RESUMO
Micro and nanoscale patterning of surface features and biochemical cues have emerged as tools to precisely direct neurite growth into close proximity with next generation neural prosthesis electrodes. Biophysical cues can exert greater influence on neurite pathfinding compared to the more well studied biochemical cues; yet the signaling events underlying the ability of growth cones to respond to these microfeatures remain obscure. Intracellular Ca2+ signaling plays a critical role in how a growth cone senses and grows in response to various cues (biophysical features, repulsive peptides, chemo-attractive gradients). Here, we investigate the role of inositol triphosphate (IP3) and ryanodine-sensitive receptor (RyR) signaling as sensory neurons (spiral ganglion neurons, SGNs, and dorsal root ganglion neurons, DRGNs) pathfind in response to micropatterned substrates of varied geometries. We find that IP3 and RyR signaling act in the growth cone as they navigate biophysical cues and enable proper guidance to biophysical, chemo-permissive, and chemo-repulsive micropatterns. In response to complex micropatterned geometries, RyR signaling appears to halt growth in response to both topographical features and chemo-repulsive cues. IP3 signaling appears to play a more complex role, as growth cones appear to sense the microfeatures in the presence of xestospongin C but are unable to coordinate turning in response to them. Overall, key Ca2+ signaling elements, IP3 and RyR, are found to be essential for SGNs to pathfind in response to engineered biophysical and biochemical cues. These findings inform efforts to precisely guide neurite regeneration for improved neural prosthesis function, including cochlear implants.
Assuntos
Neuritos , Canal de Liberação de Cálcio do Receptor de Rianodina , Transdução de Sinais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Neuritos/metabolismo , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Cones de Crescimento/metabolismo , Cones de Crescimento/efeitos dos fármacos , Sinalização do Cálcio , Ratos , Propriedades de Superfície , Células Cultivadas , Oxazóis , Compostos MacrocíclicosRESUMO
Mechanosensitive ion channels, particularly Piezo channels, are widely expressed in various tissues. However, their role in immune cells remains underexplored. Therefore, this study aimed to investigate the functional role of Piezo1 in the human eosinophil cell line AML14.3D10. We detected Piezo1 mRNA expression, but not Piezo2 expression, in these cells, confirming the presence of the Piezo1 protein. Activation of Piezo1 with Yoda1, its specific agonist, resulted in a significant calcium influx, which was inhibited by the Piezo1-specific inhibitor Dooku1, as well as other nonspecific inhibitors (Ruthenium Red, Gd3+, and GsMTx-4). Further analysis revealed that Piezo1 activation modulated the expression and secretion of both pro-inflammatory and anti-inflammatory cytokines in AML14.3D10 cells. Notably, supernatants from Piezo1-activated AML14.3D10 cells enhanced capsaicin and ATP-induced calcium responses in the dorsal root ganglion neurons of mice. These findings elucidate the physiological role of Piezo1 in AML14.3D10 cells and suggest that factors secreted by these cells can modulate the activity of transient receptor potential 1 (TRPV1) and purinergic receptors, which are associated with pain and itch signaling. The results of this study significantly advance our understanding of the function of Piezo1 channels in the immune and sensory nervous systems.
Assuntos
Eosinófilos , Canais Iônicos , Humanos , Canais Iônicos/metabolismo , Canais Iônicos/genética , Animais , Eosinófilos/metabolismo , Eosinófilos/imunologia , Camundongos , Linhagem Celular , Cálcio/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Citocinas/metabolismo , Rutênio Vermelho/farmacologia , Trifosfato de Adenosina/metabolismo , Tiadiazóis/farmacologia , PirazinasRESUMO
The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here, we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression program at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C, promoter-capture Hi-C, CUT&Tag for H3K27ac and RNA-seq. We find that genes involved in axonal regeneration form long-range, complex chromatin loops, and that cohesin is required for the full induction of the regenerative transcriptional program. Importantly, loss of cohesin results in disruption of chromatin architecture and severely impaired nerve regeneration. Complex enhancer-promoter loops are also enriched in the human fetal cortical plate, where the axonal growth potential is highest, and are lost in mature adult neurons. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent long-range promoter interactions in nerve regeneration.
Assuntos
Axônios , Cromatina , Coesinas , Regeneração Nervosa , Regiões Promotoras Genéticas , Células Receptoras Sensoriais , Animais , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Camundongos , Regiões Promotoras Genéticas/genética , Cromatina/metabolismo , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Axônios/metabolismo , Axônios/fisiologia , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Elementos Facilitadores Genéticos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Nervo Isquiático/metabolismoRESUMO
Traumatic spinal cord injury (tSCI) has complex pathophysiological events that begin after the initial trauma. One such event is fibroglial scar formation by fibroblasts and reactive astrocytes. A strong inhibition of axonal growth is caused by the activated astroglial cells as a component of fibroglial scarring through the production of inhibitory molecules, such as chondroitin sulfate proteoglycans or myelin-associated proteins. Here, we used neural precursor cells (aldynoglia) as promoters of axonal growth and a fibrin hydrogel gelled under alkaline conditions to support and guide neuronal cell growth, respectively. We added Tol-51 sulfoglycolipid as a synthetic inhibitor of astrocyte and microglia in order to test its effect on the axonal growth-promoting function of aldynoglia precursor cells. We obtained an increase in GFAP expression corresponding to the expected glial phenotype for aldynoglia cells cultured in alkaline fibrin. In co-cultures of dorsal root ganglia (DRG) and aldynoglia, the axonal growth promotion of DRG neurons by aldynoglia was not affected. We observed that the neural precursor cells first clustered together and then formed niches from which aldynoglia cells grew and connected to groups of adjacent cells. We conclude that the combination of alkaline fibrin with synthetic sulfoglycolipid Tol-51 increased cell adhesion, cell migration, fasciculation, and axonal growth capacity, promoted by aldynoglia cells. There was no negative effect on the behavior of aldynoglia cells after the addition of sulfoglycolipid Tol-51, suggesting that a combination of aldynoglia plus alkaline fibrin and Tol-51 compound could be useful as a therapeutic strategy for tSCI repair.
Assuntos
Axônios , Fibrina , Gânglios Espinais , Animais , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Axônios/metabolismo , Axônios/efeitos dos fármacos , Fibrina/metabolismo , Hidrogéis/química , Hidrogéis/farmacologia , Ratos , Glicolipídeos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/citologia , Movimento Celular/efeitos dos fármacosRESUMO
Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins.
Assuntos
Caenorhabditis elegans , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Optogenética , Proteínas RGS , Transdução de Sinais , Peixe-Zebra , Animais , Optogenética/métodos , Caenorhabditis elegans/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas RGS/metabolismo , Proteínas RGS/genética , Peixe-Zebra/embriologia , Neurônios/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Domínios Proteicos , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Drosophila/metabolismoRESUMO
BACKGROUND INFORMATION: The purinergic ligand-gated ion channel 7 receptor (P2X7R) is an ATP-gated ion channel that transmits extracellular signals and induces corresponding biological effects, transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that maintains normal physiological functions; numerous studies showed that P2X7R and TRPV1 are associated with inflammatory reactions. RESULTS: The effect of P2X7R knockdown in satellite glial cells (SGCs) on neuronal TRPV1 expression under high glucose and high free fat (HGHF) environment was investigated. P2X7 short hairpin RNA (shRNA) was utilized to downregulate P2X7R in SGCs, and treated and untreated SGCs were co-cultured with neuronal cell lines. The expression levels of inflammatory factors and signaling pathways in SGCs and neurons were measured using Western blot analysis, RT-qPCR, immunofluorescence, and enzyme-linked immunosorbent assays. Results suggested that P2X7 shRNA reduced the expression levels of P2X7R protein and mRNA in SGCs surrounding DRG neurons and downregulated the release of tumor necrosis factor-alpha and interleukin-1 beta via the Ca2+/p38 MAPK/NF-κB pathway. Additionally, the downregulation of P2X7R might decrease TRPV1 expression in neurons via the Ca2+/PKC-É/p38 MAPK pathway. CONCLUSIONS: Reducing P2X7R expression in SCGs in an HGHF environment could decrease neuronal TRPV1 expression via the Ca2+/PKC-É/p38 MAPK pathway.
Assuntos
Técnicas de Cocultura , Regulação para Baixo , Gânglios Espinais , Neuroglia , Neurônios , Receptores Purinérgicos P2X7 , Canais de Cátion TRPV , Animais , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Ratos , Neurônios/metabolismo , Neurônios/citologia , Neuroglia/metabolismo , Animais Recém-Nascidos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Econazole is a widely used imidazole derivative antifungal for treating skin infections. The molecular targets for its frequent adverse effects of skin irritation symptoms, such as pruritus, burning sensation, and pain, have not been clarified. Transient receptor potential (TRP) channels, non-selective cation channels, are mainly expressed in peripheral sensory neurons and serve as sensors for various irritants. METHODS: We investigated the effect of econazole on TRP channel activation by measuring intracellular calcium concentration ([Ca2+]i) through fluorescent ratio imaging in mouse dorsal root ganglion (DRG) neurons isolated from wild-type, TRPA1(-/-) and TRPV1(-/-) mice, as well as in heterologously TRP channel-expressed cells. A cheek injection model was employed to assess econazole-induced itch and pain in vivo. RESULTS: Econazole evoked an increase in [Ca2+]i, which was abolished by the removal of extracellular Ca2+ in mouse DRG neurons. The [Ca2+]i responses to econazole were suppressed by a TRPA1 blocker but not by a TRPV1 blocker. Attenuation of the econazole-induced [Ca2+]i responses was observed in the TRPA1(-/-) mouse DRG neurons but was not significant in the TRPV1(-/-) neurons. Econazole increased the [Ca2+]i in HEK293 cells expressing TRPA1 (TRPA1-HEK) but not in those expressing TRPV1, although at higher concentrations, it induced Ca2+ mobilization from intracellular stores in untransfected naïve HEK293 cells. Miconazole, which is a structural analog of econazole, also increased the [Ca2+]i in mouse DRG neurons and TRPA1-HEK, and its nonspecific action was larger than econazole. Fluconazole, a triazole drug failed to activate TRPA1 and TRPV1 in mouse DRG neurons and TRPA1-HEK. Econazole induced itch and pain in wild-type mice, with reduced responses in TRPA1(-/-) mice. CONCLUSIONS: These findings suggested that the imidazole derivatives econazole and miconazole may induce skin irritation by activating nociceptive TRPA1 in the sensory neurons. Suppression of TRPA1 activation may mitigate the adverse effects of econazole.
Assuntos
Antifúngicos , Cálcio , Econazol , Gânglios Espinais , Células Receptoras Sensoriais , Canal de Cátion TRPA1 , Canais de Cátion TRPV , Canais de Potencial de Receptor Transitório , Animais , Econazol/farmacologia , Canal de Cátion TRPA1/metabolismo , Canal de Cátion TRPA1/genética , Antifúngicos/toxicidade , Antifúngicos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Humanos , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/genética , Células HEK293 , Cálcio/metabolismo , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Camundongos , Masculino , Camundongos Knockout , Camundongos Endogâmicos C57BL , Prurido/induzido quimicamente , Dor/tratamento farmacológicoRESUMO
Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss.
Assuntos
Analgésicos , Gânglios Espinais , Canal de Sódio Disparado por Voltagem NAV1.7 , Neurônios , Tetrodotoxina , Animais , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/citologia , Neurônios/efeitos dos fármacos , Camundongos , Tetrodotoxina/farmacologia , Analgésicos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Venenos de Crotalídeos/toxicidade , Venenos de Crotalídeos/farmacologia , Masculino , Crotalus , Potenciais da Membrana/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , PeptídeosRESUMO
Neuroimmune cross-talk participates in intestinal tissue homeostasis and host defense. However, the matrix of interactions between arrays of molecularly defined neuron subsets and of immunocyte lineages remains unclear. We used a chemogenetic approach to activate eight distinct neuronal subsets, assessing effects by deep immunophenotyping, microbiome profiling, and immunocyte transcriptomics in intestinal organs. Distinct immune perturbations followed neuronal activation: Nitrergic neurons regulated T helper 17 (TH17)-like cells, and cholinergic neurons regulated neutrophils. Nociceptor neurons, expressing Trpv1, elicited the broadest immunomodulation, inducing changes in innate lymphocytes, macrophages, and RORγ+ regulatory T (Treg) cells. Neuroanatomical, genetic, and pharmacological follow-up showed that Trpv1+ neurons in dorsal root ganglia decreased Treg cell numbers via the neuropeptide calcitonin gene-related peptide (CGRP). Given the role of these neurons in nociception, these data potentially link pain signaling with gut Treg cell function.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Gânglios Espinais , Neuroimunomodulação , Nociceptores , Linfócitos T Reguladores , Canais de Cátion TRPV , Células Th17 , Animais , Camundongos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/genética , Neurônios Colinérgicos/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Microbioma Gastrointestinal , Intestinos/imunologia , Intestinos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Nociceptividade , Nociceptores/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
Insulin has been shown to modulate neuronal processes through insulin receptors. The ion channels located on neurons may be important targets for insulin/insulin receptor signaling. Both insulin receptors and acid-sensing ion channels (ASICs) are expressed in dorsal root ganglia (DRG) neurons. However, it is still unclear whether there is an interaction between them. Therefore, the purpose of this investigation was to determine the effects of insulin on the functional activity of ASICs. A 5 min application of insulin rapidly enhanced acid-evoked ASIC currents in rat DRG neurons in a concentration-dependent manner. Insulin shifted the concentration-response plot for ASIC currents upward, with an increase of 46.2 ± 7.6% in the maximal current response. The insulin-induced increase in ASIC currents was eliminated by the insulin receptor antagonist GSK1838705, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol-3 kinase antagonist wortmannin. Moreover, insulin increased the number of acid-triggered action potentials by activating insulin receptors. Finally, local administration of insulin exacerbated the spontaneous nociceptive behaviors induced by intraplantar acid injection and the mechanical hyperalgesia induced by intramuscular acid injections through peripheral insulin receptors. These results suggested that insulin/insulin receptor signaling enhanced the functional activity of ASICs via tyrosine kinase and phosphatidylinositol-3 kinase pathways. Our findings revealed that ASICs were targets in primary sensory neurons for insulin receptor signaling, which may underlie insulin modulation of pain.
Assuntos
Canais Iônicos Sensíveis a Ácido , Gânglios Espinais , Insulina , Receptor de Insulina , Células Receptoras Sensoriais , Animais , Canais Iônicos Sensíveis a Ácido/metabolismo , Insulina/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/citologia , Ratos , Receptor de Insulina/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Ratos Sprague-Dawley , Hiperalgesia/metabolismo , Células CultivadasRESUMO
We developed a rat dorsal root ganglion (DRG)-derived sensory nerve organotypic model by culturing DRG explants on an organoid culture device. With this method, a large number of organotypic cultures can be produced simultaneously with high reproducibility simply by seeding DRG explants derived from rat embryos. Unlike previous DRG explant models, this organotypic model consists of a ganglion and an axon bundle with myelinated A fibers, unmyelinated C fibers, and stereo-myelin-forming nodes of Ranvier. The model also exhibits Ca2+ signaling in cell bodies in response to application of chemical stimuli to nerve terminals. Further, axonal transection increases the activating transcription factor 3 mRNA level in ganglia. Axons and myelin are shown to regenerate 14 days following transection. Our sensory organotypic model enables analysis of neuronal excitability in response to pain stimuli and tracking of morphological changes in the axon bundle over weeks.
Assuntos
Axônios , Gânglios Espinais , Sistemas Microfisiológicos , Animais , Ratos , Fator 3 Ativador da Transcrição , Axônios/fisiologia , Axônios/metabolismo , Sinalização do Cálcio , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Bainha de Mielina/fisiologia , Bainha de Mielina/metabolismo , Organoides/metabolismo , Nervos Periféricos/metabolismo , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologiaRESUMO
BACKGROUND: Delivering optogenetic genes to the peripheral sensory nervous system provides an efficient approach to study and treat neurological disorders and offers the potential to reintroduce sensory feedback to prostheses users and those who have incurred other neuropathies. Adeno-associated viral (AAV) vectors are a common method of gene delivery due to efficiency of gene transfer and minimal toxicity. AAVs are capable of being designed to target specific tissues, with transduction efficacy determined through the combination of serotype and genetic promoter selection, as well as location of vector administration. The dorsal root ganglia (DRGs) are collections of cell bodies of sensory neurons which project from the periphery to the central nervous system (CNS). The anatomical make-up of DRGs make them an ideal injection location to target the somatosensory neurons in the peripheral nervous system (PNS). COMPARISON TO EXISTING METHODS: Previous studies have detailed methods of direct DRG injection in rats and dorsal horn injection in mice, however, due to the size and anatomical differences between rats and strains of mice, there is only one other published method for AAV injection into murine DRGs for transduction of peripheral sensory neurons using a different methodology. NEW METHOD/RESULTS: Here, we detail the necessary materials and methods required to inject AAVs into the L3 and L4 DRGs of mice, as well as how to harvest the sciatic nerve and L3/L4 DRGs for analysis. This methodology results in optogenetic expression in both the L3/L4 DRGs and sciatic nerve and can be adapted to inject any DRG.
Assuntos
Dependovirus , Gânglios Espinais , Técnicas de Transferência de Genes , Células Receptoras Sensoriais , Animais , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Dependovirus/genética , Camundongos , Células Receptoras Sensoriais/fisiologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Optogenética/métodos , Masculino , Camundongos Endogâmicos C57BLRESUMO
The cell intrinsic mechanisms directing peripheral nerve regeneration have remained largely understudied, thus limiting our understanding of these processes and constraining the advancement of novel clinical therapeutics. The use of primary adult rat dorsal root ganglion (DRG) neurons cultured in vitro is well established. Despite this, these cells can be challenging to culture and have so far not been amenable to robust transfection or live-cell imaging. The ability to transfect these cells with fluorescent plasmid constructs to label subcellular structures, combined with high resolution time-lapse imaging has the potential to provide invaluable insight into how peripheral neurons coordinate their regenerative response, and which specific cellular structures are involved in this process. Here we describe a protocol that facilitates transfection and subsequent live-imaging of adult rat DRG neurons.
Assuntos
Gânglios Espinais , Regeneração Nervosa , Neurônios , Animais , Gânglios Espinais/citologia , Regeneração Nervosa/fisiologia , Ratos , Neurônios/citologia , Neurônios/fisiologia , Neurônios/metabolismo , Células Cultivadas , Transfecção/métodos , Imagem com Lapso de Tempo/métodosRESUMO
Isolation and culture of dorsal root ganglion (DRG) neurons from adult animals is a useful experimental system for evaluating neural plasticity after axonal injury, as well as the neurological dysfunction resulting from aging and various types of disease. In this chapter, we will introduce a detailed method for the culture of mature rat DRG neurons. About 30-40 ganglia are dissected from a rat and mechanically and enzymatically digested. Subsequently, density gradient centrifugation of the digested tissue using 30% Percoll efficiently eliminates myelin debris and non-neuronal cells, to afford neuronal cells with a high yield and purity.
Assuntos
Técnicas de Cultura de Células , Separação Celular , Gânglios Espinais , Regeneração Nervosa , Neurônios , Animais , Gânglios Espinais/citologia , Ratos , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Cultura de Células/métodos , Regeneração Nervosa/fisiologia , Separação Celular/métodos , Degeneração Neural/patologia , Células Cultivadas , Centrifugação com Gradiente de Concentração/métodosRESUMO
The cell-adhesion molecule NEPH1 is required for maintaining the structural integrity and function of the glomerulus in the kidneys. In the nervous system of Drosophila and C. elegans, it is involved in synaptogenesis and axon branching, which are essential for establishing functional circuits. In the mammalian nervous system, the expression regulation and function of Neph1 has barely been explored. In this study, we provide a spatiotemporal characterization of Neph1 expression in mouse dorsal root ganglia (DRGs) and spinal cord. After the neurogenic phase, Neph1 is broadly expressed in the DRGs and in their putative targets at the dorsal horn of the spinal cord, comprising both GABAergic and glutamatergic neurons. Interestingly, we found that PRRXL1, a homeodomain transcription factor that is required for proper establishment of the DRG-spinal cord circuit, prevents a premature expression of Neph1 in the superficial laminae of the dorsal spinal cord at E14.5, but has no regulatory effect on the DRGs or on either structure at E16.5. By chromatin immunoprecipitation analysis of the dorsal spinal cord, we identified four PRRXL1-bound regions within the Neph1 introns, suggesting that PRRXL1 directly regulates Neph1 transcription. We also showed that Neph1 is required for branching, especially at distal neurites. Together, our work showed that Prrxl1 prevents the early expression of Neph1 in the superficial dorsal horn, suggesting that Neph1 might function as a downstream effector gene for proper assembly of the DRG-spinal nociceptive circuit.
Assuntos
Gânglios Espinais , Proteínas de Homeodomínio , Neuritos , Corno Dorsal da Medula Espinal , Fatores de Transcrição , Animais , Camundongos , Corno Dorsal da Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/citologia , Neuritos/metabolismo , Neuritos/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas do Tecido NervosoRESUMO
Local translation in growth cones plays a critical role in responses to extracellular stimuli, such as axon guidance cues. We previously showed that brain-derived neurotrophic factor activates translation and enhances novel protein synthesis through the activation of mammalian target of rapamycin complex 1 signaling in growth cones of dorsal root ganglion neurons. In this study, we focused on 40S ribosomal protein S6 (RPS6), 60S ribosomal protein P0/1/2 (RPP0/1/2), and actin filaments to determine how localization of ribosomal proteins changes with overall protein synthesis induced by neurotrophins. Our quantitative analysis using immunocytochemistry and super-resolution microscopy indicated that RPS6, RPP0/1/2, and actin tend to colocalize in the absence of stimulation, and that these ribosomal proteins tend to dissociate from actin and associate with each other when local protein synthesis is enhanced. We propose that this is because stimulation causes ribosomal subunits to associate with each other to form actively translating ribosomes (polysomes). This study further clarifies the role of cytoskeletal components in local translation in growth cones.
Assuntos
Citoesqueleto de Actina , Gânglios Espinais , Cones de Crescimento , Biossíntese de Proteínas , Proteínas Ribossômicas , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Cones de Crescimento/metabolismo , Proteínas Ribossômicas/metabolismo , Citoesqueleto de Actina/metabolismo , Biossíntese de Proteínas/fisiologia , Células Cultivadas , Neurônios/metabolismo , RatosRESUMO
GRT-X, which targets both the mitochondrial translocator protein (TSPO) and the Kv7.2/3 (KCNQ2/3) potassium channels, has been shown to efficiently promote recovery from cervical spine injury. In the present work, we investigate the role of GRT-X and its two targets in the axonal growth of dorsal root ganglion (DRG) neurons. Neurite outgrowth was quantified in DRG explant cultures prepared from wild-type C57BL6/J and TSPO-KO mice. TSPO was pharmacologically targeted with the agonist XBD173 and the Kv7 channels with the activator ICA-27243 and the inhibitor XE991. GRT-X efficiently stimulated DRG axonal growth at 4 and 8 days after its single administration. XBD173 also promoted axonal elongation, but only after 8 days and its repeated administration. In contrast, both ICA27243 and XE991 tended to decrease axonal elongation. In dissociated DRG neuron/Schwann cell co-cultures, GRT-X upregulated the expression of genes associated with axonal growth and myelination. In the TSPO-KO DRG cultures, the stimulatory effect of GRT-X on axonal growth was completely lost. However, GRT-X and XBD173 activated neuronal and Schwann cell gene expression after TSPO knockout, indicating the presence of additional targets warranting further investigation. These findings uncover a key role of the dual mode of action of GRT-X in the axonal elongation of DRG neurons.
Assuntos
Axônios , Gânglios Espinais , Receptores de GABA , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Camundongos , Axônios/metabolismo , Receptores de GABA/metabolismo , Receptores de GABA/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ2/genética , Camundongos Knockout , Camundongos Endogâmicos C57BL , Células Cultivadas , Células de Schwann/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/citologia , Técnicas de Cocultura , Neurônios/metabolismo , Neurônios/efeitos dos fármacosRESUMO
Silicone-based devices have the potential to achieve an ideal interface with nervous tissue but suffer from scalability, primarily due to the mechanical mismatch between established electronic materials and soft elastomer substrates. This study presents a novel approach using conventional electrode materials through multifunctional nanomesh to achieve reliable elastic microelectrodes directly on polydimethylsiloxane (PDMS) silicone with an unprecedented cellular resolution. This engineered nanomesh features an in-plane nanoscale mesh pattern, physically embodied by a stack of three thin-film materials by design, namely Parylene-C for mechanical buffering, gold (Au) for electrical conduction, and Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) for improved electrochemical interfacing. Nanomesh elastic neuroelectronics are validated using single-unit recording from the small and curvilinear epidural surface of mouse dorsal root ganglia (DRG) with device self-conformed and superior recording quality compared to plastic control devices requiring manual pressing is demonstrated. Electrode scaling studies from in vivo epidural recording further revealed the need for cellular resolution for high-fidelity recording of single-unit activities and compound action potentials. In addition to creating a minimally invasive device to effectively interface with DRG sensory afferents at a single-cell resolution, this study establishes nanomeshing as a practical pathway to leverage traditional electrode materials for a new class of elastic neuroelectronics.
Assuntos
Gânglios Espinais , Ouro , Polímeros , Xilenos , Animais , Camundongos , Gânglios Espinais/citologia , Ouro/química , Polímeros/química , Xilenos/química , Microeletrodos , Poliestirenos/química , Dimetilpolisiloxanos/química , Elasticidade , Nanoestruturas/química , Potenciais de Ação/fisiologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Tiofenos/químicaRESUMO
Recent work demonstrated that activation of spinal D1 and D5 dopamine receptors (D1/D5Rs) facilitates non-Hebbian long-term potentiation (LTP) at primary afferent synapses onto spinal projection neurons. However, the cellular localization of the D1/D5Rs driving non-Hebbian LTP in spinal nociceptive circuits remains unknown, and it is also unclear whether D1/D5R signaling must occur concurrently with sensory input in order to promote non-Hebbian LTP at these synapses. Here we investigate these issues using cell-type-selective knockdown of D1Rs or D5Rs from lamina I spinoparabrachial neurons, dorsal root ganglion (DRG) neurons, or astrocytes in adult mice of either sex using Cre recombinase-based genetic strategies. The LTP evoked by low-frequency stimulation of primary afferents in the presence of the selective D1/D5R agonist SKF82958 persisted following the knockdown of D1R or D5R in spinoparabrachial neurons, suggesting that postsynaptic D1/D5R signaling was dispensable for non-Hebbian plasticity at sensory synapses onto these key output neurons of the superficial dorsal horn (SDH). Similarly, the knockdown of D1Rs or D5Rs in DRG neurons failed to influence SKF82958-enabled LTP in lamina I projection neurons. In contrast, SKF82958-induced LTP was suppressed by the knockdown of D1R or D5R in spinal astrocytes. Furthermore, the data indicate that the activation of D1R/D5Rs in spinal astrocytes can either retroactively or proactively drive non-Hebbian LTP in spinoparabrachial neurons. Collectively, these results suggest that dopaminergic signaling in astrocytes can strongly promote activity-dependent LTP in the SDH, which is predicted to significantly enhance the amplification of ascending nociceptive transmission from the spinal cord to the brain.
Assuntos
Astrócitos , Potenciação de Longa Duração , Receptores de Dopamina D1 , Receptores de Dopamina D5 , Sinapses , Animais , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/genética , Potenciação de Longa Duração/fisiologia , Astrócitos/metabolismo , Astrócitos/fisiologia , Camundongos , Masculino , Receptores de Dopamina D5/metabolismo , Receptores de Dopamina D5/agonistas , Receptores de Dopamina D5/genética , Feminino , Sinapses/fisiologia , Sinapses/metabolismo , Gânglios Espinais/citologia , Corno Dorsal da Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/citologia , Camundongos Transgênicos , Camundongos Endogâmicos C57BLRESUMO
The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.