Este trabajo es apoyado por fondos de puesta en marcha del Laboratorio Jackson.

1. Preparación de Cultura del ratón para Glia acondicionado Cultura Neuronal <ol><li> Matraces T75 se recubren con una solución de colágeno (1:3 dilución de Purecol en ddH 2 O). Los matraces se colocará en posición vertical y se dejó secar durante la noche en una campana de cultivo de tejidos. </li><li> Tampón de disección (LCRa: 119 mM NaCl, 5 mM KCl, 1 mM de MgCl 2, 30 mM dextrosa, 25 mM HEPES, pH 7,4, sin calcio) y los medios de comunicación glía (DMEM suplementado con FBS al 10%, 10 unidades / ml de penicilina, 10 g / ml de estreptomicina, y Glutamax) se preparan y se almacenan a 4 ° C hasta que esté listo para su uso. </li><li> En el día de la cultura glia, los medios de comunicación glía se calienta durante al menos 30 min en un baño a 37 ° C antes de la disección de los cerebros de ratón. Ratones embrionarios o neonatal son sacrificados por decapitación rápida. Cortezas cerebrales se diseccionan bajo un microscopio de disección. </li><li> Disecados tejidos cerebrales son digeridos en solución de papaína (100 ml papaína y 20 l 1% de DNasa I en tampón de disección 2 ml) a 37 ° C for 20 min. </li><li> Después de la digestión con papaína, los tejidos del cerebro se aclaró con 10 ml de medio glía precalentado. Medios frescos glía (2 ml) se añade entonces a los tejidos del cerebro digeridos, y los tejidos se trituraron con un pulido al fuego pipeta Pasteur de vidrio. </li><li> Medios frescos glía (8 ml) se añade a los tejidos disociados. A 70 m de células colador se utiliza para filtrar la suspensión de tejido disociado. Después las células se sembraron en frascos T75 (usualmente 2-3 embriones pueden sembrar una matraz T75) y se colocaron en un 37 ° C incubadora de células de cultivo. </li><li> El día siguiente, los medios de comunicación de los matraces T75 se aspira. Los matraces se enjuagaron con 10 ml de PBS. Medios frescos glía (10-15 ml) se añade a continuación a cada matraz. </li><li> Dentro de 10-12 días, en las células de los matraces T75 debe ser confluente. Las células gliales se lavan con PBS, y 5 ml de tripsina al 0,05% se añade a cada matraz T75. </li><li> Después de la incubación de tripsina a 37 º C durante 5 min, las células gliales son células disociadas y gliales de cada matraz T75 se dividen en dosdiferentes recubiertas con colágeno matraces T75. </li><li> Insertos de cultivo (Millipore PICM03050) se preparan colocándolos en placas de 6-y, recubrimiento con colágeno, y secado con aire durante la noche. Medios de comunicación glía (2 ml cada uno) se coloca debajo de cada inserto de cultivo. </li><li> Glia de matraces T75 confluentes se tratan con tripsina y se siembran en los insertos de cultivo (un matraz T75 por 6-así placa, 2 ml cada pieza de inserción). Estos insertos de cultivo de células gliales se utiliza para acondicionar los cultivos neuronales. </li></ol> 2. Cultura Neuronal <ol><li> Los cubreobjetos de vidrio para el cultivo neuronal se limpian por inmersión en 70% de HNO 3 al menos durante la noche. Los cubreobjetos se lavan a continuación con ddH 2 O por lo menos 3 veces, y se secó en un horno Isotemp (&gt; 200 ° C) durante la noche. </li><li> Cubreobjetos limpios se colocan en una placa de 6-así, un cubreobjetos por pocillo. Los cubreobjetos se recubren con poli-L-lisina solución (2 ml, 0,1% en ácido bórico 100 mM, pH = 8,5) durante la noche en una incubadora de 37 ° C. </li><li> Lapoli-L-lisina solución se elimina de los cubreobjetos y los cubreobjetos se lavaron 3 veces con ddH autoclave 2 O. Los cubreobjetos se incuban a continuación en un 37 ° C durante la noche en medios galvánicos (Neurobasal suplementado con suero de caballo al 5% inactivado por calor, 10 unidades / ml de penicilina, 10 mg / ml de estreptomicina, Glutamax incubadora; 2% suplemento B27 se añade al medio de chapado el día en que se utiliza). </li><li> Al día siguiente, los medios de deposición se retira y 2 ml de medio fresco con enchapado de B-27 se añadieron a cada pocillo que tiene un cubreobjetos de vidrio. </li><li> Ratones embarazadas (E15-18) son sacrificados con el CO 2, y embriones de ratones son sacrificados por decapitación. El hipocampo o estriado se disecciona el tejido cerebral y se colocó en un tubo cónico de 15 ml con tampón enfriado con hielo disección, para el cultivo de neuronas del hipocampo o estriatal neuronas espinosas medias, respectivamente. </li><li> Disecados tejidos cerebrales se disocian con papaína como se describe en los pasos 1,4) y 1,5). </li><li> Fespués de disociación, el número de células en cada suspensión neuronal se contaron con un hemocitómetro. Las neuronas se sembraron entonces sobre cada cubreobjetos a una densidad de 0,4 x 10 6 células por pocillo de una placa de 6 pocillos y se cultivaron en una incubadora de 37 ° C durante la noche (las neuronas son DIV 0). </li><li> Para preparar insertos glía de cultivo para el acondicionamiento de los cultivos neuronales, los medios de comunicación glía se elimina de los insertos de cultivo de células gliales y medianas recubrimiento fresco se añade (2 ml en el inserto y 2 ml debajo de la pieza de inserción). </li><li> El día siguiente (DIV 1), los cubreobjetos con las neuronas se colocan debajo de los insertos de cultivo glia, un cubreobjetos en cada pocillo. </li><li> Las neuronas se alimentan con medio fresco que el volumen de los medios de alimentación pre-calentado (con B-27) cada 3-4 días hasta que están listas para experimentos de transfección y de imagen. </li></ol> 3. Transfección <ol><li> Neuronal transfección se realiza usando Lipofectamine 2000. Para cada cultivo neuronal (un cultivo por cubreobjetos), 2 lLipofectamine 2000 y 1 ug de ADN se utilizan. Medio fresco Neurobasal sin suplemento se usa para diluir Lipofectamine y ADN. </li><li> ADN y Lipofectamine se incuban durante 20 min a temperatura ambiente después de la mezcla. </li><li> Guardar 1 ml de los medios de debajo de cada inserto de cultivo glia, y 1 ml desde el interior de cada inserto, y transferir los medios de comunicación a un tubo cónico. Añadir el mismo volumen de medios de alimentación + B27 al medio de salvado y se incuba a 37 ° C. Este medio se utiliza para el cultivo neuronal después de la transfección. </li><li> Después de la 20-min Lipofectamine / ADN incubación, insertos glía de cultivo se retira momentáneamente de las placas 6-y. El 200 l Lipofectamine / ADN mezcla se añade a cada cubreobjetos con las neuronas. Los insertos de cultivo de células gliales se devuelven luego a cada pocillo, por encima de las neuronas. Generación de burbujas debajo de la membrana de los insertos de cultivo debe ser evitado. Las neuronas se incubaron con la mezcla de Lipofectamine / ADN a 37 ° C durante 2-4 h. </li><li> Después de the 2-4 hr período de incubación, las inserciones glía se retiran de nuevo. Los medios de cultivo con la mezcla de Lipofectamine / ADN se retiraron de cada pocillo. Los medios guardados en el paso 3,3) se añade a las neuronas (2 ml por pocillo). Glia insertos de cultivo se devuelven a cada pocillo, y los medios de comunicación glía de cada inserto se retira, y 2 ml de los medios de paso 3,3) se añadió a cada inserto. </li><li> Las neuronas se cultivaron durante 24-72 horas para permitir la expresión de cDNA transfectadas antes TIRF formación de imágenes se realiza en estas neuronas. </li></ol> 4. TIRF imágenes <ol><li> Líquido cefalorraquídeo artificial (LCRa: 119 mM NaCl, 2,5 mM KCl, 30 mM de glucosa, 2 mM CaCl 2, 1 mM MgCl 2, 25 mM de HEPES, pH = 7,4) se utiliza para los experimentos de formación de imágenes en vivo. Nuestro sistema de imagen TIRF es costumbre puesta a punto con un láser de 100 mW Cian para la excitación, y una carga del electrón Multiplicando Coupled Device (EMCCD) de la cámara por un detector. </li><li> Antes de los experimentos de imagen, la LCRa se advirtió en un 37 &amp; dpor ejemplo, baño de agua C durante al menos 30 min. El inserto de calefacción para formación de imágenes en vivo se coloca en la platina del microscopio, y el inserto de calefacción, el láser y la cámara se calienta durante al menos 30 min antes del inicio de los experimentos de imagen. </li><li> Un cubreobjetos con neuronas transfectadas se monta en la cámara de formación de imágenes en vivo. El LCRa pre-calentado se coloca en la cámara después de que la cámara está montada, este paso debe ser completado tan pronto como sea posible. </li><li> Una vez que la cámara se coloca en el inserto de calefacción en la platina del microscopio, las neuronas transfectadas se identifican visualmente bajo el modo de formación de imágenes epi-fluorescente. </li><li> Después sanos neuronas transfectadas se identifican (consulte las Figuras 1 y 2), se realizan típicamente 1 min de foto-blanqueador para eliminar la pre-existente pHluorin fluorescencia en la membrana plasmática, como pHluorin-receptores en la membrana plasmática tienen niveles de fluorescencia muy alta cuando TIRF imágenes por primera vez, lo que interfiere con la visualización de ins individuoertion eventos. </li><li> Después de fotoblanqueador, el ajuste de ganancia del multiplicador de electrones en la cámara EMCCD se ajusta al máximo, y la grabación se realiza durante 1 min a 10 Hz. </li><li> Cada registro contendrá 600 imágenes. Los eventos individuales de inserción se identifican por jugar la parte de atrás de grabación usando ImageJ. Los eventos individuales de inserción se analizaron manualmente usando ImageJ. </li></ol> 5. Los resultados representativos Cultivo neuronal consistente es la clave para el éxito de formación de imágenes en vivo-experimentos. Figura 1 muestra las neuronas del hipocampo de ratón cultivadas usando el protocolo de DIV 11 a DIV 18. Es evidente a partir de las imágenes que las neuronas han desarrollado procesos extensos y que los procesos dendríticos son cubiertos densamente con las espinas dendríticas, las indicaciones de que estas neuronas son saludables en cultivo. Nuestro protocolo de cultivo también podría ser utilizado para otros tipos de neuronas en el cerebro. Por ejemplo, recientemente hemos usado este protocolo a la culturaestriatal neuronas espinosas medias en un estudio para examinar los receptores de dopamina D2 (DRD2) inserción en cultivos de ratón estriatal neuronas espinosas medias 9. Figura 2 muestra ratón estriatal neuronas espinosas medias cultivadas utilizando nuestro protocolo en DIV 8. También hemos realizado con éxito TIRF imágenes de superecliptic DRD2s pHluorin-etiquetados en este tipo de neurona. Figura 3 muestra la expresión de pHluorin-etiquetados DRD2 (pH-DRD2) en una neurona espinosa medio y visualización de pH-DRD2 inserción en esta neurona. <img alt="Figura 1" src="/files/ftp_upload/4450/4450fig1.jpg" /> Figura 1. Hipocampo de ratones de cultivo utilizando el método de cultivo de interfaz. DIV: días in vitro, el día de la cultura se considera como DIV 0. De las imágenes, es evidente que las neuronas del hipocampo maduros en este tipo de cultivo entre 11 DIV y DIV 15. En DIV 11 y 13, las dendritas neuronales están cubiertos con filipodia y espinas inmaduras. En DIV 15 y18, las dendritas neuronales están cubiertos por espinas grandes, una indicación de más neuronas maduras. Paneles izquierdo: bajo aumento imágenes de las neuronas del hipocampo de ratones de diferentes edades. Paneles de la derecha: imágenes de gran aumento (zoom en las dendritas neuronales del panel de la izquierda) de los procesos neuronales dendríticas con las espinas dendríticas en las diferentes edades. <img alt="Figura 2" src="/files/ftp_upload/4450/4450fig2.jpg" /> Figura 2. Ratón espinoso neurona estriatal medio de cultivo utilizando el método de cultivo de interfaz. Las neuronas de las imágenes están en DIV 8. <img alt="Figura 3" src="/files/ftp_upload/4450/4450fig3.jpg" /> Figura 3. Un ratón medio estriatal neurona espinosa transfectadas con super eclíptica pHluorin marcada con receptor de dopamina D2 (DRD2 pH). Imagen de la izquierda es la proyección de intensidad máxima de la grabación de lapso de tiempo (600 imágenes, 100 ms por imagen). Puntas de flechas blancas indican inserción individual de pH vesicular-DRD2. Esta imagen retiene la información de la inserción en la dimensión XY pero pierde la información de tiempo. Imagen de la derecha muestra la proyección yt intensidad máxima, en el que la pila XYT se gira 90 grados alrededor del eje y, y el máximo de píxeles en cada línea del eje x se proyecta sobre un único eje y pixel. Esta imagen retiene la información de inserción a lo largo del eje yt pero pierde la información eje x.

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Por razones desconocidas, las neuronas de ratón son siempre más difíciles de cultivar que las neuronas de rata. En nuestra experiencia, un cultivo mixto de las neuronas y las células gliales funciona bien para los principales cultivos de neuronas de ratón. Sin embargo, como un cultivo mixto no es adecuado para TIRF experimentos de imagen, como en este tipo de neuronas de cultivo y sus procesos tienden a crecer en la parte superior de células de la glia, situando la somata neuronal y procesos dendríticos más all?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nombre del reactivo</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;<td<strong&gt; Comentarios</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Purificado solución de colágeno bovino (Purecol)</td&gt;<td&gt; Advanced Biomatrix</td&gt;<td&gt; 5005-B</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Hank solución 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 sodio</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 11360-070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM alto en glucosa</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 10313-021</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Suero fetal 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; I de páncreas bovino desoxirribonucleasa (DNasa I)</td&gt;<td&gt; SIGMA</td&gt;<td&gt; DN25-10MG</td&gt;<td</td&gt;</tr&gt;<tr&gt;<tdFosfato&gt; Dulbecco Buffered Saline (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 hidrobromuro</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 Scientific-</td&gt;<td&gt; BP 168-500</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Medio 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 libre de suero Suplemento</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 17504-044</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Calor inactiva suero de caballo</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 Scientific-</td&gt;<td&gt; BP310-500</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Cultura Insertar</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

Una mejor comprensión de los mecanismos que regulan el tráfico del receptor entre la membrana plasmática (PM) y los compartimentos intracelulares requiere un enfoque experimental con excelentes resoluciones espaciales y temporales. Además, este enfoque también debe tener la capacidad de distinguir los receptores localizados en el PM de aquellos en los compartimentos intracelulares. Lo más importante, la detección de los receptores en una sola vesícula requiere sensibilidad de detección relevante, ya que cada vesícula lleva sólo un pequeño número de receptores. Enfoques estándar para examinar el tráfico de receptores incluyen biotinilación superficie seguido por la detección bioquímica, que carece tanto de las resoluciones espaciales y temporales necesarias, y examen de microscopía de fluorescencia de receptores de superficie inmunomarcadas, lo que requiere la fijación química de las células y por lo tanto carece de suficiente resolución temporal 1-6. Para superar estas limitaciones, nosotros y otros han desarrollado y empleado una nueva estrategia que permite la visualización de la inserción dinámica de los receptores en el PM con excelentes resoluciones 7-17 espaciales y temporales. El enfoque incluye el etiquetado de un GFP sensible al pH, el superecliptic pHluorin 18, para el dominio extracelular N-terminal de los receptores. Superecliptic pHluorin tiene la propiedad única de ser fluorescente a pH neutro y no fluorescentes a pH ácido (pH &lt;6,0). Por lo tanto, los receptores son etiquetados no fluorescente cuando está dentro del lumen de las vesículas ácidas de tráfico intracelular o los compartimentos endosomales, y se convierten fácilmente visualizadas sólo cuando se expone al entorno extracelular pH neutro, en la superficie exterior de la PM. Nuestra estrategia en consecuencia nos permite distinguir los receptores de superficie de las PM en vesículas intracelulares de tráfico. Para lograr suficientes resoluciones espaciales y temporales, así como la sensibilidad requerida para estudiar el tráfico dinámico de los receptores, se emplearon interna total reflejarmicroscopía fluorescente ion (TIRFM), lo que nos permitió lograr la resolución espacial óptima de imagen óptica (~ 170 nm), la resolución temporal de microscopía de vídeo-rate (30 fotogramas / segundo), y la sensibilidad para detectar la fluorescencia de GFP una sola molécula. Por pHluorin imagen marcada con receptores de bajo TIRFM, hemos sido capaces de visualizar directamente los receptores individuales de los eventos de inserción en el PM en las neuronas cultivadas. Este enfoque de formación de imágenes potencialmente se puede aplicar a cualquier proteína de la membrana con un dominio extracelular que podría etiquetarse con pHluorin superecliptic, y permitirá a la disección de los mecanismos clave modalidades de inserción de proteínas de membrana diferentes (receptores, canales iónicos, transportadores, etc) a la 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

Mediante el etiquetado de los dominios extracelulares de los receptores de membrana con pHluorin superecliptic, y por imágenes de estos receptores de fusión en las neuronas de ratón en cultivo, se puede visualizar directamente los eventos de inserción individuales vesiculares de los receptores a la membrana plasmática. Esta técnica será fundamental para elucidar los mecanismos moleculares que regulan la inserción del receptor en la membrana plasmática.

Formación de imágenes pHluorin-etiquetados Inserción del receptor a la membrana plasmática en neuronas primarias de ratón en cultivo

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 vistas.  The Jackson Laboratory. Mediante el etiquetado de los dominios extracelulares de los receptores de membrana con pHluorin superecliptic, y por imágenes de estos receptores de fusión en las neuronas de ratón en cultivo, se puede visualizar directamente los eventos de inserción individuales vesiculares de los receptores a la membrana plasmática. Esta técnica será fundamental para elucidar los mecanismos moleculares que regulan la inserción del receptor en la membrana plasmática.

Video: 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.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

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

Investigación

Neurociencias

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.

Análisis cuantitativo de Reposición Synaptic piscina vesículas en cultivos de neuronas granulares del cerebelo con tintes 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.

Inducir el crecimiento dendríticas en las neuronas simpáticas cultivadas

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.

Difusión lateral y exocitosis de proteínas de membrana en neuronas cultivadas evaluó a través de la recuperación de fluorescencia y la pérdida de fluorescencia photobleaching

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.

Cerebro Slice Biotinilación: Una<em&gt; Ex Vivo</em&gt; Enfoque medir tráfico de proteínas específicas Región membrana plasmática en adultos las neuronas

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.

Cultivo primario de ratón neuronas dopaminérgicas

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 metodologías combinadas para definir el papel de la membrana plasmática de entrega Durante Axon ramas y morfogénesis neuronal

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.

Modelado de la muerte Neuronal y la degeneración en las neuronas del gránulo cerebeloso primario de ratón

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.

Imágenes en vivo de la proteína GLUT4 trata de ratón las neuronas hipotalámica primaria utilizando microscopía de deconvolución

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.

Cultivo primario de neuronas aisladas del cerebelo del ratón embrionario

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 ...