Este trabalho é apoiado por fundos de inicialização do Laboratório Jackson.

1. Preparando Cultura Glia Rato para o condicionamento de Cultura Neuronal <ol><li> Frascos T75 são revestidos com uma solução de colagénio (1:3 diluição de Purecol em DDH 2 O). Os frascos são erguidas e deixa-se secar durante a noite numa câmara de cultura de tecidos. </li><li> Tampão dissecção (aCSF: 119 mM de NaCl, 5 mM KCl, 1 mM de MgCl2, 30 mM de glicose, 25 mM de HEPES, pH 7,4, sem cálcio) e os meios da glia (DMEM suplementado com 10% de FBS, 10 unidades / ml de penicilina, 10 ug / ml de estreptomicina, e Glutamax) são preparadas e armazenadas a 4 ° C até que esteja pronto para uso. </li><li> No dia da cultura as células gliais, os meios de comunicação glia é aquecida durante pelo menos 30 min em banho de água a 37 ° C antes da dissecação de cérebros de ratos. Ratos embrionários ou neonatais são sacrificados por decapitação rápida. Córtex cerebral são dissecados sob um microscópio de dissecação. </li><li> Tecidos cerebrais dissecados são digeridas em solução de papaína (100 ml de papaína e 20 ul de 1% de DNase I, em 2 ml de tampão de dissecção) a 37 ° C for 20 min. </li><li> Após a digestão com papaína, tecidos cerebrais são lavados com 10 ml de meio pré-aquecido da glia. Media glia frescas (2 ml) é então adicionado aos tecidos cerebrais digeridos, e os tecidos são triturados com uma chapa de vidro polido ao fogo pipeta de Pasteur. </li><li> Meios frescos glia (8 ml) é adicionado aos tecidos dissociados. A 70 uM de células-filtro é usado para filtrar a suspensão de tecido dissociado. As células são então semeadas em frascos T75 (geralmente 2-3 embriões pode semear um frasco T75), e colocados em um de 37 ° C célula incubadora de cultura. </li><li> No dia seguinte, os meios de comunicação a partir dos frascos T75 é aspirado. Os balões são lavados com 10 ml de PBS. Media glia frescos (10-15 ml) é então adicionado a cada frasco. </li><li> Dentro de 10-12 dias, as células gliais nos frascos T75 deve ser confluente. As células gliais são lavadas com PBS, e de 5 ml de tripsina a 0,05% é adicionado a cada frasco T75. </li><li> Após incubação com tripsina a 37 ° C durante 5 minutos, as células gliais são células dissociadas e glial de cada frasco T75 são divididos em doisdiferentes de colagénio revestidos frascos T75. </li><li> Inserções de cultura (Millipore PICM03050) são preparados colocando-as em placas de 6 poços, revestindo-os com o colagénio, e o ar de secagem durante a noite. Media glia (2 ml cada) é colocado por baixo de cada inserção de cultura. </li><li> Glia de frascos T75 confluentes são tripsinizadas e semeadas sobre as inserções de cultura (de um frasco T75 por placa de 6 poços, 2 ml cada inserção). Estas inserções de cultura glia será utilizada para condicionar as culturas neuronais. </li></ol> 2. Cultura neuronal <ol><li> As lamelas de vidro para cultura neuronal são limpos por imersão em 70% HNO 3, pelo menos durante a noite. As lamelas são em seguida lavadas com DDQ 2 O, pelo menos, 3 vezes, e secou-se num forno Isotemp (&gt; 200 ° C) durante a noite. </li><li> Lamelas limpos são colocados numa placa de 6 poços, uma lamela por poço. As lamelas são revestidas com poli-L-Lisina a solução (2 ml, 0,1% em 100 mM de ácido bórico, pH = 8,5) durante a noite numa incubadora a 37 ° C. </li><li> Opoli-L-lisina solução é removida e as lamelas lamelas foram lavadas 3 vezes com autoclavado DDH 2 O. As lamelas são em seguida incubadas numa incubadora a 37 ° C durante a noite em meio de plaqueamento (Neurobasal suplementado com soro de cavalo a 5% inactivado pelo calor, a 10 unidades / ml de penicilina, estreptomicina 10 ug / ml, Glutamax, 2% B27 suplemento é adicionado ao meio de plaqueamento no dia em que é usado). </li><li> No dia seguinte, o meio de revestimento é removida e 2 ml de meio fresco com chapeamento B-27 é adicionada a cada poço que tem uma lamela de vidro. </li><li> Ratas grávidas (E15-18) são sacrificados com CO 2, e os ratos embrionárias são sacrificados por decapitação. O hipocampo ou estriado é dissecado a partir do tecido cerebral e colocados em um tubo de 15 ml com tampão arrefecido com gelo de dissecação, para a cultura de neurónios do hipocampo ou do estriado neurónios espinhosos médios, respectivamente. </li><li> Tecidos cerebrais dissecados são dissociados com papaína, tal como descrito nas etapas 1.4) e 1.5). </li><li> Fepois de dissociação, o número de células em cada suspensão neuronal é contado com um hemocitómetro. Os neurónios são então semeadas em cada lamela, a uma densidade de 0,4 x 10 6 células por poço de uma placa de 6 poços, e cultivadas numa incubadora a 37 ° C durante a noite (neurónios são DIV 0). </li><li> Para preparar inserções de cultura condicionado por células gliais as culturas neuronais, os meios de comunicação glia é removido das inserções de cultura de células da glia e meio de revestimento fresco é adicionado (2 ml na inserção e 2 ml por baixo da inserção). </li><li> No dia seguinte (DIV 1), com lamelas de neurónios são colocadas por baixo das inserções de cultura da glia, um lamínulas em cada poço. </li><li> Os neurónios são alimentados com metade do volume de meio fresco pré-aquecido com alimentação (B-27) a cada 3-4 dias até que estejam prontos para experiências de transfecção e de imagem. </li></ol> 3. Transfecção <ol><li> Neuronal transfecção é realizada utilizando Lipofectamine 2000. Para cada cultura neuronal (uma cultura por cada lamela), 2 ulLipofectamine 2000 e 1 ug de ADN são utilizados. Meio Neurobasal fresco, sem suplemento é usado para diluir a Lipofectamina e DNA. </li><li> DNA Lipofectamine e são incubadas durante 20 min à temperatura ambiente após a mistura. </li><li> Salve 1 ml dos meios de comunicação de baixo cada inserção da cultura glia, e 1 ml de dentro de cada inserção, e transferir a mídia para um tubo cônico. Adicionar o mesmo volume de meio de alimentação + B27 para o meio de salva, e incubar a 37 ° C. Este meio é utilizado para a cultura neuronal após transfecção. </li><li> Seguindo a incubação de 20 min Lipofectamine / DNA, as inserções de cultura são removidas da glia momentaneamente a partir das placas de 6 poços. A mistura de Lipofectamine 200 / ul de ADN é adicionado a cada lamela com neurónios. As inserções de cultura de células gliais são então devolvidos a cada poço, acima dos neurónios. Geração de bolhas sob a membrana das inserções de cultura deve ser evitado. Os neurónios são incubadas com a mistura de Lipofectamine / DNA a 37 ° C durante 2-4 hr. </li><li> Depois ªe 2-4 hr período de incubação, as inserções são removidas novamente da glia. Os meios de cultura com a mistura de Lipofectamine / DNA é removida de cada poço. Os meios de comunicação guardados no passo 3.3) é adicionada a neurónios (2 ml por poço). Inserções de cultura glia são devolvidos a cada poço, e os meios de comunicação glia de cada inserção é removido, e 2 ml de meios de comunicação a partir do passo 3.3) é adicionado em cada pastilha. </li><li> Os neurónios são cultivados durante uma hora adicional a 24-72 permitir a expressão de ADNc transfectado antes TIRF imagem é executada no estes neurónios. </li></ol> 4. TIRF imagem <ol><li> Fluido cerebrospinal artificial (aCSF: 119 mM de NaCl, 2,5 mM KCl, 30 mM de glicose, 2 mM de CaCl2, 1 mM de MgCl2, 25 mM de HEPES, pH = 7,4) será utilizado para geração de imagens ao vivo experimentos. Nosso sistema de imagem TIRF é costume set-up com um 100-mW Ciano laser para excitação, e uma carga do elétron Multiplicando Juntamente câmera do dispositivo (EMCCD) para um detector. </li><li> Antes de experimentos com imagens, o aCSF é advertido em um 37 &amp; dpor exemplo, C banho de água durante pelo menos 30 min. O inserto para geração de imagens ao vivo de aquecimento é colocada na platina do microscópio, e a inserção de aquecimento, laser, câmara e são aquecidos durante pelo menos 30 minutos antes do início das experiências de imagiologia. </li><li> A lamela com os neurônios transfectadas é montado na câmara ao vivo de imagens. O aCSF pré-aquecido é colocado na câmara depois da câmara é montada, esta etapa deve ser completado tão rapidamente quanto possível. </li><li> Uma vez que a câmara é colocada sobre o inserto de aquecimento sobre a platina do microscópio, os neurónios transfectadas são identificadas visualmente sob epi-fluorescente modo de imagem. </li><li> Após saudáveis ​​neurônios transfectadas são identificadas (ver as Figuras 1 e 2), que normalmente executam 1 ​​min de foto-branqueamento para eliminar preexistente pHluorin fluorescência na membrana plasmática, tal como pHluorin-receptores na membrana plasma ter níveis muito elevados de fluorescência quando TIRF imagem é iniciado, o que interfere com a visualização de ins indivíduoertion eventos. </li><li> Seguindo Fotobranqueador, o ajuste de ganho do multiplicador de electrões na câmara EMCCD é ajustado para o máximo, e a gravação é realizada durante 1 min a 10 Hz. </li><li> Cada gravação irá conter 600 imagens. Eventos de inserção individuais são identificadas por jogar nas costas de gravação usando ImageJ. Eventos de inserção individuais são analisados ​​manualmente usando ImageJ. </li></ol> 5. Resultados representativos Cultura neuronal consistente é a chave para o sucesso de imagem ao vivo experimentos. Figura 1 mostra os neurônios do hipocampo de rato cultivadas utilizando nosso protocolo de DIV DIV 11 a 18. É claro a partir das imagens que os neurônios se desenvolveram processos extensos e que os processos dendríticas são cobertos densamente com espinhas dendríticas, indicações de que esses neurônios são saudáveis ​​na cultura. O protocolo de cultura pode também ser utilizado para outros tipos de neurónios no cérebro. Por exemplo, recentemente utilizado este protocolo para culturaestriado neurônios espinhosos médios em um estudo para examinar receptor de dopamina D2 (DRD2) inserção na cultura de neurônios de rato médio estriado espinhosas. 9 Figura 2 mostra rato estriado neurônios espinhosos médios cultivadas utilizando nosso protocolo em DIV 8. Temos também realizada com sucesso TIRF imagiologia de superecliptic DRD2s pHluorin-tagged neste tipo de neurónio. Figura 3 mostra a expressão de pHluorin-tagged DRD2 (pH-DRD2) num neurónio spiny médio e visualização de pH-DRD2 inserção neste neurónio. <img alt="Figura 1" src="/files/ftp_upload/4450/4450fig1.jpg" /> Figura 1. Cultura do hipocampo do rato pelo método de cultura de interface. DIV: dias in vitro, o dia de cultura é considerada como DIV 0. A partir das imagens, é evidente que os neurónios do hipocampo maduros neste tipo de cultura entre 11 e DIV DIV 15. Às 11 e 13 DIV, dendrites neuronais são cobertas com espinhas filipodia e imaturas. Às 15 e DIV18, dendritos neuronais são cobertos com espinhos grandes, uma indicação de mais neurônios maduros. Painéis esquerdo: ampliação de imagens de baixa de neurônios do hipocampo de rato em diferentes idades. Painéis direito: ampliação de imagens de alta (zoom em dendritos neuronais do painel esquerdo) de processos neuronais dendríticas com espinhas dendríticas em diferentes idades. <img alt="Figura 2" src="/files/ftp_upload/4450/4450fig2.jpg" /> Figura 2. Rato estriatal média spiny cultura neuronal utilizando o método de cultura de interface. Neurônios nas imagens estão em DIV 8. <img alt="Figura 3" src="/files/ftp_upload/4450/4450fig3.jpg" /> Figura 3. Um rato estriatal meio neurônio espinhoso transfectadas com super eclíptica pHluorin-marcado receptor de dopamina D2 (pH-DRD2). Imagem à esquerda é a projeção de intensidade máxima de uma gravação de lapso de tempo (600 imagens, 100 ms por imagem). Setas brancas indicam a inserção vesicular individual de pH-DRD2. Esta imagem retém informação de inserção na dimensão xy, mas perde a informação da hora. Imagem da direita mostra a intensidade de projecção yt máximo, em que a pilha XYT é girada em 90 graus em torno do eixo y, e o pixel máximo em cada linha do eixo x é projectada para um pixel único eixo y. Esta imagem retém informação de inserção ao longo do eixo yt mas perde a informação eixo x.

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Por razões desconhecidas, os neurônios de rato são sempre mais difíceis de cultura do que neurônios de rato. Em nossa experiência, uma cultura mista de neurônios e da glia funciona bem para os neurônios primários de rato cultivadas. No entanto, uma tal cultura mista não é adequado para as experiências de imagiologia TIRF, como neste tipo de cultura de neurónios e seus processos tendem a crescer em cima de células da glia, situando o somata neuronal e processos dendríticos para além do alcance de microsco...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nome do Reagente</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;<td<strong&gt; Comentários</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Purificada solução de colágeno bovino (Purecol)</td&gt;<td&gt; Avançado Biomatrix</td&gt;<td&gt; 5005-B</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Hank solução salina equilibrada (HBSS)</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 14185-045</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Penicilina-estreptomicina (Pen Strep)</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 15140-122</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Piruvato de sódio</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 11360-070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Glucose DMEM High</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 10313-021</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; De soro fetal de bovino (FBS)</td&gt;<td</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; GlutaMAX</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 35050-061</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Papaína</td&gt;<td&gt; Worthington Biochemical Corp</td&gt;<td&gt; LS003126</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Desoxirribonuclease I a partir de pâncreas de bovino (DNase I)</td&gt;<td&gt; SIGMA</td&gt;<td&gt; DN25-10mg</td&gt;<td</td&gt;</tr&gt;<tr&gt;<tdFosfato&gt; Dulbecco tamponado salino (DPBS)</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 14190-144</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Tripsina 0,05%</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 25300-054</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Poli-L-lisina bromidrato</td&gt;<td&gt; SIGMA</td&gt;<td&gt; P2636-1G</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Ácido bórico</td&gt;<td&gt; Fisher-Científico</td&gt;<td&gt; BP 168-500</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Médio Neurobasal</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 21103-049</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; B-27 Suplemento Serum-Free</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 17504-044</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Calor soro de cavalo inativo</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 26050-070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Lipofectamine 2000</td&gt;<td&gt; Invitrogen</td&gt;<td&gt; 11668 019</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; HEPES</td&gt;<td&gt; Fisher-Científico</td&gt;<td&gt; BP310-500</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Inserir Cultura</td&gt;<td&gt; Millipore</td&gt;<td&gt; PICM03050</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

imaging phluorin-tagged receptor insertion to the plasma membrane in primary cultured mouse neurons

Uma melhor compreensão dos mecanismos que regem o tráfico receptor entre a membrana do plasma (PM) e compartimentos intracelulares requer uma abordagem experimental, com excelentes resoluções espaciais e temporais. Além disso, esta abordagem também deve ter a capacidade de distinguir os receptores localizados na PM daqueles em compartimentos intracelulares. Mais importante ainda, a detecção de receptores de uma vesícula única requer excelente sensibilidade de detecção, uma vez que cada vesícula leva apenas um pequeno número de receptores. Abordagens normalizadas para examinar o tráfico receptor incluem a biotinilação da superfície seguido de detecção bioquímica, que carece tanto as resoluções necessárias espaciais e temporais, e exame de microscopia de fluorescência de receptores de superfície, o que requer immunolabeled fixação química de células e, portanto, carece de resolução temporal suficiente 1-6. Para superar essas limitações, nós e outros têm desenvolvido e utilizado um novo stratégia que permite a visualização da inserção dinâmica de receptores para a PM, com excelentes resoluções espaciais e temporais 7-17. A abordagem inclui a marcação de uma GFP sensível ao pH, o pHluorin superecliptic 18, para o domínio N-terminal extracelular de receptores. Superecliptic pHluorin tem a propriedade única de ser fluorescente a pH neutro e não fluorescente a pH ácido (pH &lt;6,0). Portanto, os receptores marcados são não-fluorescente quando dentro do lúmen ácida tráfico de vesículas intracelulares ou compartimentos endossomais e tornam-se facilmente visualizado apenas quando exposto ao ambiente extracelular pH neutro, na superfície exterior do PM. Nossa estratégia, consequentemente, permite-nos distinguir receptores de superfície PM daqueles dentro de vesículas tráfico intracelular. Suficientes para alcançar resoluções espaciais e temporais, bem como a sensibilidade necessária para estudar o tráfico dinâmica dos receptores, foram empregados interna total reflectemion microscopia fluorescente (TIRFM), o que nos permitiu obter a melhor resolução espacial da imagem óptica (~ 170 nm), a resolução temporal do vídeo de taxa de microscopia (30 quadros / seg), e a sensibilidade para a detecção de fluorescência de uma única GFP molécula. Pela imagem pHluorin-marcado receptores sob TIRFM, fomos capazes de visualizar diretamente eventos receptores individuais inserção na PM em neurônios cultivados. Esta abordagem de imagem pode, potencialmente, ser aplicada a qualquer proteína de membrana com um domínio extracelular que poderiam ser marcadas com pHluorin superecliptic, e permitirá a dissecação dos mecanismos chave de pormenor que regem a inserção de proteínas de membrana diferentes (receptores, canais de iões, transportadores, etc) para da PM.

Watch this Scientific Journal Video about Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons at JoVE.com

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

Ao marcar os domínios extracelulares dos receptores de membrana com pHluorin superecliptic, e por imagiologia estes receptores de fusão em neurónios de rato em cultura, pode-se visualizar directamente os eventos de inserção individuais vesiculares dos receptores na membrana plasmática. Esta técnica será útil para elucidar os mecanismos moleculares que regulam a inserção do receptor para a membrana plasmática.

Imagiologia pHluorin-tagged Inserção receptor para a membrana plasmática em primários de rato cultivadas Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

imaging-phluorin-tagged-receptor-insertion-to-plasma-membrane-primary

Research

JoVE Journal

Neuroscience

13.1K visualizações.  The Jackson Laboratory. Ao marcar os domínios extracelulares dos receptores de membrana com pHluorin superecliptic, e por imagiologia estes receptores de fusão em neurónios de rato em cultura, pode-se visualizar directamente os eventos de inserção individuais vesiculares dos receptores na membrana plasmática. Esta técnica será útil para elucidar os mecanismos moleculares que regulam a inserção do receptor para a membrana plasmática.

Vídeo: Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Neurociência

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análise quantitativa de Reposição da vesícula sináptica Piscina em Cultured neurônios granulares do cerebelo usando Dyes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Induzir o crescimento dendrítico no cultivadas neurônios simpáticos

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusão lateral e Exocitose de proteínas de membrana em neurônios cultivados avaliada através da recuperação de fluorescência e fluorescência perda de fotobranqueamento

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Cérebro Fatia Biotinilação: Uma<em&gt; Ex Vivo</em&gt; Abordagem medir Região específicas do tráfego de proteínas de membrana plasmática em adultos Neurônios

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultura primária de neurônios dopaminérgicos mouse

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologias combinadas para definir o papel do Plasma Membrane entrega durante Axon Ramificação e Neuronal morfogênese

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Modelagem de morte Neuronal e a degeneração no Mouse grânulo cerebelar primária de neurônios

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Imagens ao vivo da proteína GLUT4 tráfico de Mouse de neurônios hipotálamo primário usando microscopia de deconvolução

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultura primária de neurônios isolados do cerebelo embrionário do rato

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...