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1.
Genes (Basel) ; 10(10)2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561430

RESUMO

Transcriptional responses to the appropriate temporal pattern of action potential firing are essential for long-term adaption of neuronal properties to the functional activity of neural circuits and environmental experience. However, standard transcriptome analysis methods can be too limited in identifying critical aspects that coordinate temporal coding of action potential firing with transcriptome response. A Pearson correlation analysis was applied to determine how pairs of genes in the mouse dorsal root ganglion (DRG) neurons are coordinately expressed in response to stimulation producing the same number of action potentials by two different temporal patterns. Analysis of 4728 distinct gene-pairs related to calcium signaling, 435,711 pairs of transcription factors, 820 pairs of voltage-gated ion channels, and 86,862 pairs of calcium signaling genes with transcription factors indicated that genes become coordinately activated by distinct action potential firing patterns and this depends on the duration of stimulation. Moreover, a measure of expression variance revealed that the control of transcripts abundances is sensitive to the pattern of stimulation. Thus, action potentials impact intracellular signaling and the transcriptome in dynamic manner that not only alter gene expression levels significantly (as previously reported) but also affects the control of their expression fluctuations and profoundly remodel the transcriptional networks.


Assuntos
Potenciais de Ação , Redes Reguladoras de Genes , Neurônios/metabolismo , Transcriptoma , Animais , Sinalização do Cálcio , Células Cultivadas , Gânglios Espinais/citologia , Camundongos , Neurônios/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio/genética , Canais de Sódio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Elife ; 82019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31500698

RESUMO

Patients with liver diseases often suffer from chronic itch, yet the pruritogen(s) and receptor(s) remain largely elusive. Here, we identify bile acids as natural ligands for MRGPRX4. MRGPRX4 is expressed in human dorsal root ganglion (hDRG) neurons and co-expresses with itch receptor HRH1. Bile acids elicited Ca2+ responses in cultured hDRG neurons, and bile acids or a MRGPRX4 specific agonist induced itch in human subjects. However, a specific agonist for another bile acid receptor TGR5 failed to induce itch in human subjects and we find that human TGR5 is not expressed in hDRG neurons. Finally, we show positive correlation between cholestatic itch and plasma bile acids level in itchy patients and the elevated bile acids is sufficient to activate MRGPRX4. Taken together, our data strongly suggest that MRGPRX4 is a novel bile acid receptor that likely underlies cholestatic itch in human, providing a promising new drug target for anti-itch therapies.


Assuntos
Ácidos e Sais Biliares/metabolismo , Prurido/induzido quimicamente , Receptores de Superfície Celular/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Gânglios Espinais/citologia , Humanos , Receptores Histamínicos H4/metabolismo
3.
Neurochem Res ; 44(9): 2215-2229, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31422522

RESUMO

The ability to regrow their axons after an injury is a hallmark of neurons in peripheral nervous system which distinguish them from central nervous system neurons. This ability is influenced by their intrinsic capacity to regrow and by the extracellular environment which needs to be supportive of regrowth. CXCL1 [Chemokine (C-X-C motif) Ligand 1] and CXCL2 [Chemokine (C-X-C motif) Ligand 2] are two low-molecular-weight chemokines which can influence neuronal proliferation, differentiation and neurogenesis, but which are also upregulated by injury or inflammation. In this study we investigated the effects of long-term incubation (24, 48 and 72 h) with different concentrations of CXCL1 (0.4, 4 or 40 nM) or CXCL2 (0.36, 3.6 or 36 nM) on the axon outgrowth of adult rat dorsal root ganglia neurons in culture. The results showed that both chemokines significantly inhibited the axon outgrowth, with large and medium NF200 (NeuroFilament 200) (+) dorsal root ganglia neurons affected quicker, compared to small IB4 (Isolectin B4) (+) dorsal root ganglia neurons which were affected after longer exposure. Blocking CXCR2 (C-X-C motif chemokine receptor 2) which mediates the effects of CXCL1 and CXCL2 prevented these effects, suggesting that CXCR2 may represent a new therapeutic target for promoting the axon outgrowth after a peripheral nerve injury.


Assuntos
Axônios/efeitos dos fármacos , Quimiocina CXCL1/farmacologia , Quimiocina CXCL2/farmacologia , Gânglios Espinais/citologia , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Masculino , Ratos Wistar
4.
Adv Exp Med Biol ; 1155: 923-934, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468457

RESUMO

Diabetic neuropathy (DN) is the most common chronic complication of DM and its major pathological changes show axonal dysfunction, atrophy and loss. However, there are few reports that taurine promotes neurite growth of dorsal root ganglion (DRG) cells. In current study, DRG neurons were exposed to high glucose (HG) with or without taurine. The neurite outgrowth of DRG neurons was observed by fluorescent immunohistochemistry method. Expression of Gap-43, Akt, phosphorylated Akt, mTOR and phosphorylated mTOR was determined by Western blot assay. Our results showed that HG significantly decreased the neurite outgrowth and expression of Gap-43 in DRG neurons. Moreover, phosphorylated levels of Akt and mTOR were downregulated in DRG neurons exposed to HG. On the contrary, taurine supplementation significantly reversed the decreased neurite outgrowth and Gap-43 expression, and the downregulated phosphorylated levels of Akt and mTOR. However, the protective effects of taurine were blocked in the presence of PI3K antagonists LY294002 or Akt antagonists Perifosine. These results indicate that taurine promotes neurite outgrowth of DRG neurons exposed to HG via activating Akt/mTOR signal pathway.


Assuntos
Gânglios Espinais/citologia , Neurônios/efeitos dos fármacos , Taurina/farmacologia , Células Cultivadas , Proteína GAP-43/metabolismo , Glucose , Humanos , Neuritos/efeitos dos fármacos , Neurônios/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
5.
Artif Cells Nanomed Biotechnol ; 47(1): 2918-2929, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31317777

RESUMO

We investigated the occurrence of mesenchymal stem cell (MSC)-derived exosome uptake and retrograde transport at peripheral nerve endings using bone marrow MSCs (bMSCs) transduced with recombinant CD63-green fluorescent protein (GFP) lentiviral plasmid. GFP was used to track the release of bMSC-derived exosomes and the uptake and transport at peripheral nerve terminals, the dorsal root ganglion (DRG), and the spinal cord. In vitro cell culture and injection of a CD63-GFP exosome suspension into the right gastrocnemius muscle of an in vivo rat model were also performed. Fluorescence microscopy of co-cultured CD63-GFP exosomes and SH-SY5Y or BV2 cell lines and primary cultured DRG cells in a separate experiment demonstrated exosome uptake into DRG neurons and glia. Moreover, we observed both retrograde axoplasmic transport and hematogenous transport of exosomes injected into rat models at the DRG and the ipsilateral side of the anterior horn of the spinal cord using fluorescence microscopy, immunohistochemistry, and Western blot analyses. In conclusion, we showed that exosome uptake at peripheral nerve endings and retrograde transport of exosomes to DRG neurons and spinal cord motor neurons in the anterior horn can occur. In addition, our findings propose a novel drug delivery approach for treating neuronal diseases.


Assuntos
Exossomos/metabolismo , Células-Tronco Mesenquimais/citologia , Terminações Nervosas/metabolismo , Animais , Transporte Biológico , Linhagem Celular Tumoral , Gânglios Espinais/citologia , Humanos , Masculino , Neurônios/citologia , Ratos
6.
Artif Cells Nanomed Biotechnol ; 47(1): 2948-2956, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31317779

RESUMO

Neurotoxicity of local anesthetics is often reported in the clinic, more and more people pay attention to them. CaMKIIß, a subtype of CaMKII, is detected in the central nervous system. Previous study found that CaMKIIß mRNA are up-regulated in DRG neurons treated with ropivacaine hydrochloride, as well as inhibition of Cav3.2 and Cav3.3 expression can improve the local anesthetics neurotoxicity. In this study, we observed the effect of CaMKIIß on neurotoxicity injury induced by ropivacaine hydrochloride with DRG cell in vitro. We first constructed the pAd-shRNA-CaMKIIß-DRG to inhibit CaMKIIß mRNA expression and detected the cell viability, cell apoptosis rate, CaMKIIß, Cav3.2 and Cav3.3 expression. The results showed that ropivacaine hydrochloride caused the DRG cell injury with cell viability decreased and cell apoptosis rate increased, CaMKIIß, Cav3.2 and Cav3.3 expression up-regulated. Interestingly, inhibition of CaMKIIß expression protected the DRG cell from the neurotoxicity injury induced by ropivacaine hydrochloride, increased the cell viability and decreased the apoptosis rate, as well as inhibition of CaMKIIß expression down-regulated Cav3.2 and Cav3.3 expression. In other words, CaMKIIß is involved with the DRG injury induced by ropivacaine hydrochloride. Inhibition CaMKIIß expression improved DRG injury, increased the cell viability and decreased cell apoptosis rate.


Assuntos
Anestésicos Locais/toxicidade , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Gânglios Espinais/citologia , Neurotoxinas/toxicidade , Ropivacaina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Sobrevivência Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley
7.
Cell Prolif ; 52(5): e12660, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31264327

RESUMO

OBJECTIVES: Muscle spindles are proprioceptive receptors in the skeletal muscle. Peripheral nerve injury results in a decreased number of muscle spindles and their morphologic deterioration. However, the muscle spindles recover when skeletal muscles are reinnervated with surgical procedures, such as nerve suture or nerve transfer. Morphological changes in muscle spindles by cell transplantation procedure have not been reported so far. Therefore, we hypothesized that transplantation of embryonic sensory neurons may improve sensory neurons in the skeletal muscle and reinnervate the muscle spindles. MATERIALS AND METHODS: We collected sensory neurons from dorsal root ganglions of 14-day-old rat embryos and prepared a rat model of peripheral nerve injury by performing sciatic nerve transection and allowing for a period of one week before which we performed the cell transplantations. Six months later, the morphological changes of muscle spindles in the cell transplantation group were compared with the naïve control and surgical control groups. RESULTS: Our results demonstrated that transplantation of embryonic dorsal root ganglion cells induced regeneration of sensory nerve fibre and reinnervation of muscle spindles in the skeletal muscle. Moreover, calbindin D-28k immunoreactivity in intrafusal muscle fibres was maintained for six months after denervation in the cell transplantation group, whereas it disappeared in the surgical control group. CONCLUSIONS: Cell transplantation therapies could serve as selective targets to modulate mechanosensory function in the skeletal muscle.


Assuntos
Gânglios Espinais/transplante , Fusos Musculares/metabolismo , Traumatismos dos Nervos Periféricos/terapia , Animais , Calbindinas/metabolismo , Embrião de Mamíferos/citologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Masculino , Fibras Nervosas/fisiologia , Ratos , Ratos Endogâmicos F344 , Regeneração , Nervo Tibial/metabolismo , Nervo Tibial/patologia
8.
Nat Commun ; 10(1): 3087, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300648

RESUMO

The dorsal root ganglia (DRG) contain the somas of first-order sensory neurons critical for somatosensation. Due to technical difficulties, DRG neuronal activity in awake behaving animals remains unknown. Here, we develop a method for imaging DRG at cellular and subcellular resolution over weeks in awake mice. The method involves the installation of an intervertebral fusion mount to reduce spinal movement, and the implantation of a vertebral glass window without interfering animals' motor and sensory functions. In vivo two-photon calcium imaging shows that DRG neuronal activity is higher in awake than anesthetized animals. Immediately after plantar formalin injection, DRG neuronal activity increases substantially and this activity upsurge correlates with animals' phasic pain behavior. Repeated imaging of DRG over 5 weeks after formalin injection reveals persistent neuronal hyperactivity associated with ongoing pain. The method described here provides an important means for in vivo studies of DRG functions in sensory perception and disorders.


Assuntos
Gânglios Espinais/diagnóstico por imagem , Microscopia Intravital/métodos , Percepção da Dor/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Técnicas de Observação do Comportamento/instrumentação , Técnicas de Observação do Comportamento/métodos , Comportamento Animal/fisiologia , Cálcio/química , Feminino , Formaldeído/administração & dosagem , Formaldeído/toxicidade , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Microscopia Intravital/instrumentação , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Dor/induzido quimicamente , Dor/fisiopatologia , Fótons , Vigília
9.
Neuron ; 103(3): 412-422.e4, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31221560

RESUMO

Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease.


Assuntos
Axônios/patologia , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Axônios/enzimologia , Caspase 3/fisiologia , Células Cultivadas , Ativação Enzimática , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Camundongos , Camundongos Knockout , Degeneração Neural/enzimologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/deficiência , Fosforilação , Processamento de Proteína Pós-Traducional , Receptor trkA/fisiologia , Células Receptoras Sensoriais/fisiologia , Relação Estrutura-Atividade
10.
Neuron ; 103(4): 598-616.e7, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31248728

RESUMO

Dorsal root ganglion (DRG) sensory neuron subtypes defined by their in vivo properties display distinct intrinsic electrical properties. We used bulk RNA sequencing of genetically labeled neurons and electrophysiological analyses to define ion channel contributions to the intrinsic electrical properties of DRG neuron subtypes. The transcriptome profiles of eight DRG neuron subtypes revealed differentially expressed and functionally relevant genes, including voltage-gated ion channels. Guided by these data, electrophysiological analyses using pharmacological and genetic manipulations as well as computational modeling of DRG neuron subtypes were undertaken to assess the functions of select voltage-gated potassium channels (Kv1, Kv2, Kv3, and Kv4) in shaping action potential (AP) waveforms and firing patterns. Our findings show that the transcriptome profiles have predictive value for defining ion channel contributions to sensory neuron subtype-specific intrinsic physiological properties. The distinct ensembles of voltage-gated ion channels predicted to underlie the unique intrinsic physiological properties of eight DRG neuron subtypes are presented.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Canais Iônicos/fisiologia , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação , Vias Aferentes/fisiologia , Animais , Simulação por Computador , Gânglios Espinais/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Canais Iônicos/biossíntese , Canais Iônicos/genética , Mecanorreceptores/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Neurológicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , RNA/genética , Células Receptoras Sensoriais/química , Células Receptoras Sensoriais/classificação , Transcriptoma
11.
BMC Neurosci ; 20(1): 21, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036074

RESUMO

BACKGROUND: The dorsal root ganglia (DRG) are a critical component of the peripheral nervous system, and function to relay somatosensory information from the body's periphery to sensory perception centres within the brain. The DRG are primarily comprised of two cell types, sensory neurons and glia, both of which are neural crest-derived. Notch signalling is known to play an essential role in defining the neuronal or glial fate of bipotent neural crest progenitors that migrate from the dorsal ridge of the neural tube to the sites of the DRG. However, the involvement of Notch ligands in this process and the timing at which neuronal versus glial fate is acquired has remained uncertain. RESULTS: We have used tissue specific knockout of the E3 ubiquitin ligase mindbomb1 (Mib1) to remove the function of all Notch ligands in neural crest cells. Wnt1-Cre; Mib1fl/fl mice exhibit severe DRG defects, including a reduction in glial cells, and neuronal cell death later in development. By comparing formation of sensory neurons and glia with the expression and activation of Notch signalling in these mice, we define a critical period during embryonic development in which early migrating neural crest cells become biased toward neuronal and glial phenotypes. CONCLUSIONS: We demonstrate active Notch signalling between neural crest progenitors as soon as trunk neural crest cells delaminate from the neural tube and during their early migration toward the site of the DRG. This data brings into question the timing of neuroglial fate specification in the DRG and suggest that it may occur much earlier than originally considered.


Assuntos
Diferenciação Celular , Movimento Celular , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Crista Neural/citologia , Neuroglia/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Morte Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Crista Neural/metabolismo , Neurogênese/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Fatores de Transcrição SOXE/metabolismo , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética
12.
Andrologia ; 51(8): e13302, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31074030

RESUMO

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is the prostate gland inflammation characterised as genitourinary pain in the pelvic region. The rat experimental autoimmune prostatitis (EAP) was achieved to mimic CP/CPPS. The expressions of transient receptor potential vanilloid 1 (TRPV1) in the prostate, bladder and spinal dorsal root ganglion (DRG) were analysed by Western blotting. Tropomyosin receptor kinase A (TrkA) and nerve growth factor (NGF) in the DRG were also analysed by Western blotting. Measurements of inflammatory cytokines were carried out according to the instructions of the corresponding kits. The expressions of TRPV1 in the prostate, bladder and DRG in the EAP group were significantly higher than those in the control group. The expressions of NGF and TrkA in the DRG in the EAP group were significantly higher than those in the control group. The levels of serum TNF-α and IL-1ß in the EAP group were significantly higher than those in the control group. We conclude that CP/CPPS may participate in the pathological activation of neurons in the L5-S1 segment of DRG by activating NGF-TrkA pathway and cause pelvic organ cross-sensitisation by upregulating the expression of TRPV1 in the prostate, bladder and DRG.


Assuntos
Gânglios Espinais/patologia , Prostatite/patologia , Canais de Cátion TRPV/metabolismo , Animais , Vacina contra Difteria, Tétano e Coqueluche/administração & dosagem , Vacina contra Difteria, Tétano e Coqueluche/imunologia , Modelos Animais de Doenças , Gânglios Espinais/citologia , Humanos , Injeções Intraperitoneais , Masculino , Fator de Crescimento Neural/metabolismo , Neurônios/patologia , Próstata/imunologia , Próstata/inervação , Próstata/patologia , Prostatite/imunologia , Ratos , Ratos Sprague-Dawley , Receptor trkA/metabolismo , Transdução de Sinais , Regulação para Cima , Bexiga Urinária/inervação , Bexiga Urinária/patologia
13.
J Mol Neurosci ; 68(4): 603-619, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31077084

RESUMO

Treatment with cannabidiol (CBD) or KLS-13019 (novel CBD analog), has previously been shown to prevent paclitaxel-induced mechanical allodynia in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN). The mechanism of action for CBD- and KLS-13019-mediated protection now has been explored with dissociated dorsal root ganglion (DRG) cultures using small interfering RNA (siRNA) to the mitochondrial Na+ Ca2+ exchanger-1 (mNCX-1). Treatment with this siRNA produced a 50-55% decrease in the immunoreactive (IR) area for mNCX-1 in neuronal cell bodies and a 72-80% decrease in neuritic IR area as determined with high-content image analysis. After treatment with 100 nM KLS-13019 and siRNA, DRG cultures exhibited a 75 ± 5% decrease in protection from paclitaxel-induced toxicity; whereas siRNA studies with 10 µM CBD produced a 74 ± 3% decrease in protection. Treatment with mNCX-1 siRNA alone did not produce toxicity. The protective action of cannabidiol and KLS-13019 against paclitaxel-induced toxicity during a 5-h test period was significantly attenuated after a 4-day knockdown of mNCX-1 that was not attributable to toxicity. These data indicate that decreases in neuritic mNCX-1 corresponded closely with decreased protection after siRNA treatment. Pharmacological blockade of mNCX-1 with CGP-37157 produced complete inhibition of cannabinoid-mediated protection from paclitaxel in DRG cultures, supporting the observed siRNA effects on mechanism.


Assuntos
Canabidiol/farmacologia , Gânglios Espinais/citologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Animais , Células Cultivadas , Hiperalgesia , Neurônios/metabolismo , Paclitaxel/toxicidade , Interferência de RNA , Ratos , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo
14.
Int J Mol Sci ; 20(11)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31141907

RESUMO

Melatonin is a neurohormone produced and secreted at night by pineal gland. Many effects of melatonin have already been described, for example: Activation of potassium channels in the suprachiasmatic nucleus and inhibition of excitability of a sub-population of neurons of the dorsal root ganglia (DRG). The DRG is described as a structure with several neuronal populations. One classification, based on the repolarizing phase of the action potential (AP), divides DRG neurons into two types: Without (N0) and with (Ninf) inflection on the repolarization phase of the action potential. We have previously demonstrated that melatonin inhibits excitability in N0 neurons, and in the present work, we aimed to investigate the melatonin effects on the other neurons (Ninf) of the DRG neuronal population. This investigation was done using sharp microelectrode technique in the current clamp mode. Melatonin (0.01-1000.0 nM) showed inhibitory activity on neuronal excitability, which can be observed by the blockade of the AP and by the increase in rheobase. However, we observed that, while some neurons were sensitive to melatonin effect on excitability (excitability melatonin sensitive-EMS), other neurons were not sensitive to melatonin effect on excitability (excitability melatonin not sensitive-EMNS). Concerning the passive electrophysiological properties of the neurons, melatonin caused a hyperpolarization of the resting membrane potential in both cell types. Regarding the input resistance (Rin), melatonin did not change this parameter in the EMS cells, but increased its values in the EMNS cells. Melatonin also altered several AP parameters in EMS cells, the most conspicuously changed was the (dV/dt)max of AP depolarization, which is in coherence with melatonin effects on excitability. Otherwise, in EMNS cells, melatonin (0.1-1000.0 nM) induced no alteration of (dV/dt)max of AP depolarization. Thus, taking these data together, and the data of previous publication on melatonin effect on N0 neurons shows that this substance has a greater pharmacological potency on Ninf neurons. We suggest that melatonin has important physiological function related to Ninf neurons and this is likely to bear a potential relevant therapeutic use, since Ninf neurons are related to nociception.


Assuntos
Potenciais de Ação , Depressores do Sistema Nervoso Central/farmacologia , Gânglios Espinais/efeitos dos fármacos , Melatonina/farmacologia , Neurônios/efeitos dos fármacos , Animais , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Masculino , Neurônios/fisiologia , Ratos , Ratos Wistar
15.
J Nippon Med Sch ; 86(4): 215-221, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31061251

RESUMO

BACKGROUND: Neuropathic pain is an intractable chronic pain condition caused by damage to the somatosensory system. Although non-coding RNAs such as microRNAs are important regulators of neuropathic pain, the role of long non-coding RNAs (lncRNAs) is poorly understood. METHODS: This study used a rat model of neuropathic pain induced by lumbar fifth spinal nerve ligation (SNL). Microarray analysis of lncRNAs in the lumbar fifth dorsal root ganglion was performed at day 14 after SNL. Expression levels of H19 were examined by using quantitative PCR. In situ hybridization was used to determine the distribution of H19 at day 14 after SNL. Schwann cells were isolated from peripheral nerves at day 14 after SNL. RESULTS: H19 lncRNA was greatly increased in the L5 dorsal root ganglion at day 14 after SNL and was significantly higher at and after day 4. In the dorsal root ganglion, H19 was detected mainly in non-neuronal cells but not in primary sensory neurons. Consistent with this, H19 expression was upregulated in Schwann cells isolated from peripheral nerves after SNL. CONCLUSION: Increased H19 lncRNA in Schwann cells might be involved in neuropathic pain.


Assuntos
Gânglios Espinais , Expressão Gênica , Neuralgia/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células de Schwann/metabolismo , Animais , Doença Crônica , Modelos Animais de Doenças , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Masculino , Neuralgia/metabolismo , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real
16.
Gastroenterology ; 157(2): 522-536.e2, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31075226

RESUMO

BACKGROUND & AIMS: Proper colon function requires signals from extrinsic primary afferent neurons (ExPANs) located in spinal ganglia. Most ExPANs express the vanilloid receptor TRPV1, and a dense plexus of TRPV1-positive fibers is found around myenteric neurons. Capsaicin, a TRPV1 agonist, can initiate activity in myenteric neurons and produce muscle contraction. ExPANs might therefore form motility-regulating synapses onto myenteric neurons. ExPANs mediate visceral pain, and myenteric neurons mediate colon motility, so we investigated communication between ExPANs and myenteric neurons and the circuits by which ExPANs modulate colon function. METHODS: In live mice and colon tissues that express a transgene encoding the calcium indicator GCaMP, we visualized levels of activity in myenteric neurons during smooth muscle contractions induced by application of capsaicin, direct colon stimulation, stimulation of ExPANs, or stimulation of preganglionic parasympathetic neuron (PPN) axons. To localize central targets of ExPANs, we optogenetically activated TRPV1-expressing ExPANs in live mice and then quantified Fos immunoreactivity to identify activated spinal neurons. RESULTS: Focal electrical stimulation of mouse colon produced phased-locked calcium signals in myenteric neurons and produced colon contractions. Stimulation of the L6 ventral root, which contains PPN axons, also produced myenteric activation and contractions that were comparable to those of direct colon stimulation. Surprisingly, capsaicin application to the isolated L6 dorsal root ganglia, which produced robust calcium signals in neurons throughout the ganglion, did not activate myenteric neurons. Electrical activation of the ganglia, which activated even more neurons than capsaicin, did not produce myenteric activation or contractions unless the spinal cord was intact, indicating that a complete afferent-to-efferent (PPN) circuit was necessary for ExPANs to regulate myenteric neurons. In TRPV1-channel rhodopsin-2 mice, light activation of ExPANs induced a pain-like visceromotor response and expression of Fos in spinal PPN neurons. CONCLUSIONS: In mice, ExPANs regulate myenteric neuron activity and smooth muscle contraction via a parasympathetic spinal circuit, linking sensation and pain to motility.


Assuntos
Colo/fisiopatologia , Neurônios Aferentes/fisiologia , Peristaltismo/fisiologia , Dor Visceral/fisiopatologia , Animais , Técnicas Biossensoriais/métodos , Capsaicina/administração & dosagem , Colo/efeitos dos fármacos , Colo/inervação , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/inervação , Músculo Liso/fisiopatologia , Plexo Mientérico/citologia , Plexo Mientérico/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Optogenética , Peristaltismo/efeitos dos fármacos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Dor Visceral/induzido quimicamente
17.
ACS Appl Mater Interfaces ; 11(20): 18254-18267, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31034196

RESUMO

Poly(3,4-ethylene dioxythiophene) (PEDOT) is a promising conductive material widely used for interfacing with tissues in biomedical fields because of its unique properties. However, obtaining high charge injection capability and high stability remains challenging. In this study, pristine carbon nanotubes (CNTs) modified by dopamine (DA) self-polymerization on the surface polydopamine (PDA@CNTs) were utilized as dopants of PEDOT to prepare hybrid films through electrochemical deposition on the indium tin oxide (ITO) electrode. The PDA@CNTs-PEDOT film of the nanotube network topography exhibited excellent stability and strong adhesion to the ITO substrate compared with PEDOT and PEDOT/ p-toulene sulfonate. The PDA@CNTs-PEDOT-coated ITO electrodes demonstrated lower impedance and enhanced charge storage capacity than the bare ITO. When applying exogenous electrical stimulation (ES), robust long neurites sprouted from the dorsal root ganglion (DRG) neurons cultured on the PDA@CNTs-PEDOT film. Moreover, ES promoted Schwann cell migration out from the DRG spheres and enhanced myelination. The PDA@CNTs-PEDOT film served as an excellent electrochemical sensor for the detection of DA in the presence of biomolecule interferences. Results would shed light into the advancement of conducting nanohybrids for applications in the multifunctional bioelectrode in neuroscience.


Assuntos
Técnicas Biossensoriais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Dopamina/análise , Gânglios Espinais/metabolismo , Nanotubos/química , Neuritos/metabolismo , Polímeros/química , Animais , Eletrodos , Gânglios Espinais/citologia , Ratos , Ratos Sprague-Dawley
18.
Pharmazie ; 74(3): 147-149, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30961679

RESUMO

Mirogabalin, which is a novel ligand for the α2δ subunit of voltage-gated calcium channels, is being developed for treating neuropathic pain including diabetic peripheral neuropathy and postherpetic neuralgia. Mirogabalin possesses unique α2δ subunit binding characteristics and has potent and long-lasting analgesic effects in neuropathic pain models. In the present study, we investigated the effects of mirogabalin on N-type calcium channel currents of the rat dorsal root ganglion (DRG) culture neurons using the whole-cell patch clamp technique. Small or medium DRG neurons were isolated from Sprague-Dawley rats and were incubated for 20 to 24 h with mirogabalin or pregabalin. The DRG neurons were depolarised from a holding potential of -40 mV to +40 mV in steps of 10 mV for 220 ms, and elicited N-type calcium channel currents were recorded. The N-type calcium channel currents were verified by sensitivity to ω-conotoxin GVIA, a selective N-type calcium channel blocker. Mirogabalin inhibited the calcium channel currents of rat DRG neurons at 50 µM, and pregabalin inhibited them at 200 µM. Mirogabalin and pregabalin showed significant differences in the peak current densities at depolarisation to -20 and -10 mV when compared with that shown by the vehicle control. In conclusion, mirogabalin inhibits N-type calcium channel currents in rat DRG culture neurons. The potent and long-lasting analgesic effects of mirogabalin are thought to be associated with its potent and selective binding to α2δ-1 subunits and following functional inhibition of calcium channel currents.


Assuntos
Compostos Bicíclicos com Pontes/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/metabolismo , Gânglios Espinais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Compostos Bicíclicos com Pontes/química , Bloqueadores dos Canais de Cálcio/química , Células Cultivadas , Conotoxinas/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Pregabalina/farmacologia , Ratos , Ratos Sprague-Dawley
19.
Int J Mol Sci ; 20(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013625

RESUMO

The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.


Assuntos
Proteína Semelhante a ELAV 2/genética , Proteína Semelhante a ELAV 3/genética , Proteína Semelhante a ELAV 4/genética , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Células Receptoras Sensoriais/metabolismo , Animais , Biomarcadores , Glicemia , Peso Corporal , Diabetes Mellitus Experimental , Proteína Semelhante a ELAV 2/metabolismo , Proteína Semelhante a ELAV 3/metabolismo , Proteína Semelhante a ELAV 4/metabolismo , Gânglios Espinais/fisiopatologia , Imuno-Histoquímica , Camundongos , Proteínas de Ligação a RNA
20.
Pain ; 160(5): 1103-1118, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31008816

RESUMO

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.


Assuntos
Gânglios Espinais/citologia , Neurônios/metabolismo , Receptor A3 de Adenosina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Antagonistas do Receptor A1 de Adenosina/farmacologia , Agonistas do Receptor A3 de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/farmacologia , Células Cultivadas , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Masculino , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor A3 de Adenosina/genética , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia
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