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The three peripheral sensory neuron (SN) subtypes, nociceptors, mechanoreceptors, and proprioceptors, localize to dorsal root ganglia and convey sensations such as pain, temperature, pressure, and limb movement/position. Despite previous reports, to date no protocol is available allowing the generation of all three SN subtypes at high efficiency and purity from human pluripotent stem cells (hPSCs). We describe a chemically defined differentiation protocol that generates all three SN subtypes from the same starting population, as well as methods to enrich for each individual subtype. The protocol yields high efficiency and purity cultures that are electrically active and respond to specific stimuli. We describe their molecular character and maturity stage and provide evidence for their use as an axotomy model; we show disease phenotypes in hPSCs derived from patients with familial dysautonomia. Our protocol will allow the modeling of human disorders affecting SNs, the search for treatments, and the study of human development., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the distribution of nociceptive nerve fibers in the cervical intervertebral discs of patients with chronic neck pain and determine whether these nociceptive nerve fibers are related to discogenic neck pain., Methods: We collected 43 samples of cervical intervertebral discs from 34 patients with severe chronic neck pain (visual analog scale [VAS] ≥ 70 mm), 42 samples from 36 patients who suffered cervical spondylotic radiculopathy or myelopathy without neck pain or with mild neck pain (VAS ≤ 30 mm) and 32 samples from eight donators to investigate their innervation immunohistochemically using an antibody against neuropeptide substance P., Results: The immunohistochemical investigation revealed that substance P-positive nerve fibers were obviously increased in number and deeply ingrown into the inner anulus fibrosus and even into the nucleus pulposus in the degenerative cervical discs of patients with severe neck pain in comparison with the discs of patients with cervical spondylotic radiculopathy or myelopathy and normal control discs (P<0.01)., Conclusions: The current study may indicate a key role of nociceptive nerve fibers in the pathogenesis of neck pain of cervical disc origin., (© 2019 American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In athletic horses, diseases leading to lameness are of great importance due to the loss of performance and the resultant economic concerns. Although stifle lesions are frequent in the hindlimb, due to the large size and complexity of the joint, and although meniscal tears have been identified as the most common soft tissue injuries in this joint, little is known about the mechanism that causes the painful sensation and thus the lameness. The aim of our study was to highlight any peripheral fibres involved in meniscal nociception in five macroscopically sound cranial horns of the equine medial meniscus, which has been one of the most common sites reported for equine meniscal injuries. Immunohistochemical stainings were performed using antibodies against Substance P in order to identify nociceptive fibres; against tyrosine hydroxylase for detecting postganglionic sympathetic fibres; and against glial fibrillary acidic proteins in order to identify Schwann cells. Our work highlights for the first time the presence of nociceptive and sympathetic fibres in equine menisci. They were found in the abaxial part of the cranial horn of the equine medial meniscus. This study suggests that when the abaxial part is injured, the meniscus itself could be the source of pain. These findings could provide a better understanding of the clinical presentation of horses with meniscal injury and contribute towards improving therapeutic strategies to alleviate pain in cases of equine meniscal injury., (© 2016 Anatomical Society.)

Aims: To test the hypothesis that prolonged jaw opening, as can occur during routine dental procedures, increases nociceptive sensitivity of the masseter muscle and increases cytokine expression., Methods: Sprague-Dawley rats were used to investigate behavioral and cellular changes in response to prolonged jaw opening. A surgical retractor was placed around the maxillary and mandibular incisors, and the jaw was held at near maximal opening for 20 minutes. Head-withdrawal responses to mechanical stimuli applied to the facial skin overlying the left and right masseter muscles were determined following jaw opening. Cytokine levels in the upper cervical spinal cord containing the caudal part of the spinal trigeminal nucleus were evaluated using protein antibody microarrays (n = 3). Statistical analysis was performed using a nonparametric Mann-Whitney U test., Results: Prolonged jaw opening significantly increased nocifensive head withdrawal to mechanical stimuli at 2 hours, and days 3 and 7 postinduction (P < .05). The increase in nociceptive response resolved after 14 days. Sustained jaw opening also stimulated differential cytokine expression in the trigeminal ganglion and upper cervical spinal cord that persisted 14 days postprocedure (P < .05)., Conclusion: These findings provide evidence that near maximal jaw opening can lead to activation and prolonged sensitization of trigeminal neurons that results in nociceptive behavior evoked by stimulation of the masseter muscle, a physiologic event often associated with temporomandibular disorders (TMD). Results from this study may provide a plausible explanation for why some patients develop TMD after routine dental procedures that involve prolonged jaw opening.

A subpopulation of nociceptors, the glial cell line-derived neurotrophic factor (GDNF)-dependent, non-peptidergic C-fibers, expresses a cell-surface glycoconjugate that can be selectively labeled with isolectin B4 (IB4 ), a homotetrameric plant lectin from Griffonia simplicifolia. We show that versican is an IB4 -binding molecule in rat dorsal root ganglion neurons. Using reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and immunofluorescence experiments on rat lumbar dorsal root ganglion, we provide the first demonstration that versican is produced by neurons. In addition, by probing Western blots with splice variant-specific antibodies we show that the IB4 -binding versican contains only the glycosaminoglycan alpha domain. Our data support V2 as the versican isoform that renders this subpopulation of nociceptors IB4 -positive (+). A subset of nociceptors, the GDNF-dependent non-peptidergic C-fibers can be characterized by its reactivity for isolectin B4 (IB4), a plant lectin from Griffonia simplicifolia. We have previously demonstrated that versican V2 binds IB4 in a Ca2 + -dependent manner. However, given that versican is thought to be the product of glial cells, it was questionable whether versican V2 can be accountable for the IB4-reactivity of this subset of nociceptors. The results presented here prove - for the first time - a neuronal origin of versican and suggest that versican V2 is the molecule that renders GDNF-dependent non-peptidergic C-fibers IB4-positive., (© 2015 International Society for Neurochemistry.)

Backgrounds: Non-traumatic hip dislocation in children is most often observed in the course of developmental dysplasia of the hip (DDH) and infantile cerebral palsy. The risk of pain sensations from dislocated hip joint differentiates the discussed groups of patients. Will every painless hip joint in children with cerebral palsy painful in the future?, Methods: Material included 34 samples of joint capsule and 34 femoral head ligaments, collected during open hip joint reduction from 19 children with CP, GMFCS level V and from 15 children with DDH and unilateral hip dislocation. All the children were surgically treated.The density of nociceptive fibres was compared between the children with CP and DDH, using S-100 and substance P monoclonal antibodies., Results: More frequent positive immunohistochemical reaction to S-100 protein concerned structures of the femoral head ligaments in children with CP and cartilage losses on the femoral head, when compared to the same structures in children with DDH (p = 0.010). More frequent were found positive immunohistochemical reactions for S-100 protein in the joint capsules of children with cartilage losses (p = 0.031) and pain ailments vs. the children with DDH (p = 0.027). More frequent positive reaction to substance P concerned in femoral head ligaments in CP children and cartilage lesions (p = 0.002) or with pain ailments (p = 0.001) vs. the DDH children., Conclusions: Surgical treatment of hip joint dislocation should be regarded as a prophylactics of pain sensations, induced by tissue sensitisation, inflammatory process development or articular cartilage defects.

The discovery of new drug targets represents a real opportunity for developing fresh strategies against pain. Ion channels are interesting targets because they are directly involved in the detection and the transmission of noxious stimuli by sensory fibres of the peripheral nervous system and by neurons of the spinal cord. Acid-Sensing Ion Channels (ASICs) have emerged as important players in the pain pathway. They are neuronal, voltage-independent depolarizing sodium channels activated by extracellular protons. The ASIC family comprises several subunits that need to associate into homo- or hetero-trimers to form a functional channel. The ASIC1 and ASIC3 isoforms are particularly important in sensory neurons, whereas ASIC1a, alone or in association with ASIC2, is essential in the central nervous system. The potent analgesic effects associated with their inhibition in animals (which can be comparable to those of morphine) and data suggesting a role in human pain illustrate the therapeutic potential of these channels., (© Société de Biologie, 2014.)

Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia.

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.)

The word "nociception" is derived from the Latin "nocere," which means "to harm." Nociception refers to the sensory perception of noxious stimuli that have the potential to cause tissue damage. Since the perception of such potentially harmful stimuli often results in behavioral escape responses, nociception provides a protective mechanism that allows an organism to avoid incipient (or further) damage to the tissue. It appears to be universal in metazoans as a variety of escape responses can be observed in both mammalian and non-mammalian vertebrates, as well as diverse invertebrates such as leeches, nematodes, and fruit flies (Sneddon [2004] Brain Research Review 46:123-130; Tobin and Bargmann [2004] Journal of Neurobiology 61:161-174; Smith and Lewin [2009] Journal of Comparative Physiology 195:1089-1106). Several types of stimuli can trigger nociceptive sensory transduction, including noxious heat, noxious chemicals, and harsh mechanical stimulation. Such high-threshold stimuli induce the firing of action potentials in peripheral nociceptors, the sensory neurons specialized for their detection (Basbaum et al. [2009] Cell 139:267-284). In vertebrates, these action potentials can either be relayed directly to a spinal motor neuron to provoke escape behavior (the so-called monosynaptic reflex) or can travel via spinal cord interneurons to higher-order processing centers in the brain. This review will cover the establishment of Drosophila as a system to study various aspects of nociceptive sensory perception. We will cover development of the neurons responsible for detecting noxious stimuli in larvae, the assays used to assess the function(s) of these neurons, and the genes that have been found to be required for both thermal and mechanical nociception. Along the way, we will highlight some of the genetic tools that make the fly such a powerful system for studies of nociception. Finally, we will cover recent studies that introduce new assays employing adult Drosophila to study both chemical and thermal nociception and provide an overview of important unanswered questions in the field., (Copyright © 2011 Wiley Periodicals, Inc.)

There is increasing evidence for estrogenic modulation of neurotransmission within the trigeminal pain pathway. It is also likely that the effects of estrogens may be influenced by the presence and localization of estrogen receptors (ERs) in a given brain area. To date, human data on the localization of ERs in the spinal trigeminal nucleus (STN), a key brain region in craniofacial nociception, are lacking. To ascertain whether ERs are expressed in the human STN, we performed immunohistochemical analysis on medulla oblongata samples taken from eight adult subjects (three men and five women; age range, 23-71 years) who had died from causes unrelated to neurologic or endocrine diseases. Paraffin-embedded sections at the level of the subnucleus caudalis and interpolaris were incubated with anti-estrogen receptor alpha (ERα) and anti-estrogen receptor beta (ERβ) antibodies. ERα immunoreactivity was detected in the nucleus and cytoplasm of neuronal and glial cells in the STN and in the nerve fibers within the spinal trigeminal tract in all eight subjects; ERβ immunoreactivity was observed in the cytoplasm of neuronal cells in five subjects. This study is the first to provide evidence in humans that ER immunoreactivity is detectable on neuronal and glial cells of the STN. The two ER subtypes exhibited different expression patterns, with higher expression levels of ERα than ERβ. The presence of ER-containing cells in the STN suggests that estrogens may directly affect trigeminal neuron excitability in humans., (Copyright © 2011 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.)

The cannabinoid receptor CB1 is involved in modulation of neuronal hypersensitivity and pain. The aim of this study was to evaluate CB1 receptor levels for the first time in dental pain. A total of 19 patients due for molar extraction were divided into two groups, those with existing dental pain (n=9), and those with no history of pain (n=10). Immunohistochemistry and computer image analysis was used to evaluate CB1-positive nerve fibres in tooth pulp, with neurofilament-immunostaining as a structural nerve marker. CB1-immunoreactive nerve fibres were scattered throughout the tooth pulp and often seen in nerve bundles, but the fibres did not penetrate the subodontoblastic layer. There was no statistically significant change in the CB1 nerve fibre percentage area in the painful group compared to the non-painful group (p=0.146); the neurofilament fibres were significantly reduced in the painful group compared to the controls (p=0.028), but there was no difference in the ratio of CB1 to neurofilaments between the two groups. Thus, CB1 expression is maintained by nerve fibres in painful human dental pulp, and peripherally-restricted CB1 agonists currently in development may advance the treatment of dental pain., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We have previously described a rat model for the contribution of neuroplastic changes in nociceptors to the transition from acute to chronic pain. In this model a prior injury activates protein kinase C epsilon (PKCepsilon), inducing a chronic state characterized by marked prolongation of the hyperalgesia induced by inflammatory cytokines, prototypically prostaglandin E(2) (PGE(2)), referred to as hyperalgesic priming. In this study we evaluated the population of nociceptors involved in priming, by lesioning isolectin B4-positive (IB4(+)) nociceptors with intrathecal administration of a selective neurotoxin, IB4-saporin. To confirm that the remaining, TrkA(+)/IB4(-), nociceptors are still functional, we evaluated if nerve growth factor (NGF) induced hyperalgesia. While pretreatment with IB4-saporin eliminated the acute mechanical hyperalgesia induced by glia-derived neurotrophic factor (GDNF), NGF and PsiepsilonRACK, a highly selective activator of PKCepsilon, induced robust hyperalgesia. After injection of NGF, GDNF or PsiepsilonRACK, at a time at which hyperalgesia induced by PGE(2) is markedly prolonged (hyperalgesic priming) in control rats, in IB4-saporin-pretreated rats PGE(2) failed to produce this prolonged hyperalgesia. Thus, while PKCepsilon is present in most dorsal root ganglion neurons, where it can contribute to acute mechanical hyperalgesia, priming is restricted to IB4(+)-nociceptors, including those that are TrkA(+). While PKCepsilon activation can induce acute hyperalgesia in the IB4(+) population, it fails to induce priming. We suggest that hyperalgesic priming occurs only in IB4(+) nociceptors, and that in the peripheral terminals of nociceptors separate intracellular pools of PKCepsilon mediate nociceptor sensitization and the induction of hyperalgesic priming., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Pain arises from activation of peripheral nociceptors, and strong noxious stimuli may cause an increase in spinal excitability called central sensitization, which is likely involved in many pathological pain states. So far, it has not been achieved to simultaneously visualize in vivo both the temporal and spatial aspects of spinal activity, including central sensitization. Using autofluorescent flavoprotein imaging (AFI), an optical technique suitable for mapping activity in nervous tissue, we demonstrate a close temporal and spatial correlation of electrically evoked nociceptive input with the spinal AFI signal, representing spinal neuronal activity. The AFI signal increases linearly with stimulation intensity. Furthermore, we found that the AFI signal was much larger in intensity and size when the same electrical stimulation was applied after the induction of central sensitization by a subcutaneous capsaicin injection. Finally, innocuous palpation of the hindpaw did not evoke an AFI response in naive animals, but after capsaicin injection a strong response was obtained. This is the first report demonstrating simultaneously the temporal and spatial propagation of spinal nociceptive activity in vivo.

Study Design: Immunohistochemical study on fresh cadaver specimens., Objective: Assessment of mechanoreceptor and nociceptor levels and distribution in iliolumbar ligament., Summary and Background Data: The function of iliolumbar ligament and its role in low back pain has not been yet fully clarified. Understanding the innervation of this ligament should provide a ground which enables formation of stronger hypotheses., Methods: Bilateral 30 iliolumbar ligaments of 15 fresh cadavers were included in the study. Morphologic properties were recorded and the ligaments were examined by focusing on 3 main parts: ligament, bone insertions, and tendon body. Assessment of mechanoreceptor and nociceptor levels and their distribution in iliolumbar ligament were performed on the basis of immunohistochemistry using the S-100 antibody specific for nerve tissue., Results: Iliac wing insertion was found to be the richest region of the ligament in terms of mechanoreceptors and nociceptors. Pacinian (type II) mechanoreceptor was determined to be the most common (66.67%) receptor followed by Ruffini (type I) (19.67%) mechanoreceptor, whereas free nerve endings (type IV) and Golgi tendon organs (type III) were found to be less common, 10.83% and 2.83%, respectively., Conclusion: Immunohistochemical staining has shown that iliolumbar ligament had a rich nerve tissue. Those results indicate that ILL plays an important role in proprioceptive coordination of lumbosacral region alongside its known biomechanic support function. Moreover, the presence of type IV nerve endings suggest that the injury of this ligament might contribute to the low back pain.

The primary afferent nociceptive neuron has recently attracted major research interest because of the cloning of very selectively expressed and well-conserved ion channel genes. All parts of the neuron, sensory terminals, axon and cell body, are accessible to validated research techniques in vitro using various isolated tissues or cells taken from laboratory animals. Single-unit recording and measuring stimulated calcitonin gene-related peptide (CGRP) release as well as patch-clamping and calcium imaging of cultured sensory neurons provide different kinds of information, and no model alone answers all questions. In combination, however, consistent results and complementary evidence form a solid basis for translational research to follow.

Sodium channel Na(v)1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Na(v)1.7 produce painful disorders, whereas loss-of-function Na(v)1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Na(v)1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Na(v)1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Na(v)1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Na(v)1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Na(v)1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Na(v)1.7.

In this study, we investigated postnatal changes in Rexed's laminae and distribution of nociceptive afferents in the dorsal horn of the rat lumbar spinal cord at postnatal days 0, 5, 10, 15, 20, and 60. Transverse sections of the L4-L5 segments were processed for triple labeling with isolectin B4 (IB4)-binding as a marker of nonpeptidergic C-fibers, calcitonin gene-related peptide (CGRP) immunoreactivity to label peptidergic nociceptive afferents, and a fluorescent Nissl stain to visualize cells and lamination at different stages of postnatal development. The Nissl staining revealed that the thickness of lamina I (LI) and outer lamina II remained mostly unchanged from birth until adulthood. CGRP afferents terminated mostly in LI and the outer two-thirds of lamina II, whereas the termination area of fibers binding IB4 was centered on the middle one-third of lamina II at all ages studied. In absolute values, the overall width of the bands of intense CGRP and IB4 labeling increased with age but decreased as a percentage of the overall thickness of the dorsal horn with maturation. The overlap of CGRP termination area with that of IB4 afferents increased with age. The consequences of these findings are twofold. First, the size of the different laminae does not grow evenly across the dorsal horn. Second, CGRP and IB4 labeling cannot be considered per se to be reliable markers of lamination during development. These findings have implications for comparing data obtained in immature and mature tissues with respect to localization of structures in the dorsal horn.

Protease-activated receptor (PAR(2)) is expressed by nociceptive neurons and activated during inflammation by proteases from mast cells, the intestinal lumen, and the circulation. Agonists of PAR(2) cause hyperexcitability of intestinal sensory neurons and hyperalgesia to distensive stimuli by unknown mechanisms. We evaluated the role of the transient receptor potential vanilloid 4 (TRPV4) in PAR(2)-induced mechanical hyperalgesia of the mouse colon. Colonic sensory neurons, identified by retrograde tracing, expressed immunoreactive TRPV4, PAR(2), and calcitonin gene-related peptide and are thus implicated in nociception. To assess nociception, visceromotor responses (VMR) to colorectal distension (CRD) were measured by electromyography of abdominal muscles. In TRPV4(+/+) mice, intraluminal PAR(2) activating peptide (PAR(2)-AP) exacerbated VMR to graded CRD from 6-24 h, indicative of mechanical hyperalgesia. PAR(2)-induced hyperalgesia was not observed in TRPV4(-/-) mice. PAR(2)-AP evoked discharge of action potentials from colonic afferent neurons in TRPV4(+/+) mice, but not from TRPV4(-/-) mice. The TRPV4 agonists 5',6'-epoxyeicosatrienoic acid and 4alpha-phorbol 12,13-didecanoate stimulated discharge of action potentials in colonic afferent fibers and enhanced current responses recorded from retrogradely labeled colonic dorsal root ganglia neurons, confirming expression of functional TRPV4. PAR(2)-AP enhanced these responses, indicating sensitization of TRPV4. Thus TRPV4 is expressed by primary spinal afferent neurons innervating the colon. Activation of PAR(2) increases currents in these neurons, evokes discharge of action potentials from colonic afferent fibers, and induces mechanical hyperalgesia. These responses require the presence of functional TRPV4. Therefore, TRPV4 is required for PAR(2)-induced mechanical hyperalgesia and excitation of colonic afferent neurons.

An intensely painful stimulus may lead to hyperalgesia, the enhanced sensation of subsequent painful stimuli. This is commonly believed to involve facilitated transmission of sensory signals in the spinal cord, possibly by a long-term potentiation-like mechanism. However, plasticity of identified synapses in intact hyperalgesic animals has not been reported. Here, we show, using neuronal tracing and postembedding immunogold labeling, that after acute noxious stimulation (hindpaw capsaicin injections), immunolabeling of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and of CaMKII phosphorylated at Thr(286/287) (pCaMKII) are upregulated postsynaptically at synapses established by peptidergic primary afferent fibers in the superficial dorsal horn of intact rats. In contrast, postsynaptic pCaMKII immunoreactivity was instead downregulated at synapses of nonpeptidergic primary afferent C-fibers; this loss of pCaMKII immunolabel occurred selectively at distances greater than approximately 20 nm from the postsynaptic membrane and was accompanied by a smaller reduction in total CaMKII contents of these synapses. Both pCaMKII and CaMKII immunogold labeling were unaffected at synapses formed by presumed low-threshold mechanosensitive afferent fibers. Thus, distinct molecular modifications, likely indicative of plasticity of synaptic strength, are induced at different populations of presumed nociceptive primary afferent synapse by intense noxious stimulation, suggesting a complex modulation of parallel nociceptive pathways in inflammatory hyperalgesia. Furthermore, the activity-induced loss of certain postsynaptic pools of autophosphorylated CaMKII at previously unmanipulated synapses supports a role for the kinase in basal postsynaptic function.

We contrasted the physiology and peripheral targets of subclassified nociceptive and nonnociceptive afferents that express acid-sensing ion channel (ASIC)-like currents. The threshold for current activation was similar in eight distinct cell subclasses regardless of functional modality (pH 6.8). When potency was determined from concentration-response curves, nonnociceptors exhibited currents with significantly greater potency than that of all but one class of nociceptors (pH50 = 6.54 and 6.75 vs. 6.20-6.34). In nonnociceptive cells, acid transduction was also confined to a very narrow range (0.1-0.3 vs. 0.8-1.4 pH units for nociceptors). Simultaneous whole cell recording and ratiometric imaging of three peptidergic nociceptive classes were consistent with the expression of Ca2+ -permeable ASICs. Sensitivity to psalmotoxin and flurbiprofen indicated the presence of Ca2+ -permeable ASIC1a. Immunocytochemistry on these subclassified populations revealed a differential distribution of five ASIC proteins consistent with Ca2+ permeability and differential kinetics of proton-gated currents (type 5: ASIC1a, 1b, 2a, 2b, 3; type 8a: ASIC1a, 1b, 3; type 8b: ASIC1a, 1b, 2a, 2b, 3). Using DiI tracing, we found that nociceptive classes had discrete peripheral targets. ASIC-expressing types 8a and 9 projected to hairy skin, but only types 8a and 13 projected to glabrous skin. Non-ASIC-expressing types 2 and 4 were present only in hairy skin. We conclude that ASIC-expressing nociceptors differ from ASIC-expressing nonnociceptors mainly by range of proton reactivity. ASIC- as well as non-ASIC-expressing nociceptors have highly distinct cutaneous targets, and only one class was consistent with the existence of a generic C polymodal nociceptor (type 8a).

In this study we examined the superior colliculus of the midbrain of the one-humped (dromedary) camel, Camelus dromedarius, using Nissl staining and anti-neuronal-specific nuclear protein (NeuN) immunohistochemistry for total neuronal population as well as for the enkephalins, somatostatin (SOM) and substance P (SP). It was found that, unlike in most mammals, the superior colliculus is much larger than the inferior colliculus. The superior colliculus is concerned with visual reflexes and the co-ordination of head, neck and eye movements, which are certainly of importance to this animal with large eyes, head and neck, and apparently good vision. The basic neuronal architecture and lamination of the superior colliculus are similar to that in other mammals. However, we describe for the first time an unusually large content of neurons in the superior colliculus with strong immunoreactivity for met-enkephalin, an endogenous opioid. We classified the majority of these neurons as small (perimeters of 40-50 microm), and localized diffusely throughout the superficial grey and stratum opticum. In addition, large pyramidal-like neurons with perimeters of 100 microm and above were present in the intermediate grey layer. Large unipolar cells were located immediately dorsal to the deep grey layer. By contrast, small neurons (perimeters of 40-50 microm) immunopositive to SOM and SP were located exclusively in the superficial grey layer. We propose that this system may be associated with a pain-inhibiting pathway that has been described from the periaqueductal grey matter, juxtaposing the deep layers of the superior colliculus, to the lower brainstem and spinal cord. Such pain inhibition could be important in relation to the camel's life in the harsh environment of its native deserts, often living in very high temperatures with no shade and a diet consisting largely of thorny branches.

Previous reports have shown the expression of several mechanosensitive ionic channels on the plasma membrane in odontoblasts, which are the cells responsible for dentin formation. The membrane characteristics of odontoblasts imply that they could play critical roles in the mechano-transduction of fluid displacement within dentinal tubules into the electrical cell signals, to carry dentin sensation to the central nervous system. However, the direct ionic mechanism underlying such a dentin nociceptive function remains unclear. In the present study, we investigated the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1) channel--which essentially contributes to the detection of pain sensation--in rat odontoblasts by immunohistochemical and nystatin perforated patch-clamp techniques. Immunohistochemical observation showed the localization of TRPV1-immunoreactions on the distal regions of odontoblast membranes. In the patch-clamp experiments, we observed capsaicin-induced inward currents that were inhibited by capsazepine, a TRPV1 channel antagonist. Our results indicate a significant expression of TRPV1 channels in odontoblasts, suggesting that odontoblasts may directly respond to noxious stimuli such as a thermal-heat stimulus, and point to the necessity for a reconsideration of the cellular mechanisms of dentin sensation based on the transmembrane ionic signals in odontoblasts.

Vanilloid receptor 1 (TRPV1) has been proposed to be the principal heat-responsive channel for nociceptive neurons. The skin of both rat and mouse receives major projections from primary sensory afferents that bind the plant lectin isolectin B4 (IB4). The majority of IB4-positive neurons are known to be heat-responsive nociceptors. Previous studies suggested that, unlike rat, mouse IB4-positive cutaneous afferents did not express TRPV1 immunoreactivity. Here, multiple antisera were used to confirm that mouse and rat have different distributions of TRPV1 and that TRPV1 immunoreactivity is absent in heat-sensitive nociceptors. Intracellular recording in TRPV1(-/-) mice was then used to confirm that TRPV1 was not required for detecting noxious heat. TRPV1(-/-) mice had more heat-sensitive neurons, and these neurons had normal temperature thresholds and response properties. Moreover, in TRPV1(-/-) mice, 82% of heat-responsive neurons did not express immunoreactivity for TRPV2, another putative noxious heat channel.

A variety of seemingly diverse pain syndromes are characterized by movement-induced pain radiating in the distribution of a peripheral nerve or nerve root. This could be explained by the induction of ectopic mechanical sensitivity in intact sensory axons. Here we show that inflammation led to mechanical sensitivity of the axons of a subset of mechanically sensitive primary sensory neurons. Dorsal root recordings were made from 194 mechanically sensitive neurons that innervated deep and cutaneous structures and had C, Adelta, and Aalphabeta conduction velocities. No axons of any category were mechanically sensitive in control experiments. However, the axons of neurons innervating deep structures and having C- or Adelta-conduction velocities became mechanically sensitive during the neuritis, and also exhibited an increased incidence of spontaneous discharge. The incidence of mechanical sensitivity followed a distinct time course. In some cases, paw withdrawal thresholds were obtained after neuritis induction. The time course of the resultant hypersensitivity was not directly related to the time course of the axonal mechanical sensitivity. Ectopic axonal mechanical sensitivity could explain some types of radiating, nerve-related pain coexisting with diseases of seemingly diverse etiologies.

Vasoneuroactive substances were applied through intradermal microdialysis membranes and characterized as itch- or pain-inducing in psychophysical experiments. Histamine always provoked itching and rarely pain, capsaicin always pain but never itching. Prostaglandin E(2) (PGE(2)) led preferentially to moderate itching. Serotonin, acetylcholine, and bradykinin induced pain more often than itching. Subsequently the same substances were used in microneurography experiments to characterize the sensitivity profile of human cutaneous C-nociceptors. The responses of 89 mechanoresponsive (CMH, polymodal nociceptors), 52 mechanoinsensitive, histamine-negative (CMi(His-)), and 24 mechanoinsensitive, histamine-positive (CMi(His+)) units were compared. CMi(His+) units were most responsive to histamine and to PGE(2) and less to serotonin, ACh, bradykinin, and capsaicin. CMH units (polymodal nociceptors) and CMi(His-) units showed significantly weaker responses to histamine, PGE(2), and acetylcholine. Capsaicin and bradykinin responses were not significantly different in the two classes of mechano-insensitive units. We conclude that CMi(His+) units are "selective," but not "specific" for pruritogenic substances and that the pruritic potency of a mediator increases with its ability to activate CMi(His+) units but decreases with activation of CMH and CMi(His-) units.

A remarkable feature of opioids is that they inhibit pain that persists from previous injuries without eliminating either the initial pain of a new injury or the protective reflexes triggered by it. Here we ask whether selective expression of the mu-opioid receptor (MOR) gene in primary nociceptors (pain-sensing neurons) might contribute to this aspect of opioid specificity. We quantified single-cell levels of MOR mRNA and measured opioid inhibition of Ca channels on identified nociceptors and low-threshold mechanosensors (non-nociceptors) isolated from rats. Negligibly few non-nociceptors express MOR mRNA, thereby rendering nonpain sensations insensitive to opioids. Nearly half of nociceptors of all size classes also fail to express MOR mRNA or to respond to opioids. Among the opioid-responsive nociceptors, a gene dose-response relationship exists such that maximal opioid inhibition occurs when the MOR mRNA concentration of a cell is >15 pm. Almost all large, myelinated nociceptors express MOR mRNA below this level, whereas small, unmyelinated nociceptors are likely to express above it. Because myelinated nociceptors mediate anti-nociceptive reflexes, the data suggest that fine control of the MOR mRNA level contributes to a complex neural trait: the ability of opioids to suppress persistent pain without preventing response to a new injury.

The co-expression of osteocalcin (OC) with the capsaicin receptor (VR1) and vanilloid receptor 1-like receptor (VRL-1) was examined in the dorsal root (DRG) and trigeminal ganglia (TG). Virtually all OC-immunoreactive (ir) DRG neurons were devoid of VR1- and VRL-1-immunoreactivity (ir). In the TG, 14.1% of OC-ir neurons were also immunoreactive for VR1. Only 1.7% of OC-ir TG neurons co-expressed VRL-1-ir. The distribution of OC-ir was also examined in the spinal cord and trigeminal sensory nuclei. In the spinal cord, the superficial laminae of the dorsal horn were devoid of OC-ir. The neuropil was weakly stained in other regions of the spinal horns. The medullary dorsal horn (MDH) contained numerous OC-ir varicose fibers in laminae I and II. These fibers were occasionally observed originating from the spinal trigeminal tract. The neuropil was weakly stained in deeper laminae of the MDH, and the rostral parts of the trigeminal sensory nuclei. The present study suggests that OC-ir TG nociceptors send their unmyelinated axons to the superficial laminae of the MDH., (Copyright 2002 Elsevier Science B.V.)

In vertebrates, peripheral chemosensory neurons express large families of G protein-coupled receptors (GPCRs), reflecting the diversity and specificity of stimuli they detect. However, somatosensory neurons, which respond to chemical, thermal, or mechanical stimuli, are more broadly tuned. Here we describe a family of approximately 50 GPCRs related to Mas1, called mrgs, a subset of which is expressed in specific subpopulations of sensory neurons that detect painful stimuli. The expression patterns of mrgs thus reveal an unexpected degree of molecular diversity among nociceptive neurons. Some of these receptors can be specifically activated in heterologous cells by RFamide neuropeptides such as NPFF and NPAF, which are analgesic in vivo. Thus, mrgs may regulate nociceptor function and/or development, including the sensation or modulation of pain.

Glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) are neuroprotective for subpopulations of sensory neurons and thus are candidates for pain treatment. However, delivering these factors to damaged neurons will invariably result in undamaged systems also being treated, with possible consequences for sensory processing. In sensory neurons the purinergic receptor P2X(3) is found predominantly in GDNF-sensitive nociceptors. ATP signalling via the P2X(3) receptor may contribute to pathological pain, suggesting an important role for this receptor in regulating nociceptive function. We therefore investigated the effects of intrathecal GDNF or NGF on P2X(3) expression in adult rat spinal cord and dorsal root ganglia (DRG). In control spinal cords, P2X(3) expression was restricted to a narrow band of primary afferent terminals within inner lamina II (II(i)). Glial cell line-derived neurotrophic factor treatment increased P2X(3) immunoreactivity within lamina II(i) but not elsewhere in the cord. Nerve growth factor treatment, however, induced novel P2X(3) expression, with intense immunoreactivity in axons projecting to lamina I and outer lamina II and to the ventro-medial afferent bundle beneath the central canal. In the normal DRG, we found a greater proportion of P2X(3)-positive neurons at cervical levels, many of which were large-diameter and calcitonin gene-related peptide-positive. In both cervical and lumbar DRG, the number of P2X(3)-positive cells increased following GDNF or NGF treatment. De novo expression of P2X(3) in NGF-sensitive nociceptors may contribute to chronic inflammatory pain.

Patients with peripheral neuropathy have symptoms involving small-diameter nociceptive nerves and elevated thermal thresholds. Nociceptive nerves terminate in the epidermis of the skin and are readily demonstrated with the neuronal marker, protein gene product 9.5 (PGP 9.5). To investigate the pathological characteristics of elevated thermal thresholds, we performed PGP 9.5 immunocytochemistry on 3 mm punch skin biopsies (the forearm and the leg) from 55 normal subjects and 35 neuropathic patients. Skin innervation was evaluated by quantifying epidermal nerve densities. Epidermal nerve densities were reduced in neuropathic patients compared to normal subjects. Epidermal nerve densities were variably correlated with thermal thresholds. The proportion of neuropathic patients with reduced epidermal nerve densities was larger than the proportion of neuropathic patients with elevated thermal thresholds. These results indicated that degeneration of epidermal nerve terminals preceded the elevation of thermal thresholds. Skin biopsy together with immunocytochemical demonstration of epidermal innervation offers a new approach to evaluate small-fiber sensory neuropathy.

Recent cloning efforts have identified families of ion channels that may be involved in signaling noxious proton accumulation in tissue. Some conduct potassium ions outward and are closed by excess protons, others are opened under this condition carrying cations inward and their putative function is in their name ('acid sensing'), and again another channel is truly polymodal, the capsaicin receptor, sensing acid and heat. Further heat-activated channels, not yet cloned, may not be gated by protons but sensitized so strongly that they open at the command of body temperature. In either case, the result may be pain from tissue acidosis.

We determined the co-expression of immunoreactivity (IR) for ATP-receptor subunits (P2X1, P2X2, and P2X3), neuropeptides, neurofilament (NF), and binding of the isolectin B(4) from Griffonia simplicifolia type one (GS-I-B(4)) in adult dorsal root ganglion neurons. P2X1-IR was expressed primarily in small DRG neurons. Most P2X1-IR neurons expressed neuropeptides and/or GS-I-B(4)-binding, but lacked NF-IR. P2X1-IR overlapped with P2X3-IR, though each was also found alone. P2X2-IR was expressed in many P2X3-IR small neurons, as well as a group of medium to large neurons that lacked either P2X3-IR or GS-I-B(4)-binding. A novel visible four-channel fluorescence technique revealed a unique population of P2X2/3-IR neurons that lacked GS-I-B(4)-binding but expressed NF-IR. Co-expression of P2X1, and P2X3 in individual neurons was also demonstrated. We examined P2X subunit-IR on individual recorded neurons that had been classified by current signature in vitro. Types 1, 2, 4 5, and 7 expressed distinct patterns of P2X-IR that corresponded to patterns identified in DRG sections, and had distinct responses to ATP. Types with rapid ATP currents (types 2, 5, and 7) displayed P2X3-IR and/or P2X1-IR. Types with slow ATP currents (types 1 and 4) displayed P2X2/3-IR. Type 1 neurons also displayed P2X1-IR. This study demonstrates that the correlation between physiological responses to ATP and the expression of particular P2X receptor subunits derived from expression systems is also present in native neurons, and also suggests that novel functional subunit combinations likely exist.

It has been reported that calcium-binding proteins are good markers for different sets of neurons in various brain regions. We examined expression of the main calcium-binding proteins in projection neurons in the rat medullary dorsal horn (MDH) by combining immunofluorescence histochemistry for calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) with the retrograde tract-tracing method. A fluorescence tracer, tetramethylrhodamine-dextran amine (TMR-DA), was injected into the parabrachial, thalamic or hypothalamic region. After such injections, a number of PV-, CR-, and/or CB-immunoreactive MDH neurons were labeled retrogradely with TMR-DA. Triple-immunofluorescence histochemistry further revealed that a number of CB-, CR-, or PV-immunoreactive TMR-DA-labeled MDH neurons showed immunoreactivity for substance P receptor (NK1), and that they expressed immunoreactivity for c-fos protein in the rats which were injected with formalin into the lips. Thus, it was indicated that some of CB-, CR-, or PV-containing projection neurons in the MDH might be involved in the transmission of nociceptive stimuli.

This study was performed to examine the anesthetic action of halothane on the spinal cord of rats by using fos immunoreactivity, a marker for neuronal activity following noxious stimulation. Sprague-Dawley rats were injected with 150 microliters of 5% formalin subcutaneously into the left hindpaw. Control group (n = 5) received 100% oxygen for 3 hours after injection. 1 MAC group (n = 5) and 1.5 MAC group (n = 5) of rats were anesthetized with 1 MAC or 1.5 MAC halothane, respectively, for 3 hours after injection. The number of fos immunoreactive cells was counted in the lumbar spinal cord of each rat. All rats showed escape reactions against noxious stimulation in the control group. In the 1 MAC and 1.5 MAC groups, two of five rats showed response to noxious stimulation and the another three showed no response, respectively. There was profound relation between the responding rats and the expression of fos immunoreactivity in the spinal dorsal horn. The number of fos immunoreactive cells decreased in the cord of rats that showed no response to noxious stimulation by halothane 1 or 1.5 MAC. These findings suggest that halothane has analgesic action on spinal nociceptive neurons in the rats on the condition that its reactions to noxious stimulation are suppressed by halothane of any concentration.

Evidence suggests that nerve growth factor (NGF) is an important mediator in inflammatory pain states: NGF levels increase in inflamed tissue, and neutralization of endogenous NGF prevents the hyperalgesia which normally develops during inflammation of the skin. Here we asked whether NGF contributes to sensitization of primary afferent nociceptors, which are an important component of pain and hyperalgesia in inflamed tissue. An in vitro skin nerve preparation of the rat was used to directly record the receptive properties of thin myelinated (Adelta) and unmyelinated (C) nociceptors innervating normal hairy skin, carrageenan-inflamed skin and carrageenan-inflamed skin where endogenous NGF had been neutralized by application of a trkA-IgG (tyrosine kinase Aimmunoglobulin G) fusion molecule. Following carrageenan inflammation, there was a marked increase in the proportion of nociceptors which displayed ongoing activity (50% of nociceptors developed spontaneous activity compared to 4% of nociceptors innervating normal uninflamed skin), and this was reflected in a significant increase in the average ongoing discharge activity. Spontaneously active fibres were sensitized to heat and displayed a more than twofold increase in their discharge to a standard noxious heat stimulus. Furthermore, the number of nociceptors responding to the algesic mediator bradykinin increased significantly from 28% to 58%. By contrast, the mechanical threshold of nociceptive afferents did not change during inflammation. When the NGF-neutralizing molecule trkA-IgG was coadministered with carrageenan at the onset of the inflammation, primary afferent nociceptors did not sensitize and displayed essentially normal response properties, although the inflammation as evidenced by tissue oedema developed normally. We therefore conclude that NGF is a crucial component for the sensitization of primary afferent nociceptors associated with tissue inflammation.

Most of the smaller diameter neurons of dorsal root and trigeminal ganglia in adult rats expressed latexin, which has the inhibitor activity of carboxypeptidase A. Most of the dorsal root ganglion (DRG) neurons containing either calcitonin gene-related peptide (CGRP), substance P (SP) or somatostatin (SST) coexpressed latexin. Latexin was widely distributed in the cytoplasm of the cell body and in axonal fibers of cultured DRG neurons which were sensitive to capsaicin. In addition, latexin-immunoreactivity was observed throughout lamina II of the spinal cord in normal animals, but was lost following sciatic nerve-axotomy, suggesting the presence of latexin-immunoreactive axonal fibers and/or terminals from DRG neurons. Immunoelectron microscopy indeed revealed latexin-immunoreactive axonal terminals and thinly myelinated and unmyelinated axonal fibers within the dorsal horn. These observations suggest that latexin may be involved in nociceptive information transmission or its modulation., (Copyright 1998 Elsevier Science B.V.)

Serotoninergic projections to the spinal dorsal horn are implicated in the modulation of nociceptive transmission. However, morphological evidence indicating that serotoninergic projection fibers make synapses on nociceptive neurons in the medullary dorsal horn is still meager. Thus, we examined whether axonal varicosities with serotonin (5-HT)-like immunoreactivity (5-HT-LI) might make synapses on nociceptive projection neurons in the caudal spinal trigeminal nucleus (Vc) of the rat. Projection neurons were retrogradely labeled with tetramethylrhodamine-dextran amine (TMR-DA) or wheat germ agglutinin-horseradish peroxidase (WGA-HRP) that was injected into the parabrachial or thalamic region. Vc neurons in which c-fos protein-like immunoreactivity (Fos-LI) was induced by subcutaneous injection of formalin into the lip were considered nociceptive. Vc neurons in direct contact with axonal varicosities that bind isolectin I-B4 were also considered nociceptive. Triple labeling for 5-HT, TMR-DA, and Fos as well as that for 5-HT, TMR-DA, and I-B4 were done by using the immunofluorescence and fluorescence histochemical techniques. Confocal laser-scanning microscopy revealed that axonal varicosities with 5-HT-LI were in close apposition to TMR-DA-labeled neurons showing Fos-LI in lamina I and the outer part of lamina II (lamina IIo), and that both axonal varicosities with 5-HT-LI and those binding I-B4 were in close apposition to single neuronal profiles labeled with TMR-DA. The presumed nociceptive neuronal profiles in close apposition to axon terminals with 5-HT-LI were mainly those of laminae I and II neurons as well as dendrites of lamina III neurons. Electron microscopy confirmed that axon terminals with 5-HT-LI and those with I-B4 binding activity in laminae I and II made synapses on somatic and dendritic profiles that were labeled with WGA-HRP. The results indicate that serotoninergic neurons project directly on nociceptive projection neurons in the Vc.

An endogenous ligand for the opioid receptor-like (ORL1) receptor has been characterized recently. This 17 amino acid peptide, nociceptin, has potent effects on pain processing in experimental animals. In the present study, we describe the development of a radio-immunoassay for nociceptin, suitable for quantification of immunoreactivity in the rat spinal cord. in the dorsal spinal cord we found 641 fmol nociceptin-like immunoreactivity (IR)/mg protein, while in the ventral spinal cord the amount was below the detection limit of the assay (11 fmol/mg protein). High pressure liquid chromatography (HPLC) showed that the immunoreactive material was heterogeneous with the major part eluting at the position of synthetic nociceptin. The presence of nociceptin in the dorsal spinal cord is compatible with its proposed involvement in modulating afferent transmission.

Objective: Studies have proved the importance of substance P (SP) in the development and perpetuation of inflammation in joints, and describe the abundant SP neural network in the soft tissue structures of various articulations. Less information is available on the innervation of the bony structures of joints, and there is a paucity of data describing the changes associated with disease conditions. Our objectives were to evaluate changes in the distribution of sensory nerve fibers in the bony structures of naturally osteoarthritic joints., Methods: Five osteoarthritic metacarpophalangeal (MCP) articulations were removed from 4 horses euthanized because of clinically severe and persistent lameness. The articulations were examined grossly and by high detail radiographs, and substance P containing nociceptive fibers were identified on thin sections by immunoreaction., Results: Gross examination and radiographs revealed evidence of osteoarthritis (OA), including thin, eroded, and fibrillated articular cartilage and, in the most severe cases, periarticular osteophytes and palmar metacarpal flattening with cystic cavitations. Histologically, there was a generalized loss of cartilage matrix basophilia, with chondrocyte clustering or death. SP nerve fibers were evident in the articular capsule and periosteum, and their appearance and frequency were similar to nonarthritic MCP articulations. Abnormal cartilage structures such as erosion channels and osteophytes on the dorsal proximal phalangeal perimeter contained short, tortuous, immunoreactive nerve fibers. Areas of chondrocyte cloning and abnormal fibrillated or eroded articular cartilage had increased generalized SP peptide staining, but no nerve fibers were identified. Additionally, hypercellular infiltrates in cystic cavitations in the subchondral bone stained intensely for SP, but true neurofilaments were absent., Conclusion: Combined, the findings of SP innervation in areas of articular remodeling such as erosion channels and osteophytes suggest that SP plays a role in the signaling and maintenance of pain associated with OA.

In five species of decapod crustaceans--Cherax destructor (crayfish), Carcinus maenas (crab), Homarus americanus (clawed lobster), Eriocheir sinensis (crab), Macrobrachium rosenbergii (shrimp)--immunocytochemical stainings revealed the presence of sensory afferents with FMRFamide-like immunoreactivity in the central nervous system. These afferents were extremely thin, very numerous, and innervated all sensory neuropils except the optic and olfactory lobes. In their target neuropils they gave rise to condensed net- or ball-like terminal structures. Only in Homarus americanus but not in any other studied species immunocytochemistry revealed a separate, non-overlapping class of sensory afferents with substance P-like immunoreactivity. Also the afferents with substance P-like immunoreactivity were very thin and numerous, innervated all sensory neuropils except optic and olfactory lobes, and gave rise to condensed terminal structures. From their morphological characteristics it can be concluded that likely both classes of afferents are chemosensory. The substance P-like immunoreactivity suggests a link with the nociceptor afferents of vertebrates, with which both classes of afferents share several other morphological features.

Study Design: The present study investigated the distribution of sensory and autonomic nerves to the posterior longitudinal ligament of the rat lumbar vertebra., Objective: The results were analyzed to provide a new insight into the pathogenesis of low back pain., Summary of Background Data: Previous studies that have descriptively reported on posterior longitudinal ligament innervation have attributed important roles to the sinuvertebral nerve as an output of nociceptive sensation. Meanwhile, development of immunohistochemistry has made it possible to identify numerous neurotransmitters, and thus various suggested pathophysiologic roles of the nerve fibers in the posterior longitudinal ligament. However, such microscopic identification of the nerve fibers has not been able to reveal comprehensive distributions of the various fibers, which are presumed to be derived from the sinuvertebral nerve., Methods: Rat posterior longitudinal ligaments of whole thickness was immunohistochemically stained for calcitonin gene-related peptide and tyrosine hydroxylase, which have been considered markers of nociceptive and noradrenergic fibers respectively. Light-microscopic and electron-microscopic observation was reinforced with another experimental study in which resection of the dorsal root ganglia induced characteristic denervation patterns to the rat posterior longitudinal ligament., Results: The present study revealed that the lumbar. PLL was dually innervated by two distinctive systems of nociceptive fibers. One of the systems was polysegmentally innervated and closely associated with autonomic innervation, whereas the other was unisegmentally innervated and not associated with autonomic fibers., Conclusion: Pathogenesis of low back pain can be better understood if the presence of the two distinctive nerve systems innervating the posterior longitudinal ligament is taken into account.

Locations and morphological characteristics of nociceptive specific (NS) and wide dynamic range (WDR) neurons of the ventrobasal (VB) thalamic complex were studied using an intracellular HRP injection technique in cats anesthetized with urethane and chloralose. Both NS and WDR neurons were found within the marginal zone of the VB complex. Their somata were confined to the VB complex, but dendrites were extended into the structures of the thalamus surrounding the VB complex. NS neurons were found in the caudal part of the VB complex, whereas WDR neurons were found more rostrally. Examinations of their locations within the marginal zone of the VB complex confirmed the somatotopic organization, as found previously. We also confirmed the previous reports that there are 3 different types of low-threshold mechanoreceptive (LTM) neurons within the VB complex. They are type I, type II and type III neurons. Both NS and WDR neurons were either type I or type II neurons. Obvious morphological differences were not found between LTM neurons and two classes of nociceptive VB neurons investigated.

There is now good evidence that amino acids act as neurotransmitters in primary afferent neurons of dorsal root ganglia. Glutamate is the primary candidate for such a role, and there are reasons to believe that release of glutamate may be accompanied by the release of other neuroactive substances. Using immunocytochemical techniques, we have tested the hypothesis that some dorsal root ganglion neurons contain elevated levels of aspartate as well as glutamate. Antisera raised against conjugates of aspartate or glutamate were used for this purpose. Blocking experiments confirmed that these antibodies were specific to their antigens in cryostat sections of dorsal root ganglia. Aspartate immunoreactivity was found in approximately 30% of neurons in cervical dorsal root ganglia. The relation between cell size and staining intensity for aspartate was examined using quantitative video microscopy; the great majority of cells immunopositive for aspartate were small (15-30 microns in diameter); about 85% of these cells stained for aspartate, although staining intensities varied over a wide range. By reacting consecutive sections with anti-aspartate and anti-glutamate it was shown that elevated levels of aspartate were found in the same cells which contained elevated levels of glutamate. By measuring the staining intensity of individual cells for both aspartate and glutamate, it was also shown that there was a positive correlation between staining intensity and, presumably, concentration of the two amino acids. The presence of high levels of aspartate in terminals located in the superficial laminae of the dorsal horn was verified by pre- and post-embedding immunocytochemistry with the electron microscope. Aspartate was demonstrated in scalloped terminals, including dark scalloped terminals believed to be associated with unmyelinated fibers of nociceptors. This evidence supports the hypothesis that aspartate as well as glutamate is present in the cell bodies and terminals of nociceptive primary afferents, and may be released by the terminals of these afferents to activate neurons in the superficial laminae of the dorsal horn.