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Dynamic regulation of G-protein-coupled receptor (GPCR) kinase 2 (GRK2) expression restores cellular function by protecting from overstimulation via GPCR and non-GPCR signaling. In the primary afferent neurons, GRK2 negatively regulates nociceptive tone. The present study tested the hypothesis that induction of GRK2 in the primary afferent neurons contributes to the resolution of acute pain after tissue injury. GRK2 expression in the dorsal root ganglion (DRG) was analyzed at 1 and 7 days after the incision. Intraperitoneal administration of a GRK2 inhibitor was performed 7 days post-incision in male Sprague-Dawley rats who underwent plantar incisions to analyze the pain-related behavioral effect of the GRK2 inhibitor. Separately, GRK2 expression was analyzed after injecting insulin-like growth factor 1 (IGF1) into the rat hind paw. In addition, an IGF1 receptor (IGF1R) inhibitor was administered in the plantar incision rats to determine its effect on the incision-induced hyperalgesia and GRK2 expression. Plantar incision induced an increase in GRK2 in the DRG at 7 days, but not at 1 day post-incision. Acute hyperalgesia after the plantar incision disappeared by 7 days post-incision. Intraperitoneal injection of the GRK2 inhibitor at this time reinstated mechanical hyperalgesia, although the GRK2 inhibitor did not produce hyperalgesia in naive rats. After the incision, IGF1 expression increased in the paw, but not in the DRG. Intraplantar injection of IGF1 increased GRK2 expression in the ipsilateral DRG. IGF1R inhibitor administration prevented both the induction of GRK2 and resolution of hyperalgesia after the plantar incision. These findings demonstrate that induction of GRK2 expression driven by tissue IGF1 has potent analgesic effects and produces resolution of hyperalgesia after tissue injury. Dysregulation of IGF1-GRK2 signaling could potentially lead to failure of the spontaneous resolution of acute pain and, hence, development of chronic pain after surgery., (Copyright © 2021. Published by Elsevier Inc.)

cAMP (Cyclic Adenosine monophosphate), one of the most highly studied second messengers, is regulated by a family of adenylyl cyclase (AC) enzymes. Type 3 adenylyl cyclase (abbreviated as AC3), a subtype of adenylyl cyclase, is reported to be expressed in cilia in the olfactory and central nervous system and plays an important role in many physiological functions such as olfaction, development. However, expression of AC3 in the dorsal root ganglion (DRG) is not reported. In the present study, using immunohistochemical method, we discovered that AC3 immunoreactivity (IR) is predominantly expressed in the cytoplasm of small to medium sized DRG neurons. Double labelling revealed that the majority of AC3 IR are colocalized with CGRP (a peptidergic nociceptor marker), rarely with NF200 (a myelinated neuronal marker) or IB4 (a nonpeptidergic nociceptor marker). Furthermore, dense AC3 IR exists in the superficial dorsal horn, especially in laminaⅠand dorsal part of lamina II, where CGRP-positive DRG neurons terminate. The expression pattern of AC3 is similar between C57/BL6 J mouse and Sprague Dawley rat. For instance, AC3 is primarily expressed in the cell bodies of small to medium sized DRG neurons and the majority of AC3 IR is also in CGRP-containing neurons in rat. Taken together, our data suggest that AC3 is primarily expressed in the small to medium sized cell bodies and central terminals of CGRP-positive DRG neurons, implying AC3 enzyme might potentially function in nociception., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The main aim of the present review is to provide at first a short survey of the basic anatomical description of sensory ganglion neurons in relation to cell size, conduction velocity, thickness of myelin sheath, and functional classification of their processes. In addition, we have focused on discussing current knowledge about the distribution pattern of neuronal nitric oxide synthase containing sensory neurons especially in the dorsal root ganglia in different animal species; hence, there is a large controversy in relation to interpretation of the results dealing with this interesting field of research.

Acoustic information propagates from the ear to the brain via spiral ganglion neurons that innervate hair cells in the cochlea. These afferents include unmyelinated type II fibers that constitute 5 % of the total, the majority being myelinated type I neurons. Lack of specific genetic markers of type II afferents in the cochlea has been a roadblock in studying their functional role. Unexpectedly, type II afferents were visualized by reporter proteins induced by tyrosine hydroxylase (TH)-driven Cre recombinase. The present study was designed to determine whether TH-driven Cre recombinase (TH-2A-CreER) provides a selective and reliable tool for identification and genetic manipulation of type II rather than type I cochlear afferents. The "TH-2A-CreER neurons" radiated from the spiral lamina, crossed the tunnel of Corti, turned towards the base of the cochlea, and traveled beneath the rows of outer hair cells. Neither the processes nor the somata of TH-2A-CreER neurons were labeled by antibodies that specifically labeled type I afferents and medial efferents. TH-2A-CreER-positive processes partially co-labeled with antibodies to peripherin, a known marker of type II afferents. Individual TH-2A-CreER neurons gave off short branches contacting 7-25 outer hair cells (OHCs). Only a fraction of TH-2A-CreER boutons were associated with CtBP2-immunopositive ribbons. These results show that TH-2A-CreER provides a selective marker for type II versus type I afferents and can be used to describe the morphology and arborization pattern of type II cochlear afferents in the mouse cochlea., Competing Interests: The authors declare that they have no competing interests.

At its most fundamental level, touch sensation requires the translation of mechanical energy into mechanosensitive ion channel opening, thereby generating electro-chemical signals. Our understanding of this process, especially how the cytoskeleton influences it, remains unknown. Here we demonstrate that mice lacking the α-tubulin acetyltransferase Atat1 in sensory neurons display profound deficits in their ability to detect mechanical stimuli. We show that all cutaneous afferent subtypes, including nociceptors have strongly reduced mechanosensitivity upon Atat1 deletion, and that consequently, mice are largely insensitive to mechanical touch and pain. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, which when absent leads to a decrease in cellular elasticity. By mimicking α-tubulin acetylation genetically, we show both cellular rigidity and mechanosensitivity can be restored in Atat1 deficient sensory neurons. Hence, our results indicate that by influencing cellular stiffness, α-tubulin acetylation sets the force required for touch., Competing Interests: The authors declare that no competing interests exist.

Background: Neuropathic pain and sensory abnormalities are a debilitating secondary consequence of spinal cord injury (SCI). Maladaptive structural plasticity is gaining recognition for its role in contributing to the development of post SCI pain syndromes. We previously demonstrated that excitotoxic induced SCI dysesthesias are associated with enhanced dorsal root ganglia (DRG) neuronal outgrowth. Although glycogen synthase kinase-3β (GSK-3β) is a known intracellular regulator neuronal growth, the potential contribution to primary afferent growth responses following SCI are undefined. We hypothesized that SCI triggers inhibition of GSK-3β signaling resulting in enhanced DRG growth responses, and that PI3K mediated activation of GSK-3β can prevent this growth and the development of at-level pain syndromes., Results: Excitotoxic SCI using intraspinal quisqualic acid (QUIS) resulted in inhibition of GSK-3β in the superficial spinal cord dorsal horn and adjacent DRG. Double immunofluorescent staining showed that GSK-3β(P) was expressed in DRG neurons, especially small nociceptive, CGRP and IB4-positive neurons. Intrathecal administration of a potent PI3-kinase inhibitor (LY294002), a known GSK-3β activator, significantly decreased GSK-3β(P) expression levels in the dorsal horn. QUIS injection resulted in early (3 days) and sustained (14 days) DRG neurite outgrowth of small and subsequently large fibers that was reduced with short term (3 days) administration of LY294002. Furthermore, LY294002 treatment initiated on the date of injury, prevented the development of overgrooming, a spontaneous at-level pain related dysesthesia., Conclusions: QUIS induced SCI resulted in inhibition of GSK-3β in primary afferents and enhanced at-level DRG intrinsic growth (neurite elongation and initiation). Early PI3K mediated activation of GSK-3β attenuated QUIS-induced DRG neurite outgrowth and prevented the development of at-level dysesthesias.

Glucagon is released from the pancreatic islets postprandially and under hypoglycemic and cold conditions, and regulates glucose metabolism, feeding, energy expenditure and heat production, the functions partly controlled by the brain. Peripheral glucagon could signal to the brain via passing through the blood-brain barrier and/or acting on the vagal afferent. However, the latter remains to be determined. The present study aimed to clarify whether glucagon directly interacts with the nodose ganglion (NG) neurons of vagal afferent nerves in mice. In vivo study showed that intraperitoneal injection of glucagon induced phosphorylation of extracellular signal regulated kinase 1 and 2 (ERK1/2), cellular activation makers, in NG neurons. In fura-2 microfluorometric studies, glucagon increased cytosolic Ca(2+) concentration ([Ca(2+)]i) in single NG neurons. The glucagon-induced [Ca(2+)]i increases were suppressed by a glucagon receptor antagonist, des-His(1)-[Glu(9)]-Glucagon (1-29) amide, and the glucagon receptor mRNA was expressed in NG neurons. The majority of glucagon-responsive NG neurons exhibited [Ca(2+)]i responses to insulin and cholecystokinin-8, the hormones that are secreted postprandially and implicated in satiety. These results demonstrate that glucagon, by interacting with the glucagon receptor, directly activates vagal afferent nerves, possibly being relayed to the signaling to the brain and formation of satiety., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Increasing evidence indicates that the pathogenesis of neuropathic pain is mediated through spinal cord microglia activation. The intracellular protease caspase-6 (CASP6) is known to regulate neuronal apoptosis and axonal degeneration; however, the contribution of microglia and CASP6 in modulating synaptic transmission and pain is unclear. Here, we found that CASP6 is expressed specifically in C-fiber axonal terminals in the superficial spinal cord dorsal horn. Animals exposed to intraplantar formalin or bradykinin injection exhibited CASP6 activation in the dorsal horn. Casp6-null mice had normal baseline pain, but impaired inflammatory pain responses. Furthermore, formalin-induced second-phase pain was suppressed by spinal injection of CASP6 inhibitor or CASP6-neutralizing antibody, as well as perisciatic nerve injection of CASP6 siRNA. Recombinant CASP6 (rCASP6) induced marked TNF-α release in microglial cultures, and most microglia within the spinal cord expressed Tnfa. Spinal injection of rCASP6 elicited TNF-α production and microglia-dependent pain hypersensitivity. Evaluation of excitatory postsynaptic currents (EPSCs) revealed that rCASP6 rapidly increased synaptic transmission in spinal cord slices via TNF-α release. Interestingly, the microglial inhibitor minocycline suppressed rCASP6 but not TNF-α-induced synaptic potentiation. Finally, rCASP6-activated microglial culture medium increased EPSCs in spinal cord slices via TNF-α. Together, these data suggest that CASP6 released from axonal terminals regulates microglial TNF-α secretion, synaptic plasticity, and inflammatory pain.

Sciatic nerve (SN) transection-induced apoptosis of dorsal root ganglion neurons (DRGN) is one factor determining the efficacy of peripheral axonal regeneration and the return of sensation. Here, we tested the hypothesis that caspase-2 (CASP2) orchestrates apoptosis of axotomised DRGN both in vivo and in vitro by disrupting the local neurotrophic supply to DRGN. We observed significantly elevated levels of cleaved CASP2 (C-CASP2), compared to cleaved caspase-3 (C-CASP3), within TUNEL+DRGN and DRG glia (satellite and Schwann cells) after SN transection. A serum withdrawal cell culture model, which induced 40% apoptotic death in DRGN and 60% in glia, was used to model DRGN loss after neurotrophic factor withdrawal. Elevated C-CASP2 and TUNEL were observed in both DRGN and DRG glia, with C-CASP2 localisation shifting from the cytosol to the nucleus, a required step for induction of direct CASP2-mediated apoptosis. Furthermore, siRNA-mediated downregulation of CASP2 protected 50% of DRGN from apoptosis after serum withdrawal, while downregulation of CASP3 had no effect on DRGN or DRG glia survival. We conclude that CASP2 orchestrates the death of SN-axotomised DRGN directly and also indirectly through loss of DRG glia and their local neurotrophic factor support. Accordingly, inhibiting CASP2 expression is a potential therapy for improving both the SN regeneration response and peripheral sensory recovery.

Isolectin B(4)-positive [IB(4)(+)] primary afferent nociceptors challenged with an inflammatory or neuropathic insult develop a PKCε-dependent long-lasting hyperalgesic response to a subsequent challenge by the proinflammatory cytokine prostaglandin E(2) (PGE(2)), a phenomenon known as hyperalgesic priming. Here we demonstrate that the neuroplasticity underlying nociceptor priming requires 72 h to be established; rats that have been challenged with the inflammatory mediator TNFα 24 or 48 h ahead of PGE(2) do not show the enhanced and prolonged hyperalgesic response by which primed IB(4)(+)-nociceptors are being characterized. Moreover, as the underlying plasticity can be interrupted by the peripheral administration of the protein translation inhibitor anisomycin it is reflected by changes in the peripheral protein expression pattern. Finally, the induction of priming by the selective PKCε agonist, psi ε receptor for activated c kinase (ψεRACK) can be prevented, but not reversed by intrathecal injections of antisense oligodeoxynucleotides for the cytoplasmic polyadenylation element binding protein (CPEB) mRNA, a master regulator of protein translation that coimmunoprecipitated with PKCε and is almost exclusively expressed by IB(4)(+)-nociceptors. Our results suggest that CPEB is downstream of PKCε in the cellular signaling cascade responsible for the induction of priming, raising the intriguing possiblity that prion-like misfolding could be a responsible mechanism for the chronification of pain.

Background: Migraine headache is one of the most common neurological disorders, but the pathophysiology contributing to migraine is poorly understood. Intracranial interleukin-6 (IL-6) levels have been shown to be elevated during migraine attacks, suggesting that this cytokine may facilitate pain signaling from the meninges and contribute to the development of headache., Methods: Cutaneous allodynia was measured in rats following stimulation of the dura with IL-6 alone or in combination with the MEK inhibitor, U0126. The number of action potentials and latency to the first action potential peak in response to a ramp current stimulus as well as current threshold were measured in retrogradely-labeled dural afferents using patch-clamp electrophysiology. These recordings were performed in the presence of IL-6 alone or in combination with U0126. Association between ERK1 and Nav1.7 following IL-6 treatment was also measured by co-immunoprecipitation., Results: Here we report that in awake animals, direct application of IL-6 to the dura produced dose-dependent facial and hindpaw allodynia. The MEK inhibitor U0126 blocked IL-6-induced allodynia indicating that IL-6 produced this behavioral effect through the MAP kinase pathway. In trigeminal neurons retrogradely labeled from the dura, IL-6 application decreased the current threshold for action potential firing. In response to a ramp current stimulus, cells treated with IL-6 showed an increase in the numbers of action potentials and a decrease in latency to the first spike, an effect consistent with phosphorylation of the sodium channel Nav1.7. Pretreatment with U0126 reversed hyperexcitability following IL-6 treatment. Moreover, co-immunoprecipitation experiments demonstrated an increased association between ERK1 and Nav1.7 following IL-6 treatment., Conclusions: Our results indicate that IL-6 enhances the excitability of dural afferents likely via ERK-mediated modulation of Nav1.7 and these responses contribute to migraine-related pain behavior in vivo. These data provide a cellular mechanism by which IL-6 in the meninges causes sensitization of dural afferents therefore contributing to the pathogenesis of migraine headache.

Thiamine (Vitamin B1) is an essential vitamin that must be obtained from the diet for proper neurological function. At higher doses, thiamine and benfotiamine (S-benzoylthiamine O-monophosphate, BT)-a phosphorylated derivative of thiamine-have antinociceptive effects in animals and humans, although how these compounds inhibit pain is unknown. Here, we found that Prostatic acid phosphatase (PAP, ACPP) can dephosphorylate BT in vitro, in dorsal root ganglia (DRG) neurons and in primary-afferent axon terminals in the dorsal spinal cord. The dephosphorylated product S-benzoylthiamine (S-BT) then decomposes to O-benzoylthiamine (O-BT) and to thiamine in a pH-dependent manner, independent of additional enzymes. This unique reaction mechanism reveals that BT only requires a phosphatase for conversion to thiamine. However, we found that the antinociceptive effects of BT, thiamine monophosphate (TMP) and thiamine-a compound that is not phosphorylated-were entirely dependent on PAP at the spinal level. Moreover, pharmacokinetic studies with wild-type and Pap(-/-) mice revealed that PAP is not required for the conversion of BT to thiamine in vivo. Taken together, our study highlights an obligatory role for PAP in the antinociceptive effects of thiamine and phosphorylated thiamine analogs, and suggests a novel phosphatase-independent function for PAP.

Specific sensory neuronal subpopulations show contrasting responses to peripheral nerve injury, as shown by the axotomy-induced death of many cutaneous sensory neurons whilst muscular sensory afferents survive an identical insult. We used a novel combination of retrograde neuronal tracing with immunohistochemistry and laser microdissection techniques, in order to describe the neurochemistry of medial gastrocnemius (muscular sensory afferents) and sural (cutaneous sensory afferents) branches of the rat sciatic nerve and relate this to the pro-apoptotic caspase-3 gene expression following nerve transection. Our results demonstrated distinctions in medial gastrocnemius and sural neuron populations with the most striking difference in the respective proportions of isolectin B4 (IB4) staining neurons (3.7 V 32.8%). The mean neuronal area of the medial gastrocnemius (MG) neurons was larger than that of the sural (SUR) neurons (1,070.8 V 646.2 μm²) and each phenotypic group was significantly smaller in sural neurons than in MG neurons. At 1 week post-axotomy, MG neurons markedly downregulated caspase-3, whilst SUR neurons upregulated caspase-3 gene expression; this may be attributable to the differing IB4-positive composition of the subpopulations. These findings provide further clarification in the understanding of two distinct neuronal populations used increasingly in nerve injury models.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca(2+) and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca(2+)-independent. The atypical PKCs are activated by neither Ca(2+) nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation (1-2). Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail (3). In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article (4), expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

The Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is expressed in sensory neurons where it accumulates intracellular Cl(-) and facilitates primary afferent depolarization. Depolarization of primary afferent fibre terminals interferes with the gating of incoming sensory signals to the spinal cord. The cotransporter belongs to a family of ion transporters which are sensitive to changes in cell volume. Cell shrinkage, through mechanisms that are still unknown, leads to the phosphorylation and activation of NKCC1. Similarly, axotomy results in increased NKCC1 phosphorylation in dorsal root ganglion (DRG) neurons. This review summarizes the work on the kinases that directly mediate NKCC1 activation. These are the sterile-20-like kinases SPAK and OSR1. Upon their activation through phosphorylation by upstream kinases, SPAK and OSR1 bind to specific peptides located in the cytosolic N-terminal tail of NKCC1, phosphorylate, and stimulate cotransport activity. Expression of SPAK and OSR1 varies from tissue to tissue, but in DRG neurons and in spinal cord, SPAK and OSR1 expression levels are similar. In DRG neurons, both kinases participate in the modulation of NKCC1, as the knockdown of one kinase only results in a partial decrease of NKCC1 function, while the knockdown of both kinases is additive. The identity of the kinases (e.g. WNK kinases) that possibly act upstream of SPAK and OSR1 is also discussed.

Migraine is a common neurological disorder often treated with triptans. Triptan overuse can lead to increased frequency of headache in some patients, a phenomenon termed medication overuse headache. Previous preclinical studies have demonstrated that repeated or sustained triptan administration for several days can elicit persistent neural adaptations in trigeminal ganglion cells innervating the dura, prominently characterized by increased labelling of neuronal profiles for calcitonin gene related peptide. Additionally, triptan administration elicited a behavioural syndrome of enhanced sensitivity to surrogate triggers of migraine that was maintained for weeks following discontinuation of drug, a phenomenon termed 'triptan-induced latent sensitization'. Here, we demonstrate that triptan administration elicits a long-lasting increase in identified rat trigeminal dural afferents labelled for neuronal nitric oxide synthase in the trigeminal ganglion. Cutaneous allodynia observed during the period of triptan administration was reversed by NXN-323, a selective inhibitor of neuronal nitric oxide synthase. Additionally, neuronal nitric oxide synthase inhibition prevented environmental stress-induced hypersensitivity in the post-triptan administration period. Co-administration of NXN-323 with sumatriptan over several days prevented the expression of allodynia and enhanced sensitivity to stress observed following latent sensitization, but not the triptan-induced increased labelling of neuronal nitric oxide synthase in dural afferents. Triptan administration thus promotes increased expression of neuronal nitric oxide synthase in dural afferents, which is critical for enhanced sensitivity to environmental stress. These data provide a biological basis for increased frequency of headache following triptans and highlight the potential clinical utility of neuronal nitric oxide synthase inhibition in preventing or treating medication overuse headache.

Sensory neurons in the dorsal root ganglion express two kinds of tetrodotoxin resistant (TTX-R) isoforms of voltage-gated sodium channels, Na(V)1.8 and Na(V)1.9. These isoforms play key roles in the pathophysiology of chronic pain. Of special interest is Na(V)1.9: our previous studies revealed a unique property of the Na(V)1.9 current, i.e., the Na(V)1.9 current shows a gradual and notable up-regulation of the peak amplitude during recording ("spontaneous augmentation of Na(V)1.9"). However, the mechanism underlying the spontaneous augmentation of Na(V)1.9 is still unclear. In this study, we examined the effects of protein kinases A and C (PKA and PKC), on the spontaneous augmentation of Na(V)1.9. The spontaneous augmentation of the Na(V)1.9 current was significantly suppressed by activation of PKA, whereas activation of PKA did not affect the voltage dependence of inactivation for the Na(V)1.9 current. On the contrary, the finding that activation of PKC can affect the voltage dependence of inactivation for Na(V)1.9 in the perforated patch recordings, where the augmentation does not occur, suggests that the effects of PMA are independent of the augmentation process. These results indicate that the spontaneous augmentation of Na(V)1.9 was regulated directly by PKA, and indirectly by PKC.

Structural elements of afferent innervations of brain arteries in rats such as receptors and nervous fibers, neurons of ganglia jugularis unit and the nucleus of a single way were investigated with the help of histochemical and electron cytochemical methods. The presence of three types of receptors and afferent fibers has been established in vessels. Neurons with positive reaction to NADPH-diaphorase have been allocated in the ganglia jugularis unit and the nucleus of a single way (17.4 and 24.5% accordingly).

In the present study, we examined whether activation of p-38alpha MAPK modulates mechanical allodynia and neuronal hyperexcitability, and if propentofylline (PPF, a glial modulator) modulates specifically localized activated p-38alpha MAPK expression in caudal regions remote from a low thoracic hemisection injury in rats. T13 spinal hemisection produces bilateral mechanical allodynia in hindpaws with evoked (in response to mechanical stimuli) neuronal hyperexcitability in lumbar spinal wide dynamic range (WDR) neurons compared to sham controls. The mechanical allodynia and the evoked activity of WDR neurons is attenuated by intrathecal and topical administration of SB203580, an inhibitor of p-38 MAPK activation, dose dependently (p<0.05); however, the spontaneous activity showed no significant differences compared to sham controls. After T13 spinal hemisection, significantly increased phosphorylated (activated form) p-38alpha MAPK expression was present in both superficial and deep dorsal horn neurons as well as in microglia, but not in astrocytes, in the lumbar spinal cord compared to sham controls (p<0.05). Intrathecal application of PPF significantly attenuated the expression of phosphorylated p-38alpha MAPK in superficial dorsal horn neurons (10 mM) and in microglia (1 and 10 mM) in the lumbar spinal cord compared to the hemisection group (p<0.05). In conclusion, our present data demonstrate that activated neuronal and microglial, but not astrocytic, p-38alpha MAPK contributes to the maintenance of neuronal hyperexcitability in caudal regions following spinal cord injury.

Unlabelled: Pig has been used recently as an animal model for studying diseases of human urinary tract, however, the sensory innervations of urinary bladder in this species has not been yet described., Objective: The present study was aimed at neurochemical characterization of sensory neurons of dorsal root ganglia (DRGs) supplying porcine urinary bladder., Methods: Retrograde tracer Fast Blue (FB) was injected into the right half of the urinary bladder wall of six juvenile female pigs. Three weeks later ipsi- and contralateral DRGs of interest were harvested from all animals and a neurochemical characterization of retrogradely-labeled neurons was performed using routine single-immunofluorescence labeling technique on 10 microm-thick cryostat sections., Results: 85% of spinal sensory neurons supplying porcine urinary bladder was located in ipsilateral sacral S3-S4 ganglia and in first coccygeal ganglion (Cq1),whereas rest of FB-positive (FB+) nerve cells were found in lumbar L3-L6 DRGs. FB+ neurons belonged mostly to the medium-sized (54%) and small-sized afferent perikarya (45%). Bladder sensory neurons contained substance P (SP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating peptide (PACAP), galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM) and/or calbindin-28k (CB), and these neurons constituted 45%, 36%, 26%, 6%, 6%, 4% and 3% of all retrogradely traced DRGs perikarya, respectively. Distinct differences in the number of traced cells and their neuropeptide content were observed between the lumbar and sacral/coccygeal division of bladder-projecting DRG neurons. Thus, FB+ neurons expressing CGRP, GAL, nNOS or SOM were more numerous in lumbar DRGs (44%, 9%, 9% and 6%, respectively), than in sacral/coccygeal ganglia (23%, 2%, 1.5% and 0.3%, respectively). On the other hand, more FB+ cells expressed PACAP in sacral (31%) than in lumbar DRGs (23%). However, fractions of SP-IR or CB-IR bladder sensory neurons were similar in lumbar and sacral/coccygeal DRGs., Conclusions: This novel description of both spatial and neurochemical organization pattern of porcine urinary bladder sensory innervation constitutes a basis for further functional studies aimed at unraveling neurogenic mechanisms of urinary bladder diseases.

The mechanisms that generate itch are poorly understood at both the molecular and cellular levels despite its clinical importance. To explore the peripheral neuronal mechanisms underlying itch, we assessed the behavioral responses (scratching) produced by s.c. injection of various pruritogens in PLCbeta3- or TRPV1-deficient mice. We provide evidence that at least 3 different molecular pathways contribute to the transduction of itch responses to different pruritogens: 1) histamine requires the function of both PLCbeta3 and the TRPV1 channel; 2) serotonin, or a selective agonist, alpha-methyl-serotonin (alpha-Me-5-HT), requires the presence of PLCbeta3 but not TRPV1, and 3) endothelin-1 (ET-1) does not require either PLCbeta3 or TRPV1. To determine whether the activity of these molecules is represented in a particular subpopulation of sensory neurons, we examined the behavioral consequences of selectively eliminating 2 nonoverlapping subsets of nociceptors. The genetic ablation of MrgprD(+) neurons that represent approximately 90% of cutaneous nonpeptidergic neurons did not affect the scratching responses to a number of pruritogens. In contrast, chemical ablation of the central branch of TRPV1(+) nociceptors led to a significant behavioral deficit for pruritogens, including alpha-Me-5-HT and ET-1, that is, the TRPV1-expressing nociceptor was required, whether or not TRPV1 itself was essential. Thus, TRPV1 neurons are equipped with multiple signaling mechanisms that respond to different pruritogens. Some of these require TRPV1 function; others use alternate signal transduction pathways.

Background: Peripheral nerve injury leads to a persistent neuropathic pain state in which innocuous stimulation elicits pain behavior (tactile allodynia), but the underlying mechanisms have remained largely unknown. We have previously shown that spinal nerve injury induces the activation of cytosolic phospholipase A2 (cPLA2) in injured dorsal root ganglion (DRG) neurons that contribute to tactile allodynia. However, little is known about the signaling pathway that activates cPLA2 after nerve injury. In the present study, we sought to determine the mechanisms underlying cPLA2 activation in injured DRG neurons in an animal model of neuropathic pain, focusing on mitogen-activated protein kinases (MAPKs) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)., Results: Pharmacological inhibition of either p38 or extracellular signal-regulated kinase (ERK) in the injured DRG, which led to suppression of the development of tactile allodynia, did not affect cPLA2 phosphorylation and translocation after nerve injury. By contrast, a CaMKII inhibitor prevented the development and expression of nerve injury-induced tactile allodynia and reduced both the level of cPLA2 phosphorylation and the number of DRG neurons showing translocated cPLA2 in response to nerve injury. Applying ATP to cultured DRG neurons increased the level of both phosphorylated cPLA2 and CaMKII in the vicinity of the plasma membrane and caused physical association of these two proteins. In addition, ATP-stimulated cPLA2 and CaMKII phosphorylation were inhibited by both a selective P2X3R/P2X2+3R antagonist and a nonselective voltage-dependent Ca2+ channel (VDCC) blocker., Conclusion: These results suggest that CaMKII, but not MAPKs, has an important role in cPLA2 activation following peripheral nerve injury, probably through P2X3R/P2X2+3R and VDCCs in primary afferent neurons.

Processing of sound in the cochlea involves both afferent and efferent innervation. The Na,K-ATPase (NKA) is essential for cells that maintain hyperpolarized membrane potentials and sodium and potassium concentration gradients. Heterogeneity of NKA subunit expression is one mechanism that tailors physiology to particular cellular demands. Therefore, to provide insight into molecular differences that distinguish the various innervation pathways in the cochlea, we performed a variety of double labeling experiments with antibodies against three of the alpha isoforms of the NKA (NKA alpha 1-3) and markers identifying particular subsets of neurons or supporting cells in whole mount preparations of the organ of Corti and spiral ganglion. We found that the NKA alpha 3 is abundantly expressed within the membranes of the spiral ganglion somata, the type I afferent terminals contacting the inner hair cells, and the medial efferent terminals contacting the outer hair cells. We also found expression of the NKA alpha 1 in the supporting cells that neighbor the inner hair cells and express the glutamate transporter GLAST. These findings suggest that both the NKA alpha 1 and NKA alpha 3 are poised to play an essential role in the regulation of the type I afferent synapses, the medial efferent synapses, and also glutamate transport from the afferent-inner hair cell synapse.

In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKCepsilon, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.

The Na(V)1.7 tetrodotoxin-sensitive voltage-gated sodium channel isoform plays a critical role in nociception. In rodent models of diabetic neuropathy, increased Na(V)1.7 in dorsal root ganglia (DRG) neurons correlates with the emergence of pain-related behaviors characteristic of painful diabetic neuropathy (PDN). We examined the effect of transgene-mediated expression of enkephalin on pain-related behaviors and their biochemical correlates in DRG neurons. Transfection of DRG neurons by subcutaneous inoculation of a herpes simplex virus-based vector expressing proenkephalin reversed nocisponsive behavioral responses to heat, cold, and mechanical pressure characteristic of PDN. Vector-mediated enkephalin production in vivo prevented the increase in DRG Na(V)1.7 observed in PDN, an effect that correlated with inhibition of phosphorylation of p38 MAPK (mitogen-activated protein kinase) and protein kinase C (PKC). Primary DRG neurons in vitro exposed to 45 mm glucose for 18 h also demonstrated an increase in Na(V)1.7 and increased phosphorylation of p38 and PKC; these changes were prevented by transfection in vitro with the enkephalin-expressing vector. The effect of hyperglycemia on Na(V)1.7 production in vitro was mimicked by exposure to PMA and blocked by the myristolated PKC inhibitor 20-28 or the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole]; the effect of vector-mediated enkephalin on Na(V)1.7 levels was prevented by naltrindole. The results of these studies suggest that activation of the presynaptic delta-opioid receptor by enkephalin prevents the increase in neuronal Na(V)1.7 in DRG through inhibition of PKC and p38. These results establish a novel interaction between the delta-opioid receptor and voltage-gated sodium channels.

Patch-clamp techniques and Ca2+ imaging were used to examine the interaction between neurokinins (NK) and the capsaicin(CAPS)-evoked transient receptor potential vanilloid receptor 1 (TRPV1) responses in rat dorsal root ganglia neurons. Substance P (SP; 0.2-0.5 microM) prevented the reduction of Ca2+ transients (tachyphylaxis) evoked by repeated brief applications of CAPS (0.5 microM). Currents elicited by CAPS were increased in amplitude and desensitized more slowly after administration of SP or a selective NK2 agonist, [Ala8]-neurokinin A (4-10) (NKA). Neither an NK1-selective agonist, [Sar9, Met11]-SP, nor an NK3-selective agonist, [MePhe7]-NKB, altered the CAPS currents. The effects of SP on CAPS currents were inhibited by a selective NK2 antagonist, MEN10,376, but were unaffected by the NK3 antagonist, SB 235,375. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C(PKC), also increased the amplitude and slowed the desensitization of CAPS responses. Phosphatase inhibitors, decamethrin and alpha-naphthyl acid phosphate (NAcPh), also enhanced the currents and slowed desensitization of CAPS currents. Facilitatory effects of SP, NKA and PDBu were reversed by bisindolylmaleimide, a PKC inhibitor, and gradually decreased in magnitude when the agents were administered at increasing intervals after CAPS application. The decrease was partially prevented by prior application of NAcPh. These data suggest that activation of NK2 receptors in afferent neurons leads to PKC-induced phosphorylation of TRPV1, resulting in sensitization of CAPS-evoked currents and slower desensitization. Thus, activation of NK2 autoreceptors by NKs released from the peripheral afferent terminals or by mast cells during inflammatory responses may be a mechanism that sensitizes TRPV1 channels and enhances afferent excitability.

Arachidonic acid and its metabolites produced via cyclooxygenase (COX) and lipoxygenase pathways have been reported to contribute to the cardiovascular reflexes evoked by stimulating thin fibre muscle afferents during muscle contraction. 20-Hydroxyeicosatetraenoic acid (20-HETE), a primarily metabolized product of arachidonic acid by cytochrome P450 enzymes, can be accumulated in contracting muscles. Thus, the purpose of this study was to determine the role of 20-HETE in modulating the reflex sympathetic responses to activation of chemically and mechanically sensitive muscle afferents. The renal sympathetic nerve activity (RSNA) and cardiovascular responses were examined after injections of 20-HETE into the arterial blood supply of the hindlimb muscles of decerebrated rats. This induced a dose-dependent increases in RSNA and mean arterial pressure (MAP). We also tested the hypothesis that 20-HETE would sensitize muscle afferents and, thereby, augment the RSNA and blood pressure response to muscle stretch. The results show that arterial infusion of 20-HETE significantly enhanced the RSNA and MAP responses to muscle stretch. In contrast, N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine, a potent inhibitor of 20-HETE production, attenuated the reflex muscle responses. Furthermore, the sensitizing effect of 20-HETE on the muscle reflex was significantly attenuated after blocking COX activity with indomethacin. Our data suggest that 20-HETE plays a role in modulating muscle afferent-mediated sympathetic responses, probably through engagement of a COX-dependent mechanism.

Background: Animal models show us that specific activation of the p38 mitogen-activated protein kinase (MAPK) may be a pivotal step in lidocaine neurotoxicity, but this has not been investigated in the case of two very widely used local anesthetics, bupivacaine and ropivacaine. We investigated the hypotheses that these drugs (A) are less neurotoxic than the prototype local anesthetic, lidocaine (B) are selectively toxic for subcategories of dorsal root ganglion neurons and (C) induce activation of either p38 MAPK or related enzymes, such as the c-jun terminal N-kinase (JNK) and extracellular signal-regulated kinase (ERK)., Methods: We incubated primary sensory neuron cultures with doses of lidocaine, bupivacaine, and ropivacaine equipotent at blocking sodium currents. Next, we sought to determine potential selectivity of bupivacaine and ropivacaine toxicity on neuron categories defined by immunohistochemical staining, or size. Subsequently, the involvement of p38 MAPK, JNK, and ERK was tested using enzyme-linked immunosorbent assays. Finally, the relevance of MAPK pathways in bupivacaine- and ropivacaine-induced neurotoxicity was determined by selectively inhibiting activity of p38 MAPK, JNK, and ERK., Results: We found that the neurotoxic potency of bupivacaine and ropivacaine is dose-dependent and similar in vitro, but is not selective for any of the investigated subgroups of neurons. Neurotoxicity of bupivacaine and ropivacaine was mediated, at least in part, by MAPKs. Specifically, we demonstrated the relevance of both p38 MAPK and JNK pathways for the neurotoxicity of bupivacaine and characterized the involvement of the p38 MAPK pathway in the neurotoxicity of ropivacaine., Conclusions: Given equipotent doses, the neurotoxic potential of lidocaine does not appear to be significantly different from that of bupivacaine and ropivacaine in vitro. Moreover, bupivacaine and ropivacaine do not exert their neurotoxicity differently on specific subsets of dorsal root ganglion neurons. Their neurotoxic effects are brought about through the activation of specific MAPKs; the specific pharmacologic inhibition of these kinases attenuates toxicity in vitro.

Background and Purpose: The endogenous cannabinoid anandamide (AEA) acts at cannabinoid (CB(1)) and vanilloid (TRPV(1)) receptors. AEA also shows antinociceptive properties; although the underlying mechanism for this is not fully understood, both CB(1) and TRPV(1) may be involved. Voltage-activated Ca(2+) channels in rat-cultured dorsal root ganglion (DRG) neurons are modulated by AEA. However, AEA in different populations of neurons enhanced or attenuated KCl-evoked Ca(2+) influx; these effects were linked with soma size. The aim of this study was to determine how AEA or its metabolites might produce these variable responses., Experimental Approach: The whole cell patch-clamp technique and fura-2 Ca(2+) imaging were used to characterize the actions of AEA on action potential firing and voltage-activated K(+) currents and to determine whether AEA metabolism plays any role in its effects on cultured DRG neurons., Key Results: AEA attenuated multiple action potential firing evoked by 300 ms depolarizing current commands in a subpopulation of DRG neurons. Application of 1 microM AEA attenuated voltage-activated K(+) currents and the recovery of KCl-evoked Ca(2+) transients. The insensitivity of these responses to the CB(1) receptor antagonist rimonabant (100 nM) and preincubation of DRG neurons with pertussis toxin suggested that these actions are not CB(1) receptor-mediated. Preincubating DRG neurons with the fatty acid amide hydrolase (FAAH) inhibitor phenylmethylsulphonyl fluoride (PMSF) attenuated the inhibitory actions of AEA on K(+) currents and Ca(2+) influx., Conclusion and Implications: These data suggest that the products of AEA metabolism by FAAH contribute to the attenuation of K(+) conductances and altered excitability of cultured sensory neurons.

Unlabelled: Skeletal muscle injuries can induce chronic pain, but the underlying mechanism is unknown. One possible cause has been suggested to be an increased sensitivity to inflammatory mediators. We demonstrate that self-limited inflammatory hyperalgesia induced by intramuscular carrageenan (lasting approximately 5 days) results in a state of chronic-latent hyperalgesia, revealed by injection of prostaglandin E(2) (PGE(2)) 10 days after carrageenan at the same site. In carrageenan-pretreated muscle, PGE(2) produced hyperalgesia that was unattenuated even 14 days after injection, markedly longer than the 4-hour hyperalgesia induced by PGE(2) in naive rats. This chronic-latent hyperalgesia was reversed as well as prevented by spinal intrathecal injection of oligodeoxynucleotide antisense to protein kinase Cepsilon, a second messenger implicated in long-lasting plasticity in cutaneous nociceptors., Perspective: We describe a novel experimental model for chronic muscle pain, produced by mild acute muscle inflammation, that has clinical significance since it has the potential to reveal cellular processes by which acute inflammation or muscle trauma underlies chronic muscle pain.

Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.

Aims: Activation of the transient receptor potential vanilloid receptor 1 (TRPV1) leads to release of potent microvascular vasodilator neuropeptides. This study was designed to investigate in vivo mechanisms involved in TRPV1-mediated peripheral vasodilatation., Methods and Results: Wildtype (WT) and TRPV1 knockout (KO) mice were investigated in a model of peripheral vasodilatation. Blood flow was measured by laser Doppler flowmetry under anaesthesia and following local application of the TRPV1 agonist capsaicin. A sustained (60 min) increase in blood flow was observed in WT but not TRPV1 KO mouse ears. This response was resistant to blockers of classic vasodilators but inhibited in pharmacogenetic experiments that targeted blockade of the substance P (SP) and calcitonin gene-related peptide (CGRP) pathways. The TRPV1-mediated vasodilatation was also attenuated by treatment with superoxide dismutase and the hydrogen peroxide scavenger catalase, but not by deactivated enzymes, supporting a novel role for reactive oxygen species (ROS) generation. Furthermore, neurogenic vasodilatation was observed neither in the presence of the selective NADPH inhibitor apocynin, nor in gp91 phox KO mice, under conditions where prostaglandin E1-induced vasodilatation occurred. Finally, a role of neuropeptides in initiating a ROS-dependent component was verified as superoxide dismutase, catalase, and apocynin inhibited SP and CGRP vasodilatation., Conclusion: These studies provide in vivo evidence that ROS are involved in mediating TRPV1- and neuropeptide-dependent neurogenic vasodilatation. An essential role of NADPH oxidase-dependent ROS is revealed that may be of fundamental importance to the neurogenic vasodilator component involved in circulatory homeostasis and the pathophysiology of certain cardiovascular diseases.

Background & Aims: Changes in the properties of visceral sensory neurons contribute to the development of gastrointestinal pain. However, little is known about the molecules involved in mechanosensation from the gastrointestinal tract. We investigated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase cascade, in dorsal root ganglion (DRG) and nodose ganglion (NG) neurons by noxious gastric distention (GD) and its involvement in acute visceral pain in rats., Methods: Electromyographic responses to gastric balloon distention through gastrostomy were recorded from the acromiotrapezius muscle in rats after splanchnic nerve resection or vagotomy and in control rats. We then examined the phosphorylated-ERK1/2 (p-ERK1/2) labeling in the DRG and NG after GD using immunohistochemistry., Results: Gastric distention induced p-ERK1/2 in DRG and NG neurons with a peak at 2 minutes after stimulation. We found a stimulus intensity-dependent increase in the number of activated neurons, and this activation corresponded well with the incidence of the visceromotor response. Most of these p-ERK1/2-labeled neurons were small- and medium-sized neurons that coexpressed transient receptor potential vanilloid 1 ion channel and acid-sensing ion channel 3. Splanchnic nerve resection, but not vagotomy, affected the visceromotor response, and attenuated the ERK1/2 activation in DRG neurons produced by GD. Furthermore, intrathecal administration of the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126, altered the response to noxious GD., Conclusions: The activation of ERK1/2 pathways in DRG neurons by noxious GD may be correlated with functional activity, and may be involved in acute visceral pain.

Hexokinase is known as the first enzyme and rate-limiting step in glycolysis. The role of hexokinase activity and localization in regulating the rate of axonal regeneration was studied in cultured adult sensory neurons of dorsal root ganglia (DRG). Immunofluorescent staining of DRG demonstrated that small-medium neurons and satellite cells exhibited high levels of expression of hexokinase I. Large neurons had negative staining for hexokinase I. Intracellular localization and biochemical studies in cultured adult rat sensory neurons revealed that hexokinase I was almost exclusively found in the mitochondrial compartment. The hypothesis that neurotrophic factor dependent activation of Akt would regulate hexokinase association with the mitochondria was tested and quantitative Western blotting showed no effect of blockade of the phosphoinositide 3-kinase (PI 3-kinase)/Akt pathway using the inhibitor LY294002, indicating this interaction of hexokinase with mitochondria was not neurotrophic factor or Akt-dependent. Finally, pharmacological blockade of hexokinase activity and inhibition of localization to the mitochondrial compartment with hexokinase II VDAC binding domain (Hxk2VBD) peptide caused a significant inhibition of neurotrophic factor-directed axon outgrowth. The results support a key role for hexokinase activity and/or localization to the mitochondria in the regulation of neurite outgrowth in cultured adult sensory neurons.

To define the somatotopic arrangement of neurons in the trigeminal spinal subnucleus caudalis and upper cervical cord activated by acute noxious stimulation of various orofacial sites, pERK expression was analyzed in these neurons. After capsaicin injection into the tongue, lower gum, upper and lower lips, or mental region, pERK-like immunoreactive (pERK-LI) cells were distributed mainly in the dorsal half of the trigeminal spinal nucleus interporalis (Vi) and caudalis (Vc) transition zone (Vi/Vc zone), middle Vc, and Vc and upper cervical cord transition zone (Vc/C2 zone). pERK-LI cells were distributed throughout the dorsal to ventral portion of the Vi/Vc zone, middle Vc, and Vc/C2 zone following capsaicin injection into the anterior hard palate, upper gum, buccal mucosa, or vibrissal pad and in the ventral portion of the Vi/Vc zone, middle Vc, and Vc/C2 zone following snout, ophthalmic, or ocular injection of capsaicin. The rostrocaudal distribution area of pERK-LI cells was more extensive from the Vi/Vc zone to the Vc/C2 zone after intraoral injection than that after facial injection, and the rostrocaudal distribution of pERK-LI cells from the Vi/Vc zone to the Vc/C2 zone had a somatotopic arrangement, with the snout being represented most rostrally and ophthalmic, ocular, or mental regions represented most caudally. These findings suggest that the pERK-LI cells expressed from the Vi/Vc zone to the Vc/C2 zone following injection of capsaicin in facial and intraoral structures may be differentially involved in pain perception in facial and intraoral sites., (Copyright 2008 Wiley-Liss, Inc.)

The aim of the present study was to investigate whether direct activation of protein kinase C (PKC) in the spinal cord could change brain activation using a functional magnetic resonance imaging (fMRI) analysis in mice that lack the PKCgamma gene. The activation of spinal PKC by intrathecal (i.t.) injection with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused a time-dependent decrease in paw-withdrawal latency to the heat thermal stimulus. In contrast, i.t. injection of PDBu failed to cause thermal hyperalgesia in mice which lacked the PKCgamma gene. We found that the i.t. injection with PDBu caused a remarkable increase in the activity of several brain regions in wild-type mice compared with vehicle injection. In the somatosensory cortex and lateral and medial thalamus, i.t. injection of PDBu produced a dramatic and time-dependent increase in signal intensity at 1-6h after i.t. PDBu injection. In contrast, i.t. injection of PDBu produced a delayed but significant increase in signal intensity at 3-6h in the cingulate cortex, at 4-6h in the nucleus accumbens and at 3-6h in the ventral tegmental area. In addition, all effects of PDBu were abolished in mice that lacked the PKCgamma gene. These results suggest that the activation of spinal PKCgamma associated with the activation of ascending pain transmission may be an important factor in chronic pain-like hyperalgesia with changes in emotionality.

The distribution of NADPH-diaphorase-reactive (NADPH-dr) neurons and neuronal processes in the cerebral cortex and basal forebrain and their association with parenchymal vessels were studied in normal adult rats using NADPH-d histochemical protocol. The intensely stained cortical interneurons and reactive subcortically originating afferents, and stained microvessels were examined through a light microscope at law (x250) and high (x630) magnifications. NADPH-dr interneurons were concentrated in layers 2-6 of the M1 and M2 areas. However, clear predominance in their concentration (14 +/- 0.8 P < 0.05 per section) was found in layer 6. A mean number of labeled neurons in auditory (AuV), granular and agranular (GI, AIP) areas of the insular cortex was calculated to reach 12.3 +/- 0.7, 18.5 +/- 1.0 and 23.3 +/- 1.7 units per section, respectively (P < 0.05). The distinct apposition of labelled neurons to intracortical vessels was found in the M1, M2. The order of frequency of neurovascular coupling in different zones of the cerebral cortex was as following sequence: AuV (31.2%, n = 1040) > GI (18.0%, n = 640) > S1 (13.3%, n = 720) > M1 (6.3%, n = 1360). A large number of structural associations between labeled cells and vessels in the temporal and insular cortex indicate that NADPH-d-reactive interneurons can contribute to regulation of the cerebral regional blood flow in these areas.

Although it is commonly appreciated that spaced training is superior to massed training in memory formation, the molecular mechanisms underlying this feature of memory are largely unknown. We previously described the selective benefit of multiple spaced (vs massed) training trials in the induction of long-term memory (LTM) for sensitization in Aplysia californica. We now report that LTM can be induced with only two spaced training trials [tail shocks (TSs)] when the second TS is administered 45 min after the first. In contrast, spacing intervals of 15 and 60 min are ineffective. This surprisingly narrow permissive training window for two-trial LTM is accompanied by an equally narrow window of transient mitogen-activated protein kinase (MAPK) activation, a necessary signaling molecule for LTM induction, at 45 min after a single TS. Thus, the transient recruitment of MAPK following a single TS may provide a narrow molecular window for two-trial LTM formation.

Background: All local anesthetics (LAs) are, to some extent, neurotoxic. Toxicity studies have been performed in dissociated neuron cultures, immersing both axon and soma in LA. This approach, however, does not accurately reflect the in vivo situation for peripheral nerve blockade, where LA is applied to the axon alone., Methods: We investigated lidocaine neurotoxicity in compartmental sensory neuron cultures, which are composed of one central compartment containing neuronal cell bodies and a peripheral compartment containing their axons, allowing for selective incubation. We applied lidocaine +/- neuroprotective drugs to neuronal somata or axons, and assessed neuron survival and axonal outgrowth., Results: Lidocaine applied to the peripheral compartment led to a decreased number of axons (to 59% +/- 9%), without affecting survival of cell bodies. During axonal incubation with lidocaine, the p38 mitogen-activated protein kinase inhibitor SB203580 (10 microM) attenuated axonal injury when applied to the axon (insignificant reduction of maximal axonal distance to 93% +/- 9%), but not when applied to the cell body (deterioration of maximal axonal length to 48% +/- 6%). Axonal co-incubation of lidocaine with the caspase inhibitor z-vad-fmk (20 microM) was not protective., Conclusions: Whereas inhibition of either p38 mitogen-activated protein kinase or caspase activity promote neuronal survival after LA treatment of dissociated neuronal cultures, axonal degeneration induced by lidocain (40 mM) is prevented by p38 MAP kinase but not by caspase inhibition. We conclude that processes leading to LA-induced neurotoxicity in dissociated neuronal culture may be different from those observed after purely axonal application.

Neurotrophin receptors of the Trk family play a vital role in the survival of developing neurons and the process of axonogenesis. The Trk family are receptor protein tyrosine kinases (RTKs) and their signalling in response to neurotrophins is critically dependent upon their ability to transphosphorylate and act as signalling centres for multiple adaptor proteins and distinct, downstream pathways. Such phosphotyrosine signalling also depends upon the appropriate counter-regulation by phosphatases. A large family of receptor-like protein tyrosine phosphatases (RPTPs) are also expressed in developing neurons and in this study we have examined the ability of the phosphatase PTPsigma to interact with and regulate Trk proteins in transfected HEK 293T cells. PTPsigma can bind differentially to Trk proteins, binding stably in complexes with TrkA and TrkC, but not TrkB. The transmembrane domains of PTPsigma and TrkA appear to be sufficient for the direct or indirect interaction between these two receptors. Furthermore, PTPsigma is shown to dephosphorylate all three Trk receptors and suppress their phosphorylation in the presence of neurotrophins. In addition, overexpression of PTPsigma in primary sensory neurons in culture inhibits neurite outgrowth without affecting the short-term survival of these neurons. PTPsigma can thus show differential complex formation with different Trk family members and in neurons can selectively target the neurite-forming signalling pathway driven by TrkA.

Activation of P2X(3) and P2X(2/3) receptors (P2X(3)R/P2X(2/3)R), ionotropic ATP receptor subtypes, in primary sensory neurons is involved in neuropathic pain, a debilitating chronic pain that occurs after peripheral nerve injury. However, the underlying mechanisms remain unknown. We investigated the role of cytosolic phospholipase A(2) (cPLA(2)) as a downstream molecule that mediates the P2X(3)R/P2X(2/3)R-dependent neuropathic pain. We found that applying ATP to cultured dorsal root ganglion (DRG) neurons increased the level of Ser505-phosphorylated cPLA(2) and caused translocation of Ser505-phosphorylated cPLA(2) to the plasma membrane. The ATP-induced cPLA(2) activation was inhibited by a selective antagonist of P2X(3)R/P2X(2/3)R and by a selective inhibitor of cPLA(2). In the DRG in vivo, the number of cPLA(2)-activated neurons was strikingly increased after peripheral nerve injury but not after peripheral inflammation produced by complete Freund's adjuvant. Pharmacological blockade of P2X(3)R/P2X(2/3)R reversed the nerve injury-induced cPLA(2) activation in DRG neurons. Moreover, administering the cPLA(2) inhibitor near the DRG suppressed nerve injury-induced tactile allodynia, a hallmark of neuropathic pain. Our results suggest that P2X(3)R/P2X(2/3)R-dependent cPLA(2) activity in primary sensory neurons is a key event in neuropathic pain and that cPLA(2) might be a potential target for treating neuropathic pain.

The effect of streptozotocin (STZ)-induced diabetes on expression and activity of hexokinase, the first enzyme and rate-limiting step in glycolysis, was studied in sensory neurons of lumbar dorsal root ganglia (DRG). The DRG and sciatic nerve of adult rats expressed the hexokinase I isoform only. Immunofluorescent staining of lumbar DRG demonstrated that small-medium neurons and satellite cells exhibited high levels of expression of hexokinase I. Large, mainly proprioceptive neurons, had very low or negative staining for hexokinase I. Intracellular localization and biochemical studies on intact DRG from adult rats and cultured adult rat sensory neurons revealed that hexokinase I was almost exclusively found in the mitochondrial compartment. Duration of STZ-diabetes of 6 or 12 weeks diminished hexokinase activity by 28% and 30%, respectively, in lumbar DRG compared with age matched controls (P<0.05). Quantitative Western blotting showed no effect of diabetes on hexokinase I protein expression in homogenates or mitochondrial preparations from DRG. Immunofluorescent staining for hexokinase I showed no diabetes-dependent change in small-medium neuron expression in DRG, however, large neurons became positive for hexokinase I (P<0.05). Such complex effects of diabetes on hexokinase I expression in the DRG may be due to glucose-driven up-regulation of expression or the result of impaired axonal transport and perikaryal accumulation in the large neuron sub-population. Because hexokinase is the rate-limiting enzyme of glycolysis these results imply that metabolic flux through the glycolytic pathway is reduced in diabetes. This finding, therefore, questions the role of high glucose-induced metabolic flux as a key driving force in reactive oxygen species generation by mitochondria.

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.

Peripheral tissue injury causes the release of various mediators from damaged and inflammatory cells, which in turn activates and sensitizes primary sensory neurons and thereby produces persistent pain. The present study investigated the role of platelet-activating factor (PAF), a phospholipid mediator, in pain signaling using mice lacking PAF receptor (pafr-/- mice). Here we show that pafr-/- mice displayed almost normal responses to thermal and mechanical stimuli but exhibit attenuated persistent pain behaviors resulting from tissue injury by locally injecting formalin at the periphery as well as capsaicin pain and visceral inflammatory pain without any alteration in cytoarchitectural or neurochemical properties in dorsal root ganglion (DRG) neurons and a defect in motor function. However, pafr-/- mice showed no alterations in spinal pain behaviors caused by intrathecally administering agonists for N-methyl-d-aspartate (NMDA) and neurokinin(1) receptors. A PAFR agonist evoked an intracellular Ca(2+) response predominantly in capsaicin-sensitive DRG neurons, an effect was not observed in pafr-/- mice. By contrast, the PAFR agonist did not affect C- or Adelta-evoked excitatory post-synaptic currents in substantia gelatinosa neurons in the dorsal horn. Interestingly, mice lacking PAFR showed reduced phosphorylation of extracellular signal-related protein kinase (ERK), an important kinase for the sensitization of primary sensory neurons, in their DRG neurons after formalin injection. Furthermore, U0126, a specific inhibitor of the ERK pathway suppressed the persistent pain by formalin. Thus, PAFR may play an important role in both persistent pain and the sensitization of primary sensory neurons after tissue injury.

Neuropathic pain that occurs after peripheral nerve injury is poorly controlled by current therapies. Increasing evidence shows that mitogen-activated protein kinase (MAPK) play an important role in the induction and maintenance of neuropathic pain. Here we show that activation of extracellular signal-regulated protein kinases 5 (ERK5), also known as big MAPK1, participates in pain hypersensitivity caused by nerve injury. Nerve injury increased ERK5 phosphorylation in spinal microglia and in both damaged and undamaged dorsal root ganglion (DRG) neurons. Antisense knockdown of ERK5 suppressed nerve injury-induced neuropathic pain and decreased microglial activation. Furthermore, inhibition of ERK5 blocked the induction of transient receptor potential channels and brain-derived neurotrophic factor expression in DRG neurons. Our results show that ERK5 activated in spinal microglia and DRG neurons contributes to the development of neuropathic pain. Thus, blocking ERK5 signaling in the spinal cord and primary afferents has potential for preventing pain after nerve damage.

Background: Drosophila flies explore the environment very efficiently in order to colonize it. They explore collectively, not individually, so that when a few land on a food spot, they attract the others by signs. This behaviour leads to aggregation of individuals and optimizes the screening of mates and egg-laying on the most favourable food spots., Results: Flies perform cycles of exploration/aggregation depending on the resources of the environment. This behavioural ecology constitutes an excellent model for analyzing simultaneous processing of neurosensory information. We reasoned that the decision of flies to land somewhere in order to achieve aggregation is based on simultaneous integration of signals (visual, olfactory, acoustic) during their flight. On the basis of what flies do in nature, we designed laboratory tests to analyze the phenomenon of neuronal coincidence. We screened many mutants of genes involved in neuronal metabolism and the synaptic machinery., Conclusion: Mutants of NO-dependent cyclase show a specifically-marked behaviour phenotype, but on the other hand they are associated with moderate biochemical defects. We show that these mutants present errors in integrative and/or coincident processing of signals, which are not reducible to the functions of the peripheral sensory cells.

Several lines of evidence indicate that phosphatidylinositol 3-kinase (PI3K) and PI3K-protein kinase B/Akt (PKB/Akt) signal pathway mediate the pain hypersensitivity induced by intradermal injection of capsaicin or nerve growth factor. However, the role of PI3K and PI3K-PKB/Akt signal pathway activation in neuropathic pain is still unclear. Using L5 spinal nerve ligation (L5 SNL) and immunohistochemistry, we found that the numbers of phospho-PKB/Akt-immunoreactive (p-PKB/Akt IR) positive neurons were significantly increased in ipsilateral L5 dorsal root ganglia (DRG) and adjacent L4 DRG started at 12 h after surgery and maintained to the 3rd day. Meanwhile, L5 SNL also induced an increased expression of p-PKB/Akt in ipsilateral L5 spinal dorsal horn. Double immunofluorescence staining showed that p-PKB/Akt expressed entirely in DRG neurons, especially in IB4-positive neurons. Intrathecal injection of PI3K inhibitor wortmannin or LY294002 and PKB/Akt inhibitor Akt inhibitor IV or (-)-Deguelin, started before L5 SNL, reduced the behavioral signs of neuropathic pain. Intraperitoneal injection of wortmannin or (-)-Deguelin as above also reduced the pain hypersensitivity. Post-treatment with wortmannin, started at the 1st day or the 3rd day after L5 SNL, decreased abnormal pain behaviors. Whereas the inhibitory effect of Akt inhibitor IV on established neuropathic pain was observed only in those rats that received the drug treatment started at the 1st day. Immunohistochemistry revealed that intrathecal injection of wortmannin significantly inhibited the activation of PKB/Akt in L5 DRG and L5 spinal cord. The data suggested that PI3K and PI3K-PKB/Akt signal pathway activation might contribute to the development of neuropathic pain.

Objectives: The objective of this study was to examine the effects of rofecoxib, meloxicam, both cyclooxygenase-2 (COX-2) inhibitors and aminoguanidine hydrochloride, an inducible nitric oxide synthase (iNOS) inhibitor and their combinations in neuropathic pain in rats., Methods: Neuropathy was induced by chronic constriction injury (CCI) of right sciatic nerve under ketamine anesthesia in rats. Effect of ED(50) of aminoguanidine hydrochloride, rofecoxib and meloxicam administered orally was investigated using behavioral tests. Effect of combinations of aminoguanidine hydrochloride with rofecoxib and meloxicam was also investigated in neuropathic pain employing behavioral tests., Results: Behavioral tests, mechanical, thermal and cold stimuli confirmed the development of neuropathic pain after CCI. Aminoguanidine hydrochloride, rofecoxib and meloxicam when administered alone, produced significant increase in paw withdrawal threshold to mechanical stimuli at 6 h in ipsilateral hind paw after CCI. Co-administration of aminoguanidine hydrochloride (30 mg/kg) with rofecoxib (1.31 mg/kg) and meloxicam (1.34 mg/kg) was also found to produce significant increase in paw withdrawal latencies to mechanical stimuli at 6 h. Combined administration of aminoguanidine hydrochloride with meloxicam and rofecoxib produced significant rise in pain threshold for mechanical hyperalgesia in ipsilateral hind paw when compared with the groups treated with aminoguanidine hydrochloride, meloxicam and rofecoxib alone., Conclusion: Co-administration of meloxicam and rofecoxib with aminoguanidine hydrochloride may be an alternative approach for the treatment of neuropathic pain.

Mutations in the thymidine kinase gene (tk) of herpes simplex virus type 1 (HSV-1) explain most cases of virus resistance to acyclovir (ACV) treatment. Mucocutaneous lesions of patients with ACV resistance contain mixed populations of tk mutant and wild-type virus. However, it is unknown whether human ganglia also contain mixed populations since the replication of HSV tk mutants in animal neurons is impaired. Here we report the detection of mutated HSV tk sequences in human ganglia. Trigeminal and dorsal root ganglia were obtained at autopsy from an immunocompromised woman with chronic mucocutaneous infection with ACV-resistant HSV-1. The HSV-1 tk open reading frames from ganglia were amplified by PCR, cloned, and sequenced. tk mutations were detected in a seven-G homopolymer region in 11 of 12 ganglia tested, with clonal frequencies ranging from 4.2 to 76% HSV-1 tk mutants per ganglion. In 8 of 11 ganglia, the mutations were heterogeneous, varying from a deletion of one G to an insertion of one to three G residues, with the two-G insertion being the most common. Each ganglion had its own pattern of mutant populations. When individual neurons from one ganglion were analyzed by laser capture microdissection and PCR, 6 of 14 HSV-1-positive neurons were coinfected with HSV tk mutants and wild-type virus, 4 of 14 were infected with wild-type virus alone, and 4 of 14 were infected with tk mutant virus alone. These data suggest that diverse tk mutants arise independently under drug selection and establish latency in human sensory ganglia alone or together with wild-type virus.

Glutamatergic inputs arising from the parabrachial nucleus to neurons in the lateral sector of the central amygdala were studied in vitro. Tetanic stimulation of these inputs led to LTP that did not require activation of NMDA receptors or a rise of postsynaptic calcium. LTP was accompanied by a reduction in the paired-pulse ratio, indicating that LTP results from an increase in transmitter release probability. Activation of adenylyl cyclase with forskolin potentiated these inputs with a similar reduction in paired-pulse facilitation and occluded LTP induction. LTP was inhibited by the protein kinase A blocker H89. Low-frequency stimulation led to LTD that required activation of postsynaptic NMDA receptors and a rise in postsynaptic calcium. There was no change in paired-pulse facilitation with LTD. LTD was blocked by protein phosphatase blockers calyculin and okadaic acid. We conclude that parabrachial inputs to the lateral sector of the central amygdala show presynaptic LTP that requires activation of a presynaptic protein kinase A via a calcium-dependent adenylyl cyclase while LTD at the same synapses is postsynaptic and requires a rise in postsynaptic calcium and activation of protein phosphatase.