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1.
Nat Commun ; 12(1): 5916, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625548

RESUMO

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.


Assuntos
Mesencéfalo/citologia , Microglia/citologia , Neurogênese/genética , Rombencéfalo/citologia , Colículos Superiores/citologia , Transcriptoma , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Catepsina B/genética , Catepsina B/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Mesencéfalo/crescimento & desenvolvimento , Mesencéfalo/imunologia , Microglia/imunologia , Neurogênese/imunologia , Neurônios/citologia , Neurônios/imunologia , Fagocitose , Rombencéfalo/crescimento & desenvolvimento , Rombencéfalo/imunologia , Análise de Célula Única , Colículos Superiores/crescimento & desenvolvimento , Colículos Superiores/imunologia , Sinapses/imunologia , Sinapses/metabolismo , Sinapses/ultraestrutura , Peixe-Zebra , Proteínas de Peixe-Zebra/imunologia
2.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502063

RESUMO

α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.


Assuntos
Corpo Estriado/metabolismo , Substância Negra/metabolismo , Sinapses/metabolismo , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Corpo Estriado/ultraestrutura , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Pessoa de Meia-Idade , Substância Negra/ultraestrutura , Sinapses/ultraestrutura
4.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34213535

RESUMO

The extracellular matrix has emerged as an active component of chemical synapses regulating synaptic formation, maintenance, and homeostasis. The heparan sulfate proteoglycan (HSPG) syndecans are known to regulate cellular and axonal migration in the brain. They are also enriched at synapses, but their synaptic functions remain more elusive. Here, we show that SDN-1, the sole orthologue of syndecan in C. elegans, is absolutely required for the synaptic clustering of homomeric α7-like acetylcholine receptors (AChRs) and regulates the synaptic content of heteromeric AChRs. SDN-1 is concentrated at neuromuscular junctions (NMJs) by the neurally secreted synaptic organizer Ce-Punctin/MADD-4, which also activates the transmembrane netrin receptor DCC. Those cooperatively recruit the FARP and CASK orthologues that localize α7-like-AChRs at cholinergic NMJs through physical interactions. Therefore, SDN-1 stands at the core of the cholinergic synapse organization by bridging the extracellular synaptic determinants to the intracellular synaptic scaffold that controls the postsynaptic receptor content.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Sinapses/metabolismo , Sindecanas/metabolismo , Acetilcolina/metabolismo , Proteína 4 Semelhante a Angiopoietina/genética , Proteína 4 Semelhante a Angiopoietina/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Receptor DCC/genética , Receptor DCC/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Proteínas do Tecido Nervoso/genética , Junção Neuromuscular/ultraestrutura , Neurônios/citologia , Neurônios/metabolismo , Receptores Colinérgicos/genética , Sinapses/ultraestrutura , Transmissão Sináptica/genética , Sindecanas/genética
5.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34241635

RESUMO

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Neurônios/metabolismo , Via Secretória/genética , Sinapses/metabolismo , Transmissão Sináptica/genética , Animais , Animais Recém-Nascidos , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Feminino , Corantes Fluorescentes/química , Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Hipocampo/citologia , Hipocampo/metabolismo , Luz , Masculino , Imagem Molecular/métodos , Neurônios/citologia , Cultura Primária de Células , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/ultraestrutura , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
6.
Elife ; 102021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34085637

RESUMO

Neuroendocrine systems in animals maintain organismal homeostasis and regulate stress response. Although a great deal of work has been done on the neuropeptides and hormones that are released and act on target organs in the periphery, the synaptic inputs onto these neuroendocrine outputs in the brain are less well understood. Here, we use the transmission electron microscopy reconstruction of a whole central nervous system in the Drosophila larva to elucidate the sensory pathways and the interneurons that provide synaptic input to the neurosecretory cells projecting to the endocrine organs. Predicted by network modeling, we also identify a new carbon dioxide-responsive network that acts on a specific set of neurosecretory cells and that includes those expressing corazonin (Crz) and diuretic hormone 44 (Dh44) neuropeptides. Our analysis reveals a neuronal network architecture for combinatorial action based on sensory and interneuronal pathways that converge onto distinct combinations of neuroendocrine outputs.


Assuntos
Conectoma , Drosophila melanogaster/ultraestrutura , Interneurônios/ultraestrutura , Sistemas Neurossecretores/ultraestrutura , Células Receptoras Sensoriais/ultraestrutura , Sinapses/ultraestrutura , Animais , Animais Geneticamente Modificados , Dióxido de Carbono/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Hormônios de Inseto/genética , Hormônios de Inseto/metabolismo , Interneurônios/metabolismo , Microscopia Eletrônica de Transmissão , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Sistemas Neurossecretores/metabolismo , Células Receptoras Sensoriais/metabolismo , Sinapses/metabolismo
7.
Elife ; 102021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34018922

RESUMO

Neurofibrillary tangles composed of hyperphosphorylated tau and synaptic dysfunction are characteristics of Alzheimer's disease (AD). However, the underlying molecular mechanisms remain poorly understood. Here, we identified Amphiphysin I mediates both tau phosphorylation and synaptic dysfunction in AD. Amphiphysin I is cleaved by a cysteine proteinase asparagine endopeptidase (AEP) at N278 in the brains of AD patients. The amount of AEP-generated N-terminal fragment of Amphiphysin I (1-278) is increased with aging. Amphiphysin I (1-278) inhibits clathrin-mediated endocytosis and induces synaptic dysfunction. Furthermore, Amphiphysin I (1-278) binds p35 and promotes its transition to p25, thus activates CDK5 and enhances tau hyperphosphorylation. Overexpression of Amphiphysin I (1-278) in the hippocampus of Tau P301S mice induces synaptic dysfunction, tau hyperphosphorylation, and cognitive deficits. However, overexpression of the N278A mutant Amphiphysin I, which resists the AEP-mediated cleavage, alleviates the pathological and behavioral defects. These findings suggest a mechanism of tau hyperphosphorylation and synaptic dysfunction in AD.


Assuntos
Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Cisteína Endopeptidases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Sinapses/enzimologia , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Comportamento Animal , Encéfalo/fisiopatologia , Encéfalo/ultraestrutura , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , Cognição , Quinase 5 Dependente de Ciclina/metabolismo , Cisteína Endopeptidases/genética , Modelos Animais de Doenças , Células HEK293 , Humanos , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/ultraestrutura , Fosforilação , Ratos , Sinapses/ultraestrutura , Proteínas tau/genética
8.
Turk Neurosurg ; 31(4): 623-633, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33978223

RESUMO

AIM: To investigate neurogenesis in both adult and 3-week-old genetic absence epilepsy rats from Strasbourg (GAERS) to determine if newly formed neurons within the dentate gyrus (DG) form synaptic contacts with GABAergic (gamma aminobutyric acid) and glutamatergic nerve terminals and compared to the control (non-GAERS) Wistar rats. MATERIAL AND METHODS: Brain tissue was processed for electron microscopic assessment. Thin sections from the hippocampal DG were double-labelled for anti-GABA or anti-VGLUT1 (vesicular glutamate transporter 1) and anti-doublecortin (DCX) antibodies using immunogold methodology and examined with the transmission electron microscope for morphological changes and to quantify the density of gold labeling. RESULTS: DCX immunoreactivity was demonstrated within axon terminals, dendrites and somata in all groups. DCX and GABA or VGLUT1 were found to be co-localized in the axon terminals in all groups. We observed that DCX-immunoreactive (-ir) profiles formed synaptic contacts with GABAergic and glutamatergic terminals. The percentage of DCX labeling in dendrites, compared to axons, and the percentage of DCX-ir terminal profiles forming asymmetrical synapses, compared to those forming symmetrical synapses, were increased in all groups compared to the control group. DCX immunoreactivity in the 21-day-old GAERS group was found to be increased compared to the Wistar group. CONCLUSION: We conclude that newly born neurons are incorporated into the local hippocampal network in both the GAERS and the control Wistar rats. The results suggest that the neurogenesis taking place in the hippocampus may also be involved in the mechanism underlying absence seizures in GAERS.


Assuntos
Epilepsia Tipo Ausência/genética , Epilepsia Tipo Ausência/fisiopatologia , Neurogênese/fisiologia , Animais , Epilepsia Tipo Ausência/diagnóstico , Epilepsia Tipo Ausência/metabolismo , Hipocampo/diagnóstico por imagem , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/ultraestrutura , Imuno-Histoquímica/métodos , Masculino , Microscopia Eletrônica/métodos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Ratos , Ratos Transgênicos , Ratos Wistar , Sinapses/fisiologia , Sinapses/ultraestrutura , Ácido gama-Aminobutírico/metabolismo
9.
Science ; 372(6539)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33859005

RESUMO

Protocadherin-19 (PCDH19) mutations cause early-onset seizures and cognitive impairment. The PCDH19 gene is on the X-chromosome. Unlike most X-linked disorders, PCDH19 mutations affect heterozygous females (PCDH19HET♀ ) but not hemizygous males (PCDH19HEMI♂ ); however, the reason why remains to be elucidated. We demonstrate that PCDH19, a cell-adhesion molecule, is enriched at hippocampal mossy fiber synapses. Pcdh19HET♀ but not Pcdh19HEMI♂ mice show impaired mossy fiber synaptic structure and physiology. Consistently, Pcdh19HET♀ but not Pcdh19HEMI♂ mice exhibit reduced pattern completion and separation abilities, which require mossy fiber synaptic function. Furthermore, PCDH19 appears to interact with N-cadherin at mossy fiber synapses. In Pcdh19HET♀ conditions, mismatch between PCDH19 and N-cadherin diminishes N-cadherin-dependent signaling and impairs mossy fiber synapse development; N-cadherin overexpression rescues Pcdh19HET♀ phenotypes. These results reveal previously unknown molecular and cellular mechanisms underlying the female-specific PCDH19 disorder phenotype.


Assuntos
Caderinas/metabolismo , Disfunção Cognitiva/fisiopatologia , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Fibras Musgosas Hipocampais/fisiopatologia , Sinapses/fisiologia , Animais , Região CA3 Hipocampal/fisiopatologia , Região CA3 Hipocampal/ultraestrutura , Caderinas/genética , Disfunção Cognitiva/genética , Modelos Animais de Doenças , Epilepsia/genética , Epilepsia/fisiopatologia , Feminino , Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Potenciação de Longa Duração , Masculino , Camundongos , Fibras Musgosas Hipocampais/ultraestrutura , Mutação , Caracteres Sexuais , Sinapses/ultraestrutura , beta Catenina/metabolismo
10.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802775

RESUMO

Silver nanoparticles (AgNPs) are the one of the most extensively used nanomaterials. The strong antimicrobial properties of AgNPs have led to their use in a wide range of medical and consumer products. Although the neurotoxicity of AgNPs has been confirmed, the molecular mechanisms have not been extensively studied, particularly in immature organisms. Based on information gained from previous in vitro studies, in the present work, we examine whether ionotropic NMDA glutamate receptors contribute to AgNP-induced neurotoxicity in an animal model of exposure. In brains of immature rats subjected to a low dose of AgNPs, we identified ultrastructural and molecular alterations in the postsynaptic region of synapses where NMDA receptors are localized as a multiprotein complex. We revealed decreased expression of several NMDA receptor complex-related proteins, such as GluN1 and GluN2B subunits, scaffolding proteins PSD95 and SynGAP, as well as neuronal nitric oxide synthase (nNOS). Elucidating the changes in NMDA receptor-mediated molecular mechanisms induced by AgNPs, we also identified downregulation of the GluN2B-PSD95-nNOS-cGMP signaling pathway which maintains LTP/LTD processes underlying learning and memory formation during development. This observation is accompanied by decreased density of NMDA receptors, as assessed by a radioligand binding assay. The observed effects are reversible over the post-exposure time. This investigation reveals that NMDA receptors in immature rats are a target of AgNPs, thereby indicating the potential health hazard for children and infants resulting from the extensive use of products containing AgNPs.


Assuntos
Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Nanopartículas Metálicas/toxicidade , Receptores de N-Metil-D-Aspartato/metabolismo , Prata/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/ultraestrutura , GMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Ligantes , Masculino , Nanopartículas Metálicas/ultraestrutura , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Subunidades Proteicas/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/ultraestrutura
11.
Neurochem Res ; 46(7): 1659-1673, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33770320

RESUMO

Parvalbumin-immunoreactive (Parv+) interneurons is an important component of striatal GABAergic microcircuits, which receive excitatory inputs from the cortex and thalamus, and then target striatal projection neurons. The present study aimed to examine ultrastructural synaptic connection features of Parv+ neruons with cortical and thalamic input, and striatal projection neurons by using immuno-electron microscopy (immuno-EM) and immunofluorescence techniques. Our results showed that both Parv+ somas and dendrites received numerous asymmetric synaptic inputs, and Parv+ terminals formed symmetric synapses with Parv- somas, dendrites and spine bases. Most interestingly, spine bases targeted by Parv+ terminals simultaneously received excitatory inputs at their heads. Electrical stimulation of the motor cortex (M1) induced higher proportion of striatal Parv+ neurons express c-Jun than stimulation of the parafascicular nucleus (PFN), and indicated that cortical- and thalamic-inputs differentially modulate Parv+ neurons. Consistent with that, both Parv + soma and dendrites received more VGlut1+ than VGlut2+ terminals. However, the proportion of VGlut1+ terminal targeting onto Parv+ proximal and distal dendrites was not different, but VGlut2+ terminals tended to target Parv+ somas and proximal dendrites than distal dendrites. These functional and morphological results suggested excitatory cortical and thalamic glutamatergic inputs differently modulate Parv+ interneurons, which provided inhibition inputs onto striatal projection neurons. To maintain the balance between the cortex and thalamus onto Parv+ interneurons may be an important therapeutic target for neurological disorders.


Assuntos
Córtex Cerebral/ultraestrutura , Dendritos/ultraestrutura , Interneurônios/ultraestrutura , Núcleos Intralaminares do Tálamo/ultraestrutura , Parvalbuminas/metabolismo , Sinapses/ultraestrutura , Animais , Córtex Cerebral/metabolismo , Dendritos/metabolismo , Interneurônios/metabolismo , Núcleos Intralaminares do Tálamo/metabolismo , Masculino , Ratos Sprague-Dawley , Sinapses/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo
12.
Life Sci Alliance ; 4(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33563652

RESUMO

Increasing levels of the cold-shock protein, RNA-binding motif 3 (RBM3), either through cooling or by ectopic over-expression, prevents synapse and neuronal loss in mouse models of neurodegeneration. To exploit this process therapeutically requires an understanding of mechanisms controlling cold-induced RBM3 expression. Here, we show that cooling increases RBM3 through activation of TrkB via PLCγ1 and pCREB signaling. RBM3, in turn, has a hitherto unrecognized negative feedback on TrkB-induced ERK activation through induction of its specific phosphatase, DUSP6. Thus, RBM3 mediates structural plasticity through a distinct, non-canonical activation of TrkB signaling, which is abolished in RBM3-null neurons. Both genetic reduction and pharmacological antagonism of TrkB and its downstream mediators abrogate cooling-induced RBM3 induction and prevent structural plasticity, whereas TrkB inhibition similarly prevents RBM3 induction and the neuroprotective effects of cooling in prion-diseased mice. Conversely, TrkB agonism induces RBM3 without cooling, preventing synapse loss and neurodegeneration. TrkB signaling is, therefore, necessary for the induction of RBM3 and related neuroprotective effects and provides a target by which RBM3-mediated synapse-regenerative therapies in neurodegenerative disorders can be used therapeutically without the need for inducing hypothermia.


Assuntos
Glicoproteínas de Membrana/metabolismo , Neuroproteção , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Temperatura Baixa , Glicoproteínas de Membrana/agonistas , Camundongos , Fosforilação , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Príons/metabolismo , Ligação Proteica , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/ultraestrutura
13.
Oxid Med Cell Longev ; 2021: 6643171, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33628369

RESUMO

Background: Perinatal hypoxia is a universal cause of death and neurological deficits in neonates worldwide. Activation of microglial NADPH oxidase 2 (NOX2) leads to oxidative stress and neuroinflammation, which may contribute to hypoxic damage in the developing brain. Dexmedetomidine has been reported to exert potent neuroprotection in several neurological diseases, but the mechanism remains unclear. We investigated whether dexmedetomidine acts through microglial NOX2 to reduce neonatal hypoxic brain damage. Methods: The potential role of microglial NOX2 in dexmedetomidine-mediated alleviation of hypoxic damage was evaluated in cultured BV2 microglia and neonatal rats subjected to hypoxia. In vivo, neonatal rats received dexmedetomidine (25 µg/kg, i.p.) 30 min before or immediately after hypoxia (5% O2, 2 h). Apocynin-mediated NOX inhibition and lentivirus-mediated NOX2 overexpression were applied to further assess the involvement of microglial NOX2 activation. Results: Pre- or posttreatment with dexmedetomidine alleviated hypoxia-induced cognitive impairment, restored damaged synapses, and increased postsynaptic density-95 and synaptophysin protein expression following neonatal hypoxia. Importantly, dexmedetomidine treatment suppressed hypoxia-induced microglial NOX2 activation and subsequent oxidative stress and the neuroinflammatory response, as reflected by reduced 4-hydroxynonenal and ROS accumulation, and decreased nuclear NF-κB p65 and proinflammatory cytokine levels in cultured BV2 microglia and the developing hippocampus. In addition, treating primary hippocampal neurons with conditioned medium (CM) from hypoxia-activated BV2 microglia resulted in neuronal damage, which was alleviated by CM from dexmedetomidine-treated microglia. Moreover, the neuroprotective effect of dexmedetomidine was reversed in NOX2-overexpressing BV2 microglia and diminished in apocynin-pretreated neonatal rats. Conclusion: Dexmedetomidine targets microglial NOX2 to reduce oxidative stress and neuroinflammation and subsequently protects against hippocampal synaptic loss following neonatal hypoxia.


Assuntos
Disfunção Cognitiva/enzimologia , Disfunção Cognitiva/etiologia , Dexmedetomidina/farmacologia , Hipocampo/patologia , Hipóxia/complicações , Microglia/enzimologia , NADPH Oxidase 2/metabolismo , Sinapses/patologia , Acetofenonas/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Disfunção Cognitiva/patologia , Citocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Hipocampo/ultraestrutura , Mediadores da Inflamação/metabolismo , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/ultraestrutura , Modelos Biológicos , NADPH Oxidase 2/antagonistas & inibidores , NF-kappa B/metabolismo , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura
14.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530380

RESUMO

Three-dimensional (3D) reconstruction from electron microscopy (EM) datasets is a widely used tool that has improved our knowledge of synapse ultrastructure and organization in the brain. Rearrangements of synapse structure following maturation and in synaptic plasticity have been broadly described and, in many cases, the defective architecture of the synapse has been associated to functional impairments. It is therefore important, when studying brain connectivity, to map these rearrangements with the highest accuracy possible, considering the affordability of the different EM approaches to provide solid and reliable data about the structure of such a small complex. The aim of this work is to compare quantitative data from two dimensional (2D) and 3D EM of mouse hippocampal CA1 (apical dendrites), to define whether the results from the two approaches are consistent. We examined asymmetric excitatory synapses focusing on post synaptic density and dendritic spine area and volume as well as spine density, and we compared the results obtained with the two methods. The consistency between the 2D and 3D results questions the need-for many applications-of using volumetric datasets (costly and time consuming in terms of both acquisition and analysis), with respect to the more accessible measurements from 2D EM projections.


Assuntos
Região CA1 Hipocampal/ultraestrutura , Espinhas Dendríticas/ultraestrutura , Células Piramidais/ultraestrutura , Animais , Imageamento Tridimensional , Camundongos , Microscopia Eletrônica , Sinapses/ultraestrutura
15.
Cell ; 184(3): 759-774.e18, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33400916

RESUMO

To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.


Assuntos
Envelhecimento/fisiologia , Drosophila melanogaster/ultraestrutura , Microscopia Eletrônica de Transmissão , Neurônios Motores/ultraestrutura , Células Receptoras Sensoriais/ultraestrutura , Animais , Automação , Conectoma , Extremidades/inervação , Nervos Periféricos/ultraestrutura , Sinapses/ultraestrutura
16.
Nature ; 591(7848): 111-116, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33442056

RESUMO

In 1986, electron microscopy was used to reconstruct by hand the entire nervous system of a roundworm, the nematode Caenorhabditis elegans1. Since this landmark study, high-throughput electron-microscopic techniques have enabled reconstructions of much larger mammalian brain circuits at synaptic resolution2,3. Nevertheless, it remains unknown how the structure of a synapse relates to its physiological transmission strength-a key limitation for inferring brain function from neuronal wiring diagrams. Here we combine slice electrophysiology of synaptically connected pyramidal neurons in the mouse somatosensory cortex with correlated light microscopy and high-resolution electron microscopy of all putative synaptic contacts between the recorded neurons. We find a linear relationship between synapse size and strength, providing the missing link in assigning physiological weights to synapses reconstructed from electron microscopy. Quantal analysis also reveals that synapses contain at least 2.7 neurotransmitter-release sites on average. This challenges existing release models and provides further evidence that neocortical synapses operate with multivesicular release4-6, suggesting that they are more complex computational devices than thought, and therefore expanding the computational power of the canonical cortical microcircuitry.


Assuntos
Neocórtex/citologia , Neocórtex/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura , Transmissão Sináptica , Animais , Tamanho Celular , Fenômenos Eletrofisiológicos , Masculino , Camundongos , Microscopia , Microscopia Eletrônica , Neurotransmissores/metabolismo , Células Piramidais/citologia , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Córtex Somatossensorial/citologia , Córtex Somatossensorial/ultraestrutura
17.
Mol Brain ; 14(1): 23, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33494786

RESUMO

N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.


Assuntos
Caderinas/metabolismo , Hipocampo/metabolismo , Aprendizagem Espacial , Sinapses/metabolismo , Análise e Desempenho de Tarefas , Proteína ADAM10/metabolismo , Alelos , Animais , Comportamento Animal , Células CHO , Membrana Celular/metabolismo , Cricetulus , Medo , Técnicas de Introdução de Genes , Memória , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Mutação/genética , Estabilidade Proteica , Células Piramidais/metabolismo , Sinapses/patologia , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Sinaptossomos/metabolismo , Sinaptossomos/ultraestrutura
18.
Science ; 371(6525)2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33414193

RESUMO

The ability to perceive and interact with the world depends on a diverse array of neural circuits specialized for carrying out specific computations. Each circuit is assembled using a relatively limited number of molecules and common developmental steps, from cell fate specification to activity-dependent synaptic refinement. Given this shared toolkit, how do individual circuits acquire their characteristic properties? We explore this question by comparing development of the circuitry for seeing and hearing, highlighting a few examples where differences in each system's sensory demands necessitate different developmental strategies.


Assuntos
Vias Auditivas/embriologia , Núcleo Coclear/embriologia , Neurogênese , Retina/embriologia , Vias Visuais/embriologia , Animais , Audição/fisiologia , Camundongos , Células Receptoras Sensoriais/ultraestrutura , Sinapses/ultraestrutura , Visão Ocular/fisiologia
19.
Nat Commun ; 12(1): 677, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514725

RESUMO

Neurotransmitter is released synchronously and asynchronously following an action potential. Our recent study indicates that the release sites of these two phases are segregated within an active zone, with asynchronous release sites enriched near the center in mouse hippocampal synapses. Here we demonstrate that synchronous and asynchronous release sites are aligned with AMPA receptor and NMDA receptor clusters, respectively. Computational simulations indicate that this spatial and temporal arrangement of release can lead to maximal membrane depolarization through AMPA receptors, alleviating the pore-blocking magnesium leading to greater activation of NMDA receptors. Together, these results suggest that release sites are likely organized to activate NMDA receptors efficiently.


Assuntos
Hipocampo/fisiologia , Modelos Neurológicos , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Astrócitos , Células Cultivadas , Simulação por Computador , Feminino , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/ultraestrutura , Masculino , Camundongos , Microscopia Eletrônica , Neurônios , Cultura Primária de Células , Análise Espaço-Temporal , Sinapses/metabolismo , Sinapses/ultraestrutura
20.
Neuron ; 109(1): 27-41, 2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33098763

RESUMO

Neurons are highly polarized cells with a single axon and multiple dendrites derived from the cell body to form tightly associated pre- and postsynaptic compartments. As the biosynthetic machinery is largely restricted to the somatodendritic domain, the vast majority of presynaptic components are synthesized in the neuronal soma, packaged into synaptic precursor vesicles, and actively transported along the axon to sites of presynaptic biogenesis. In contrast with the significant progress that has been made in understanding synaptic transmission and processing of information at the post-synapse, comparably little is known about the formation and dynamic remodeling of the presynaptic compartment. We review here our current understanding of the mechanisms that govern the biogenesis, transport, and assembly of the key components for presynaptic neurotransmission, discuss how alterations in presynaptic assembly may impact nervous system function or lead to disease, and outline key open questions for future research.


Assuntos
Neurogênese/fisiologia , Terminações Pré-Sinápticas/metabolismo , Transporte Proteico/fisiologia , Sinapses/metabolismo , Animais , Humanos , Terminações Pré-Sinápticas/ultraestrutura , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura
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