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1.
Stimulation of Cyclic GMP Production via a Nitrosyl Factor in Sensory Neuronal Cultures by Algesic or Inflammatory Agents 总被引:2,自引:0,他引:2
Abstract: Capsaicin stimulates cyclic GMP production via nitric oxide (NO) (or another nitrosyl factor) in dorsal root ganglion (DRG) neurons maintained in culture. The purpose of the present study was to characterize further capsaicin stimulation of cyclic GMP production in DRG cells maintained in culture, investigate other algesic and/or inflammatory agents for effects on cyclic GMP production, and examine cells responsible for NO production and cyclic GMP production. Capsaicin stimulation of cyclic GMP production in DRG cells was dose dependent, receptor mediated, and attenuated by hemoglobin. Prostaglandin E2 , substance P, and calcitonin gene-related peptide did not affect basal, capsaicin-stimulated, or bradykinin-stimulated cyclic GMP production. Other inflammatory or algesic agents, including serotonin, histamine, ATP, glutamate, aspartate, and NMDA, did not affect cyclic GMP production. Pretreatment of DRG cells with lipopolysaccharide increased basal cyclic GMP production in neuronal but not in nonneuronal cultures and facilitated stimulation of cyclic GMP production by l -arginine. Capsaicin pretreatment of neuronal DRG cultures, which destroys capsaicin-sensitive (small diameter) afferent neurons, attenuated capsaicin- and bradykinin-stimulated cyclic GMP production but did not affect basal or sodium nitroprusside-stimulated cyclic GMP production. These results indicate that capsaicin elicits production of a nitrosyl factor via capsaicin-sensitive (small diameter) neurons. Capsaicin evoked cyclic GMP production in nonneuronal DRG cultures in the presence but not in the absence of apposed neuronal DRG cultures. Overall, these findings suggest that specific exogenous (or endogenous) substances may stimulate production of a nitrosyl factor(s) by a subset of DRG neurons, and nitrosyl factors produced by these neurons may affect cyclic GMP production in neighboring neuronal or non-neuronal cells. 相似文献
2.
Studies on rat sympathetic neurons developing in cell culture. I. Growth characteristics and electrophysiological properties 总被引:3,自引:0,他引:3
In this series of three papers, we describe electrophysiological and pharmacological studies on sympathetic principal neurons developing in cell culture. This paper is concerned with the methods for growing and recording from the neurons and with observations on some of their electrical properties. The succeeding papers are concerned with functional synapses which the neurons form with one another. Superior cervical ganglia of newborn rats were dissociated into single cells and small cell clusters, and the resulting cell suspension of principal neurons and a much smaller number of non-neuronal cells was cultured at low density in medium containing nerve growth factor (D. Bray, 1970, Proc. Nat. Acad. Sci. USA.65, 905–910;R. E. Mains and P. H. Patterson, 1973a, J. Cell Biol.59, 329–345). As in the previous studies the multiplication of the non-neuronal cells could be controlled so that the neurons grew in the presence of an increasing number of non-neuronal cells or in the virtual absence of other cell types. Another method for obtaining mixed cultures was to plate the initial cell suspension onto a preexisting layer of cells dissociated from some other tissue (e.g., heart). Neurons grown for 3 weeks or longer in the presence of non-neuronal cells had resting potentials, passive electrical properties, and action potentials generally similar to those reported for principal neurons of the superior cervical ganglia of adult rats. Through the use of tetrodotoxin, tetraethylammonium, and cobalt, evidence was obtained for the presence of potential-sensitive sodium, potassium, and calcium channels. Frequently the action potential was followed by a prolonged after-hyperpolarization whose properties suggested the presence of potassium channels controlled by calcium ions. When the neurons were grown in the absence of non-neuronal cells, the action potentials were similar, but the prolonged after-hyperpolarization was rarely seen, and the neurons usually discharged repetitively in response to a steady depolarization. 相似文献
3.
Background
The dorsal root ganglia (DRG) neuron is an invaluable tool in axon growth, growth factor regulation, myelin formation and myelin-relevant researches. The purification of DRG neurons is a key step in these studies. Traditionally, purified DRG neurons were obtained in two weeks after exposure to several rounds of anti-mitotic reagent.Methods and Results
In this report, a novel, simple and efficient method for DRG purification is presented. DRG cultures were treated once with a high-dose anti-mitotic reagent cocktail for 72 hours. Using this new method, DRG neurons were obtained with 99% purification within 1 week. We confirmed that the neurite growth and the viability of the purified DRG neurons have no difference from the DRG neurons purified by traditional method. Furthermore, P0 and MBP expression was observed in myelin by immunocytochemistry in the DRG/SC co-culture system. The formation of mature node of Ranvier in DRG-Schwann cell co-culture system was observed using anti-Nav 1.6 and anti-caspr antibody.Conclusion and Significance
The results indicate that this high dose single treatment did not compromise the capacity of DRG neurons for myelin formation in the DRG/SC co-culture system. In conclusion, a convenient approach for purifying DRG neurons was developed which is time-saving and high-efficiency. 相似文献4.
Jessica Erriquez Silvia Bernascone Monica Ciarletta Nicoletta Filigheddu Andrea Graziani Carla Distasi 《Cell calcium》2009,46(3):197-208
Ghrelin is a hormone regulating energy homeostasis via interaction with its receptor, GHSR-1a. Ghrelin activities in dorsal root ganglia (DRG) cells are unknown. Herein we show that ghrelin induces a change of cytosolic calcium concentration in both glia and neurons of embryonic chick DRG. Both RT-PCR and binding studies performed with fluorescent ghrelin in the presence of either unlabeled ghrelin or GHSR-1a antagonist D-Lys3-GHRP-6, indicate that DRG cells express GHSR-1a. In glial cells the response is characterized by a rapid transient rise in [Ca2+]i followed by a long lasting rise. The calcium elevation is dependent on calcium release from thapsigargin-sensitive intracellular stores and on activation of two distinct Ca2+ entry pathways, a receptor activated calcium entry and a store operated calcium entry. Surprisingly, D-Lys3-GHRP-6 exerts several activities in the absence of exogenous ghrelin: (i) it activates calcium release from thapsigargin-sensitive intracellular stores and calcium entry via voltage-operated channels in non-neuronal cells; (ii) it inhibits calcium oscillations in non-neuronal cells exhibiting spontaneous Ca2+ activity and iii) it promotes apoptosis of DRG cells, both neurons and glia. In summary, we provide the first evidence for ghrelin activity in DRG, and we also demonstrate that the widely used D-Lys3-GHRP-6 ghrelin antagonist features ghrelin independent activities. 相似文献
5.
Jerzy Kazimierczak Ernst W. Sommer Eric Philippe Dr. Bernard Droz 《Cell and tissue research》1986,245(3):487-495
Summary Subpopulations of primary sensory neurons in mammalian dorsal root ganglion (DRG) exhibit carbonic anhydrase (CA) activity. To identify these subpopulations in DRG cells of mouse and chicken, the reliability of the cytochemical localization of the enzyme requires that several conditions be fulfilled:(1) Preservation of the enzyme activity in glutaraldehyde-containing fixative; (2) accessibility of the cytoenzymatic reaction throughout 20-m thick Vibratome sections; (3) retention of the reaction product in situ during OsO4 post-fixation; (4) specificity of the cytoenzymatic reaction for CA activity as corroborated by the immunocytochemical detection with antibodies anti-CA II in mouse DRG; (5) strict correlation between the CA activity and the cytological characteristics in a given subclass of neurons. On the basis of these criteria, it is concluded that the CA activity may be used as a cell marker to identify cytologically defined neuronal subpopulations and their axons in mouse DRG. In chicken DRG, CA activity is not consistently expressed in a given subclass of ganglion cells and their axons. Hence, it is assumed that the expression of CA activity by DRG cells in chicken is modulated by functional or environmental conditions. 相似文献
6.
In co-cultured dorsal root ganglion (DRG) neurons and spinal cord neurons from newborn rats, using a voltage-clamp technique in the whole-cell configuration enabled us to observe in DRG neurons the effects evoked by extracellular local electrical stimulation of cells corresponding to spinal cord neurons in their morphological characteristics. Such stimulation caused the appearance of postsynaptic currents (PSC) in DRG neurons in 9% of the cases. The mean delay of these currents (measured from the stimulus leading edge) was 4.7 ± 0.29 msec, the mean time to peak was 2.6 ± 0.77 msec, and the decay time constant = 14.5 ± 1.04 msec. The reversal potential of evoked PSC (ePSC) was close to the equilibrium potential for chloride ions estimated by the Nernst equation. Application of 20 M bicuculline induced practically complete and reversible ePSC block. The conclusion was drawn that these currents arise due to activation of the chloride channels operated by GABA receptors and, hence, represent an inhibitory PSC. Thus, one may deem it proved that spinal cord neurons can establish functional inhibitory synapses with DRG neurons. 相似文献
7.
Efficient delivery of RNA Interference to peripheral neurons in vivo using herpes simplex virus 总被引:1,自引:0,他引:1
Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge limiting its applications. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors. HSV-mediated delivery of short-hairpin RNA (shRNA) targeting reporter genes resulted in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo including in a transgenic mouse model. We further establish proof of concept by demonstrating in vivo silencing of the endogenous trpv1 gene. These data are the first to show silencing in DRG neurons in vivo by vector-mediated delivery of shRNA. Our results support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies. 相似文献
8.
Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors. 相似文献
9.
The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG ("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accom-panied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP- 1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword. 相似文献
10.
Heterogeneity of the Functional Expression of P2X3 and P2X2/3 Receptors in the Primary Nociceptive Neurons of Rat 总被引:3,自引:0,他引:3
Pankratov Yu. V. Lalo U. V. Dashkin A. N. Krishtal O. A. 《Neurochemical research》2001,26(8-9):993-1000
The properties and functional expression of the purinergic receptors in small (nociceptive) neurons acutely isolated from the DRG of rat were studied using whole-cell patch-clamp recording. The responses of small DRG neurons to ATP exhibited diverse kinetics and could be subdivided into three types: rapid, slow and mixed kinetics responses. Their affinities to agonists allowed to identify the responsible receptors as P2X3 (fast) and heteromeric P2X2/3 (slow) subtypes. The expression of different responses dramatically varied both on the neuron-to-neuron and animal-to-animal basis. Out of 744 neurons tested 24% of cells demonstrated predominance of functional P2X2/3 receptors, 44% had mixed representation and in 32% of cells P2X3 receptors dominated. All the animals tested (110) could be subdivided into 3 groups: in 19% of animals the response of each cell to ATP was mediated by P2X2/3 receptors, both types of ATP-evoked currents were found in 58% of animals and only in 23% of the animals P2X3 receptors dominated. Our results argue with exclusive role of P2X3 receptors in purinergic signaling in primary nociceptive neurons. 相似文献
11.
Nicotinic receptor alpha7-subunits are coupled to the stimulation of nitric oxide synthase in rat dorsal root ganglion neurons 总被引:3,自引:1,他引:2
In dorsal root ganglia (DRG) intraganglionic communication takes place both among neurons and between neurons and satellite cells. One diffusible substance involved in this signalling is nitric oxide (NO), and acetylcholine (ACh) is a candidate for the stimulation of intraganglionic NO synthesis. DRG neurons react to ACh-receptor stimulation with NO-dependent cGMP production. Here, we investigated the role of the 7-subunit containing Ca2+-permeable nicotinic ACh receptors (nAChR) in this process. The 7-nAChR mRNA and the protein were expressed in virtually all lumbar DRG neurons as evidenced by laser-assisted cell picking and oligo cell RT-PCR, in situ hybridisation and immunohistochemistry. Strong 7-nAChR immunoreactivity was present in vanilloid receptor 1-immunoreactive, i.e. nociceptive, neurons. A neuronal production of NO in response to nicotine could be demonstrated in DRG slice preparations utilising the NO-sensitive fluorescent indicator diaminofluorescein diacetate (DAF-2DA). This stimulation of NO production was sensitive to inhibition of 7-nAChR by mecamylamine and -bungarotoxin, to inhibition of nitric oxide synthase (NOS) with L-NAME and L-NMMA, and to the blockade of voltage-operated Ca2+ channels by verapamil. The results show the presence of the 7-nAChR subunit in nociceptive rat DRG neurons and provide evidence for its coupling to NOS activation, indicating a role of this pathway in the intraganglionic communication in sensory ganglia. 相似文献
12.
P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy 总被引:4,自引:0,他引:4
ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X3-receptor subtype showed P2X3- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X3-receptor-immunoreactive DRG neurons to the cervix. Some P2X3-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X3-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition. 相似文献
13.
Primary cultures of rat dorsal root ganglionic (DRG) cells were stained with isoform-specific antibodies against non-muscle myosin II. Antibodies against the brain type myosin (MIIB) stained the peripheries of growth cones and non-neuronal cells. Double staining of the cells with the anti-myosin antibodies and rhodamine-phalloidin or anti-actin antibodies indicated that MIIB co-exists, with F-actin, at the leading edge. Antibodies against platelet myosin stained neither leading edges nor neurites, but stained the cell bodies of neurons and the stress fibers of non-neuronal cells. These results suggest that MIIB functions in the motility of the leading edge of DRG cells. 相似文献
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16.
Hongwei Yu Greg Fischer Guangfu Jia Jakob Reiser Frank Park Quinn H Hogan 《Molecular pain》2011,7(1):1-17
Background
Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K+ channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K2P channels after peripheral axotomy in mammals.Results
Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo.Conclusions
In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability. 相似文献17.
Expression and histochemical localization of ciliary neurotrophic factor in cultured adult rat dorsal root ganglion neurons 总被引:4,自引:3,他引:1
Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not
in neurons. After peripheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival
and axonal regeneration. In the present study, we examined the expression and histochemical localization of CNTF in adult
rat DRG in vivo and in vitro. In contrast to the restricted expression in Schwann cells in vivo, we observed abundant CNTF
mRNA and protein expression in DRG neurons after 3 h, 2, 7, and 15 days in dissociated cell culture. At later stages (7 and
15 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and regenerating neurites. These results
suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF immunoreactivity
in DRG neurons in explant culture, disruption of cell–cell interactions, rather than the culture itself, may be an inducible
factor for localization of CNTF in the neurons. 相似文献
18.
ALCAM is a member of the cell adhesion molecule (CAM) family which plays an important role during nervous system formation. We here show that the two neuron populations of developing dorsal root ganglia (DRG) display ALCAM transiently on centrally and peripherally projecting axons during the two phases of axon outgrowth. To analyze the impact of ALCAM on cell adhesion and axon growth, DRG single cells were cultured on ALCAM-coated coverslips or on nanopatterns where ALCAM is presented in physiological amino-carboxyl terminal orientation at highly defined distances (29, 54, 70, 86, and 137 nm) and where the interspaces are passivated to prevent unspecific protein deposition. Some axonal features (branching, lateral deviation) showed density dependence whereas others (number of axons per neuron, various axon growth parameters) turned out to be an all-or-nothing reaction. Time-lapse analyses revealed that ALCAM density has an impact on axon velocity and advance efficiency. The behavior of the sensory axon tip, the growth cone, partially depended on ALCAM density in a dose-response fashion (shape, dynamics, detachment) while other features did not (size, complexity). Whereas axon growth was equally promoted whether ALCAM was presented at high (29 nm) or low densities (86 nm), the attachment of non-neuronal cells depended on high ALCAM densities. The attachment of non-neuronal cells to the rather unspecific standard proteins presented by conventional implants designed to enhance axonal regeneration is a severe problem. Our findings point to ALCAM, presented as 86 nm pattern, for a promising candidate for the improvement of such implants since this pattern drives axon growth to its full extent while at the same time non-neuronal cell attachment is clearly reduced. 相似文献
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