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

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

Ingrowth of Nociceptive Receptors into Diseased Cervical Intervertebral Disc Is Associated with Discogenic Neck Pain.

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.

Nociceptive and sympathetic innervations in the abaxial part of the cranial horn of the equine medial meniscus: an immunohistochemical approach.

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.

Prolonged Jaw Opening Promotes Nociception and Enhanced Cytokine Expression.

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.

Neuronally produced versican V2 renders C-fiber nociceptors IB4 -positive.

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.

[Acid-Sensing Ion Channels (ASICs) in pain]. / Les canaux ioniques ASIC dans la douleur.

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.

Hip joint pain in children with cerebral palsy and developmental dysplasia of the hip: why are the differences so huge?

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.

Black mamba venom peptides target acid-sensing ion channels to abolish pain.

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.

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

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

Pokes, sunburn, and hot sauce: Drosophila as an emerging model for the biology of nociception.

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.

Estrogen receptors localization in the spinal trigeminal nucleus: an immunohistochemical study in humans.

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 (ERß) 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; ERß 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 ERß. The presence of ER-containing cells in the STN suggests that estrogens may directly affect trigeminal neuron excitability in humans.

Cannabinoid receptor CB1-immunoreactive nerve fibres in painful and non-painful human tooth pulp.

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.

Hyperalgesic priming is restricted to isolectin B4-positive nociceptors.

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.

Autofluorescent flavoprotein imaging of spinal nociceptive activity.

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.

Electrophysiological and neurochemical techniques to investigate sensory neurons in analgesia research.

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.

Immunohistochemical demonstration of nerve endings in iliolumbar ligament.

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.

A pore-blocking hydrophobic motif at the cytoplasmic aperture of the closed-state Nav1.7 channel is disrupted by the erythromelalgia-associated F1449V mutation.

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.

Postnatal changes in the Rexed lamination and markers of nociceptive afferents in the superficial dorsal horn of the rat.

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.

Transient receptor potential vanilloid 4 mediates protease activated receptor 2-induced sensitization of colonic afferent nerves and visceral hyperalgesia.

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.

Pathway-specific bidirectional regulation of Ca2+/calmodulin-dependent protein kinase II at spinal nociceptive synapses after acute noxious stimulation.

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.