RESUMO
This study aimed to investigate and compare cell growth manners and functional differences of primary cortical neurons cultured on either poly-d-lysine (PDL) and or Matrigel, to delineate the role of extracellular matrix on providing resemblance to in vivo cellular interactions in nervous tissue. Primary cortical neurons, obtained from embryonic day 15 mice pups, seeded either on PDL- or Matrigel-coated culture ware were investigated by DIC/bright field and fluorescence/confocal microscopy for their morphology, 2D and 3D structure, and distribution patterns. Patch clamp, western blot, and RT-PCR studies were performed to investigate neuronal firing thresholds and sodium channel subtypes Nav1.2 and Nav1.6 expression. Cortical neurons cultured on PDL coating possessed a 2D structure composed of a few numbers of branched and tortuous neurites that contacted with each other in one to one manner, however, neurons on Matrigel coating showed a more complicated dimensional network that depicted tight, linear axonal bundles forming a 3D interacted neuron-astrocyte construction. This difference in growth patterns also showed a significant alteration in neuronal firing threshold which was recorded between 80 < Iinj > 120 pA on PDL and 2 < Iinj > 160 pA on Matrigel. Neurons grown up on Matrigel showed increased levels of sodium channel protein expression of Nav1.2 and Nav1.6 compared to neurons on PDL. These results have demonstrated that a 3D interacted neuron-astrocyte construction on Matrigel enhances the development of Nav1.2 and Nav1.6 in vitro and decreases neuronal firing threshold by 40 times compared to conventional PDL, resembling in vivo neuronal networks and hence would be a better in vitro model of adult neurons.
Assuntos
Astrócitos/fisiologia , Astrócitos/ultraestrutura , Colágeno , Laminina , Neurônios/fisiologia , Neurônios/ultraestrutura , Proteoglicanas , Canais de Sódio Disparados por Voltagem/biossíntese , Animais , Córtex Cerebral/citologia , Combinação de Medicamentos , Fenômenos Eletrofisiológicos , Embrião de Mamíferos/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Canal de Sódio Disparado por Voltagem NAV1.2/biossíntese , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.6/biossíntese , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Neuritos/fisiologia , Técnicas de Patch-Clamp , Gravidez , Cultura Primária de CélulasRESUMO
In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos-Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para Despite being the only NaV channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (NaV) channel in the fly, para, and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally, para is only expressed in 23 ± 1% of third instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the third instar larval brain shows that para expression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ± 1% of third instar larval neurons may be able to actively fire NaV-dependent APs.
Assuntos
Segmento Inicial do Axônio/metabolismo , Proteínas de Drosophila/biossíntese , Drosophila/metabolismo , Neurônios/metabolismo , Canais de Sódio/biossíntese , Canais de Sódio Disparados por Voltagem/biossíntese , Potenciais de Ação/fisiologia , Animais , Axônios/fisiologia , Dendritos/metabolismo , Proteínas de Drosophila/genética , Fenômenos Eletrofisiológicos , Eletrorretinografia , Expressão Gênica/genética , Larva , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiologia , Técnicas de Patch-Clamp , Canais de Sódio/genética , Transcriptoma , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
Objectives: Despite the etiology of trigeminal neuralgia has been verified by microvascular decompression as vascular compression of the trigeminal root, very few researches concerning its underlying pathogenesis has been reported in the literature. The present study focused on those voltage-gated sodium channels, which are the structural basis for generation of ectopic action potentials. Methods: The trigeminal neuralgia modeling was obtained with infraorbital nerve chronic constriction injury (ION-CCI) in rats. Two weeks postoperatively, the infraorbital nerve (TN), the trigeminal ganglion (TG), and the brain stem (BS) were removed and analyzed with a series of molecular biological techniques. Results: Western blot depicted a significant up-regulation of Nav1.3 in TN and TG but not in BS, while none of the other isoforms (Nav1.6, Nav1.7, Nav1.8, or Nav1.9) presented a statistical change. The Nav1.3 from ION-CCI group was quantified as 2.5-fold and 1.7-fold than that from sham group in TN and TG, respectively (p < .05). Immunocytochemistry showed the Nav1.3-IR from ION-CCI group accounted for 21.2 ± 2.3% versus 6.1 ± 1.2% from sham group in TN, while the Nav1.3-positive neurons from ION-CCI group accounted for 34.1 ± 3.5% versus 11.2 ± 1.8% from sham group in TG. Immunohistochemical labeling showed the Nav1.3 was co-localized with CGRP and IB4 but not with GFAP or NF-200 in TG. Conclusion: ION-CCI may give rise to an up-regulation of Nav1.3 in trigeminal nerve as well as in C-type neurons at the trigeminal ganglion. It implied that the ectopic action potential may generate from both the compressed site of the trigeminal nerve and the ganglion rather than from the trigeminal nuclei.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.3/biossíntese , Nervo Trigêmeo/metabolismo , Neuralgia do Trigêmeo/metabolismo , Animais , Constrição , Expressão Gênica , Masculino , Canal de Sódio Disparado por Voltagem NAV1.3/genética , Fibras Nervosas Amielínicas/metabolismo , Ratos , Ratos Sprague-Dawley , Gânglio Trigeminal/metabolismo , Neuralgia do Trigêmeo/genética , Canais de Sódio Disparados por Voltagem/biossíntese , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of the avian lumbosacral spinal cord. Histological evidence indicates that neuron-like cells gather in the ALs, and behavioral evidence suggests that the ALs act as a sensory organ of equilibrium during bipedal walking. Recently, using an electrophysiological method, we reported that cells showing Na+ currents and action potentials exist among cells that were dissociated from the ALs. However, it was unclear which isoforms of the voltage-gated sodium channel (VGSC) are expressed in the ALs and whether cells having neuronal morphology in the ALs express VGSCs. To elucidate these points, RT-PCR and immunohistochemical experiments were performed. In RT-PCR analysis, PCR products for Nav 1.1-1.7 were detected in the ALs. The signal intensities of the Nav 1.1 and 1.6 isoforms were stronger than those of the other isoforms. We confirmed that an antibody raised against an epitope peptide of the rat VGSC had cross-reactivity to chick tissues by Western blotting, and we performed immunofluorescence staining using the antibody. The AL contained cells having neuron-like morphology and VGSC-like immunoreactivity at their cytoplasm and/or cell membranes. Filament-like structures showing GFAP-like immunoreactivity infilled intercellular spaces. The VGSC- and GFAP-like immunoreactivities did not overlap. These results indicate that the neuronal isoforms of the VGSC are mainly expressed in the AL and that the neuron-like cells in the ALs express VGSCs. Our findings indicate that AL neurons generate action potentials and send sensory information to the motor systems on the contralateral side of the spinal segment.
Assuntos
Proteínas Aviárias/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Medula Espinal/embriologia , Canais de Sódio Disparados por Voltagem/biossíntese , Animais , Embrião de Galinha , Masculino , Neurônios/citologia , Ratos , Ratos Wistar , Medula Espinal/citologiaRESUMO
This study aimed to obtain the coding cDNA sequences of voltage-gated Na+ channel (scn) α-subunit (scna) and ß-subunit (scnb) isoforms from, and to quantify their transcript levels in, the main electric organ (EO), Hunter's EO, Sach's EO and the skeletal muscle (SM) of the electric eel, Electrophorus electricus, which can generate both high and low voltage electric organ discharges (EODs). The full coding sequences of two scna (scn4aa and scn4ab) and three scnb (scn1b, scn2b and scn4b) were identified for the first time (except scn4aa) in E. electricus. In adult fish, the scn4aa transcript level was the highest in the main EO and the lowest in the Sach's EO, indicating that it might play an important role in generating high voltage EODs. For scn4ab/Scn4ab, the transcript and protein levels were unexpectedly high in the EOs, with expression levels in the main EO and the Hunter's EO comparable to those of scn4aa. As the key domains affecting the properties of the channel were mostly conserved between Scn4aa and Scn4ab, Scn4ab might play a role in electrogenesis. Concerning scnb, the transcript level of scn4b was much higher than those of scn1b and scn2b in the EOs and the SM. While the transcript level of scn4b was the highest in the main EO, protein abundance of Scn4b was the highest in the SM. Taken together, it is unlikely that Scna could function independently to generate EODs in the EOs as previously suggested. It is probable that different combinations of Scn4aa/Scn4ab and various Scnb isoforms in the three EOs account for the differences in EODs produced in E. electricus. In general, the transcript levels of various scn isoforms in the EOs and the SM were much higher in adult than in juvenile, and the three EOs of the juvenile fish could be functionally indistinct.
Assuntos
Electrophorus/metabolismo , Isoformas de Proteínas/biossíntese , RNA Mensageiro/biossíntese , Canais de Sódio Disparados por Voltagem/biossíntese , Animais , Órgão Elétrico/enzimologia , Electrophorus/genética , Regulação Enzimológica da Expressão Gênica , Músculo Esquelético/enzimologia , Isoformas de Proteínas/genética , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
L1 (also known as L1CAM) is a trans-membrane glycoprotein mediating neuron-neuron adhesion through homophilic and heterophilic interactions. Although experimental evidence has implicated L1 in axonal outgrowth, fasciculation and pathfinding, its contribution to voltage-gated Na(+) channel function and membrane excitability has remained unknown. Here, we show that firing rate, single cell spiking frequency and Na(+) current density are all reduced in hippocampal excitatory neurons from L1-deficient mice both in culture and in slices owing to an overall reduced membrane expression of Na(+) channels. Remarkably, normal firing activity was restored when L1 was reintroduced into L1-deficient excitatory neurons, indicating that abnormal firing patterns are not related to developmental abnormalities, but are a direct consequence of L1 deletion. Moreover, L1 deficiency leads to impairment of action potential initiation, most likely due to the loss of the interaction of L1 with ankyrin G that produces the delocalization of Na(+) channels at the axonal initial segment. We conclude that L1 contributes to functional expression and localization of Na(+) channels to the neuronal plasma membrane, ensuring correct initiation of action potential and normal firing activity.
Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Canais de Sódio Disparados por Voltagem/biossíntese , Animais , Membrana Celular/genética , Hipocampo/citologia , Camundongos , Camundongos Knockout , Molécula L1 de Adesão de Célula Nervosa/genética , Neurônios/citologia , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
Multiple isoforms of voltage-gated Na(+) channels (NaChs) have been identified in sinoatrial node (SAN) and contribute to a rapid intrinsic heart rate. However, their roles in aging remain unclear. Here, we sought to clarify whether the age-related expression of NaChs contributes to the impaired SAN function during aging. Blockade of the tetrodotoxin (TTX)-sensitive Na(+) current with nanomolar concentrations of TTX prolonged the cycle length (CL) in both the rat intact heart and SAN. The effect of nanomolar concentrations of TTX on SAN pacemaking was lessened in adulthood compared with that in youth. Interestingly, the pacemaking became more sensitive to TTX and TTX-induced sinus arrhythmias occurred more frequently in the senescent group. The presences of NaCh α subunit isoforms Nav1.1, Nav1.6 as well as ß subunit isoforms Navß1 and Navß3 in SAN were confirmed by immunohistochemistry. Western blot revealed a declination of Nav1.1, Nav1.6, Navß1 and Navß3 proteins during aging. Furthermore, laser captured SAN cells were used for further real-time quantitative RT-PCR analysis, which also confirmed the presences of Nav1.1, Nav1.6, Navß1 and Navß3 mRNA and their reduced levels in rat SAN during aging. These results indicated an age-dependent alterations in expression and relative function of NaCh in rat SAN.
Assuntos
Envelhecimento/metabolismo , Regulação da Expressão Gênica , Canais de Sódio Disparados por Voltagem/biossíntese , Animais , Masculino , Isoformas de Proteínas/biossíntese , Ratos , Ratos Sprague-Dawley , Nó SinoatrialRESUMO
Voltage dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming α-subunit, encoded in mammals by up to 9 different genes, and 4 different ancillary ß-subunits. The expression pattern of the α subunit isoforms confers the distinctive functional and pharmacological properties to different excitable tissues. ß subunits are important modulators of channel function and expression. Mutation C121W of the ß1-subunit causes an autosomal dominant epileptic syndrome without cardiac symptoms. The C121W mutation may act by a dominant-competition, modifying the expression of α-subunit proteins. To test this hypothesis, we transfected GH3 cells, from neuro-ectoderm origin, with wild-type or mutant ß1 subunits and compared them to native cells. To examine the tissue specificity of the C121W-ß1 mutation, we compared the effects of the mutation on neural cells with those of H9C2 cells of cardiac origin. We found that in GH3 cells the over-expression of the ß1 subunit augments the α subunit mRNA and protein levels, while in the H9C2 cells the enhanced level of ß1 subunit not only increases but also qualitatively modifies the sodium channel α isoform expression pattern. Interestingly, the introduction of the epileptogenic C121W-ß1 subunit does not alter the sodium channel isoform composition of GH3 cells, while produces additional changes in the α-subunit expression pattern of H9C2 cells. Electrophysiological measurements confirm these molecular results. The expression differences observed could be correlated to the tissue-specific regulatory action of the ß1 subunit and to the nervous system specificity of the C121W mutation. Our findings could be helpful for the comprehension of the molecular mechanism of generalised epileptic with febrile seizures plus in patients with identified ß1 subunit mutations.
Assuntos
Epilepsia/genética , Epilepsia/metabolismo , Mutação , Canais de Sódio Disparados por Voltagem/biossíntese , Canais de Sódio Disparados por Voltagem/genética , Animais , Linhagem Celular , Potenciais da Membrana/fisiologia , Subunidades Proteicas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.
Assuntos
Canais Iônicos/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Animais , Carbocianinas/química , Corantes Fluorescentes/química , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Canais Iônicos/biossíntese , Canais Iônicos/química , Músculos/inervação , Técnicas de Patch-Clamp , Ratos , Canais de Sódio Disparados por Voltagem/biossíntese , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
The functional expression of voltage-gated sodium channels (Na(v)) in cancer cells is associated with an increase of metastatic potential. The activity of Na(v) channels modulates different cellular processes related to the development of the malignant phenotype, such as adhesion, galvanotaxis, motility and invasiveness. Among the great diversity of cancerous phenotypes, Na(v) channels expression is common in highly metastatic cells with their distribution following a primary tumor-specific pattern. The purpose of this paper is to review the literature, regarding to: the types of Na(v) channels expressed by different types of cancer cells, the cancer cellular processes in which they play important roles, and the molecular mechanisms by which these channels promote metastasis.