptic labeling in all regions studied (dorsal horn, dorsolateral nucleus, and lamina X). The morphological heterogeneity of labeled dorsal horn cell bodies suggests that they belong to functionally distinct neurons (projection neurons and interneurons). In the lumbar dorsolateral nucleus, the labeling is preferentially localized on dendrites, suggesting that in this nucleus 5-HT preferentially acts at the dendritic level. Finally, the dense labeling of postsynaptic specializations suggests that the receptor may be in stock before being addressed to the synaptic differentiation.

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.

In the rat experimental model, molar tooth movement induced by Waldo's method is known to cause a temporally and spatially defined pattern of brain neuronal activation. Since orthodontic correction usually involves the entire dental arch, we used a spring-activated appliance to extend the investigation to incisors, and we included brain regions related to antinociception. Adjustment of the non-activated appliance on incisors resulted in c-fos expression in the dorsal raphe, peri-aqueductal gray matter, and the locus coeruleus, in addition to trigeminal sensory subnuclei and the parabrachial nucleus, where neuronal activation has already been detected in previous studies on molar tooth movement. Appliance activation with a 70-g force resulted in a further increase in Fos-immunoreactive neurons in the trigeminal sensory subnucleus caudalis and in the dorsal raphe. This result suggests that there is a recruitment of neurons related to nociception and to antinociception when tooth movement is increased.

In this study the ultrastructural appearance of primary sensory neurones labelled by the injection of the plant lectin Bandeiraea simplicifolia I-isolectin B(4) (BSI-B(4)) into a peripheral nerve has been examined in the rat. Electron microscopy of the somata of retrogradely labelled neurones showed the lectin to be associated with the inner surface of cytoplasmic vesicles, supporting the premise that the uptake of BSI-B(4) into sensory neurones is by the process of receptor-mediated endocytosis. Light and electron microscopic analysis of the spinal cord revealed transganglionically transported lectin in unmyelinated axons in the dorsolateral funiculus and axon terminals concentrated mainly within lamina II of the dorsal horn. Detailed analysis of 1377 of these axon terminals revealed that the majority were glomerular in shape and surrounded by up to 14 other unlabelled profiles. These findings suggest that primary sensory neurones which transganglionically transport BSI-B(4) have a synaptic ultrastructure similar to that which has been previously reported for unmyelinated primary sensory neurones. Moreover, it appears that the axon terminals of these neurones are subjected to extensive modulation. Examination of the vesicle content of lectin labelled axon terminals revealed that the majority contained small agranular vesicles while large granular vesicles were observed only occasionally. These findings support the suggestion that the populations of neurones expressing binding sites for BSI-B(4) are fairly distinct from those containing neuroactive peptides. In conclusion, the results of the current study suggest that the lectin BSI-B(4) can be used as a histological marker for a subpopulation of small diameter primary sensory neurones and provide further evidence for the potential of this lectin as a useful tool in the study of pain.

The receptive fields (RFs) of polymodal nociceptor units of canine testis consist of small fascicles of branching axons ending as clusters within the thin vascular layer overlying the seminiferous tubules. This propitious arrangement enabled serial reconstruction of electron micrographs of a flat, punctate zone, identified during recording of impulse discharge of a single physiologically characterized nociceptor "unit." The RFs showed multiple axons, with some branching and sequential vesicle-containing swellings, similar to what have been called terminals and assumed to constitute impulse generating sites in other tissues. Consistent with this interpretation is the local presence of: (1) clusters of small mitochondria, (2) glycogen granules, and (3) sectors of axolemma denuded of Schwann cell processes and thus "free" endings directly contacting the epithelial basal lamina. The most distal sectors showed additional morphologic properties: (1) zones of vesicles embedded in an extensive granular matrix, (2) preterminal arrays of "axonal reticulum" derived from the smooth endoplasmic reticulum displacing axonal cytoskeletal elements, (3) a variety of sizes and electron lucency in clear, spherical vesicles and a few granular or dense-core vesicles, and (4) specialization in the last Schwann cell of the fascicle. These latter features may reflect differences in the specialized receptor mechanisms of nociceptors that are difficult to detect without extensive serial electron microscopic analysis. Alternatively, these features may constitute a regional specialization of the testis. The term free nerve ending is perhaps an insufficient and inaccurate descriptor of the morphology of nociceptors. These findings are considered in the context of their possible relation to the sensitized vanilloid receptor mechanism unique to nociceptors., (Copyright 2003 Wiley-Liss, Inc.)

Neurotomy is widely used as a model of chronic, intractable pain, the proverbial "crux medicorum". Immunohistochemical aspects of this chronic pain model are discussed in this paper, with the aim of shedding new light on the pathomechanism and possible therapeutical consequences. Central terminals of nociceptive neurons contain substance P, somatostatin and calcitonin generelated peptide or exhibit fluoride resistant acid phosphatase and thiamine monophosphatase enzyme reaction in the superficial dorsal horn of the spinal cord and in analogous structures of the brain stem. These neuropeptides and neuroproteins are expressed by the related dorsal root ganglion cells and transported via orthograde axoplasmic transport via dorsal roots to the central nervous system. Transection of the ipsilateral, segmentally related peripheral sensory nerve results in transganglionic degenerative atrophy of central terminals of primary nociceptive neurons. Transganglionic degenerative atrophy is characterized by marked ultrastructural alterations superficially similar to, but essentially differing from the signs of Wallerian degeneration which ensue after dorsal rhizotomy. Transganglionic degenerative atrophy is accompanied by depletion of marker neuropeptides and enzymes, and later by the expression of vicarious neuropeptides such as vasoactive intestinal polypeptide, neuropeptide Y and galanin and of the enzyme choline acetyl transferase. Consequences of blockade of retrograde axoplasmic transport of the nerve growth factor elicited either by perineural application of microtubule inhibitors or by perineural administration of anti-nerve growth factor are similar to peripheral neurotomy. According to recent studies described in this paper, the blockade of nerve growth factor supply to primary nociceptive neurons induces activation of c-jun in nuclei of primary nociceptive neurons probably responsible for the plasticity of the neuropeptide and neuroprotein synthesizing machinery. In contrast, invasion of and formation of pericellular baskets by noradrenergic axons can be elicited only by axotomy and not by blockade of retrograde axoplasmic transport. Involvement of nerve growth factor and the nerve growth factor-dependent immediate early genes in neuroplasticity of neuropeptidergic primary sensory neurons raise the possibility of a gene therapy of chronic intractable pain.

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.

A direct action of mu-opioid agonists on neurons in the spinal dorsal horn is thought to contribute to opiate-induced analgesia. In this study we have investigated neurons that express the mu-opioid receptor MOR-1 in rat spinal cord to provide further evidence about their role in nociceptive processing. MOR-1-immunoreactive cells were largely restricted to lamina II, where they comprised approximately 10% of the neuronal population. The cells received few contacts from nonpeptidergic unmyelinated afferents, but many from substance P-containing afferents. However, electron microscopy revealed that most of these contacts were not associated with synapses. None of the MOR-1 cells in lamina II expressed the neurokinin 1 receptor; however, the mu-selective opioid peptide endomorphin-2 was present in the majority (62-82%) of substance P axons that contacted them. Noxious thermal stimulation of the foot induced c-Fos expression in approximately 15% of MOR-1 cells in the medial third of the ipsilateral dorsal horn at mid-lumbar level. However, following pinching of the foot or intraplantar injection of formalin very few MOR-1 cells expressed c-Fos, and for intraplantar formalin injection this result was not altered significantly by pretreatment with systemic naloxone. Although these findings indicate that at least some of the neurons in lamina II with MOR-1 are activated by noxious thermal stimulation, the results do not support the hypothesis that the cells have a role in transmitting nociceptive information following acute mechanical or chemical noxious stimuli.

The vanilloid receptor VR1 and the vanilloid receptor-like protein VRL-1 are associated with polymodal nociceptors, and may be important for pain processing in normal and injured teeth. Using immunohistochemistry, we have studied the distribution of these receptors in rat pulpal or gingival trigeminal ganglion neurons that were identified through retrograde labeling with fluoro-gold. Twenty-one percent to 34% of tooth pulp-innervating neurons were VRl-positive, while 32%-51% were VRL-1-immunoreactive. However, double-labeling experiments revealed that VR1 and VRL-1 rarely co-existed in the same cells, but rather seemed to be confined to separate subpopulations. Among the gingival neurons, about 25% were VR1-positive and about 41% were VRL-1-immunoreactive. A lesion of the inferior alveolar nerve, which supplies mandibular teeth and gingiva, resulted in a marked down-regulation of VR1 in the affected trigeminal ganglion cells. A down-regulation of VRL-l was also indicated. The results suggest that both VR1 and VRL-1 could have significant roles in pulpal and gingival nociceptive transduction.

Nociceptive dorsal root ganglion (DRG) neurones have fibres that conduct in the C, Adelta and Aalpha/beta conduction velocity range. The properties of nociceptive compared with non-nociceptive somatic afferent dorsal root ganglion neurones appear to fall into two patterns, A and B. Pattern A properties of nociceptive neurones, the more common type, include longer action potential duration and slower maximum rate of fibre firing, as well as a greater expression of substance P and calcitonin gene-related peptide immunoreactivity. The values of pattern A properties appear to be graded according to the conduction velocity group (C, Adelta or Aalpha/beta) of the fibres. The most pronounced forms of A-type properties are expressed by nociceptive neurones with C-fibres, and these become less pronounced in nociceptive neurones with Adelta-fibres and least pronounced in those with Aalpha/beta fibres (C > Adelta > Aalpha/beta). Some of these properties are also expressed in a less extreme but similarly graded manner through C, Adelta and Aalpha/beta groups of non-nociceptive low threshold mechanoreceptive (LTM) neurone. The less common pattern B properties of nociceptive neurones have similar values in C-, Adelta- and Aalpha/beta-fibre nociceptive neurones but these clearly differ from LTM units with C-, Adelta- and Aalpha/beta-fibre conduction velocities. These features of nociceptive neurones include consistently larger action potential overshoots and longer after-hyperpolarisation durations in nociceptive than in LTM neurones.

The expression of capsaicin (VR1) receptor by A-fiber primary afferent neurons has been investigated by double immunohistochemical staining with VR1 and 200 kD neurofilament (NF200; an A-fiber marker) antibodies, and by VR1 immunohistochemical staining in combination with cholera toxin B subunit (CTB; also an A-fiber marker) retrograde tracing. Approximately 30% of the VR1-positive dorsal root ganglion (DRG) neurons were NF200-positive. Intra-sciatic nerve injection of CTB labeled over 30% of the VR1-positive neurons in the L5 DRG. Size frequency distribution analysis revealed that these VR1 and NF200, or VR1 and CTB, double-labeled neurons were predominantly small and medium sized. These results suggest that capsaicin receptors are likely to be expressed by Adelta-fiber neurons as well as C-fiber neurons.

Several lines of evidence show that endogenous and exogenous cannabinoids modulate pain transmission at the spinal level through specific cannabinoid-1 (CB1) receptors. Since anatomical data concerning spinal CB1 receptors are rather contradictory, we studied the cellular and subcellular localizations of the CB1 receptors by immunocytochemistry. Results show a dual pre- and postsynaptic localization of CB1 receptors. Presynaptic receptors are evidenced by the labeling of (1) heterogeneous dorsal root ganglion neurons and (2) axons of Lissauer's tract. Postsynaptic receptors are shown by the labeling of numerous interneurons in the outer part of lamina II. Double immunolabelings show that lamina II outer CB1 neurons, probably islet cells, may also contain GABA or nitric oxide synthase. Numerous CB1-containing neurons in lamina X are also immunostained with anti-nitric oxide synthase (NOS) antibody. Under the electron microscope, CB1 immunoreactivity is exclusively localized postsynaptically in both somatic and dendritic compartments. The absence of labeling on primary afferent axon terminals is discussed and compared to the absence of labeling on terminals or vesicle-containing dendrites of islet cells, where a presynaptic localization was expected according to data of the literature.

The synaptic connections between gamma-aminobutyric acid (GABA)- and glycine-immunoreactive terminals and neurons projecting to the lateral parabrachial region were examined by a combination of retrograde tracing and immunohistochemical staining in the rat medullary dorsal horn. After injection of horseradish peroxidase (HRP) into the right lateral parabrachial region, HRP retrogradely labeled neurons were observed bilaterally in laminae I, II and III of the medullary dorsal horn with an ipsilateral predominance. GABA- and glycine-like immunoreactive terminals were found in laminae I, II and III. Some of these GABA- and glycine-like immunoreactive terminals were observed chiefly to make symmetric synapses with HRP-labeled neuronal cell bodies and dendritic processes. The present results indicate that neurons in the medullary dorsal horn projecting to the lateral parabrachial region might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons, which might be significantly involved in the regulation of the noxious information transmission.

Cannabinoids and opioids interact in the control of nociception at the spinal level. Likely, several mechanisms are involved, with one of them being co-localization of cannabinoid and opioid receptors. In order to validate this hypothesis, a double labeling study of CB1 cannabinoid receptors and mu-opioid receptors in the dorsal horn of the rat spinal cord was performed. A strong co-localization of CB1 and mu-opioid receptors was observed in lamina II interneurons at the ultrastructural level. The physiological consequences of the co-localization are discussed.

The capsaicin receptor (VR1) homologue, VRL1, is thought to be responsible for transducing high-threshold heat responses in Adelta-fiber neurons. In the present study, the expression of VRL1 by A- or C-fiber sensory neurons in rats was investigated by using a VRL1 and 200 kDa neurofilament (NF200, an A-fiber marker) double immunohistochemical staining method. Approximately 46% of VRL-positive neurons were NF200 positive. Though double-labeled neurons tended to be medium to large, many VRL1 single-labeled neurons were large. Dense VRL1 immunoreactivity was also found in laminae I and II of the spinal dorsal horn, where nociceptive Adelta- and C-fibers normally terminate. These results suggest that both C-fiber and Adelta-fiber primary sensory neurons express VRL1, and VRL1 may play an important role in nociception.

The afferent (excitability) and efferent functions (release of neuropeptides) of primary afferent nerve fibres are based on Ca(2+)-influx. The aim of the present study was to examine the presence of L- and N-type Ca(2+)-channels at sensory nerve fibres in the mouse knee joint capsule. Specific fluorescent labelled channel blockers and antisera against these channel subtypes were combined with an immunohistochemical staining for calcitonin gene-related peptide (CGRP), a neuropeptide that is widely distributed in primary afferents. There was a nearly complete colocalisation of CGRP immunoreactivity and the binding of omega-conotoxin GVIA (toxin VIA of Conus geographus or BODIPY-verapamil (BODIPY(R) FL verapamil, hydrochloride) demonstrating the presence of N-type and L-type Ca(2+)-channels, respectively. These data were further confirmed by identical results obtained after an immunohistochemical demonstration of the two channel subtypes at the peptidergic nerve fibres.

We examined protein kinase C gamma-immunoreactivity (PKCgamma-IR) in the substantia gelatinosa (SG) of the rat medullary dorsal horn (MDH). The density of PKCgamma-IR in the MDH was most intense in the SG. The number of neurons with PKCgamma-IR were also much larger in the SG than in the other layers of the MDH. Double-immunohistochemical studies indicated light and electron microscopically that substance P-containing fibers and I-B4 (isolectin from Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCgamma-IR, indicating that SG neurons with PKCgamma might receive nociceptive primary afferent fibers. The results support the notion that PKCgamma in the MDH may contribute to the regulation of the nociception.