DRG Neurons (drg + neuron)

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Distribution within Life Sciences

Selected Abstracts

,-Conotoxin CVIB differentially inhibits native and recombinant N- and P/Q-type calcium channels

Leonid Motin
Abstract ,-Conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist ,-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. ,-Conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 0.05 and 7.64 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (, 66% of total) appeared to be irreversible for >,30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (, 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (, 22% of total) was attributable to P/Q-type VGCCs. ,-Conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (CaV2.2) and P/Q- (CaV2.1), but not R- (CaV2.3) type VGCCs expressed in Xenopus oocytes. The ,2,1 auxiliary subunit co-expressed with CaV2.2 and CaV2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. ,-Conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information. [source]

Ultrastructural evidence for a pre- and postsynaptic localization of full-length trkB receptors in substantia gelatinosa (lamina II) of rat and mouse spinal cord

Chiara Salio
Abstract Brain-derived neurotrophic factor (BDNF) exerts its trophic effects by acting on the high-affinity specific receptor trkB. BDNF also modulates synaptic transmission in several areas of the CNS, including the spinal cord dorsal horn, where it acts as a pain modulator by yet incompletely understood mechanisms. Spinal neurons are the main source of trkB in lamina II (substantia gelatinosa). Expression of this receptor in dorsal root ganglion (DRG) cells has been a matter of debate, whereas a subpopulation of DRG neurons bears trkA receptors and contains BDNF. By the use of two different trkB antibodies we observed that 7.7% and 10.8% of DRG neurons co-expressed BDNF + trkB but not trkA, respectively, in rat and mouse. Ultrastructurally, full-length trkB (fl-trkB) receptors were present at somato-dendritic membranes of lamina II neurons (rat: 66.8%; mouse: 73.8%) and at axon terminals (rat: 33.2%; mouse: 26.2%). In both species, about 90% of these terminals were identified as primary afferent fibres (PAFs) considering their morphology and/or neuropeptide content. All fl-trkB-immunopositive C boutons in type Ib glomeruli were immunoreactive for BDNF and, at individual glomeruli and axo-dendritic synapses, fl-trkB receptors were located in a mutually exclusive fashion at pre- or postsynaptic membranes. Thus, only a small fraction of fl-trkB-immunoreactive dendrites were postsynaptic to BDNF-immunopositive PAFs. This is the first ultrastructural description of fl-trkB localization at synapses between first- and second-order sensory neurons in lamina II, and suggests that BDNF may be released by fl-trkB-immunopositive PAFs to modulate nociceptive input in this lamina of dorsal horn. [source]

Dynamic changes in glypican-1 expression in dorsal root ganglion neurons after peripheral and central axonal injury

Stefan Bloechlinger
Abstract Glypican-1, a glycosyl phosphatidyl inositol (GPI)-anchored heparan sulphate proteoglycan expressed in the developing and mature cells of the central nervous system, acts as a coreceptor for diverse ligands, including slit axonal guidance proteins, fibroblast growth factors and laminin. We have examined its expression in primary sensory dorsal root ganglion (DRG) neurons and spinal cord after axonal injury. In noninjured rats, glypican-1 mRNA and protein are constitutively expressed at low levels in lumbar DRGs. Sciatic nerve transection results in a two-fold increase in mRNA and protein expression. High glypican-1 expression persists until the injured axons reinnervate their peripheral targets, as in the case of a crushed nerve. Injury to the central axons of DRG neurons by either a dorsal column injury or a dorsal root transection also up-regulates glypican-1, a feature that differs from most DRG axonal injury-induced genes, whose regulation changes only after peripheral and not central axonal injury. After axonal injury, the cellular localization of glypican-1 changes from a nuclear pattern restricted to neurons in noninjured DRGs, to the cytoplasm and membrane of injured neurons, as well as neighbouring non-neuronal cells. Sciatic nerve transection also leads to an accumulation of glypican-1 in the proximal nerve segment of injured axons. Glypican-1 is coexpressed with robo 2 and its up-regulation after axonal injury may contribute to an altered sensitivity to axonal growth or guidance cues. [source]

Background potassium channel block and TRPV1 activation contribute to proton depolarization of sensory neurons from humans with neuropathic pain

Thomas K. Baumann
Abstract Protons cause a sustained depolarization of human dorsal root ganglion (DRG) neurons [Baumann et al. (1996) Pain, 65, 31,38]. In the present study we sought to determine which ion channels are expressed in human DRG neurons that could mediate the sustained responses observed in the patch-clamp recordings. RT-PCR of material from the DRG tissue revealed the presence of mRNAs for a nonselective cation channel that is activated by protons (TRPV1) and background potassium channels that are blocked by protons (TASK-1, TASK-3 and Kir2.3). Highly acidic solution (pH 5.4) applied to cultured DRG neurons evoked prolonged currents that were associated with a net increase in membrane conductance. Consistent with the involvement of TRPV1, these proton-evoked currents were blocked by capsazepine and were only found in neurons that responded to capsaicin with an increase in membrane conductance. Less acidic extracellular solution (pH 6.0) evoked such currents only rarely, but was able to strongly enhance the currents evoked by capsaicin. Capsazepine (1 m) blocked the currents evoked by capsaicin at pH 7.35, as well as the potentiated responses to capsaicin at pH 6.0. In neurons that were not excited by capsaicin, moderate extracellular acidification (pH 6.0) caused a sustained decrease in resting membrane conductance. The decrease in membrane conductance by protons was associated with inhibition of background potassium channels. This excitatory effect of protons was not blocked by capsazepine. We conclude that in most neurons the sustained depolarization in response to moderately acidic solutions is the result of blocked background potassium channels. In a subset of neurons, TRPV1 also contributes. [source]

c-Src kinase activation regulates preprotachykinin gene expression and substance P secretion in rat sensory ganglia

Orisa J. Igwe
Abstract Increased synthesis of substance P (SP) in the dorsal root ganglia (DRG) and enhanced axonal transport to and secretion from the primary afferent sensory neurons might enhance pain signalling in the spinal dorsal horn by modifying pronociceptive pathways. IL-1, increases SP synthesis by enhancing the expression of preprotachykinin (PPT) mRNA encoding for SP and other tachykinins in the DRG. Stimulation of IL-1 receptor by IL-1, may induce the phosphorylation of tyrosine residues in many effector proteins through the activation of p60c-src kinase. The hypothesis that the synthesis of SP in and secretion from the primary sensory ganglia are regulated by the activation of p60c-src kinase induced by IL-1, was tested. Pretreatment of DRG neurons in culture with herbimycin A, genistein or PP2, three structurally different nonreceptor tyrosine kinase inhibitors that act by different mechanisms, decreased the kinase activity of p60c-src induced by the activation of IL-1 receptor. PP3, a negative control for the Src family of tyrosine kinase inhibitor PP2 had no effect. Herbimycin A and genistein also decreased IL-1,-induced expression of PPT mRNA-encoding transcripts and the levels of SP-li synthesized in the cells and secreted into the culture medium in a concentration-dependent manner. SB 203580 [a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor] and PD 98059 (a p44/42 MAPK kinase inhibitor) were ineffective in modulating IL-1,-induced SP synthesis and secretion, and p60c-src kinase activity in DRG neurons. Whereas, IL-1 receptor antagonist and cycloheximide inhibited IL-1,-evoked secretion of SP-like immunoreactivity (SP-li), actinomycin D decreased it significantly but did not entirely abolish it. These findings show that phosphorylation of specific protein tyrosine residue(s) following IL-1 receptor activation might play a key role in IL-1, signalling to modulate PPT gene expression and SP secretion in sensory neurons. In view of the role of SP as an immunomodulator, these studies provide a new insight into neural-immune intercommunication in pain regulation in the sensory ganglia through the IL-1,-induced p60c-src activation. [source]

Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord

Lan Bao
Abstract Peripheral axotomy-induced sprouting of thick myelinated afferents (A-fibers) from laminae III,IV into laminae I,II of the spinal cord is a well-established hypothesis for the structural basis of neuropathic pain. However, we show here that the cholera toxin B subunit (CTB), a neuronal tracer used to demonstrate the sprouting of A-fibers in several earlier studies, also labels unmyelinated afferents (C-fibers) in lamina II and thin myelinated afferents in lamina I, when applied after peripheral nerve transection. The lamina II afferents also contained vasoactive intestinal polypeptide and galanin, two neuropeptides mainly expressed in small dorsal root ganglion (DRG) neurons and C-fibers. In an attempt to label large DRG neurons and A-fibers selectively, CTB was applied four days before axotomy (pre-injury-labelling), and sprouting was monitored after axotomy. We found that only a small number of A-fibers sprouted into inner lamina II, a region normally innervated by C-fibers, but not into outer lamina II or lamina I. Such sprouts made synaptic contact with dendrites in inner lamina II. Neuropeptide Y (NPY) was found in these sprouts in inner lamina II, an area very rich in Y1 receptor-positive processes. These results suggest that axotomy-induced sprouting from deeper to superficial layers is much less pronounced than previously assumed, in fact it is only marginal. This limited reorganization involves large NPY immunoreactive DRG neurons sprouting into the Y1 receptor-rich inner lamina II. Even if quantitatively small, it cannot be excluded that this represents a functional circuitry involved in neuropathic pain. [source]

Low-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitive

Antonia Marn-Burgin
Abstract The heat-transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin-insensitive (chick) and a capsaicin-sensitive (rat) species. In chick, whole-cell patch-clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at ,,43 C and ,,53 C. In cobalt uptake experiments, the proportion of neurons showing a heat-induced response increased with increasing heat stimuli. Application of capsaicin (1,10 ,m) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin-sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1,10 ,m) was effective in blocking heat-induced responses, verified by patch-clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 ,m) reduced to almost nil the proportion of heat-responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low-threshold heat-transducing receptor with a pharmacological profile distinct from the low-threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site. [source]

Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues

FEBS JOURNAL, Issue 6 2001
Sven Moosmang
Hyperpolarization-activated cation currents (Ih) have been identified in cardiac pacemaker cells and a variety of central and peripheral neurons. Four members of a gene family encoding hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN1,4) have been cloned recently. Native Ih currents recorded from different cell types exhibit distinct activation kinetics. To determine if this diversity of Ih currents may be caused by differential expression of HCN channel isoforms, we investigated the cellular distribution of the transcripts of HCN1,4 in the murine sinoatrial node, retina and dorsal root ganglion (DRG) by in situ hybridization. In the sinoatrial node, the most prominently expressed HCN channel is HCN4, whereas HCN2 and HCN1 are detected there at moderate and low levels, respectively. Retinal photoreceptors express high levels of HCN1, whereas HCN2, 3 and 4 were not found in these cells. In DRG neurons, the dominant HCN transcript is HCN1, followed by HCN2. We next determined the functional properties of recombinant HCN1,4 channels expressed in HEK293 cells. All four channel types gave rise to Ih currents but displayed marked differences in their activation kinetics. Our results suggest that the heterogeneity of native Ih currents is generated, at least in part, by the tissue-specific expression of HCN channel genes. [source]

Few cultured rat primary sensory neurons express a tolbutamide-sensitive K+ current

Violeta Ristoiu
Abstract The response of dorsal root ganglion (DRG) neurons to metabolic inhibition is known to involve calcium-activated K+ channels; in most neuronal types ATP-sensitive K+ channels (KATP) also contribute, but this is not yet established in the DRG. We have investigated the presence of a KATP current using whole-cell recordings from rat DRG neurons, classifying the neurons functionally by their "current signature" (Petruska et al, J Neurophysiol 84: 2365,2379, 2000). We clearly identified a KATP current in only 1 out of 62 neurons, probably a nociceptor. The current was activated by cyanide (2 mM NaCN) and was sensitive to 100 ,M tolbutamide; the relation between reversal potential and external K+ concentration indicated it was a K+ current. In a further two neurons, cyanide activated a K+ current that was only partially blocked by tolbutamide, which may also be an atypical KATP current. We conclude that KATP channels are expressed in normal DRG, but in very few neurons and only in nociceptors. [source]

Substance P release evoked by capsaicin or potassium from rat cultured dorsal root ganglion neurons is conversely modulated with bradykinin

He-Bin Tang
Abstract To clarify the molecular mechanism of substance P (SP) release from dorsal root ganglion (DRG) neurons, we investigated the involvement of several intracellular effectors in the regulation of SP release evoked by capsaicin, potassium or/and bradykinin. Bradykinin-evoked SP release from cultured adult rat DRG neurons was attenuated by either the mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) or cycloheximide. As the long-term exposure of DRG neurons to bradykinin (3 h) resulted in extracellular signal-regulated kinase (ERK) phosphorylation at an early stage and thereafter induced cyclooxygenase-2 (COX-2) protein expression, which both contribute to the SP release triggered by bradykinin B2 receptor. The long-term exposure of DRG neurons to bradykinin enhanced the SP release by capsaicin, but attenuated that by potassium. Interestingly, the inositol 1,4,5-triphosphate (IP3)-induced calcium release blocker [2-aminoethyl diphenylborinate (2-APB)] not only inhibited the potassium-evoked SP release, but also completely abolished the enhancement of capsaicin-induced SP release by bradykinin from cultured DRG neurons. Together, these findings suggest that the molecular mechanisms of SP release by bradykinin involve the activation of MEK, and also require the de novo protein synthesis of COX-2 in DRG neurons. The IP3 -dependent calcium release could be involved in the processes of the regulation by bradykinin of capsaicin-triggered SP release. [source]

Semaphorin 3A and neurotrophins: a balance between apoptosis and survival signaling in embryonic DRG neurons

Ayal Ben-Zvi
Abstract Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12,E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)- and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels. [source]

Cannabinoid,vanilloid receptor interactions in pain signaling

V. Di Marzo
Agents that activate cannabinoid CB1 receptors for marijuana's active principal, THC, or vanilloid VR1 receptors for red chilli peppers' pungent ingredient, capsaicin, modulate pain perception. Stimulation of presynaptic CB1 leads to inhibition of glutamate release in the spinal cord, whereas VR1 stimulation causes release of substance P and CGRP from DRG neurons. VR1 undergoes rapid desensitization by its agonists, which makes VR1-expressing neurons insensitive to subsequent stimulation and results in analgesia. Thus, both CB1 and VR1, which are coexpressed in several spinal and DRG neurons, are targets for analgesic drug development. CB1 and VR1 also share endogenous agonists, namely anandamide, NADA and some of their analogs, and may be regarded as metabotropic and ionotropic receptors for the same family of mediators, with opposing roles in pain perception. The development of ,hybrid' CB1/VR1 agonists as potent analgesics and the functional relationships between CB1 and VR1 in sensory neurons will be discussed. [source]

Oxidative stress in the pathogenesis of experimental diabetic neuropathy

P. A. Low
We evaluated the effects of chronic hyperglycemia on L5 DRG neurons. Experimental diabetic neuropathy (EDN) was induced by streptozotocin. We studied peripheral nerve after 1, 3, 12 months of diabetes. A conduction deficit was present from the first month and persisted over 12 months, affecting mainly sensory fibers. 8-Hydroxy-deoxyguanosine labeling was significantly increased at all time points in DRG neurons, indicating oxidative injury. Caspase-3 labeling was increased at all three time-points, indicating commitment to the efferent limb of the apoptotic pathway. Apoptosis was confirmed by a significant increase in the percent of neurons undergoing apoptosis (TUNEL staining) at 1 month (8%), 3 months (7%) and 12 months (11%). Morphometry of DRG showed a selective loss (42%) of the largest neurons. These findings support the concept that oxidative stress leads to oxidative injury of DRG neurons, with mitochondrium as a specific target, leading to apoptosis and a predominantly sensory neuropathy. [source]

Transient rise in intracellular calcium produces a long-lasting increase in plasma membrane calcium pump activity in rat sensory neurons

William J. Pottorf II
Abstract The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Calmodulin stimulates PMCA activity and for some isoforms this activation persists following clearance of Ca2+ owing to the slow dissociation of calmodulin. We tested the hypothesis that PMCA-mediated Ca2+ efflux from rat dorsal root ganglion (DRG) neurons in culture would remain stimulated following increases in intracellular Ca2+ concentration ([Ca2+]i). PMCA-mediated Ca2+ extrusion was recorded following brief trains of action potentials using indo-1-based photometry in the presence of cyclopiazonic acid. A priming stimulus that increased [Ca2+]i to 506 28 nm (>15 min) increased the rate constant for [Ca2+]i recovery by 47 3%. Ca2+ clearance from subsequent test stimuli remained accelerated for up to an hour despite removal of the priming stimulus and a return to basal [Ca2+]i. The acceleration depended on the magnitude and duration of the priming [Ca2+]i increase, but was independent of the source of Ca2+. Increases in [Ca2+]i evoked by prolonged depolarization, sustained trains of action potentials or activation of vanilloid receptors all accelerated Ca2+ efflux. We conclude that PMCA-mediated Ca2+ efflux in DRG neurons is a dynamic process in which intense stimuli prime the pump for the next Ca2+ challenge. [source]

Involvement of nerve injury and activation of peripheral glial cells in tetanic sciatic stimulation-induced persistent pain in rats

Lingli Liang
Abstract Tetanic stimulation of the sciatic nerve (TSS) produces long-lasting pain hypersensitivity in rats. Long-term potentiation (LTP) of C- and A-fiber-evoked field potentials in the spinal cord has been explored as contributing to central sensitization in pain pathways. However, the peripheral mechanism underlying TSS-induced pain hypersensitivity remains largely unknown. We investigated the effect of TSS on peripheral nerve and the expression of activating transcription factor 3 (ATF3) in dorsal root ganglion (DRG) as a marker of neuronal injury. TSS induced a mechanical allodynia for at least 35 days and induced ATF3 expression in the ipsilateral DRG. ATF3 is colocalized with NF200-labeled myelinated DRG neurons or CGRP- and IB4-labeled unmyelinated ones. Furthermore, we found that TSS induced Wallerian degeneration of sciatic nerve at the level of myelinisation by S100 protein (to label Schwann cells) immunohistochemistry, luxol fast blue staining, and electron microscopy. TSS also elicited the activation of satellite glial cells (SGCs) and enhanced the colocalization of GFAP and P2X7 receptors. Repeated local treatment with tetrodotoxin decreased GFAP expression in SGCs and behavioral allodynia induced by TSS. Furthermore, reactive microglia and astrocytes were found in the spinal dorsal horn after TSS. These results suggest that TSS-induced nerve injury and glial activation in the DRG and spinal dorsal horn may be involved in cellular mechanisms underlying the development of persistent pain after TSS and that TSS-induced nerve injury may be used as a novel neuropathic pain model. 2010 Wiley-Liss, Inc. [source]

Differential clustering of Caspr by oligodendrocytes and Schwann cells

Menahem Eisenbach
Abstract Formation of the paranodal axoglial junction (PNJ) requires the presence of three cell adhesion molecules: the 155-kDa isoform of neurofascin (NF155) on the glial membrane and a complex of Caspr and contactin found on the axolemma. Here we report that the clustering of Caspr along myelinated axons during development differs fundamentally between the central (CNS) and peripheral (PNS) nervous systems. In cultures of Schwann cells (SC) and dorsal root ganglion (DRG) neurons, membrane accumulation of Caspr was detected only after myelination. In contrast, in oligodendrocytes (OL)/DRG neurons cocultures, Caspr was clustered upon initial glial cell contact already before myelination had begun. Premyelination clustering of Caspr was detected in cultures of oligodendrocytes and retinal ganglion cells, motor neurons, and DRG neurons as well as in mixed cell cultures of rat forebrain and spinal cords. Cocultures of oligodendrocyte precursor cells isolated from contactin- or neurofascin-deficient mice with wild-type DRG neurons showed that clustering of Caspr at initial contact sites between OL processes and the axon requires glial expression of NF155 but not of contactin. These results demonstrate that the expression of membrane proteins along the axolemma is determined by the type of the contacting glial cells and is not an intrinsic characteristic of the axon. 2009 Wiley-Liss, Inc. [source]

Impaired inhibitory G-protein function contributes to increased calcium currents in rats with diabetic neuropathy

KE Hall
There is a growing body of evidence that sensory neuropathy in diabetes is associated with abnormal calcium signaling in dorsal root ganglion (DRG) neurons. Enhanced influx of calcium via multiple high-threshold calcium currents is present in sensory neurons of several models of diabetes mellitus, including the spontaneously diabetic BioBred/Worchester (BB/W) rat and the chemical streptozotocin (STZ)-induced rat. We believe that abnormal calcium signaling in diabetes has pathologic significance as elevation of calcium influx and cytosolic calcium release has been implicated in other neurodegenerative conditions characterized by neuronal dysfunction and death. Using electrophysiologic and pharmacologic techniques, the present study provides evidence that significant impairment of G-protein-coupled modulation of calcium channel function may underlie the enhanced calcium entry in diabetes. N- and P-type voltage-activated, high-threshold calcium channels in DRGs are coupled to mu opiate receptors via inhibitory G(o)-type G proteins. The responsiveness of this receptor coupled model was tested in dorsal root ganglion (DRG) neurons from spontaneously-diabetic BB/W rats, and streptozotocin-induced (STZ) diabetic rats. Intracellular dialysis with GTPgammaS decreased calcium current amplitude in diabetic BB/W DRG neurons compared with those of age-matched, nondiabetic controls, suggesting that inhibitory G-protein activity was diminished in diabetes, resulting in larger calcium currents. Facilitation of calcium current density (I(DCa)) by large-amplitude depolarizing prepulses (proposed to transiently inactivate G proteins), was significantly less effective in neurons from BB/W and STZ-induced diabetic DRGs. Facilitation was enhanced by intracellular dialysis with GTPgammaS, decreased by pertussis toxin, and abolished by GDPbetaS within 5 min. Direct measurement of GTPase activity using opiate-mediated GTPgamma[(35)S] binding, confirmed that G-protein activity was significantly diminished in STZ-induced diabetic neurons compared with age-matched nondiabetic controls. Diabetes did not alter the level of expression of mu opiate receptors and G-protein alpha subunits. These studies indicate that impaired regulation of calcium channels by G proteins is an important mechanism contributing to enhanced calcium influx in diabetes. [source]

Electroacupuncture attenuates visceral hyperalgesia and inhibits the enhanced excitability of colon specific sensory neurons in a rat model of irritable bowel syndrome

G.-y. Xu
Abstract, The causes of irritable bowel syndrome remain elusive and there are few effective treatments for pain in this syndrome. Electroacupunture (EA) is used extensively for treatment of various painful conditions including chronic visceral hyperalgesia (CVH). However, mechanism of its analgesic effect remains unknown. This study was designed to investigate effect of EA on colon specific dorsal root ganglion (DRG) neurons in rats with CVH. CVH was induced by intracolonic injection of acetic acid (AA) in 10-day-old rats. Electromyography and patch clamp recordings were performed at age of 8,10 weeks. Colon DRG neurons were labelled by injection of DiI into the colon wall. EA was given at ST36 in both hindlimbs. As adults, neonatal AA-injected rats displayed an increased sensitivity to colorectal distension (CRD) and an enhanced excitability of colon DRG neurons. EA treatment for 40 min significantly attenuated the nociceptive responses to CRD in these rats; this attenuation was reversed by pretreatment with naloxone. EA treatment for 40 min per day for 5 days produced a prolonged analgesic effect and normalized the enhanced excitability of colon DRG neurons. Furthermore, in vitro application of [D-Ala2, N -MePhe4, Gly5 -Ol] enkephalin (DAMGO) suppressed the enhanced excitability of colon neurons from rats with CVH. These findings suggest that EA produced-visceral analgesia, which might be mediated in a large part by endogenous opioids pathways, is associated with reversal of the enhanced excitability of colon DRG neurons in rats with CVH. [source]

5-Hydroxytryptamine selectively activates the vagal nodose C-fibre subtype in the guinea-pig oesophagus

S. Yu
Abstract, The afferent neurons innervating the oesophagus originate from two embryonic sources: neurons located in vagal nodose ganglia originate from embryonic placodes and neurons located in vagal jugular and spinal dorsal root ganglia (DRG) originate from the neural crest. Here, we address the hypothesis that 5-hydroxytryptamine (5-HT) differentially stimulates afferent nerve subtypes in the oesophagus. Extracellular recordings of single unit activity originating from nerve terminals were made in the isolated innervated guinea-pig oesophagus. Whole cell patch clamp recordings (35 C) were made from the primary afferent neurons retrogradely labelled from the oesophagus. 5-Hydroxytryptamine (10 ,mol L,1) activated vagal nodose C-fibres (70%) in the oesophagus but failed to activate overtly vagal jugular nerve fibres and oesophagus-specific spinal DRG neurons. The response to 5-HT in nodose C-fibre nerve terminals was mimicked by the selective 5-HT3 receptor agonist 2-methyl-5-HT (10 ,mol L,1) and nearly abolished by the 5-HT3 receptor antagonists ondansetron (10 ,mol L,1) and Y-25130 (10 ,mol L,1). In patch clamp studies, 2-methyl-5-HT (10 ,mol L,1) activated a proportion of isolated oesophagus-specific nodose capsaicin-sensitive neurons (putative cell bodies of nodose C-fibres). We conclude that the responsiveness to 5-HT discriminates placode-derived (vagal nodose) C-fibres from the neural crest-derived (vagal jugular and spinal DRG) afferent nerves in the oesophagus. The response to 5-HT in nodose C-fibres is mediated by the 5-HT3 receptor in their neuronal membrane. [source]

C-fiber (Remak) bundles contain both isolectin B4-binding and calcitonin gene-related peptide-positive axons

Beth Brianna Murinson
Abstract Unmyelinated nerve fibers (Remak bundles) in the rodent sciatic nerve typically contain multiple axons. This study asked whether C-fiber bundles contain axons arising from more than one type of neuron. Most small neurons of the lumbar dorsal root ganglion (DRG) are either glial cell line-derived neurotrophic factor dependent or nerve growth factor dependent, binding either isolectin B4 (IB4) or antibodies to calcitonin gene-related peptide (CGRP), respectively. Injection of IB4-conjugated horseradish peroxidase into a lumbar DRG resulted in intense labeling of IB4 axons, with very low background. Visualized by confocal fluorescence, IB4-binding and CGRP-positive nerve fibers orginating from different DRG neurons came together and remained closely parallel over long distances, suggesting that these two types of axon occupy the same Remak bundle. With double-labeling immunogold electron microscopy (EM), we confirmed that IB4 and CGRP axons were distinct and were found together in single Remak bundles. Previous studies indicate that some DRG neurons express both CGRP and IB4 binding. To ensure that our immunogold results were not a consequence of coexpression, we studied large populations of unmyelinated axons by using quantitative single-label EM. Tetramethylbenzidine, a chromogen with strong intrinsic signal amplification of IB4-horseradish peroxidase, labeled as many as 52% of unmyelinated axons in the dorsal root. Concomitantly, 97% of the Remak bundles with more than one axon contained at least one IB4-labeled axon. Probabilistic modeling using binomial distribution functions rejected the hypothesis that IB4 axons segregate into IB4-specific bundles (P < 0.00001). We conclude that most Remak bundle Schwann cells simultaneously support diverse axon types with different growth factor dependences. J. Comp. Neurol. 484:392,402, 2005. 2005 Wiley-Liss, Inc. [source]

Glial cell line-derived neurotrophic factor-responsive and neurotrophin-3-responsive neurons require the cytoskeletal linker protein dystonin for postnatal survival

Julie A. Carlsten
Abstract We have investigated the fate of different neurotrophin-responsive subpopulations of dorsal root ganglion neurons in dystonia musculorum (dt) mice. These mice have a null mutation in the cytoskeletal linker protein, dystonin. Dystonin is expressed by all sensory neurons and cross links actin filaments, intermediate filaments, and microtubules. The dt mice undergo massive sensory neurodegeneration postnatally and die at around 4 weeks of age. We assessed the surviving and degenerating neuronal populations by comparing the dorsal root ganglion (DRG) neurons and central and peripheral projections in dt mice and wildtype mice. Large, neurofilament-H-positive neurons, many of which are muscle afferents and are neurotrophin-3 (NT-3)-responsive, were severely decreased in number in dt DRGs. The loss of muscle afferents was correlated with a degeneration of muscle spindles in skeletal muscle. Nerve growth factor (NGF)-responsive populations, which were visualized using calcitonin gene-related peptide and p75, appeared qualitatively normal in the lumbar spinal cord, DRG, and hindlimb skin. In contrast, glial cell line-derived neurotrophic factor (GDNF)-responsive populations, which were visualized using the isolectin B-4 and thiamine monophosphatase, were severely diminished in the lumbar spinal cord, DRG, and hindlimb skin. Analysis of NT-3, NGF, and GDNF mRNA levels using semiquantitative reverse transcriptase-polymerase chain reaction revealed normal trophin synthesis in the peripheral targets of dt mice, arguing against decreased trophic synthesis as a possible cause of neuronal degeneration. Thus, the absence of dystonin results in the selective survival of NGF-responsive neurons and the postnatal degeneration of many NT-3- and GDNF-responsive neurons. Our results reveal that the loss of this ubiquitously expressed cytoskeletal linker has diverse effects on sensory subpopulations. Moreover, we show that dystonin is critical for the maintenance of certain DRG neurons, and its function may be related to neurotrophic support. J. Comp. Neurol. 432:155,168, 2001. 2001 Wiley-Liss, Inc. [source]

Intracellular calcium regulation among subpopulations of rat dorsal root ganglion neurons

Shao-Gang Lu
Primary afferent neurons are functionally heterogeneous. To determine whether this functional heterogeneity reflects, in part, heterogeneity in the regulation of the concentration of intracellular Ca2+ ([Ca2+]i), the magnitude and decay of evoked Ca2+ transients were assessed in subpopulations of dorsal root ganglion (DRG) neurons with voltage clamp and fura-2 ratiometric imaging. To determine whether differences in evoked Ca2+ transients among subpopulations of DRG neurons reflected differences in the contribution of Ca2+ regulatory mechanisms, pharmacological techniques were employed to assess the contribution of influx, efflux, release and uptake pathways. Subpopulations of DRG neurons were defined by cell body size, binding of the plant lectin IB4 and responsiveness to the algogenic compound capsaicin (CAP). Ca2+ transients were evoked with 30 mm K+ or voltage steps to 0 mV. There were marked differences between subpopulations of neurons with respect to both the magnitude and decay of the Ca2+ transient, with the largest and most slowly decaying Ca2+ transients in small-diameter, IB4 -positive, CAP-responsive neurons. The smallest and most rapidly decaying transients were in large-diameter, IB4 -negative and CAP-unresponsive DRG neurons. These differences were not due to a differential distribution of voltage-gated Ca2+ currents. However, these differences did appear to reflect a differential contribution of other influx, efflux, release and uptake mechanisms between subpopulations of neurons. These results suggest that electrical activity in subpopulations of DRG neurons will have a differential influence on Ca2+ -regulated phenomena such as spike adaptation, transmitter release and gene transcription. Significantly more activity should be required in large-diameter non-nociceptive afferents than in small-diameter nociceptive afferents to have a comparable influence on these processes. [source]

Localization of Vesicular Glutamate Transporter 2 mRNA in the Dorsal Root Ganglion of the Pigeon (Columba Livia)

Y. Atoji
Summary Our previous study showed localization of glutamate receptor 1 (GluR1) mRNA in neurons of the pigeon spinal cord, suggesting glutamatergic input from intrinsic and extrinsic origins. The present study examined localization of vesicular glutamate transporter 2 (VGLUT2) mRNA to confirm an extrinsic origin of glutamatergic neurons in the dorsal root ganglion (DRG). GluR1 and GluR2 mRNAs were examined in DRG and spinal cord to seek projection regions from VGLUT2 mRNA-expressing neurons. VGLUT2 mRNA was expressed in most DRG neurons and labelling intensity varied from weakly to intensely. Intense VGLUT2 mRNA expression was mainly seen in medium to large neurons. GluR1 and GluR2 mRNAs were expressed in the dorsal horn and GluR2 mRNA signal was also seen in the marginal nucleus. The results suggest that the pigeon DRG has an extrinsic glutamatergic origin that project to the dorsal horn and marginal nucleus of the spinal cord. [source]

A sodium channel gene SCN9A polymorphism that increases nociceptor excitability,

Mark Estacion PhD
Sodium channel NaV1.7, encoded by the SCN9A gene, is preferentially expressed in nociceptive primary sensory neurons, where it amplifies small depolarizations. In studies on a family with inherited erythromelalgia associated with NaV1.7 gain-of-function mutation A863P, we identified a nonsynonymous single-nucleotide polymorphism within SCN9A in the affected proband and several unaffected family members; this polymorphism (c. 3448C&T, Single Nucleotide Polymorphisms database rs6746030, which produces the amino acid substitution R1150W in human NaV1.7 [hNaV1.7]) is present in 1.1 to 12.7% of control chromosomes, depending on ethnicity. In this study, we examined the effect of the R1150W substitution on function of the hNaV1.7 channel, and on the firing of dorsal root ganglion (DRG) neurons in which this channel is normally expressed. We show that this polymorphism depolarizes activation (7.9,11mV in different assays). Current-clamp analysis shows that the 1150W allele depolarizes (6mV) resting membrane potential and increases (,2-fold) the firing frequency in response to depolarization in DRG neurons in which it is present. Our results suggest that polymorphisms in the NaV1.7 channel may influence susceptibility to pain. Ann Neurol 2009;66:862,866 [source]

Modulation of sensory neuron potassium conductances by anandamide indicates roles for metabolites

R M Evans
Background and purpose: The endogenous cannabinoid anandamide (AEA) acts at cannabinoid (CB1) and vanilloid (TRPV1) receptors. AEA also shows antinociceptive properties; although the underlying mechanism for this is not fully understood, both CB1 and TRPV1 may be involved. Voltage-activated Ca2+ 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 Ca2+ 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 Ca2+ 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 ,M AEA attenuated voltage-activated K+ currents and the recovery of KCl-evoked Ca2+ transients. The insensitivity of these responses to the CB1 receptor antagonist rimonabant (100 nM) and preincubation of DRG neurons with pertussis toxin suggested that these actions are not CB1 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 Ca2+ 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. British Journal of Pharmacology (2008) 154, 480,492; doi:10.1038/bjp.2008.93; published online 31 March 2008 [source]


Ai-Hua Pan
SUMMARY 1ATP-gated P2X receptors in nociceptive sensory neurons participate in the transmission of pain signals from the periphery to the spinal cord. The effect of formalin on the expression of P2X3 receptors in dorsal root ganglia (DRG) was characterized using molecular and immunological approaches and the patch-clamp technique. 2Adult Sprague-Dawley rats were injected with 100 L of 5% formalin in the planar surface of the hindpaw and were killed 30 min and 1, 3, 6, 12, 24 and 48 h later for in vitro analyses. The expression and distribution of P2X3 receptors in the lumbar spinal cord and in L5/L6 DRG were examined; 24 and 48 h after formalin injection, currents in neurons were examined using whole-cell patch-clamp recording. 3Western blots showed that anti-P2X3 antibody recognized a major monomer of approximately 64 kDa in DRG. Immunoreactivity for P2X3 receptors was detected predominantly in the cytoplasm and plasma membrane of small (< 25 m) and middle-sized (25,50 m) DRG neurons. Expression of the P2X3 transcript in the DRG was unchanged 30 min and 1 h after formalin injection, but increased after 12 h. There was no distinct change in P2X3 immunostaining of the spinal cord lamina at 30 min or 1 h after injection, but after 24 h P2X3 labelling increased. At 24 h after the formalin injection, currents in isolated small and middle-sized DRG neurons were increased by 1 mol/L ,,,-methylene-ATP. These currents were completely inhibited by 1 mol/L A-317491, a potent and selective P2X3 receptor antagonist. 4These data suggest that formalin injection leads to early upregulation of P2X3 expression in the spinal cord and DRG and that this may be one of the mechanisms giving rise to nociception. [source]