Este trabalho foi financiado pelo NINDS e American Heart Association para S.Ge. Esta abordagem foi originalmente criado e desenvolvido pelo autor no laboratório do Dr. Hongjun Song na Johns Hopkins University. Gostaríamos de agradecer ao Dr. Canção Hongjun por sua orientação e apoio.

1. Injeção de vírus Alto título de retrovírus engenharia (1 × 10 9 unidade / ml) é produzida por co-transfecção de vetores retrovirais em células e VSVG HEK293T, seguido por ultracentrifugação do sobrenadante viral. Para as fontes e métodos de produção, ver a demonstração JOVE excelente (3). Nota: adultos jovens (4-6 semanas de idade) do sexo feminino camundongos C57BL / 6 (Charles River) estão alojados em condições normais. Todos os procedimentos seguem o Manual do Conselho Nacional de Pesquisa para o cuidado e uso de animais de laboratório sob um protocolo aprovado pelo Stony Brook University IACUC. <ol><li> 2ul degelo dos retrovírus congeladas por animal no gelo. </li><li> Anestesiar (100 mg + 10 quetamina xilazina mcg por grama de peso corporal) e montar o animal em uma estrutura estereotáxica (Steolting). Remover o cabelo na cabeça e limpe a pele com álcool 70%. </li><li> Expor o crânio e faça quatro furos rasos usando uma broca de dentista (0,6 mm broca) na seguintes coordenadas: <ul><li> anterioposterior = mm -2 de bregma; lateral = ± 1,6 mm; ventral = 2,5 mm; anterioposterior = mm -3 de bregma; lateral = ± 2,6 mm; ventral = 3,2 mm. </li><li> anterioposterior = mm -2 de bregma; lateral = ± 1,6 mm; ventral = 2,5 mm; anterioposterior = mm -3 de bregma; lateral = ± 2,6 mm; ventral = 3,2 mm. </li></ul></li><li> Montar um Hamilton 1μl, flat ponta da seringa e injetar 0,5 mL por local de retrovírus em uma taxa de 0,25 mL / min no giro denteado. (Veja as coordenadas acima de profundidade ventral.) Pause 2 minutos após cada injecção antes de retirar a seringa lentamente para evitar o refluxo do fluido. Entre as injeções, brevemente lavar a superfície externa da ponta da seringa com PBS estéril e um aplicador para remover vestígios de sangue. </li><li> Fechar a ferida com material de sutura cirúrgica estéril (tipo P-1; Tamanho 6-0) eo retorno do animal às condições de alojamento padrão até os estágios de tempo desejado após a injeção. </li></ol> 2. Preparação fatia Para obter alta qualidade fatias aguda de camundongos adultos, usamos uma solução de corte modificada para a preparação fatia (veja abaixo). <ol><li> Pré-chill solução de corte de 0-4 ° C no gelo e bolhas com 95% de O 2 -5% CO 2 por pelo menos 30 minutos. </li><li> Anestesiar e transcardially perfundir um animal com gelado solução saturada de oxigênio de corte. </li><li> Remover rapidamente o cérebro em uma placa de Petri com papel-filtro pré-umedecido com frio, solução oxigenada de corte. Cortar o cerebelo e uma fatia superfície de montagem, pelo menos, um milímetro anterior para as coordenadas de injeção (visível). Cola do cérebro em um estágio vibrotome e montá-la na câmara de corte cheio de frio e oxigenado solução de corte. </li><li> Prepare as fatias coronal ou horizontal (300-350μm) e transferi-las com uma pipeta de grande calibre a uma câmara de incubação contendo temperatura ambiente ACSF aeradas com 95% O 2 / 5% CO 2. </li><li> Incubar as fatias, borbulhando de forma contínua, a 32 ° C por 30-60 minutos. Retorno da câmara à temperatura ambiente para a duração da gravação. </li></ol> 3. Experimentos eletrofisiológicas <ol><li> Fatias são transferidos para a câmara de gravação que é continuamente perfundidos com oxigênio saturado ACSF em 32 ° C - 34 ° C. </li><li> Um eletrodo de estimulação bipolar de tungstênio é colocado na camada molecular do giro denteado utilizando uma objetiva de microscópio de baixa ampliação (10X). </li><li> PED recém-nascido na zona sub-granular / camada de células granulares são identificados pela expressão de GFP ou dTomato. De célula inteira de gravação patchclamp é realizada em neurônios recém-nascidos sob alta ampliação (40X). </li><li> Propriedades de queima das células gravadas são obtidas por injeção de uma série de correntes sob clamp de corrente de modo. </li><li> Estímulos elétricos são entregues pelo eletrodo de estimulação através de um isolador de estimulação controlado pelo software de gravação, e evocou as correntes pós-sinápticos no PED recém-nascidos são registrados. </li></ol> 4. Análise de Dados Amplitudes de respostas evocadas pós-sinápticos são analisados ​​em diferentes estágios de desenvolvimento do adulto-nascido neurônios. 5. Resultados representante Usando o protocolo acima, GFP expressão em recém-nascidos neurônios do giro denteado semelhante à Figura 1 é comum. Note-se que os neurônios recém-nascidos são facilmente visualizadas e ambos dendritos e axônios são fortemente marcadas. Expressão em thin axônios DGC e pequenas espinhas depende da qualidade e de título do vírus, o promotor utilizado ea duração da expressão em vivo. Em nossas mãos, as células recém-nascido e sub-celular organelas são normalmente visíveis dentro de poucos dias após a injeção, e expressão da coluna pode ser rastreada desde os primeiros estágios de desenvolvimento. Wburaco células gravações dos neurônios recém-nascidos em estágios diferentes de tempo permitem o estudo das propriedades únicas células recém-nascido, por exemplo, potenciais de ação (Figura 2a), bem como o processo de integração sináptica em circuitos neurais existentes durante a maturação (Figura 2b). <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="Figura 1" /> Figura 1 imagem 2-fótons confocal de neurônios recém-nascido em uma seção de giro horizontal denteado de um rato adulto. Células verdes são 21dpi (dias pós-injeção) expressando GFP-PED recém-nascido rotulados com PUX-GFP retrovírus. <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="Figura 2" /> Figura 2 a, b) Ação potencial de disparo propriedades de um recém-nascido DGC aos 21 dpi.) Representante evocado correntes excitatórias pós-sináptico (EPSCs) gravado em um recém-nascido DGC aos 21 dpi.

<ol>
	<li>Zhao, C., Deng, W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functional+implications+of+adult+neurogenesis.">Mechanisms and functional implications of adult neurogenesis.</a> Cell. 132, 645-660 (2008).</li><li>Ge, S., Goh, E. L., Sailor, K. A., Kitabatake, Y., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+regulates+synaptic+integration+of+newly+generated+neurons+in+the+adult+brain.">GABA regulates synaptic integration of newly generated neurons in the adult brain.</a> Nature. 439, 589-593 (2006).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. , (2007).</li></ol>

Não há conflitos de interesse declarados.

Neurogênese contínua no hipocampo do cérebro de mamíferos adultos oferece um sistema único modelo experimental para estudar o desenvolvimento e integração neuronal no cérebro maduro. O protocolo aqui apresentado combina rotulagem retroviral e métodos eletrofisiológicos para estudar a integração de recém-nascidos neurônios granulares denteado no cérebro de ratos adultos. Para garantir que seus experimentos forem bem sucedidos, recomendamos o seguinte durante etapas críticas do processo: Para evitar a infecção e inflamação indesejável, todas as ferramentas devem ser esterilizados (em autoclave, qualquer tipo de esterilizador, ou etanol 70%) antes da cirurgia. Use PBS estéril para lavar a ponta da agulha da seringa antes da injeção. Para injecção de vírus, os animais devem ser bem montado no dispositivo estereotáxico e fontes de movimentos desnecessários devem ser minimizados durante a injeção. Certifique-se a cabeça está bem fixo e firme, e ajustar a posição do nariz do mouse para nível bregma e lambda antes de coordenadas de injeção de cálculo. Extrato de vírus para dentro da seringa rapidamente para minimizar a exposição a temperaturas quarto - retrovírus são particularmente sensíveis à temperatura. Injetar o vírus lentamente na dose recomendada para minimizar os danos de pressão. Usando o recomendado seringa de ponta chata (vs. ponta chanfrada comum) irá garantir uma uniformemente distribuído área infecção giro denteado. Antes de preparar fatias, o corte de solução e incubar ACSF devem ser aeradas com 95% O CO 2 -5 2% para pelo menos 30 minutos para permitir que o oxigênio para saturar as soluções. Os animais devem ser perfundidos com rapidez e tecidos mantidos em frio, solução saturada de oxigênio para a melhor qualidade fatia. Para a gravação, aumente a intensidade do estímulo progressivamente até que haja uma resposta pós-sináptica. Mudar o local do eletrodo de estimulação dentro da camada molecular do giro denteado, se necessário. Em resumo, a abordagem recomendada oferece uma maneira de explorar as propriedades distintas e possíveis papéis funcionais do adulto-nascido neurônios durante a fase inicial de integração em circuitos, ativo maduro.

<ul><li> Corte de solução (em mM): 110 cloreto de colina, 2,5 KCl, 1,3 KH 2 PO 4, 25,0 NaHCO 3, 0,5 CaCl 2, 7 MgCl 2, 10 dextrose, 1,3 de ascorbato de sódio, 0,6 piruvato de sódio, ácido 5,0 cinurênico. </li><li> Fluido cérebro-espinhal artificial (ACSF, em mM): NaCl 125,0, 25,0 NaHCO 3, 2,5 KCl, 2 CaCl 2, 1,3 KH 2 PO 4, 1,3 MgCl 2, 1,3 ascorbato de sódio, 0,6 piruvato de sódio, 10 dextrose. </li><li> Solução interna para toda célula patchclamp (em mM): 120 gluconato de potássio, KCl 15, 4 MgCl 2, 0,1 EGTA HEPES, 10.0, 4 MgATP, 0,3 Na 3 GTP, 7 de fosfato (pH 7,4, 300 mOsm). </li></ul>

studying the integration of adult-born neurons

Neurogênese ocorre em cérebros de mamíferos adultos na zona sub-ventricular (SVZ) do ventrículo lateral e na zona sub-granular (SGZ) do giro denteado do hipocampo durante toda a vida. Relatórios anteriores têm mostrado que a neurogênese do hipocampo adulto está associada a diversas doenças cerebrais, incluindo a epilepsia, esquizofrenia, depressão e ansiedade (1). Decifrando o processo de integração neurônio normal e aberrante adulto-nascido pode lançar luz sobre a etiologia destas doenças e informar o desenvolvimento de novas terapias. SGZ neurogênese adulta espelhos embrionário e pós-natal o desenvolvimento neuronal, incluindo as etapas de especificação de destino, migração, integração sináptica, e maturação. No entanto, a integração ocorre ao longo de um prolongado, período de 6 semanas. Entrada sináptica inicial para adultos-nascido SGZ células granulares denteado (PED) é GABAérgica, seguido da entrada glutamatérgica menos 14 dias (2). Os fatores específicos que regulam a formação de circuito de adulto-nascido neurônios no giro dentado são actualmente desconhecidas. Nosso laboratório utiliza uma réplica deficiente do vetor retroviral com base no vírus da leucemia murina Moloney para entregar proteínas fluorescentes e hipótese de genes reguladores a essas células a proliferar. Esta técnica fornece viral elevada especificidade e resolução para a análise da data de nascimento de células, a linhagem, morfologia e sinaptogênese. A experiência típica muitas vezes emprega dois ou três vírus contendo rótulo único, transgene, e elementos de promotor para uma única célula de análise de um processo de desenvolvimento desejado in vivo. O protocolo a seguir descreve um método para analisar a integração funcional dos neurônios recém-nascido através de um único verde (GFP) ou vermelho (dTomato) retrovírus proteína fluorescente e patch-clamp eletrofisiologia.

Watch this Scientific Journal Video about Studying the Integration of Adult-born Neurons at JoVE.com

Studying the Integration of Adult-born Neurons

Uma maneira de estudar a integração de recém-nascidos células granulares denteado em animais adultos é descrito. Esta técnica utiliza um retrovírus engenharia para rotular neurônios recém-nascidos, seguido de gravações eletrofisiológicas para determinar a integração funcional vivo.

Estudar a Integração da Adult-nascido Neurônios

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

studying-the-integration-of-adult-born-neurons

Research

JoVE Journal

Neuroscience

13.7K visualizações.  State University of New York at Stony Brook. Uma maneira de estudar a integração de recém-nascidos células granulares denteado em animais adultos é descrito. Esta técnica utiliza um retrovírus engenharia para rotular neurônios recém-nascidos, seguido de gravações eletrofisiológicas para determinar a integração funcional vivo.

Vídeo: Studying the Integration of Adult-born Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

No útero Eletroporação seguido por Cultura Neuronal primário para estudar a função do gene no subconjunto dos neurônios corticais

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Neurociência

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Transdução Viral seletiva de Adultos-nascido neurônios olfativos de crônica In vivo Estímulo Optogenetic

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

A especificação de telencefálicos neurónios glutamatérgicos de células-tronco pluripotentes humanas

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Rotulagem diferencial de superfície celular e internalizado Proteínas após Anticorpo Alimentação da Vivo Cultivadas neurônios

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Um sistema de Microbiomechanical para estudar a formação de varizes e a recuperação no axônio do neurônio Central

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

Um modelo in vitro para estudar a agregação Tau usando transdução mediada por Lentivirus de neurônios humanos

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Gravação eletrofisiológica sem fio dos neurônios por tetrodes móveis em peixes livremente da natação

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

Estudando o acúmulo de α-sinucleína induzida por fibril pré-formado em neurônios de dopamina de cérebro médio do camundongo embrionário primário

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Atividade da Linha Lateral Posterior Neurônios Diferentes durante a Natação em Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...