Reconhecemos K. Mikic, que contribuiu para a fase inicial deste filme e M. Nicolescu para a imagem miR. C. Huber foi apoiado por uma bolsa da IZKF do Universtitätsklinikum Tübingen, A. Alwin Prem Anand pelo programa fortuna do Universtitätsklinikum Tübingen.

1. Requisitos para in ovo Eletroporação <ol><li> Preparação da construção de vector: miR sentido e anti-sentido foram concebidos iniciadores após as instruções de Ambion, recozidos e ligados em Apal / EcoRI digerido pSilencer U6.1 vector. Após a transformação bem sucedida de um dos clones resultantes foram cultivadas e pSilencer plasmídeo foi isolado pelo método de lise alcalina, utilizando um kit de preparação Qiagen Midi. </li><li> Eletrodos: Eletrodos podem ser comprados ou facilmente ser construído 13. Nós usamos eletrodos self-made de fio de platina (Φ = 0,2, 0,25, ou 0,5 mm de diâmetro) ou, em alguns casos, um eletrodo de fio de tungstênio ainda menor (Φ = 0,1 mm) 8 dependendo de qual área que queremos electroporate. Para evitar escaldante de tecido, usamos como regra o mais próximo do tecido mais fino os eletrodos. Por ampla eletroporação de mesencéfalo, usamos 0,5 milímetros fios de platina soldadas a um tubular de bronze / aço inoxidáveleixo e fixados a um suporte de eléctrodo, com uma distância de 3-5 mm (Figura 1). Para electroporate áreas específicas, usamos 0,25 milímetros ânodos de platina e 0,2 milímetros de fio de platina ou de 0,1 milímetros para o cátodo de tungsténio, com uma distância de aproximadamente 1 mm ou menos. </li><li> Soluções: Alguma as seguintes soluções de pronto: <ol><li> Campainha frango autoclavado, pH 7,5 (9,0 g de NaCl, 0,42 g de KCl, 0,24 g de CaCl2, dissolve-se em 1 L de água destilada). </li><li> PBS (8 g de NaCl, 0,2 g de KCl, 0,24 g de KH 2 PO 4, 1,44 g de Na 2 HPO 4, dissolve-se em 1 litro de H2O destilada). </li><li> Verde Rápido: solução de estoque de 10 mg / ml de H2O destilada </li><li> Tinta de secagem lenta (menos de goma-laca e, portanto, menos envenenamento por embrião). Nota: Um filtro dicróico azul ligado a uma lâmpada de fibra óptica, tal como descrito no Canto-Soler e Adler 14 melhora o contraste e pode eliminar a necessidade de injectar tinta. </li></ol></li></ol><img src="/files/ftp_upload/50024/50024fig1.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50024/50024fig1highres.jpg"/> Figura 1. Diagrama esquemático de eletrodos e suporte do eletrodo. (A, B, C) ​​de fio de platina foi soldada a um lado de um eixo tubular em aço inoxidável. Um furo foi perfurado no outro lado do eixo tubular, assim, que as tomadas de pinos pode caber dentro (A) vermelho e azul fios coloridos foram ligados às tomadas de pinos. No extremo oposto do fio foi montado para conectores bananas que trabalham com as tomadas elétricas do electroporador. (A, C) O fio de platina foi dobrado em um anjo de 90 °. Uma camada de verniz de unhas isolado no eixo acima da curva. 2. Preparativos Diretamente antes em ovo Eletroporação <ol><li> Prepare o seguinte material: <ol><li> Diluir tinta com frango Ringer 1:6, adicionar solução antibiótica-antimicótica (100x de GIBCo) 1:100. </li><li> Preparar a solução de vector: 1-2 mg / mL por ADN específica em H2O suplementada com Verde Rápido (1:20). </li><li> Esterilizar fórceps, tesoura, agulha de tungstênio e eletrodos com etanol 70%. </li><li> Puxe micropipetas para injeção de ácido nucleico de capilares de vidro de borosilicato (comprimento e diâmetro externo: 100 mm x 0.9 mm). Usamos um puxador simples PUL-1 (WPI, Berlin) com as configurações mostradas na Tabela 1. </li><li> Definir os parâmetros do electroporador como mostrado na Tabela 2. </li></ol></li></ol><table border="1" ><tbody><tr><td fo:width="1in"> Parâmetros </td><td fo:width="1in"> Valores </td></tr><tr><td> Calor </td><td> 350 </td></tr><tr><td> Puxe </td><td> 100 </td></tr><tr><td> Velocidade </td&gt; <td> 50 </td></tr><tr><td> Atraso </td><td> 200 </td></tr></tbody></table> Tabela 1. Configurações para PUL-1 <table border="1"><tbody><tr><td fo:width="1.5in"> Parâmetros </td><td fo:width="1.5in"> Valores </td></tr><tr><td> Freqüência </td><td> 50 Hz. </td></tr><tr><td> Atraso </td><td> 20 - 50 ms * </td></tr><tr><td> Largura </td><td> 20 mseg </td></tr><tr><td> Tensão </td><td> 8-25 V * </td></tr><tr><td> Pulso </td><td> 3 - 5 vezes </td></tr><tr><td colspan="2"> * Dependendo do diâmetro do eletrodo e distância. Por favorlevar isso em conta quando se muda um dos parâmetros. </td></tr></tbody></table> Tabela 2. Configurações para o estimulador de pulso <ol start="2"><li> Prepare embriões de galinha para eletroporação: <ol><li> Incubar ovos de galinha fertilizados em seu lado mais longo, a 37 ° C com 65% de umidade por 39 horas para adquirir embriões entre Hamburger &amp; Hamilton (HH) encena 15 9-11. O embrião se instala na parte superior do ovo, por isso você deve marcá-lo com uma linha de lápis. </li><li> Desinfetar os ovos com etanol 70%. </li><li> Perfurar o ovo no seu lado mais largo, onde a cavidade de ar deve ser. </li><li> Remover 1-2 ml de clara de ovo. Isto irá separar o embrião da casca de ovo. </li><li> Cello-tape a casca do ovo para evitar lascas de casca caindo sobre o embrião. Utilizar um par de tesouras para cortar uma pequena janela para a casca de ovo em torno da linha de lápis. </li><li> Injetar um pouco de tintadebaixo da área opaca para visualizar o embrião. </li><li> Faça um corte na membrana vitelina com uma agulha de tungstênio afiadas para alcançar uma melhor acessibilidade do embrião para a injeção de ácido nucleico e eletroporação. </li></ol></li></ol> 3. Eletroporação e Incubação <ol><li> Adicionam-se algumas gotas de solução de Ringer quente sobre os embriões. Isto irá reduzir os embriões, evitar o sobreaquecimento, a aderência dos eléctrodos ao tecido e proporcionar um meio para a corrente entre os dois eléctrodos. </li><li> Injectar a solução vetor dentro do lúmen do tubo neural em nível mesencéfalo. Se a expressão de DNA vector não contém qualquer gene repórter (por exemplo EGFP), adicionar um vector do gene repórter ligeiramente concentrada inferior (1 ug / ul miR vector de expressão para 0,9 ug / uL vector de gene repórter) para a solução de ADN. Isso irá garantir que você reconhece as células transfectadas depois 8. Nós usamos o chamado vetor pCAX, um presente amável from C. Krull 13. </li><li> Para uma ampla transfecção do mesencéfalo, coloque os eletrodos esquerdo e direito do embrião à altura do mesencéfalo. A distância entre os eletrodos deve ser cerca de 3-5 mm. As outras configurações são 15V, 20 pulsos ms e 50 ms de atraso para HH 10. A alteração da posição dos dois eléctrodos ligeiramente ao longo da (DV) do eixo dorso-ventral do mesencéfalo assegura um mais amplo a transfecção de células a transfecção eixo DV. </li><li> Para transfecção ventral, adicione um pouco de Ringer para elevar a cabeça a partir da membrana da gema e coloque o ânodo sob mesencéfalo ventral. Se a cabeça não subir empurre o eletrodo sob a membrana que separa a gema e embrião para evitar escaldante do tubo neural. O cátodo deve ser colocado por cima do mesencéfalo dorso-lateral oposto ao ânodo. Não toque no tubo neural. Nós transfecção a metade ventral esquerdo do mesencéfalo para evitar problemas com o desenvolvimento do coração. A distância entre os eléctrodosé de cerca de 1-0,5 mm, o resto das configurações são 10V, 20 de largura e 50 milissegundos de atraso ms. </li><li> Aplique uma gota de solução salina em cima dos embriões para esfriá-la e retirar as bolhas de ar. </li><li> Selar os ovos com a fita e incubar durante pelo menos 4 horas, o tempo mínimo necessário para a expressão do vetor. </li></ol> 4. Fixação de frango Embriões <ol><li> Remover os embriões dos seus ovos. </li><li> Embriões limpa sob um microscópio estereoscópico e avaliar a extensão da transfecção com um microscópio estéreo fluorescente. </li><li> Corrigir os embriões durante a noite em PFA 4% a 4 ° C. Prossiga com o corte e / ou coloração (hibridização in situ, imuno-histoquímica) para analisar as mudanças na área transfectadas. </li></ol>

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Este vídeo demonstra um método eficaz para transfectar plasmídeo nas células neuroepiteliais de áreas específicas do cérebro médio do pintainho. Pulsos elétricos retangulares de baixa tensão pode introduzir DNA em células do tubo neural garota em ovo 6,16. No entanto, a precisão da segmentação DNA é muitas vezes dificultada pela grande campo elétrico, que sobe através das relativamente grandes eletrodos (Φ = 0,5 mm). Nós tentamos resolver esse problema usando eletrodos menores em d...

O reconhecimento de pequenos RNAs não-codificantes como jogadores adicionais para a expressão do gene de lançar uma nova complexidade à regulamentação de programação / gene do genoma. Diferentes espécies de RNAs não-codificantes tem importância funcional em células neurais, incluindo pequeno RNAs não-codificantes 1-4. MicroRNAs (MIR ou miRNA), por exemplo, mostram perfis de expressão distintas e mutáveis ​​em cérebros em desenvolvimento 5. Alvejado em eletroporação in ovo de embriões de galinha oferece uma oportunidade única para o controle temporal e espacial da expressão gênica e silenciamento durante o desenvolvimento. Este vídeo demonstra as diferentes etapas da realização de expressão ectópica da RIM em áreas específicas do mesencéfalo garota usando eletroporação in ovo 6-10. Para assegurar um efeito de longa duração destes pequenos RNAs não-codificantes nas células, a sequência de ADN de RIM, foram clonados em vectores de mono-ou bi-cistronic. Pois em eletroporação in ovo, miR contendo vector é injectado no tubo neural mesencéfalo expondo o embrião depois de fazer uma pequena janela na casca do ovo. Para transfectar as áreas específicas do cérebro médio mais pequeno (ânodo) e negativo (cátodo) eléctrodos de platina são colocados em posições específicas. Para a transfecção do mesencéfalo ventral, o ânodo é colocado por baixo do mesencéfalo ventral esquerdo e o cátodo acima da metade direita do cérebro médio, antes de aplicar uma corrente. A abertura na casca do ovo é fechado com fita adesiva e os embriões foram incubados durante o tempo necessário para qualquer análise. Este método foi originalmente descrito por Muramatsu et al. 6 e melhorado por Momose et al. 8 para transfecção área específica. <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> Visão esquemática. <ol><li> O embrião no ovo é exposta pelo corte de uma pequena janela em the casca de ovo. </li><li> O vector dissolvido (s) é injectado no mesencéfalo usando um tubo capilar de micro. </li><li> Dois eletrodos colocados - paralelo ou por baixo e por cima do embrião - gerar um campo elétrico pulsado. </li><li> O campo eléctrico temporalmente cria poros na membrana da célula, que facilitam a entrada na célula de ADN carregadas negativamente (ou RNA) atraídos para o ânodo 11,12. </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

RNAs não-codificantes são jogadores adicionais na regulação da expressão gênica. Alvejado em eletroporação in ovo de áreas específicas fornece uma ferramenta única para o controle espacial e temporal da expressão do microRNA ectópica. No entanto, as estruturas cerebrais ventral como mesencéfalo ventral são bastante difíceis de alcançar por quaisquer manipulações. Aqui, nós demonstramos uma forma eficiente de electroporate miRNA em mesencéfalo ventral utilizando eletrodos de platina finas. Este método oferece uma maneira confiável de transfecção de áreas específicas do cérebro médio e uma ferramenta útil para estudos in vivo.

A extensão da área mesencéfalo transfectadas com miR é visível através da expressão de GFP a partir de um vector repórter injectado, juntamente com o vector de expressão de miR (Figura 2). Usando eléctrodos de platina com um diâmetro de 0,5 mm e colocado paralelamente ao eixo de AP de ligações mesencéfalo normalmente para a transfecção de uma área ampla e ao longo do eixo-DV AP, incluindo a parte posterior do cérebro, e por vezes mesencéfalo diencéfalo (Figuras 2A e B).</stro...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

A expressão ectópica é uma técnica para elucidar o papel microRNAs no desenvolvimento do cérebro. No entanto, em áreas específicas usando a eletroporação in ovo é um desafio. Aqui, vamos mostrar uma maneira eficiente para regiões do mesencéfalo ventral e dorsal seletiva electroporate.

Em Expression in ovo de microRNA em Ventral Pintinho Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

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Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

10.4K visualizações.  University of Tübingen.  A expressão ectópica é uma técnica para elucidar o papel microRNAs no desenvolvimento do cérebro. No entanto, em áreas específicas usando a eletroporação in ovo é um desafio. Aqui, vamos mostrar uma maneira eficiente para regiões do mesencéfalo ventral e dorsal seletiva electroporate. 

Vídeo: In ovo Expression of MicroRNA in Ventral Chick Midbrain

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotípicas culturas de mesencéfalo ventral Slice Embrionárias: Um Sistema para o Estudo de Desenvolvimento Neuronal dopaminérgicos In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

Neurociência

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Na eletroporação in ovo em Midbrain pintainho para estudar a função do gene em dopaminérgico Desenvolvimento Neuron

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Profiling abrangente do Regulamento dopamina em substância negra e área tegmental ventral

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq Análise de Gene Expression Diferencial em electroporados Pintainho Embryonic Medular

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Modulações Ambientais do número de Midbrain dopamina Neurônios no Adulto Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

A identificação fiável de Vida neurônios dopaminérgicos em culturas mesencéfalo Usando RNA Sequenciamento e TH-driven-promotor eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Um modelo em Ovo para testar compostos insulino-mimética

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Rastreamento Anatômico e Funcional In Vivo dos Terminais de Glutamato da Área Tegmental Ventral no Hipocampo

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Quantificando fluxos espontâneos ca2+ e seus efeitos a jusante em neurônios midcérebros do rato primário

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...