RESUMO
Mice heterozygously deficient for the myelin protein P0 gene (P0+/-) develop a slowly progressing neuropathy modeling demyelinating Charcot-Marie-Tooth disease (CMT1B). The aim of the study was to investigate the long-term progression of motor dysfunction in P0+/- mice at 3, 7, 12 and 20months. By comparison with WT littermates, P0+/- showed a decreasing motor performance with age. This was associated with a progressive reduction in amplitude and increase in latency of the plantar compound muscle action potential (CMAP) evoked by stimulation of the tibial nerve at ankle. This progressive functional impairment was in contrast to the mild demyelinating neuropathy of the tibial nerve revealed by histology. "Threshold-tracking" studies showed impaired motor axon excitability in P0+/- from 3months. With time, there was a progressive reduction in threshold deviations during both depolarizing and hyperpolarizing threshold electrotonus associated with increasing resting I/V slope and increasing strength-duration time constant. These depolarizing features in excitability in P0+/- as well as the reduced CMAP amplitude were absent in P0+/- NaV1.8 knockouts, and could be acutely reversed by selective pharmacologic block of NaV1.8 in P0+/-. Mathematical modeling indicated an association of altered passive cable properties with a depolarizing shift in resting membrane potential and increase in the persistent Na(+) current in P0+/-. Our data suggest that ectopic NaV1.8 expression precipitates depolarizing conduction failure in CMT1B, and that motor axon dysfunction in demyelinating neuropathy is pharmacologically reversible.
Assuntos
Axônios/patologia , Doença de Charcot-Marie-Tooth/patologia , Neurônios Motores/patologia , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Animais , Axônios/metabolismo , Doença de Charcot-Marie-Tooth/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Proteína P0 da Mielina/genética , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Condução Nervosa/fisiologia , Nervo Tibial/metabolismo , Nervo Tibial/patologiaRESUMO
BACKGROUND: The dental pulp is a common source of pain and is used to study peripheral inflammatory pain mechanisms. Results show most fibers are unmyelinated, yet recent findings in experimental animals suggest many pulpal afferents originate from fibers that are myelinated at more proximal locations. Here we use the human dental pulp and confocal microscopy to examine the staining relationships of neurofilament heavy (NFH), a protein commonly expressed in myelinated afferents, with other markers to test the possibility that unmyelinated pulpal afferents originate from myelinated axons. Other staining relationships studied included myelin basic protein (MBP), protein gene product (PGP) 9.5 to identify all nerve fibers, tyrosine hydroxylase (TH) to identify sympathetic fibers, contactin-associated protein (caspr) to identify nodal sites, S-100 to identify Schwann cells and sodium channels (NaChs). RESULTS: Results show NFH expression in most PGP9.5 fibers except those with TH and include the broad expression of NFH in axons lacking MBP. Fibers with NFH and MBP show NaCh clusters at nodal sites as expected, but surprisingly, NaCh accumulations are also seen in unmyelinated fibers with NFH, and in fibers with NFH that lack Schwann cell associations. CONCLUSIONS: The expression of NFH in most axons suggests a myelinated origin for many pulpal afferents, while the presence of NaCh clusters in unmyelinated fibers suggests an inherent capacity for the unmyelinated segments of myelinated fibers to form NaCh accumulations. These findings have broad implications on the use of dental pulp to study pain mechanisms and suggest possible novel mechanisms responsible for NaCh cluster formation and neuronal excitability.
Assuntos
Polpa Dentária/citologia , Fibras Nervosas Amielínicas/metabolismo , Proteínas de Neurofilamentos/metabolismo , Canais de Sódio/metabolismo , Contactina 1/metabolismo , Humanos , Microscopia Confocal , Proteína Básica da Mielina/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Proteínas S100/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
BACKGROUND: Animal studies and a few human studies have shown a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth as a model system to study peripheral pain mechanisms and here examine the expression of the Nav1.7 NaCh isoform in normal and painful samples. Pulpal sections were labeled with antibodies against: 1) Nav1.7, N52 and PGP9.5, and 2) Nav1.7, caspr (a paranodal protein used to identify nodes of Ranvier), and myelin basic protein (MBP), and a z-series of optically-sectioned images were obtained with the confocal microscope. Nav1.7-immunofluorescence was quantified in N52/PGP9.5-identified nerve fibers with NIH ImageJ software, while Nav1.7 expression in myelinated fibers at caspr-identified nodal sites was evaluated and further characterized as either typical or atypical as based on caspr-relationships. RESULTS: Results show a significant increase in nerve area with Nav1.7 expression within coronal and radicular fiber bundles and increased expression at typical and atypical caspr-identified nodal sites in painful samples. Painful samples also showed an augmentation of Nav1.7 within localized areas that lacked MBP, including those associated with atypical caspr-identified sites, thus identifying NaCh remodeling within demyelinating axons as the basis for a possible pulpal pain mechanism. CONCLUSION: This study identifies the increased axonal expression and augmentation of Nav1.7 at intact and remodeling/demyelinating nodes within the painful human dental pulp where these changes may contribute to constant, increased evoked and spontaneous pain responses that characterize the pain associated with toothache.
Assuntos
Polpa Dentária/metabolismo , Dor/metabolismo , Canais de Sódio/metabolismo , Adulto , Polpa Dentária/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Proteína Básica da Mielina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7RESUMO
BACKGROUND: Sodium channel (NaCh) expressions change following nerve and inflammatory lesions and this change may contribute to the activation of pain pathways. In a previous study we found a dramatic increase in the size and density of NaCh accumulations, and a remodeling of NaChs at intact and altered myelinated sites at a location just proximal to a combined partial axotomy and chromic suture lesion of the rat infraorbital nerve (ION) with the use of an antibody that identifies all NaCh isoforms. Here we evaluate the contribution of the major nodal NaCh isoform, Nav1.6, to this remodeling of NaChs following the same lesion. Sections of the ION from normal and ION lesioned subjects were double-stained with antibodies against Nav1.6 and caspr (contactin-associated protein; a paranodal protein to identify nodes of Ranvier) and then z-series of optically sectioned images were captured with a confocal microscope. ImageJ (NIH) software was used to quantify the average size and density of Nav1.6 accumulations, while additional single fiber analyses measured the axial length of the nodal gap, and the immunofluorescence intensity of Nav1.6 in nodes and of caspr in the paranodal region. RESULTS: The findings showed a significant increase in the average size and density of Nav1.6 accumulations in lesioned IONs when compared to normal IONs. The results of the single fiber analyses in caspr-identified typical nodes showed an increased axial length of the nodal gap, an increased immunofluorescence intensity of nodal Nav1.6 and a decreased immunofluorescence intensity of paranodal caspr in lesioned IONs when compared to normal IONs. In the lesioned IONs, Nav1.6 accumulations were also seen in association with altered caspr-relationships, such as heminodes. CONCLUSION: The results of the present study identify Nav1.6 as one isoform involved in the augmentation and remodeling of NaChs at nodal sites following a combined partial axotomy and chromic suture ION lesion. The augmentation of Nav1.6 may result from an alteration in axon-Schwann cell signaling mechanisms as suggested by changes in caspr expression. The changes identified in this study suggest that the participation of Nav1.6 should be considered when examining changes in the excitability of myelinated axons in neuropathic pain models.
Assuntos
Nervo Maxilar/lesões , Nervo Maxilar/metabolismo , Órbita/inervação , Dor/fisiopatologia , Nós Neurofibrosos/metabolismo , Canais de Sódio/metabolismo , Animais , Axônios/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Microscopia Confocal , Canal de Sódio Disparado por Voltagem NAV1.6 , Dor/etiologia , Isoformas de Proteínas/metabolismo , Nós Neurofibrosos/química , Ratos , Ratos Sprague-DawleyRESUMO
Na+ channel recovery from inactivation limits the maximal rate of neuronal firing. However, the properties of presynaptic Na+ channels are not well established because of the small size of most CNS boutons. Here we study the Na+ currents of the rat calyx of Held terminal and compare them with those of postsynaptic cells. We find that presynaptic Na+ currents recover from inactivation with a fast, single-exponential time constant (24 degrees C, tau of 1.4-1.8 ms; 35 degrees C, tau of 0.5 ms), and their inactivation rate accelerates twofold during development, which may contribute to the shortening of the action potential as the terminal matures. In contrast, recordings from postsynaptic cells in brainstem slices, and acutely dissociated, reveal that their Na+ currents recover from inactivation with a double-exponential time course (tau(fast) of 1.2-1.6 ms; tau(slow) of 80-125 ms; 24 degrees C). Surprisingly, confocal immunofluorescence revealed that Na+ channels are mostly absent from the calyx terminal but are instead highly concentrated in an unusually long (approximately 20-40 microm) unmyelinated axonal heminode. Outside-out patch recordings confirmed this segregation. Expression of Na(v)1.6 alpha-subunit increased during development, whereas the Na(v)1.2alpha-subunit was not present. Serial EM reconstructions also revealed a long pre-calyx heminode, and biophysical modeling showed that exclusion of Na+ channels from the calyx terminal produces an action potential waveform with a shorter half-width. We propose that the high density and polarized locus of Na+ channels on a long heminode are critical design features that allow the mature calyx of Held terminal to fire reliably at frequencies near 1 kHz.
Assuntos
Neurônios/citologia , Terminações Pré-Sinápticas/metabolismo , Canais de Sódio/fisiologia , Sinapses/fisiologia , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Vias Aferentes/fisiologia , Vias Aferentes/efeitos da radiação , Fatores Etários , Animais , Animais Recém-Nascidos , Tronco Encefálico/citologia , Tronco Encefálico/crescimento & desenvolvimento , Cloreto de Cádmio/farmacologia , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Imunofluorescência/métodos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Modelos Neurológicos , Canal de Sódio Disparado por Voltagem NAV1.6 , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/ultraestrutura , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Tempo de Reação/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologiaRESUMO
Voltage-gated sodium channels (VGSCs) ensure the saltatory propagation of action potentials along axons by acting as signal amplifiers at the nodes of Ranvier. In the retina, activity mediated by VGSCs is important for the refinement of the retinotectal map. Here, we conducted a full-field electroretinogram (ERG) study on mice null for the sodium channel NaV1.6. Interestingly, the light-activated hyperpolarization of photoreceptor cells (the a-wave) and the major "downstream" components of the ERG, the b-wave and the oscillatory potentials, are markedly reduced and delayed in these mice. The functional deficit was not associated with any morphological abnormality. We demonstrate that Scn8a is expressed in the ganglion and inner nuclear layers and at low levels in the outer nuclear layer beginning shortly before the observed ERG deficit. Together, our data reveal a previously unappreciated role for VGSCs in the physiological maturation of photoreceptors.
Assuntos
Proteínas do Tecido Nervoso/fisiologia , Células Fotorreceptoras/fisiologia , Canais de Sódio/fisiologia , Animais , Animais Recém-Nascidos , Relação Dose-Resposta à Radiação , Eletrorretinografia/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão/métodos , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/deficiência , Estimulação Luminosa/métodos , Células Fotorreceptoras/crescimento & desenvolvimento , Células Fotorreceptoras/efeitos da radiação , Células Fotorreceptoras/ultraestrutura , Canais de Sódio/deficiênciaRESUMO
Voltage-dependent sodium channels cluster at high density at axon initial segments, where propagating action potentials are thought to arise, and at nodes of Ranvier. Here, we show that the sodium channel Na(v)1.6 is precisely localized at initial segments of retinal ganglion cells (RGCs), whereas a different isoform, Na(v)1.2, is found in the neighboring unmyelinated axon. During development, initial segments first expressed Na(v)1.2, and Na(v)1.6 appeared later, approximately in parallel with the onset of repetitive RGC firing. In Shiverer mice, Na(v)1.6 localization at the initial segment was unaffected, although Na(v)1.6 expression was severely disrupted in the aberrantly myelinated optic nerve. Targeting or retention of Na(v)1.6 requires molecular interactions that normally occur only at initial segments and nodes of Ranvier. Expression at nodes but not initial segments exhibits an additional requirement for intact myelination. Because of their high density at the initial segment, Na(v)1.6 channels may be crucial in determining neuronal firing properties.
Assuntos
Axônios/metabolismo , Isoformas de Proteínas/metabolismo , Canais de Sódio/biossíntese , Animais , Anquirinas/biossíntese , Moléculas de Adesão Celular/biossíntese , Diferenciação Celular/fisiologia , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Mutantes Neurológicos , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Canal de Sódio Disparado por Voltagem NAV1.2 , Fatores de Crescimento Neural/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Nervo Óptico/patologia , Isoformas de Proteínas/genética , Ratos , Ratos Sprague-Dawley , Retina/citologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Canais de Sódio/genéticaRESUMO
We have examined the molecular organization of axons in the spinal cords of myelin-deficient (md) rats, which have profound CNS dysmyelination associated with oligodendrocyte cell death. Although myelin sheaths are rare, most large axons are at least partially surrounded by oligodendrocyte processes. At postnatal day 7 (P7), almost all node-like clusters of voltage-gated Na+ channels and ankyrinG are adjacent to axonal segments ensheathed by oligodendrocytes, but at P21, many node-like clusters are found in axonal segments that lack oligodendrocyte ensheathment. In P21 wild-type (WT) rats, the voltage-gated Na+ channels Na(v)1.2, Na(v)1.6, and Na(v)1.8, are found in different subpopulations of myelinated axons, and md rats have a similar distribution. The known molecular components of paranodes--contactin, Caspr, and neurofascin 155--are not clustered in md spinal cords, and no septate-like junctions between oligodendrocyte processes and axons are found by electron microscopy. Furthermore, Kv1.1 and Kv1.2 K+ channels are not spatially segregated from the node-like clusters of Na+ channels in md rats, in contrast to their WT littermates. These results suggest the following: node-like clusters of voltage-gated Na+ channels and ankyrinG form adjacent to ensheathed axonal segments even in the absence of a myelin sheath; these clusters persist after oligodendrocyte cell death; dysmyelination does not alter the expression of different nodal of voltage-gated Na+ channels; the absence of paranodes results in the mislocalization of neurofascin155, contactin, and Caspr, and the aberrant localization of Kv1.1 and Kv1.2.
Assuntos
Axônios/patologia , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Bainha de Mielina/patologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Medula Espinal/patologia , Animais , Anquirinas/biossíntese , Axônios/metabolismo , Axônios/ultraestrutura , Canal de Potássio Kv1.1 , Canal de Potássio Kv1.2 , Masculino , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Canais de Potássio/biossíntese , Nós Neurofibrosos/metabolismo , Nós Neurofibrosos/patologia , Nós Neurofibrosos/ultraestrutura , Ratos , Ratos Mutantes , Canais de Sódio/biossíntese , Medula Espinal/metabolismo , Medula Espinal/ultraestruturaRESUMO
The node of Ranvier is a distinct domain of myelinated axons that is highly enriched in sodium channels and is critical for impulse propagation. During development, the channel subtypes expressed at the node undergo a transition from Nav1.2 to Nav1.6. Specialized junctions that form between the paranodal glial membranes and axon flank the nodes and are candidates to regulate their maturation and delineate their boundaries. To investigate these roles, we characterized node development in mice deficient in contactin-associated protein (Caspr), an integral junctional component. Paranodes in these mice lack transverse bands, a hallmark of the mature junction, and exhibit progressive disruption of axon-paranodal loop interactions in the CNS. Caspr mutant mice display significant abnormalities at central nodes; components of the nodes progressively disperse along axons, and many nodes fail to mature properly, persistently expressing Nav1.2 rather than Nav1.6. In contrast, PNS nodes are only modestly longer and, although maturation is delayed, eventually all express Nav1.6. Potassium channels are aberrantly clustered in the paranodes; these clusters are lost over time in the CNS, whereas they persist in the PNS. These findings indicate that interactions of the paranodal loops with the axon promote the transition in sodium channel subtypes at CNS nodes and provide a lateral diffusion barrier that, even in the absence of transverse bands, maintains a high concentration of components at the node and the integrity of voltage-gated channel domains.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Nós Neurofibrosos/metabolismo , Canais de Sódio/metabolismo , Fatores Etários , Animais , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Difusão , Técnica de Fratura por Congelamento , Técnicas In Vitro , Canal de Potássio Kv1.1 , Canal de Potássio Kv1.2 , Camundongos , Camundongos Mutantes , Canal de Sódio Disparado por Voltagem NAV1.2 , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/metabolismo , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/metabolismo , Nervo Óptico/fisiologia , Canais de Potássio/biossíntese , Nós Neurofibrosos/ultraestrutura , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/metabolismo , Nervo Isquiático/fisiologiaRESUMO
The activation of voltage-gated sodium channels is necessary for action potential propagation and multiple sodium channel isoforms have been identified that show a differential distribution throughout the nervous system. An evaluation of sodium channel localization in the radicular pulp from normal human extracted third molars established the presence of the Na(v)1.8 isoform at nodes of Ranvier in a subpopulation of the myelinated axons as demonstrated with immunofluorescence confocal microscopy. A caspr antibody was used to identify the paranodal region of nodes of Ranvier and quantitative analysis revealed that 16.5% of the nodes contained significant Na(v)1.8 immunoreactivity. Since the Na(v)1.6 isoform has been described as the predominant sodium channel at essentially all nodes, the finding of Na(v)1.8 in a subpopulation of nodes suggests that multiple isoforms may coexist at some nodes of Ranvier and also suggests that this isoform may be an important nodal sodium channel type in the peripheral sensory nervous system of humans.
Assuntos
Polpa Dentária/citologia , Fatores de Crescimento Neural/metabolismo , Nós Neurofibrosos/metabolismo , Intervalos de Confiança , Humanos , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos , Canal de Sódio Disparado por Voltagem NAV1.8 , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/metabolismo , Canais de Sódio/metabolismoRESUMO
CaV1.1 acts as both the voltage sensor that triggers excitation-contraction coupling in skeletal muscle and as an L-type Ca(2+) channel. It has been proposed that, after its posttranslational cleavage, the distal C terminus of CaV1.1 remains noncovalently associated with proximal CaV1.1, and that tethering of protein kinase A to the distal C terminus is required for depolarization-induced potentiation of L-type Ca(2+) current in skeletal muscle. Here, we report that association of the distal C terminus with proximal CaV1.1 cannot be detected by either immunoprecipitation of mouse skeletal muscle or by colocalized fluorescence after expression in adult skeletal muscle fibers of a CaV1.1 construct labeled with yellow fluorescent protein (YFP) and cyan fluorescent protein on the N and C termini, respectively. We found that L-type Ca(2+) channel activity was similar after expression of constructs that either did (YFP-CaV1.11860) or did not (YFP-CaV1.11666) contain coding sequence for the distal C-terminal domain in dysgenic myotubes null for endogenous CaV1.1. Furthermore, in response to strong (up to 90 mV) or long-lasting prepulses (up to 200 ms), tail current amplitudes and decay times were equally increased in dysgenic myotubes expressing either YFP-CaV1.11860 or YFP-CaV1.11666, suggesting that the distal C-terminal domain was not required for depolarization-induced potentiation. Thus, our experiments do not support the existence of either biochemical or functional interactions between proximal CaV1.1 and the distal C terminus.
Assuntos
Canais de Cálcio Tipo L/química , Ativação do Canal Iônico , Potenciais de Ação , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
UNLABELLED: Although nerve growth factor plays a role in augmenting sodium channel expression in small dorsal root ganglion (DRG) cells, the cytochemical mediators responsible for enhanced expression in large DRG neurons are unknown. To narrow the search for mediators involved in the increased production of sodium channels in large DRG neurons, we examined the effect of cyclooxygenase inhibition on sodium channel production during inflammation. Thirty minutes before the subcutaneous injection of complete Freund's adjuvant (CFA), rats received ibuprofen (nonselective, cyclooxygenase inhibitor), NS-398 (selective, cyclooxygenase inhibitor), or vehicle. Withdrawal thresholds from thermal and mechanical stimulation were measured before and immediately after CFA injection and at selected hourly intervals after injection for the next 24 hours. Sodium channel up-regulation was then examined in DRG by using site-specific, anti-sodium channel antibodies, Na(v) 1.7 and 1.8. Both ibuprofen and NS-398 provided analgesia during the second phase of inflammatory hyperalgesia that begins 3 hours after CFA injection. The up-regulation, predominantly of Na(v) 1.7 and minimally of Na(v) 1.8 channels, seen in vehicle-treated rats was suppressed by both drugs at 24 hours after injection. By 72 hours after injection, no difference in labeling between the drug- and vehicle-treated animals was observed. Sodium channel labeling in large DRG neurons returned to baseline between 1 and 2 weeks after CFA injection, whereas small cell labeling persisted. The cytochemical signal for sodium channel up-regulation in the large DRG cells that most closely correlates with inflammatory hyperalgesia is mediated at least in part through products of the cyclooxygenase pathway. PERSPECTIVE: Expression of sodium channels in dorsal root ganglia increases dramatically during inflammation. The increase in sodium channels is thought to enhance neuronal excitability and to play a role in hyperalgesia and wound vigilance during healing. We provide evidence that prostaglandins play a role in signaling channel augmentation.
Assuntos
Ibuprofeno/farmacologia , Inflamação/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/biossíntese , Animais , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Adjuvante de Freund/toxicidade , Ibuprofeno/uso terapêutico , Inflamação/tratamento farmacológico , Canal de Sódio Disparado por Voltagem NAV1.7 , Canal de Sódio Disparado por Voltagem NAV1.8 , Proteínas do Tecido Nervoso/biossíntese , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Modulation of phosphorylation states of ion channels is a critical step in the development of hyperalgesia during inflammation. Modulatory enhancement of channel activity may increase neuronal excitability and affect downstream targets such as gene transcription. The specificity required for such regulation of ion channels quickly occurs via targeting of protein kinases and phosphatases by the scaffolding A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 has been implicated in inflammatory pain by targeting protein kinase A (PKA) and protein kinase C (PKC) to the transient receptor potential vanilloid 1 (TRPV1) channel in peripheral sensory neurons, thus lowering threshold for activation of the channel by multiple inflammatory reagents. However, the expression pattern of AKAP150 in peripheral sensory neurons is unknown. Here we identify the peripheral neuron subtypes that express AKAP150, the subcellular distribution of AKAP150, and the potential target ion channels in rat dorsal root ganglion (DRG) slices. We found that AKAP150 is expressed predominantly in a subset of small DRG sensory neurons, where it is localized at the plasma membrane of the soma, axon initial segment, and small fibers. Most of these neurons are peripherin positive and produce C fibers, although a small portion produce Aδ fibers. Furthermore, we demonstrate that AKAP79/150 colocalizes with TRPV1 and Ca(V) 1.2 in the soma and axon initial segment. Thus AKAP150 is expressed in small, nociceptive DRG neurons, where it is targeted to membrane regions and where it may play a role in the modulation of ion channel phosphorylation states required for hyperalgesia.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Canais de Cálcio/metabolismo , Gânglios Espinais/citologia , Nociceptores/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Imuno-Histoquímica , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Nociceptores/citologia , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
UNLABELLED: The expression of sodium channels (NaCh(s)) change after inflammatory and nerve lesions, and this change has been implicated in the generation of pain states. Here we examine NaCh expression within nerve fibers from normal and painful extracted human teeth with special emphasis on their localization within large accumulations, like those seen at nodes of Ranvier. Pulpal tissue sections from normal wisdom teeth and from teeth with large carious lesions associated with severe and spontaneous pain were double-stained with pan-specific NaCh antibody and caspr (paranodal protein used to visualize nodes of Ranvier) antibody, while additional sections were triple-stained with NaCh, caspr and myelin basic protein (MBP) antibodies. Z-series of images were obtained with the confocal microscope and evaluated with NIH ImageJ software to quantify the density and size of NaCh accumulations, and to characterize NaCh localization at caspr-identified typical and atypical nodal sites. Although the results showed variability in the overall density and size of NaCh accumulations in painful samples, a common finding included the remodeling of NaChs at atypical nodal sites. This remodeling of NaChs included prominent NaCh expression within nerve regions that showed a selective loss of MBP staining in a pattern consistent with a demyelinating process. PERSPECTIVE: This study identifies the remodeling of NaChs at demyelinated sites within the painful human dental pulp and suggests that the contribution of NaChs to spontaneous pulpal pain generation may be dependant not only on total NaCh density but may also be related to NaCh expression at atypical nodal sites.
Assuntos
Doenças Desmielinizantes/metabolismo , Polpa Dentária/metabolismo , Fibras Nervosas/metabolismo , Dor/metabolismo , Nós Neurofibrosos/metabolismo , Canais de Sódio/metabolismo , Adolescente , Adulto , Axônios/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Contactinas , Polpa Dentária/lesões , Polpa Dentária/inervação , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Dente Serotino , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/metabolismo , Adulto JovemRESUMO
Voltage-gated sodium channels (VGSCs) are one of the fundamental building blocks of electrically excitable cells in the nervous system. These channels are responsible for the generation of action potentials that are required for the communication of neuronal signals over long distances within a cell. VGSCs are encoded by a family of nine genes whose products have widely varying biophysical properties. In this study, we have detected the expression of two atypical VGSCs (Na(v)1.8 and Na(v)1.9) in the retina. Compared with more common VGSCs, Na(v)1.8 and Na(v)1.9 have unusual biophysical and pharmacological properties, including persistent sodium currents and resistance to the canonical sodium channel blocker tetrodotoxin (TTX). Our molecular biological and immunohistochemical data derived from mouse (Mus musculus) retina demonstrate expression of Na(v)1.8 by retinal amacrine and ganglion cells, whereas Na(v)1.9 is expressed by photoreceptors and Müller glia. The fact that these channels exist in the central nervous system (CNS) and exhibit robust TTX resistance requires a re-evaluation of prior physiological, pharmacological, and developmental data in the visual system, in which the diversity of VGSCs has been previously underestimated.
Assuntos
Neuropeptídeos/metabolismo , Retina/citologia , Retina/metabolismo , Canais de Sódio/metabolismo , Células Amácrinas/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.8 , Canal de Sódio Disparado por Voltagem NAV1.9 , Neuroglia/metabolismo , Neuropeptídeos/genética , Células Fotorreceptoras/metabolismo , Células Ganglionares da Retina/metabolismo , Canais de Sódio/deficiência , Canais de Sódio/genéticaRESUMO
Patients with hereditary neuropathies are more susceptible to vincristine (VIN)-induced neuropathy than patients without this comorbidity. The heterozygous P0(+/-) mouse is an animal model of a distinct form of inherited neuropathies. These mice produce only 50% of the major myelin protein protein zero (P0) and display signs of demyelination in motor nerves at 4 months of age. Here we investigated the development of neuropathic signs in P0(+/-) and wild-type (wt) mice after VIN treatment. Neuropathy was induced by daily intraperitoneal injections of VIN (0.5 mg/kg body weight) over 10 days. Behavioral and electrophysiological tests were performed at regular time points. Wt mice developed significant hypersensitivity to heat and mechanical stimuli between days 7 and 38 after the first VIN injection. Surprisingly, P0(+/-) mice did not show sensory or motor signs of neuropathy over the whole testing period. Immunohistochemical analysis showed an increase in macrophage numbers in sciatic nerve sections of wt mice after VIN, whereas P0(+/-) mice had higher baseline levels of macrophages without changes after VIN treatment. Semithin sections revealed a decrease in the number of small-diameter myelinated fibers in the sciatic nerves of wt mice after VIN application, whereas P0(+/-) mice had higher baseline values of this fiber subtype that did not change under treatment. Dorsal root ganglion neurons of both genotypes showed an up-regulation of voltage-gated sodium channel immunoreactivity after VIN application without differences between the genotypes. Thus, the P0(+/-) phenotype seems to be protected against VIN-induced neuropathy. The mechanism of this neuroprotection remains elusive.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Proteína P0 da Mielina/deficiência , Polineuropatias/induzido quimicamente , Vincristina/toxicidade , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Comportamento Animal/efeitos dos fármacos , Eletrofisiologia/métodos , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/metabolismo , Condução Nervosa/efeitos dos fármacos , Condução Nervosa/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Medição da Dor/métodos , Polineuropatias/patologia , Polineuropatias/fisiopatologia , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/genética , Canais de Sódio/metabolismo , Estatísticas não ParamétricasRESUMO
The localization of sodium channels (NaChs) change following nerve lesions and this change may contribute to the development of increased pain states. Here we examine the change in distribution of NaChs within the rat infraorbital nerve (ION) two weeks after a combined inflammatory/partial axotomy lesion that results in behavior showing increased sensitivity to mechanical stimuli. Sections from experimental and normal control IONs were double-stained for indirect immunofluorescence using an antibody that identifies all NaCh isoforms and caspr-antibody to identify nodes of Ranvier, and a confocal microscope z-series of optically sectioned images were then obtained. ImageJ (NIH) software was used to quantify the area of pixels showing maximum NaCh intensity within both caspr and non-caspr associated accumulations. Analysis showed that the lesioned IONs had many more split nodes, heminodes and caspr-negative "naked" accumulations, a significantly increased area of NaCh staining within typical nodes and "naked" accumulations, as well as an increased density and size of significant accumulations when compared to normal IONs. This study demonstrates a dramatic redistribution and increased immunofluorescence of NaChs especially at myelinated and demyelinated sites in fibers located just proximal to the lesion. The remodeling of NaChs seen in this study may represent an important event associated with the development of increased nerve excitability after lesions.
Assuntos
Imunofluorescência , Órbita/inervação , Nervos Periféricos/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Nós Neurofibrosos/metabolismo , Canais de Sódio/metabolismo , Animais , Axotomia/métodos , Comportamento Animal , Moléculas de Adesão Celular Neuronais/metabolismo , Doenças Desmielinizantes/etiologia , Doenças Desmielinizantes/metabolismo , Feminino , Lateralidade Funcional , Microscopia Confocal/métodos , Nervos Periféricos/patologia , Nervos Periféricos/fisiopatologia , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Estimulação Física , Nós Neurofibrosos/patologia , Ratos , Ratos Sprague-DawleyRESUMO
The sensory root entry zone demarcates the transition from the peripheral nervous system (PNS) to the central nervous system (CNS). In this study, we describe the organization of nodes of Ranvier at the trigeminal sensory and dorsal root entry zones of the rat. Caspr immunoreactivity (IR) was used to identify the paranodal region of nodes of Ranvier, while L-MAG-IR was used to identify CNS oligodendrocytes. Immunofluorescence confocal microscopy revealed a dense aggregation of nodes precisely at the PNS to CNS transition with prominent node-depleted zones on either side, while L-MAG-IR was confined to ensheathing fibers on the central side of nodes located in this dense band and identified these as transitional nodes. Morphometric analysis of the PNS and CNS sides of the trigeminal and the PNS side of the dorsal root entry zones confirmed the presence of virtually node-free domains flanking the transitional zone. Further, the reappearance of nodes on the far side of the node-free zones strongly correlated with nodal diameter, with small nodes reappearing first. These findings suggest that the PNS/CNS transition may represent the initial site of myelination of the primary afferent axon within this area.
Assuntos
Moléculas de Adesão Celular Neuronais/análise , Gânglios Espinais/química , Nós Neurofibrosos/química , Gânglio Trigeminal/química , Animais , Moléculas de Adesão Celular Neuronais/fisiologia , Agregação Celular/fisiologia , Feminino , Gânglios Espinais/fisiologia , Nós Neurofibrosos/fisiologia , Ratos , Ratos Sprague-Dawley , Gânglio Trigeminal/fisiologiaRESUMO
To elucidate the impact of myelinating Schwann cells on the molecular architecture of the node of Ranvier, we investigated the nodal expression of voltage-gated sodium channel (VGSC) isoforms and the localization of paranodal and juxtaparanodal membrane proteins in a severely affected Schwann cell mutant, the mouse deficient in myelin protein zero (P0). The abnormal myelin formation and compaction was associated with immature nodal cluster types of VGSC. Most strikingly, P0-deficient motor nerves displayed an ectopic nodal expression of the Na(v)1.8 isoform, where it is coexpressed with the ubiquitous Na(v)1.6 channel. Furthermore, Caspr was distributed asymmetrically or was even absent in the mutant nerve fibers. The potassium channel K(v)1.2 and Caspr2 were not confined to juxtaparanodes, but often protruding into the paranodes. Thus, deficiency of P0 leads to dysregulation of nodal VGSC isoforms and to altered localization of paranodal and juxtaparanodal components of the nodal complex.