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1.
Sci Adv ; 6(8): eaax4568, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32128393

RESUMO

Expression of the voltage-gated sodium channel NaV1.7 in sensory neurons is required for pain sensation. We examined the role of NaV1.7 in the dorsal horn of the spinal cord using an epitope-tagged NaV1.7 knock-in mouse. Immuno-electron microscopy showed the presence of NaV1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of NaV1.7 in the dorsal horn. Peripheral nervous system-specific NaV1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. NaV1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on NaV1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated NaV1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/genética , Células do Corno Posterior/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Fenômenos Eletrofisiológicos , Potenciais Pós-Sinápticos Excitadores , Expressão Gênica , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/ultraestrutura , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/ultraestrutura , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia
2.
Nat Neurosci ; 22(5): 828-839, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30886406

RESUMO

Electron microscopy (EM) is a powerful tool for circuit mapping, but identifying specific cell types in EM datasets remains a major challenge. Here we describe a technique enabling simultaneous visualization of multiple genetically identified neuronal populations so that synaptic interactions between them can be unequivocally defined. We present 15 adeno-associated virus constructs and 6 mouse reporter lines for multiplexed EM labeling in the mammalian nervous system. These reporters feature dAPEX2, which exhibits dramatically improved signal compared with previously described ascorbate peroxidases. By targeting this enhanced peroxidase to different subcellular compartments, multiple orthogonal reporters can be simultaneously visualized and distinguished under EM using a protocol compatible with existing EM pipelines. Proof-of-principle double and triple EM labeling experiments demonstrated synaptic connections between primary afferents, descending cortical inputs, and inhibitory interneurons in the spinal cord dorsal horn. Our multiplexed peroxidase-based EM labeling system should therefore greatly facilitate analysis of connectivity in the nervous system.


Assuntos
Córtex Cerebral/ultraestrutura , Microscopia Eletrônica/métodos , Neurônios/ultraestrutura , Células do Corno Posterior/ultraestrutura , Sinapses/ultraestrutura , Adenoviridae/fisiologia , Animais , Genes Reporter , Vetores Genéticos , Imuno-Histoquímica/métodos , Camundongos Transgênicos , Microscopia Confocal , Neurônios Aferentes/ultraestrutura , Peroxidases/química , Razão Sinal-Ruído
3.
Pain ; 160(5): 1082-1092, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30649099

RESUMO

Accumulating evidence has demonstrated that the enhanced synaptic plasticity of nociceptive interneurons in the spinal dorsal horn is the basis of central sensitization in neuropathic pain. Our previous results demonstrated that sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, alleviates neuropathic pain in type 2 diabetes mellitus rats. SIRT1 has also been reported to regulate synaptic plasticity in different brain neurons. However, the role of SIRT1 in synaptic plasticity of spinal dorsal horn neurons remains unknown. In this study, we found that in the spinal dorsal horn of diabetic neuropathic pain (DNP) rats and db/db mice, decreased SIRT1 expression was accompanied by enhanced structural synaptic plasticity. The levels of postsynaptic density protein 95 (PSD-95), growth-associated protein 43 (GAP43), and synaptophysin increased in the spinal dorsal horn of DNP rats and db/db mice and in high glucose-cultured primary spinal neurons. Upregulation of spinal SIRT1 by SIRT1 activator SRT1720 relieved pain behavior, inhibited the enhanced structural synaptic plasticity in rats and db/db mice with DNP, and decreased the levels of synapse-associated proteins in DNP rats, db/db mice, and high glucose-cultured spinal neurons. SIRT1-shRNA induced pain behavior and enhanced structural synaptic plasticity in normal rats and increased synapse-associated proteins levels in normal rats and spinal neurons. Intrathecal injection of AAV-Cre-EGFP into SIRT1 mice also induced pain behavior and enhanced synaptic plasticity of the spinal dorsal horn neurons. These results suggest that SIRT1 plays an important role in the progression of DNP by regulating synaptic plasticity of spinal dorsal horn neurons.


Assuntos
Neuropatias Diabéticas/patologia , Plasticidade Neuronal/fisiologia , Células do Corno Posterior/fisiologia , Sirtuína 1/metabolismo , Medula Espinal/patologia , Animais , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Glucose/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/ultraestrutura , RNA Interferente Pequeno/uso terapêutico , Ratos , Ratos Sprague-Dawley , Sirtuína 1/genética , Regulação para Cima/fisiologia
4.
Methods Mol Biol ; 1791: 115-129, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30006705

RESUMO

Our understanding of the processes controlling peripheral nervous system myelination have been significantly benefited by the development of an in vitro myelinating culture system in which primary Schwann cells are cocultured together with primary sensory neurons. In this chapter, we describe the protocol currently used in our laboratories to establish Schwann cells neuronal myelinating cocultures. We also include a detailed description of the various substrates that can be used to establish it.


Assuntos
Técnicas de Cocultura , Células do Corno Posterior/citologia , Cultura Primária de Células , Células de Schwann/citologia , Animais , Biomarcadores , Feminino , Camundongos , Células do Corno Posterior/metabolismo , Células do Corno Posterior/ultraestrutura , Gravidez , Cultura Primária de Células/métodos , Ratos , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura
5.
Sci Rep ; 8(1): 6809, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29717160

RESUMO

The spinal dorsal horn (SDH) is comprised of distinct neuronal populations that process different somatosensory modalities. Somatostatin (SST)-expressing interneurons in the SDH have been implicated specifically in mediating mechanical pain. Identifying the transcriptomic profile of SST neurons could elucidate the unique genetic features of this population and enable selective analgesic targeting. To that end, we combined the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) method and Fluorescence Activated Nuclei Sorting (FANS) to capture tagged SST nuclei in the SDH of adult male mice. Using RNA-sequencing (RNA-seq), we uncovered more than 13,000 genes. Differential gene expression analysis revealed more than 900 genes with at least 2-fold enrichment. In addition to many known dorsal horn genes, we identified and validated several novel transcripts from pharmacologically tractable functional classes: Carbonic Anhydrase 12 (Car12), Phosphodiesterase 11 A (Pde11a), and Protease-Activated Receptor 3 (F2rl2). In situ hybridization of these novel genes showed differential expression patterns in the SDH, demonstrating the presence of transcriptionally distinct subpopulations within the SST population. Overall, our findings provide new insights into the gene repertoire of SST dorsal horn neurons and reveal several novel targets for pharmacological modulation of this pain-mediating population and treatment of pathological pain.


Assuntos
Interneurônios/metabolismo , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , Somatostatina/genética , Corno Dorsal da Medula Espinal/metabolismo , Transcrição Gênica , 3',5'-GMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Animais , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interneurônios/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Dor Nociceptiva/genética , Dor Nociceptiva/metabolismo , Dor Nociceptiva/fisiopatologia , Células do Corno Posterior/metabolismo , Células do Corno Posterior/ultraestrutura , RNA Mensageiro/classificação , RNA Mensageiro/metabolismo , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Somatostatina/metabolismo , Corno Dorsal da Medula Espinal/citologia
6.
Exp Neurol ; 293: 144-158, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28412220

RESUMO

Peripheral inflammation induces sensitization of nociceptive spinal cord neurons. Both spinal tumor necrosis factor (TNF) and neuronal membrane insertion of Ca2+ permeable AMPA receptor (AMPAr) contribute to spinal sensitization and resultant pain behavior, molecular mechanisms connecting these two events have not been studied in detail. Intrathecal (i.t.) injection of TNF-blockers attenuated paw carrageenan-induced mechanical and thermal hypersensitivity. Levels of GluA1 and GluA4 from dorsal spinal membrane fractions increased in carrageenan-injected rats compared to controls. In the same tissue, GluA2 levels were not altered. Inflammation-induced increases in membrane GluA1 were prevented by i.t. pre-treatment with antagonists to TNF, PI3K, PKA and NMDA. Interestingly, administration of TNF or PI3K inhibitors followed by carrageenan caused a marked reduction in plasma membrane GluA2 levels, despite the fact that membrane GluA2 levels were stable following inhibitor administration in the absence of carrageenan. TNF pre-incubation induced increased numbers of Co2+ labeled dorsal horn neurons, indicating more neurons with Ca2+ permeable AMPAr. In parallel to Western blot results, this increase was blocked by antagonism of PI3K and PKA. In addition, spinal slices from GluA1 transgenic mice, which had a single alanine replacement at GluA1 ser 845 or ser 831 that prevented phosphorylation, were resistant to TNF-induced increases in Co2+ labeling. However, behavioral responses following intraplantar carrageenan and formalin in the mutant mice were no different from littermate controls, suggesting a more complex regulation of nociception. Co-localization of GluA1, GluA2 and GluA4 with synaptophysin on identified spinoparabrachial neurons and their relative ratios were used to assess inflammation-induced trafficking of AMPAr to synapses. Inflammation induced an increase in synaptic GluA1, but not GluA2. Although total GluA4 also increased with inflammation, co-localization of GluA4 with synaptophysin, fell short of significance. Taken together these data suggest that peripheral inflammation induces a PI3K and PKA dependent TNFR1 activated pathway that culminates with trafficking of calcium permeable AMPAr into synapses of nociceptive dorsal horn projection neurons.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células do Corno Posterior/metabolismo , Radiculopatia/patologia , Receptores de AMPA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Cálcio/metabolismo , Carragenina/toxicidade , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Etanercepte/uso terapêutico , Feminino , Masculino , Camundongos , Células do Corno Posterior/patologia , Células do Corno Posterior/ultraestrutura , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Radiculopatia/induzido quimicamente , Radiculopatia/tratamento farmacológico , Ratos Sprague-Dawley , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Sinaptofisina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
7.
Mol Pain ; 13: 1744806916688016, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28326929

RESUMO

Neuropathic pain is a major complication of spinal cord injury, and despite aggressive efforts, this type of pain is refractory to available clinical treatment. Our previous work has demonstrated a structure-function link between dendritic spine dysgenesis on nociceptive sensory neurons in the intermediate zone, laminae IV/V, and chronic pain in central nervous system and peripheral nervous system injury models of neuropathic pain. To extend these findings, we performed a follow-up structural analysis to assess whether dendritic spine remodeling occurs on superficial dorsal horn neurons located in lamina II after spinal cord injury. Lamina II neurons are responsible for relaying deep, delocalized, often thermally associated pain commonly experienced in spinal cord injury pathologies. We analyzed dendritic spine morphometry and localization in tissue obtained from adult rats exhibiting neuropathic pain one-month following spinal cord injury. Although the total density of dendritic spines on lamina II neurons did not change after spinal cord injury, we observed an inverse relationship between the densities of thin- and mushroom-shaped spines: thin-spine density decreased while mushroom-spine density increased. These structural changes were specifically noted along dendritic branches within 150 µm from the soma, suggesting a possible adverse contribution to nociceptive circuit function. Intrathecal treatment with NSC23766, a Rac1-GTPase inhibitor, significantly reduced spinal cord injury-induced changes in both thin- and mushroom-shaped dendritic spines. Overall, these observations demonstrate that dendritic spine remodeling occurs in lamina II, regulated in part by the Rac1-signaling pathway, and suggests that structural abnormalities in this spinal cord region may also contribute to abnormal nociception after spinal cord injury.


Assuntos
Espinhas Dendríticas/patologia , Espinhas Dendríticas/ultraestrutura , Células do Corno Posterior/ultraestrutura , Traumatismos da Medula Espinal/patologia , Análise de Variância , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Espinhas Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Nocodazol/farmacologia , Nocodazol/uso terapêutico , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/patologia , Ratos , Ratos Sprague-Dawley , Coloração pela Prata , Traumatismos da Medula Espinal/tratamento farmacológico
8.
Neuroscience ; 329: 171-81, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27185486

RESUMO

The spinal dorsal horn processes somatosensory information before conveying it to the brain. The neuronal organization of the dorsal horn is still poorly understood, although recent studies have defined several distinct populations among the interneurons, which account for most of its constituent neurons. All primary afferents, and the great majority of neurons in laminae I-III are glutamatergic, and a major factor limiting our understanding of the synaptic circuitry has been the difficulty in identifying glutamatergic synapses with light microscopy. Although there are numerous potential targets for antibodies, these are difficult to visualize with immunocytochemistry, because of protein cross-linking following tissue fixation. Although this can be overcome by antigen retrieval methods, these lead to difficulty in detecting other antigens. The aim of this study was to test whether the postsynaptic protein Homer can be used to reveal glutamatergic synapses in the dorsal horn. Immunostaining for Homer gave punctate labeling when viewed by confocal microscopy, and this was restricted to synapses at the ultrastructural level. We found that Homer puncta were colocalized with the AMPA receptor GluR2 subunit, but not with the inhibitory synapse-associated protein gephyrin. We also examined several populations of glutamatergic axons and found that most boutons were in contact with at least one Homer punctum. These results suggest that Homer antibodies can be used to reveal the great majority of glutamatergic synapses without antigen retrieval. This will be of considerable value in tracing synaptic circuits, and also in investigating plasticity of glutamatergic synapses in pain states.


Assuntos
Ácido Glutâmico/metabolismo , Proteínas de Arcabouço Homer/metabolismo , Células do Corno Posterior/metabolismo , Sinapses/metabolismo , Animais , Proteínas de Transporte/metabolismo , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica , Células do Corno Posterior/ultraestrutura , Receptores de AMPA/metabolismo , Sinapses/ultraestrutura , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
9.
Neurosci Lett ; 599: 86-91, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-26007703

RESUMO

Transmission electron microscopy (TEM) is used for three-dimensional (3-D) analysis of synaptic connections in neuroscience research. However, 3-D reconstruction of the synapses using serial ultrathin sections is a powerful but tedious approach requiring advanced technical skills. High-voltage electron microscopy (HVEM) allows examination of thicker sections of biological specimens due to the increased penetration of the more accelerated electrons, which is useful to analyze the 3-D structure of biological specimens. However, it is still difficult to visualize the neural networks and synaptic connections in 3-D using HVEM because of insufficient and non uniform heavy metal staining in the membranous structures in semi-thin sections. Here, we present the successful chemical 3-D neuroanatomy of the rat spinal dorsal horn at the ultrastructural level as a first step for effective synaptome analysis by applying a high-contrast en bloc staining method to immune-HVEM tomography. Our new approach made it possible to examine many itch-mediating synaptic connections and neural networks in the spinal cord simultaneously using HVEM tomography. This novel 3-D electron microscopy is very useful for the analysis of synaptic structure and the chemical neuroanatomy at the 3-D ultrastructural level.


Assuntos
Células do Corno Posterior/ultraestrutura , Prurido/patologia , Sinapses/fisiologia , Animais , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão/métodos , Células do Corno Posterior/metabolismo , Prurido/metabolismo , Ratos Wistar
10.
J Oral Facial Pain Headache ; 29(1): 70-82, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25635962

RESUMO

AIMS: To explore the possible relationship between protein kinase C gamma (PKCγ) and phosphorylated forms of extracellular signal-regulated kinases 1/2 (pERK1/2) in the rat medullary dorsal horn and the facial hypersensitivity indicative of dynamic mechanical allodynia (DMA) following chronic constriction of the infraorbital nerve (CCI-IoN). METHODS: A well-established rat model of trigeminal neuropathic pain involving CCI-IoN was used. Facial mechanical hypersensitivity was tested with non-noxious dynamic mechanical stimulation (air-puff), and the medullary dorsal horn was examined immunohistochemically using PKCγ and pERK1/2 as pain markers. Statistical analysis was performed using Student t test or one-way analysis of variance (ANOVA). RESULTS: Increased PKCγ and pERK1/2 expressions within the medullary dorsal horn were associated with DMA following CCI-IoN. A segmental network composed of PKCγ-positive cells located in medullary dorsal horn laminae II/III, contacting more superficially located pERK1/2-expressing cells, was identified. Ultrastructural analysis confirmed the presence of PKCγ to pERK1/2-positive cells. Moreover, intracisternal administration of the selective PKCγ inhibitor KIG31-I blocked both the DMA and pERK1/2 expression in a dose-dependent manner. Although the number of pERK1/2-positive cells was significantly elevated with air-puff stimulation, DMA rats not receiving air-puff stimulation showed significant pERK1/2 expression, suggesting they were experiencing spontaneous pain. CONCLUSION: PKCγ cells in the medullary dorsal horn may be involved in DMA following CCI-IoN through the activation of pERK1/2-expressing cells, which then may relay non-nociceptive information to lamina I cells in the medullary dorsal horn.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Nociceptividade/fisiologia , Células do Corno Posterior/enzimologia , Proteína Quinase C/fisiologia , Tato/fisiologia , Neuralgia do Trigêmeo/fisiopatologia , Animais , Biomarcadores/análise , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/análise , Dor Facial/enzimologia , Dor Facial/fisiopatologia , Hiperalgesia/enzimologia , Hiperalgesia/fisiopatologia , Imuno-Histoquímica , Isoenzimas/análise , Isoenzimas/antagonistas & inibidores , Isoenzimas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Vias Neurais/enzimologia , Vias Neurais/fisiopatologia , Vias Neurais/ultraestrutura , Órbita/inervação , Fosforilação , Células do Corno Posterior/fisiologia , Células do Corno Posterior/ultraestrutura , Proteína Quinase C/análise , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Neuralgia do Trigêmeo/enzimologia , Neuralgia do Trigêmeo/patologia
11.
J Comp Neurol ; 522(2): 393-413, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23818225

RESUMO

The γ isoform of protein kinase C (PKCγ), which is concentrated in interneurons in the inner part of lamina II (IIi ) of the dorsal horn, has been implicated in the expression of tactile allodynia. Lamina IIi PKCγ interneurons were shown to be activated by tactile inputs and to participate in local circuits through which these inputs can reach lamina I, nociceptive output neurons. That such local circuits are gated by glycinergic inhibition and that A- and C-fibers low threshold mechanoreceptors (LTMRs) terminate in lamina IIi raise the general issue of synaptic inputs to lamina IIi PKCγ interneurons. Combining light and electron microscopic immunochemistry in the rat spinal trigeminal nucleus, we show that PKCγ-immunoreactivity is mostly restricted to interneurons in lamina IIi of the medullary dorsal horn, where they constitute 1/3 of total neurons. The majority of synapses on PKCγ-immunoreactive interneurons are asymmetric (likely excitatory). PKCγ-immunoreactive interneurons appear to receive exclusively myelinated primary afferents in type II synaptic glomeruli. Neither large dense core vesicle terminals nor type I synaptic glomeruli, assumed to be the endings of unmyelinated nociceptive terminals, were found on these interneurons. Moreover, there is no vesicular glutamate transporter 3-immunoreactive bouton, specific to C-LTMRs, on PKCγ-immunoreactive interneurons. PKCγ-immunoreactive interneurons contain GABAA ergic and glycinergic receptors. At the subcellular level, PKCγ-immunoreactivity is mostly concentrated on plasma membranes, close to, but not within, postsynaptic densities. That only myelinated primary afferents were found to contact PKCγ-immunoreactive interneurons suggests that myelinated, but not unmyelinated, LTMRs play a critical role in the expression of mechanical allodynia.


Assuntos
Hiperalgesia/metabolismo , Interneurônios/enzimologia , Mecanorreceptores/enzimologia , Proteína Quinase C/metabolismo , Sinapses/ultraestrutura , Animais , Western Blotting , Imuno-Histoquímica , Interneurônios/ultraestrutura , Masculino , Mecanorreceptores/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/ultraestrutura , Nociceptores/enzimologia , Nociceptores/ultraestrutura , Células do Corno Posterior/enzimologia , Células do Corno Posterior/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapses/enzimologia
12.
Neuroscience ; 248: 180-93, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-23751177

RESUMO

The transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor regulatory proteins (TARPs) are a family of auxiliary AMPA receptor subunits that differentially modulate trafficking and many functional properties of the receptor. To investigate which TARP isoforms may be involved in AMPA receptor-mediated spinal synaptic transmission, we have mapped the localization of five of the known TARP isoforms, namely γ-2 (also known as stargazin), γ-3, γ-4, γ-7 and γ-8, in the rat spinal cord. Immunoblotting showed expression of all isoforms in the spinal cord to varying degrees. At the light microscopic level, immunoperoxidase labeling of γ-4, γ-7 and γ-8 was found throughout spinal gray matter. In white matter, γ-4 and γ-7 immunolabeling was observed in astrocytic processes and in mature oligodendrocytes. In pepsin-treated spinal cord, γ-7 often colocalized with GluA2 immunopositive puncta in the deep dorsal horn as well as in the ventral horn, but not in the superficial dorsal horn. Postembedding immunogold labeling was further used to assess the synaptic localization of γ-2, γ-7 and γ-8 in the dorsal horn. Synaptic immunogold labeling of γ-2 was sparse throughout the dorsal horn, with some primary afferent synapses weakly labeled, whereas relatively strong γ-7 immunogold labeling was found at deep dorsal horn synapses, including at synapses formed by low-threshold mechanosensitive primary afferent terminals. Prominent immunogold labeling of γ-8 was frequently detected at synapses established by primary afferent fibers. The spinal localization patterns of TARP isoforms reported here suggest that AMPA receptors at spinal synaptic populations and in glial cells may exhibit different functional characteristics owing to differences in auxiliary subunit composition.


Assuntos
Proteínas de Membrana/análise , Células do Corno Posterior/metabolismo , Subunidades Proteicas/análise , Receptores de AMPA/análise , Animais , Anticorpos , Canais de Cálcio/análise , Canais de Cálcio/imunologia , Masculino , Células do Corno Posterior/ultraestrutura , Isoformas de Proteínas/análise , Subunidades Proteicas/imunologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/imunologia
13.
J Neurosci ; 33(9): 3727-37, 2013 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-23447584

RESUMO

Endogenous acetylcholine (ACh) is a well-known modulator of nociceptive transmission in the spinal cord of rodents. It arises mainly from a sparse population of cholinergic interneurons located in the dorsal horn of the spinal cord. This population was thought to be absent from the spinal cord of monkey, what might suggest that spinal ACh would not be a relevant clinical target for pain therapy. In humans, however, pain responses can be modulated by spinal ACh, as evidenced by the increasingly used analgesic procedure (for postoperative and labor patients) consisting of the epidural injection of the acetylcholinesterase inhibitor neostigmine. The source and target of this ACh remain yet to be elucidated. In this study, we used an immunolabeling for choline acetyltransferase to demonstrate, for the first time, the presence of a plexus of cholinergic fibers in laminae II-III of the dorsal horn of the macaque monkey. Moreover, we show the presence of numerous cholinergic cell bodies within the same laminae and compared their density and morphological properties with those previously described in rodents. An electron microscopy analysis demonstrates that cholinergic boutons are presynaptic to dorsal horn neurons as well as to the terminals of sensory primary afferents, suggesting that they are likely to modulate incoming somatosensory information. Our data suggest that this newly identified dorsal horn cholinergic system in monkeys is the source of the ACh involved in the analgesic effects of epidural neostigmine and could be more specifically targeted for novel therapeutic strategies for pain management in humans.


Assuntos
Neurônios Colinérgicos/fisiologia , Células do Corno Posterior/fisiologia , Medula Espinal/citologia , Animais , Contagem de Células , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/ultraestrutura , Feminino , Imageamento Tridimensional , Macaca fascicularis , Masculino , Camundongos , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/metabolismo , Células do Corno Posterior/ultraestrutura , Proteína Quinase C/metabolismo , Especificidade da Espécie , Medula Espinal/ultraestrutura , Sinapses/metabolismo , Sinapses/ultraestrutura , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Ácido gama-Aminobutírico/metabolismo
14.
J Neurosci ; 33(13): 5590-602, 2013 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-23536074

RESUMO

Trophic support and myelination of axons by Schwann cells in the PNS are essential for normal nerve function. Herein, we show that deletion of the LDL receptor-related protein-1 (LRP1) gene in Schwann cells (scLRP1(-/-)) induces abnormalities in axon myelination and in ensheathment of axons by nonmyelinating Schwann cells in Remak bundles. These anatomical changes in the PNS were associated with mechanical allodynia, even in the absence of nerve injury. In response to crush injury, sciatic nerves in scLRP1(-/-) mice showed accelerated degeneration and Schwann cell death. Remyelinated axons were evident 20 d after crush injury in control mice, yet were largely absent in scLRP1(-/-) mice. In the partial nerve ligation model, scLRP1(-/-) mice demonstrated significantly increased and sustained mechanical allodynia and loss of motor function. Evidence for central sensitization in pain processing included increased p38MAPK activation and activation of microglia in the spinal cord. These studies identify LRP1 as an essential mediator of normal Schwann cell-axonal interactions and as a pivotal regulator of the Schwann cell response to PNS injury in vivo. Mice in which LRP1 is deficient in Schwann cells represent a model for studying how abnormalities in Schwann cell physiology may facilitate and sustain chronic pain.


Assuntos
Axônios/fisiologia , Axônios/ultraestrutura , Receptores de LDL/metabolismo , Células de Schwann/patologia , Ciática/patologia , Ciática/prevenção & controle , Proteínas Supressoras de Tumor/metabolismo , Actinas/metabolismo , Análise de Variância , Animais , Antígeno CD11b/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Hiperalgesia/etiologia , Hiperalgesia/genética , Marcação In Situ das Extremidades Cortadas , Indóis , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/genética , Proteína Básica da Mielina/metabolismo , Degeneração Neural/etiologia , Degeneração Neural/genética , Medição da Dor , Fosforilação/genética , Células do Corno Posterior/patologia , Células do Corno Posterior/ultraestrutura , Receptores de LDL/deficiência , Proteínas S100/metabolismo , Células de Schwann/ultraestrutura , Ciática/complicações , Ciática/genética , Transtornos de Sensação/etiologia , Medula Espinal/patologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
J Physiol ; 590(16): 3927-51, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22674718

RESUMO

Perception of normal bodily sensations relies on the precise regulation of sensory information entering the dorsal horn of the spinal cord. Inhibitory, axoaxonic, synapses provide a mechanism for this regulation, but the source of these important inhibitory connections remains to be elucidated. This study shows that a subpopulation of spinal interneurons that expresses parvalbumin and have specific morphological, connectivity and functional characteristics are a likely source of the inhibitory inputs that selectivity regulate non-noxious tactile input in the spinal cord. Our findings suggest that a loss of normal function in parvalbumin positive dorsal horn neurons may result in the development of tactile allodynia, where non-painful stimuli gain the capacity to evoke the sensation of pain.


Assuntos
Axônios/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Regulação da Expressão Gênica/fisiologia , Parvalbuminas/metabolismo , Células do Corno Posterior/fisiologia , Potenciais de Ação , Animais , Anticorpos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Fibras Musculares de Contração Rápida/metabolismo , Parvalbuminas/genética , Técnicas de Patch-Clamp , Células do Corno Posterior/ultraestrutura , Raios Ultravioleta
16.
J Neurosci Res ; 90(5): 1096-104, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22315169

RESUMO

Tetanic stimulation of the sciatic nerve induces long-term potentiation (LTP) of C-fiber-evoked field potentials in the spinal dorsal horn and persistent pain, suggesting that spinal LTP may be a substrate for central sensitization of the pain pathway. However, its cellular mechanism remains unclear. The present study provides electrophysiological and behavioral evidence for the involvement of ryanodine receptor (RyR) in the induction of spinal LTP and persistent pain in rats. The specific inhibitor of ryanodine receptor, ryanodine and dantrolene, dose dependently blocked the induction, but not maintenance, of spinal LTP and reduced persistent pain behaviors induced by tetanic sciatic stimulation. Both cyclic ADP ribose (cADPR), an endogenous agonist of RyR, and (±)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester (Bay K 8644), an agonist of L-type calcium channel, attenuated ryanodine-induced inhibition. Immunohistochemistry and electron microscopic observation showed that RyR subtypes RyR1 and RyR3 were located in the spinal dorsal horn. The results suggest that RyRs are involved in synaptic plasticity of the spinal pain pathway and may be a novel target for treating pain. © 2012 Wiley Periodicals, Inc.


Assuntos
Potenciação de Longa Duração/fisiologia , Dor/patologia , Células do Corno Posterior/fisiopatologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Nervo Isquiático/fisiologia , Medula Espinal/patologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Análise de Variância , Animais , Biofísica , Agonistas dos Canais de Cálcio/farmacologia , Dantroleno/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estimulação Elétrica/efeitos adversos , Potenciais Evocados/fisiologia , Lateralidade Funcional , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Transmissão , Relaxantes Musculares Centrais/farmacologia , Fibras Nervosas Amielínicas/fisiologia , Dor/etiologia , Medição da Dor , Células do Corno Posterior/ultraestrutura , Ratos , Ratos Sprague-Dawley , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/ultraestrutura
17.
J Chem Neuroanat ; 43(2): 103-11, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22119519

RESUMO

Neurons in the rostral ventromedial medulla (RVM) are thought to modulate nociceptive transmission via projections to spinal and trigeminal dorsal horns. The cellular substrate for this descending modulation has been studied with regard to projections to spinal dorsal horn, but studies of the projections to trigeminal dorsal horn have been less complete. In this study, we combined anterograde tracing from RVM with immunocytochemical detection of the GABAergic synthetic enzyme, GAD67, to determine if the RVM sends inhibitory projections to trigeminal dorsal horn. We also examined the neuronal targets of this projection using immunocytochemical detection of NeuN. Finally, we used electron microscopy to verify cellular targets. We compared projections to both trigeminal and spinal dorsal horns. We found that RVM projections to both trigeminal and spinal dorsal horn were directed to postsynaptic profiles in the dorsal horn, including somata and dendrites, and not to primary afferent terminals. We found that RVM projections to spinal dorsal horn were more likely to contact neuronal somata and were more likely to contain GAD67 than projections from RVM to trigeminal dorsal horn. These findings suggest that RVM neurons send predominantly GABAergic projections to spinal dorsal horn and provide direct input to postsynaptic neurons such as interneurons or ascending projection neurons. The RVM projection to trigeminal dorsal horn is more heavily targeted to dendrites and is only modestly GABAergic in nature. These anatomical features may underlie differences between trigeminal and spinal dorsal horns with regard to the degree of inhibition or facilitation evoked by RVM stimulation.


Assuntos
Química Encefálica/fisiologia , Bulbo/química , Bulbo/fisiologia , Células do Corno Posterior/química , Células do Corno Posterior/fisiologia , Tratos Piramidais/química , Tratos Piramidais/fisiologia , Nervo Trigêmeo/química , Animais , Química Encefálica/genética , Marcação de Genes/métodos , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Glutamato Descarboxilase/fisiologia , Masculino , Bulbo/ultraestrutura , Células do Corno Posterior/ultraestrutura , Tratos Piramidais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Medula Espinal/química , Medula Espinal/fisiologia , Medula Espinal/ultraestrutura , Nervo Trigêmeo/fisiologia , Nervo Trigêmeo/ultraestrutura
18.
Exp Neurol ; 232(2): 222-33, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21963650

RESUMO

Although prior studies have implicated maladaptive remodeling of dendritic spines on wide-dynamic range dorsal horn neurons as a contributor to pain after spinal cord injury, there have been no studies on dendritic spines after peripheral nerve injury. To determine whether dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain after peripheral nerve injury, we analyzed dendritic spine morphology and functional influence in lamina IV-V dorsal horn neurons after sham, chronic constriction injury (CCI) of the sciatic nerve, and CCI treatment with NSC23766, a selective inhibitor of Rac1, which has been implicated in dendritic spine development. 10 days after CCI, spine density increased with mature, mushroom-shaped spines preferentially distributed along dendritic branch regions closer to the cell body. Because spine morphology is strongly correlated with synaptic function and transmission, we recorded the response of single units to innocuous and noxious peripheral stimuli and performed behavioral assays for tactile allodynia and thermal hyperalgesia. Wide dynamic range dorsal horn neurons of CCI animals exhibited hyperexcitable responses to a range of stimuli. They also showed reduced nociceptive thresholds in the ipsilateral hind paw. 3-day treatment with NSC23766 significantly reduced post-CCI spine dimensions and densities, and attenuated injury-induced hyperexcitability. Drug treatment reduced behavioral measures of tactile allodynia, but not for thermal hyperalgesia. Together, our results demonstrate that peripheral nerve injury induces Rac1-regulated remodeling of dendritic spines on dorsal horn neurons, and suggest that this spine remodeling contributes to neuropathic pain.


Assuntos
Espinhas Dendríticas/fisiologia , Neuralgia/fisiopatologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Células do Corno Posterior/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Aminoquinolinas/farmacologia , Animais , Células Cultivadas , Doença Crônica , Espinhas Dendríticas/patologia , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Neuralgia/patologia , Nociceptores/fisiologia , Traumatismos dos Nervos Periféricos/patologia , Células do Corno Posterior/citologia , Células do Corno Posterior/ultraestrutura , Gravidez , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores
19.
PLoS One ; 6(8): e23275, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21858052

RESUMO

The synaptic connections between neurokinin 1 (NK1) receptor-like immunoreactive (LI) neurons and γ-aminobutyric acid (GABA)-, glycine (Gly)-, serotonin (5-HT)- or dopamine-ß-hydroxylase (DBH, a specific marker for norepinephrinergic neuronal structures)-LI axon terminals in the rat medullary dorsal horn (MDH) were examined under electron microscope by using a pre-embedding immunohistochemical double-staining technique. NK1 receptor-LI neurons were observed principally in laminae I and III, only a few of them were found in lamina II of the MDH. GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were densely encountered in laminae I and II, and sparsely in lamina III of the MDH. Some of these GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were observed to make principally symmetric synapses with NK1 receptor-LI neuronal cell bodies and dendritic processes in laminae I, II and III of the MDH. The present results suggest that neurons expressing NK1 receptor within the MDH might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons located in the MDH and 5-HT- or norepinephrine (NE)-containing descending fibers originated from structures in the brainstem.


Assuntos
Neurônios/fisiologia , Células do Corno Posterior/fisiologia , Receptores da Neurocinina-1/metabolismo , Transmissão Sináptica/fisiologia , Animais , Imunofluorescência , Glicina/metabolismo , Masculino , Bulbo/citologia , Bulbo/metabolismo , Bulbo/fisiologia , Microscopia Confocal , Microscopia Imunoeletrônica , Neurônios/metabolismo , Neurônios/ultraestrutura , Norepinefrina/metabolismo , Células do Corno Posterior/metabolismo , Células do Corno Posterior/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Wistar , Serotonina/metabolismo , Sinapses/metabolismo , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo
20.
Pain ; 152(4): 912-923, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21282008

RESUMO

Peripheral inflammation alters AMPA receptor (AMPAR) subunit trafficking and increases AMPAR Ca(2+) permeability at synapses of spinal dorsal horn neurons. However, it is unclear whether AMPAR trafficking at extrasynaptic sites of these neurons also changes under persistent inflammatory pain conditions. Using patch-clamp recording combined with Ca(2+) imaging and cobalt staining, we found that, under normal conditions, an extrasynaptic pool of AMPARs in rat substantia gelatinosa (SG) neurons of spinal dorsal horn predominantly consists of GluR2-containing Ca(2+)-impermeable receptors. Maintenance of complete Freund's adjuvant (CFA)-induced inflammation was associated with a marked enhancement of AMPA-induced currents and [Ca(2+)](i) transients in SG neurons, while, as we previously showed, the amplitude of synaptically evoked AMPAR-mediated currents was not changed 24 h after CFA. These findings indicate that extrasynaptic AMPARs are upregulated and their Ca(2+) permeability increases dramatically. This increase occurred in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibited strong adaptation. This increase was also accompanied by an inward rectification of AMPA-induced currents and enhancement of sensitivity to a highly selective Ca(2+)-permeable AMPAR blocker, IEM-1460. Electron microcopy and biochemical assays additionally showed an increase in the amount of GluR1 at extrasynaptic membranes in dorsal horn neurons 24h post-CFA. Taken together, our findings indicate that CFA-induced inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca(2+)-permeable AMPARs in tonically firing excitatory dorsal horn neurons, suggesting that the altered extrasynaptic AMPAR trafficking might participate in the maintenance of persistent inflammatory pain.


Assuntos
Potenciais de Ação/fisiologia , Inflamação/patologia , Células do Corno Posterior/metabolismo , Receptores de AMPA/metabolismo , Medula Espinal/patologia , Animais , Biotinilação/métodos , Cálcio/metabolismo , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Agonistas de Aminoácidos Excitatórios/efeitos adversos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Adjuvante de Freund/efeitos adversos , Técnicas In Vitro , Inflamação/induzido quimicamente , Ácido Caínico/efeitos adversos , Masculino , Microscopia Imunoeletrônica/métodos , Técnicas de Patch-Clamp/métodos , Células do Corno Posterior/fisiopatologia , Células do Corno Posterior/ultraestrutura , Ratos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
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