Somos gratos a Lihong Yin para assistência técnica. NVD é um destinatário de um NARSAD Young Investigator Award e também é apoiada por doações da NIH (5 K99 MH095825-02). Pesquisa no laboratório Fishell é apoiado pelo Instituto Nacional de Saúde, Instituto Nacional de Saúde Mental (5 R01 MH095147-02, 5 R01 MH071679-09), Instituto Nacional de Distúrbios Neurológicos e Derrame (5 R01 NS081297-02, 1 P01 NS074972 -01A1) e da Fundação Simons.

Todos os animais foram tratados de acordo com as normas e diretrizes do Comitê de Cuidados e Uso da NYU School of Medicine animal Institucional. Rato Cepas Swiss Webster ratinhos fêmeas fornecidos por TACONIC Foram utilizados para estas experiências. A fim de orientar especificamente interneurônios camada superficial, foram utilizados embriões E15.5. Nota: O plasmídeo utilizado no presente trabalho (Dlx5 / 6.eGFP plasmídeo 3 mg / mL) foi gerado utilizando técnicas de clonagem convencionais. O eGFP cDNA foi clonado num Dlx5 / 6-Pmin-poliA plasmídeo. Este plasmídeo está disponível mediante solicitação (demarn02@nyumc.org). 1 Preparação de microinjeção pipetas <ol><li> Defina o extrator de calor # 2-860. </li><li> Colocar e fixar o capilar de vidro perto do filamento. Verificar que o diâmetro externo (OD) da pipeta é cerca de 30 um. </li><li> Pressione o botão &quot;puxar&quot;. </li><li> Retire cuidadosamente o capilar. A parte inferior é a agulha. Descartar a parte superior. </li></ol> 2 Anestesia Procedimento <ol><li> Prepare uma câmara contendo isoflurano. Nota: O isoflurano é o anestésico preferido devido a sua ação rápida e baixa incidência de efeitos colaterais. Pentobarbital é outra opção; No entanto, a tolerância deste fármaco é variável e pode levar a uma taxa de mortalidade maior do que o isoflurano. </li><li> Definir o fluxo de isoflurano a 4-5% em 0,5-1 L / min O 2. </li><li> Certifique-se de que a válvula para a câmara é aberta enquanto que a válvula para a placa de cabeça está fechado. </li><li> Posicione o mouse grávida na câmara. </li><li> Deixe o mouse para ir sob o efeito da anestesia. Isso levará cerca de 2-3 min. Verifique a taxa de respiração. Se o rato começa a hiperventilar, reduzir a dose de isoflurano. </li><li> Após o mouse está anestesiado, imediatamente removê-lo da câmara. Coloque-o lado ventral em cima da almofada de aquecimento e iNSERT a cabeça na máscara que continuará a fornecer isoflurano durante toda a cirurgia. Certifique-se de mudar o fluxo de isoflurano da câmara para a mangueira que liga a peça adaptador cabeça. Coloque uma gota de lubrificante ocular em cada olho e fechar os olhos. </li><li> Ajuste a almofada de aquecimento a 36 ° C graus para minimizar hipotermia. </li><li> Verificar a ausência de pé e cauda reflexos, beliscar as patas traseiras ea cauda. </li></ol> 3. Cirurgia <ol><li> Limpe a pele que cobre o abdômen com etanol 70%. </li><li> Use uma pinça para puxar a pele para cima. Após as cirurgias, lavar todos os instrumentos com detergente e água, e esterilizou-se a 72 ° CO / N. </li><li> Fazer uma pequena incisão vertical (cerca de 2 cm) ao longo da linha média de partida a meio caminho entre o terceiro e quarto pares de glândulas mamarias. Use a tesoura cirurgia finas. Tome cuidado para não danificar a parede abdominal. </li><li> Separe cuidadosamente a pele do peritônio. </li><li> Visualize as artériase nervuras na parede abdominal. Adicione outro dois centímetros incisão vertical através do peritoneu (evitando os vasos) para expor a cavidade abdominal. </li><li> Prenda a pele ea parede abdominal de cada lado evitando pinçamento das artérias. Nota: Se as artérias são acidentalmente perfurada, sacrificar o animal imediatamente ao realizar deslocamento cervical ou overdose isoflurano. </li><li> Pré aqueça o PBS estéril a 37 ° C. Cubra os grampos com PBS úmidos Kim toalhetes. </li><li> Hidratar a cavidade abdominal exposta com PBS. Repita este passo várias vezes durante a realização da cirurgia. </li><li> Utilizando uma pinça redondas, puxe a cadeia embrionárias (E15.5) fora da cavidade. Deixe o resto da cadeia sobre os lenços umedecidos. Iniciar expondo metade do útero primeiro. Uma vez que os embriões em que são electroporados, trazê-lo de volta para dentro, em seguida, trabalhar no segundo semestre. </li></ol> 4. Eletroporação <ol><li> Continue a usar fórceps redondas para manipular os embriões. </li><li> Visualize os ventrículos laterais. Os ventrículos laterais correm ao longo da linha média (Figura 1b). </li><li> Adicionar Verde Rápido (Banco: 10% w / v) à solução de DNA de uma mistura 01:20 de visualizar que durante a injecção. Preparar a solução de DNA através da dissolução do sedimento obtido a partir de um protocolo padrão de purificação de maxi-prep em água destilada. </li><li> Utilize micropipetas pré puxados com um êmbolo para injetar 1 ml da solução de DNA (Dlx5 / 6-EGFP, 3 mg / mL) com Fast Green. Cortar a ponta da micropipeta com uma pinça fina # 5 (Dumont) para deixar 6 mm de comprimento a partir do início do ombro até ao final da ponta da agulha antes de colocar a pipeta. Segure a cabeça do embrião com uma pinça redondas. Entram no cérebro com a pipeta com um ângulo de 45 ° com vista para o ventrículo lateral, localizado perto da linha média. Se a agulha penetra através do ventrículo, retirar lentamente a pipeta como você usa o êmbolo para injetar DNA. O ventrículo deve ficar verde quando osolução começa a preenchê-lo. Neste ponto, param de se mover a pipeta e injectar 1 ml de ADN. Delicadamente, retire a pipeta. </li><li> Defina o electroporator CUY21 cinco 45V pulsos de 50 ms espaçadas de 950 ms. </li><li> Use 5 milímetros eletrodos de remo para os embriões E15.5. Mergulhe os eletrodos em PBS para garantir a transmissão de corrente eficiente. </li><li> Coloque as pás de eléctrodos em volta da cabeça do embrião com a pá positivo sobre o ventrículo contendo a solução de ADN. Ligeiramente inferior a pá positiva com relação à negativa para criar uma corrente ventral através das eminências ganglionares. Esta manipulação irá minimizar a expressão ectópica de células piramidais. </li><li> Após a colocação dos eletrodos, entregar um pulso, pressionando o pedal uma vez. </li><li> Retirar os eletrodos e limpe-os. Repita os passos de 4,6-4,9 com cada embrião. </li><li> Após electroporating todos os embriões, derrame 3 ml de PBS para dentro da cavidade abdominal e devolvê-las para a cavidade abdominal. </li><li>Primeiro sutura da parede abdominal com uma agulha curva e sutura de seda 5-0 e, em seguida, a pele. Aplicar lidocaína (4%) na ferida. Administrar 120 mg / kg de aspirina peritônio intra para analgesia. Nota: O procedimento completo deve ser realizado em 20 minutos ou menos. É aconselhável que os novatos só electroporate alguns embriões para manter o tempo de funcionamento curto. Cirurgias prolongadas vai diminuir a taxa de sobrevivência dos embriões. </li><li> Remover o animal da tubulação de anestesia e permitir a recuperação da almofada de aquecimento em um papel wipe. </li><li> Voltar animais para a jaula, mantê-lo no bloco de aquecimento a 37 ° C e monitorar o estado de saúde. Os animais geralmente toleram bem a cirurgia e começam a mover-se lentamente, logo depois de terem sido removidos do tubo anestesia. Se o sangramento ou letargia excessiva é observada, sacrificar o animal imediatamente realizando IACUC aprovado procedimentos de eutanásia, como a luxação cervical ou anestesia overdose. </li><li> Voltar gaiola para o vivarium. As fêmeas dão à luz naturalmente. </li></ol>

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	<li>Fishell, G., Rudy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+inhibition+within+the+telencephalon:+&amp;#34;where+the+wild+things+are&amp;#34;.">Mechanisms of inhibition within the telencephalon: &amp;#34;where the wild things are&amp;#34;.</a> Annual review of neuroscience. 34, 535-567 (2011).</li><li>Stenman, J., Toresson, H., Campbell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+distinct+progenitor+populations+in+the+lateral+ganglionic+eminence:+implications+for+striatal+and+olfactory+bulb+neurogenesis.">Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis.</a> J Neurosci. 23, 167-174 (2003).</li><li>Eisenstat, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DLX-1,+DLX-2,+and+DLX-5+expression+define+distinct+stages+of+basal+forebrain+differentiation.">DLX-1, DLX-2, and DLX-5 expression define distinct stages of basal forebrain differentiation.</a> J Comp Neurol. 414, 217-237 (1999).</li><li>Batista-Brito, R., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+integration+of+cortical+interneurons+into+a+functional+network.">The developmental integration of cortical interneurons into a functional network.</a> Curr Top Dev Biol. 87, 81-118 (2009).</li><li>Miyoshi, G., Butt, S. J., Takebayashi, H., Fishell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologically+distinct+temporal+cohorts+of+cortical+interneurons+arise+from+telencephalic+Olig2-expressing+precursors.">Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors.</a> J Neurosci. 27, 7786-7798 (2007).</li><li>Miyoshi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+fate+mapping+reveals+that+the+caudal+ganglionic+eminence+produces+a+large+and+diverse+population+of+superficial+cortical+interneurons.">Genetic fate mapping reveals that the caudal ganglionic eminence produces a large and diverse population of superficial cortical interneurons.</a> J Neurosci. 30, 1582-1594 (2010).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Dlx-2+(Tes-1)+gene+is+expressed+in+spatially+restricted+domains+of+the+forebrain,+face+and+limbs+in+midgestation+mouse+embryos.">The mouse Dlx-2 (Tes-1) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos.</a> Mechanisms of development. 40, 129-140 (1993).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+restricted+expression+of+Dlx-1,+Dlx-2+(Tes-1),+Gbx-2,+and+Wnt-3+in+the+embryonic+day+12.5+mouse+forebrain+defines+potential+transverse+and+longitudinal+segmental+boundaries.">Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries.</a> J Neurosci. 13, 3155-3172 (1993).</li><li>Robinson, G. 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(31), (2009).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> Journal of visualized experiments : JoVE. (6), e239 (2007).</li></ol>

Os autores não têm nada a revelar.

Limitações da técnica Embora esta técnica permite a célula de análise autônoma de processos celulares, não é adequado para análise da população. Os electroporations são muito escassos com menos de mil células eletroporados por cérebro. Como consequência, a técnica não pode ser utilizada para avaliar as consequências comportamentais resultantes da manipulação genética de interneurónios CGE-derivados. Enquanto electroporações realizadas em ...

A maior parte dos interneurônios GABAérgicos corticais originam de duas estruturas embrionárias transitórias nomeadas as eminências ganglionares média e caudal (MGE e CGE respectivamente) 4. Parvalbumin e somatostatina expressar interneurônios origem no MGE enquanto Calretinina (Cr), Vasointestinal peptídeo (VIP) e Reelin (Re) expressando interneurônios originam do CGE. Estes subtipos Interneuron podem ser distinguidos pelas suas datas de nascimento. MGE subtipos derivados nascem entre o dia embrionário 9.5 (E9.5) e E16.5 5,6. Em contraste, interneurônios CGE derivados nascem E12.5 através E18.5 com sua produção atingindo um máximo de 6 E15.5. A orientação genética dessa população tarde nascido, no entanto, permanece indefinida. Os (Dlx) genes distal-less murino são exclusivamente expressos no cérebro anterior ventral desenvolvimento 3. Interneurônios GABAérgicos e neurônios de projeção do estriado, mas não células piramidais corticais expressar Dlx1, 2,5, e 6 genes em estágios iniciais de desenvolvimento 3. De facto, os genes são expressos Dlx na zona MGE e CGE subventricular (SVZ), em todas as células progenitoras GABAérgicos. Expressão desses genes se torna restrita a seleccionar subtipos em fases pós-mitóticas 7-9. Evidências experimentais anteriores mostraram que o elemento potenciador Dlx5 / 6 permite o direcionamento seletivo de linhagens GABAérgicas em modelos de camundongos transgênicos 2. Foi testada a utilização de um destes elementos potenciadores no contexto da expressão episómico no cérebro do rato em desenvolvimento. Nós sub clonado o elemento potenciador Dlx5 / 6 em conjunto com um promotor mínimo e a proteína verde fluorescente melhorada (EGFP) no Bluescript (BS) plasmídeo de esqueleto (Figura 1). Nós introduzimos o plasmídeo por meio de in utero eletroporação em E15.5 para alvejar seletivamente Cr-, VIP e Re subtipos 3,8,10. A nossa técnica permite electroporação escasso, o que facilitaa reconstrução das características morfológicas de células singe. Além disso, os níveis excepcionalmente elevados de expressão de genes em neurónios GABAérgicos corticais permite estudos funcionais. Realizou-se a perda eo ganho de estudos da função utilizando vários tipo selvagem e genes dominantes negativos 11.

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subtype-selective electroporation of cortical interneurons

O estudo do sistema nervoso (CNS) central de maturação depende de orientação genética de populações neuronais. No entanto, a tarefa de restringir a expressão de genes de interesse para os subtipos neuronais específicos provou extremamente difícil devido à escassez relativa de elementos promotores específicos. Interneurônios GABAérgicos constituem uma população neuronal com ampla diversidade genética e morfológica. De facto, mais de 11 subtipos diferentes de interneurónios sensíveis a GABA têm sido caracterizadas no córtex do rato 1. Aqui apresentamos um protocolo adaptado para direcionamento seletivo das populações GABAérgicos. Conseguimos subtipo direccionamento selectivo de interneurónios sensíveis a GABA, utilizando o elemento potenciador da transcrição homeobox factores Dlx5 e DLX6, os homólogos da distal inferior (Dll) do gene de Drosophila 2,3, para dirigir a expressão de genes específicos dentro do útero por meio de electroporação.

Nós adaptamos a técnica de eletroporação in utero para atingir alvos específicos tipo de célula de neurônios vencimento. Para dirigir a expressão de GFP em interneurônios CGE derivados, foi utilizado o elemento potenciador Dlx5 / 6 e restrito nossas injeções para E15.5, a fase em que a maioria dos interneurônios CGE derivados são gerados. Realizou-se a análise em P8 e P15 11 (Figuras 1 e 2). Foi confirmada a origem ventral de neurônios eletrop...

Watch this Scientific Journal Video about Subtype-selective Electroporation of Cortical Interneurons at JoVE.com

Subtype-selective Electroporation of Cortical Interneurons

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Subtipo seletivo Eletroporação de Cortical Interneurônios

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

subtype-selective-electroporation-of-cortical-interneurons

Research

JoVE Journal

Neuroscience

8.8K visualizações.  New York University School of Medicine. This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Vídeo: Subtype-selective Electroporation of Cortical Interneurons

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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

Isolamento e Cultura de astrócitos corticais de rato

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Eletroporação Zona Neonatal subventricular

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Ex utero Eletroporação e Explantes hemisfério inteiro: um método simples Experimental de Estudos do Desenvolvimento Cortical Precoce

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Neonatal Pial Superfície Eletroporação

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-cell Recordings patch-clamp de Morphologically- e identificados Neuroquimicamente hipocampo Interneurônios

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Um Guia para<em&gt; In vivo</em&gt; Gravação única unidade de Optogenetically Identificado Cortical Inibitória Interneurônios

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Método eletrofisiológico para respostas gravação intracelular de tensão<em&gt; Drosophila</em&gt; Fotorreceptores e Interneurônios à luz Estímulos<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

Visualização de módulos corticais em córtices mamíferos achatadas

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...