IGF1-driven induction of GPCR kinase 2 in the primary afferent neuron promotes resolution of acute hyperalgesia.

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.

Expression pattern of type 3 adenylyl cyclase in rodent dorsal root ganglion and its primary afferent terminals.

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.

Distribution pattern of dorsal root ganglion neurons synthesizing nitric oxide synthase in different animal species.

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.

Tyrosine Hydroxylase Expression in Type II Cochlear Afferents in Mice.

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.

Acetylated tubulin is essential for touch sensation in mice.

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.

PI3K mediated activation of GSK-3ß reduces at-level primary afferent growth responses associated with excitotoxic spinal cord injury dysesthesias.

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-3ß (GSK-3ß) 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-3ß signaling resulting in enhanced DRG growth responses, and that PI3K mediated activation of GSK-3ß 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-3ß in the superficial spinal cord dorsal horn and adjacent DRG. Double immunofluorescent staining showed that GSK-3ß(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-3ß activator, significantly decreased GSK-3ß(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-3ß in primary afferents and enhanced at-level DRG intrinsic growth (neurite elongation and initiation). Early PI3K mediated activation of GSK-3ß attenuated QUIS-induced DRG neurite outgrowth and prevented the development of at-level dysesthesias.

Glucagon directly interacts with vagal afferent nodose ganglion neurons to induce Ca(2+) signaling via glucagon receptors.

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.

Extracellular caspase-6 drives murine inflammatory pain via microglial TNF-α secretion.

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.

Caspase-2 is upregulated after sciatic nerve transection and its inhibition protects dorsal root ganglion neurons from apoptosis after serum withdrawal.

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.

Prostatic acid phosphatase is required for the antinociceptive effects of thiamine and benfotiamine.

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.

Generation of a pain memory in the primary afferent nociceptor triggered by PKCε activation of CPEB.

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.

Sensitization of dural afferents underlies migraine-related behavior following meningeal application of interleukin-6 (IL-6).

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.

Phenotype of distinct primary sensory afferent subpopulations and caspase-3 expression following axotomy.

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.

Live-imaging of PKC translocation in Sf9 cells and in aplysia sensory neurons.

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, ßI, ßII 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.

Triptan-induced enhancement of neuronal nitric oxide synthase in trigeminal ganglion dural afferents underlies increased responsiveness to potential migraine triggers.

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.

Kinase regulation of Na+-K+-2Cl- cotransport in primary afferent neurons.

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.

Regulation of the spontaneous augmentation of Na(V)1.9 in mouse dorsal root ganglion neurons: effect of PKA and PKC pathways.

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.

[Nitric oxide in the mechanisms of afferent innervations of arteries of the brain].

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).

Activation of p-38alpha MAPK contributes to neuronal hyperexcitability in caudal regions remote from spinal cord injury.

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.

TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms.

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.