Nos gustaría dar las gracias al Dr. Jeffrey Rothstein para proporcionar GLT1 BAC ratones EGFP y anticuerpos GLT1; Tufts Center para Investigación de Neurociencia (NIH P30 NS047243, PI, Rob Jackson) para proporcionar servicios de valor básicos; Nueva facultad de contratación de subvención (NIH P30-02 5P30NS069254 , PI, Phil Haydon) en Tufts Neurociencias Departamento.

1. Asamblea de la Cámara de Cultura Microfluidic (MCP) <ol><li> MCP (Figura 1) son cámaras abiertas diseñado para culturas compartimentados de diferentes tipos de células 4. Típicamente tiene dos compartimentos que están conectados a través de los canales centrales (3 m de diámetro). Asamblea del MCP con fondo de cristal platos es necesario para la preparación de las culturas y de análisis de imágenes posteriores. </li><li> En primer lugar, capa estériles con fondo de vidrio platos con poliornitina (Sigma-Aldrich, 1 mg / ml) disuelto en tetraborato de sodio (Sigma-Aldrich, 10 mM, pH 8,5) y se incubaron durante la noche a 37 ° C. </li><li> Al día siguiente, los recubiertos con fondo de vidrio platos se lavaron tres veces con ddH 2 O y se secó bajo la campana de ventilación estéril. Los platos de vidrio de fondo fueron entonces recubiertas adicionalmente con laminina (Sigma-Aldrich, 1 mg / ml) y se secó bajo luz UV durante 1 hora. Platos recubiertos están listas para usar o se pueden almacenar a -20 ° C hasta su uso. Estos recubrimiento de unare necesario para las neuronas y astrocitos enchapado en el co-cultivo. </li><li> Para el montaje de la plataforma de la cultura, plataformas de microfluidos de cultivo se colocaron en la parte superior de los recubiertos con fondo de vidrio con platos centrales canales de conexión en su lado inferior para formar un sello hermético. Regular neurona o astrocito medios de cultivo se añadieron (a partir de las áreas de célula de recubrimiento) en ambos lados (Figura 1) en el ensamblado MCP y se incubaron durante 2-4 horas a 37 ° C, para asegurar que no hay fugas entre el MCP y de vidrio plato de fondo. Hemos probado con medio antes de células en placas para asegurar que no haya fugas. El conjunto de la MCP sobre el plato de fondo de vidrio deben estar recién preparadas antes de su uso. </li></ol> 2. Preparación de cultivo neuronal e inducción de los axones neuronales en ensamblado MCP <ol><li> Cultivos de células neuronales corticales fueron recién preparada a partir de cerebros de ratón embrionarias 14-16 días de edad. El medio de cultivo neuronal se compone de medio neurobasal, 2% B27 neurobasasuplemento de l, 2 mM de glutamina mediante la adición de 1% de 100x GlutaMAX, y 1% de penicilina-streptomysin. Después de la disección del cerebro del ratón, las meninges se eliminó de la corteza bajo el microscopio de disección. El tejido se troceó a continuación, con una hoja de afeitar para hacer pequeños bloques de tejido con el fin de aumentar el área de superficie expuesta a la tripsina. Los bloques de tejido se trataron con tripsina (Sigma-Aldrich, 0,05% de tripsina) durante 10 minutos en un baño a 37 ° C, y luego disociarse suavemente por trituración con una pipeta Pasteur pulida al fuego. Las células disociadas se filtraron a través de un filtro 70 micras para recoger suspensión clara de las células neuronales. </li><li> Las neuronas (2-3 x 10 6 / ml, 150 l) se sembraron en las áreas de célula de recubrimiento en el lado derecho (figura 1) de la MCP montado de modo que las neuronas pueden adjuntar en el área de retención celular (Figura 1). Neuronas recién preparados se sembraron con medio de chapado neurona que se complementa con 5% de suero fetal bovino (Hyclone) en la regulaciónr neurona medio de cultivo. Debido a que sólo las neuronas que se conectan en la zona de retención de células son capaces de crecer los axones en el otro lado de la MCP montado a través de los canales centrales, es importante la densidad de placa alto de neuronas en la zona de recubrimiento celular para asegurar suficientes neuronas están unidas a la célula área de retención. Una imagen representativa de alta densidad de axones neuronales se muestra en la Figura 2A. </li><li> Al día siguiente, el medio de recubrimiento neurona se sustituye por el medio de cultivo regular neurona. Cambio de medio se realizó por aspiración cuidadosamente el medio de la zona de revestimiento celular de la cámara, pero no de la zona de retención de células de la MCP montado, con el fin de evitar burbujas de aire en el área de retención de células y los canales de conexión central. Las burbujas de aire severamente inhibirá el axón y la posterior entrada en los canales centrales en el MCP. Glial de células factor neurotrófico derivado (GDNF, 10-20 ng / ml) se añadió también por el otro lado (izquierdo)de la cámara en el mismo día para facilitar la inducción del axón 5 desde el lado neuronal y transversal a través de los canales centrales de la MCP ensamblado (Figura 2A). Cantidad de crecimiento de las neuritas espontáneo sin GDNF también se observa desde el lado neuronal y transversal a través de los canales centrales de la MCP montado. Los axones que entran en el canal central se observan a menudo 2-3 días después de la siembra. Una vez que los axones entrar en el canal central, que generalmente entran en el otro lado en un día. Los axones son normalmente intacta durante al menos una semana después de entrar en el otro lado. </li></ol> 3. La adición de astrocitos en cultivo a MCP para establecer una compartimentado Co-cultura del sistema Cultivos de astrocitos primarios se prepararon a partir del cerebro P1-3 de ratón cachorro. El procedimiento de disociación cerebro es similar al procedimiento de aislamiento de células neuronales se ha descrito anteriormente. Los astrocitos se cultivaron por primera vez en placa de 10 cm que se pre-Recubiertas con poliornitina (Sigma-Aldrich, 1 mg / ml). El medio de cultivo de astrocitos (DMEM, 10% de suero bovino fetal, 1% de penicilina-streptomysin) se cambió cada día durante los siguientes dos días para eliminar los desechos. Después de eso, el medio se cambió cada tres días. <ol><li> Los astrocitos convertido en 90% confluentes y formar una monocapa después de 7 días. Astrocitos confluentes se trataron con tripsina y 150 l de re-suspendidas astrocitos (1x10 6 / ml) fueron re-chapada en la zona de recubrimiento de células de la parte izquierda de la ensamblada MCP cuando los axones están a punto de entrar o acaban de entrar en el lado izquierdo de el ensamblado MCP. Los astrocitos se sembraron por lo general 4-5 días después de que las neuronas eran plateado. GDNF se retiró antes de la primera re-recubrimiento de los astrocitos en el lado izquierdo de la MCP. Chapados Re-astrocitos se adjunta en el área de retención de células, tal como se muestra por la inmunotinción GFAP (Figura 1). Sólo flujo mínimo de medio entre ambos lados de las cámaras (determinado por fluorescencia dSí) se encontró en el MCP, como se mostró previamente 4,6. </li><li> Los astrocitos convertido en 90% confluentes y formar una monocapa después de 7 días. Astrocitos confluentes se trataron con tripsina y 150 l de suspensión re-astrocitos (1 x 10 6 / ml) fueron re-chapada en la zona de recubrimiento de células de la parte izquierda de la ensamblada MCP cuando los axones están a punto de entrar o acaban de entrar en la izquierda lado del ensamblado MCP. Los astrocitos se sembraron por lo general 4-5 días después de que las neuronas eran plateado. GDNF se retiró antes de la primera re-recubrimiento de los astrocitos en el lado izquierdo de la MCP. Chapados Re-astrocitos se adjunta en el área de retención de células, tal como se muestra por la inmunotinción GFAP (Figura 1). Sólo flujo mínimo de medio entre ambos lados de las cámaras (determinado por colorantes fluorescentes) se encontró en el MCP, como se mostró previamente 4,6. </li><li> Después de la re-recubrimiento de los astrocitos, los axones que entraron en el lado izquierdo de la MCP montado interactúan directamente con la astrocytes, ya sea por contacto directo axonal o liberación de factores solubles. La inmunotinción de GLT1 astroglial membrana plasmática transportadora de glutamato neuronal y βIII de tubulina se realizó para visualizar la interacción entre los axones y astrocitos, como se muestra en la Figura 2D-F. </li></ol>

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No hay conflictos de interés declarado.

La neurona MCP y basado en sistema de astrocitos de co-cultivo permite la disección de neurona detallada a astroglía vías de señalización permitiendo que sólo los axones pasan los canales centrales y la interacción con las células astrogliales. Este sistema de co-cultivo se puede configurar cómodamente con neurona convencional y los procedimientos de astrocitos cultura. También se describe una aplicación práctica de este sistema de co-cultivo mediante el empleo de un reportero eGFP base para demostrar axón ...

<table border="1"><tbody><tr><td> El suero fetal bovino </td><td> Hyclone </td><td> SH30070.03 </td><td> para placas neurona por neurona medio cutlure </td></tr><tr><td> El suero fetal bovino </td><td> Sigma-Aldrich </td><td> F4135 </td><td> por medio de cultivo de astrocitos </td></tr><tr><td> Glial nervio factor derivado </td><td> De I + D de sistemas </td><td> 212-GD </td><td> Aplicar 10-20 ng / ml a lado de la cámara de neurona </td></tr><tr><td> Dulbecco modificado del águila de mediano alto de glucosa </td><td> Sigma-Aldrich </td><td> 11995 </td><td></td></tr><tr><td> 70 mm filtro de células </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Vidrio estéril plato de fondo </td><td> MatTek Corporación </td><td></td><td></td></tr><tr><td> Plataformas de microfluidos cultura </td><td> Xona microfluídica LLC </td><td> SND150 </td><td></td></tr><tr><td> 6 pocillos de la placa de cultivo </td&gt; <td> Cellstar </td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> Neurona medio de cultivo <ul><li> Medio Neurobasal </li><li> 2% suplemento B27 Neurobasal </li><li> 2 mM de glutamato mediante la adición de 1% 100x GlutaMAX </li><li> 1% de penicilina-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Neurona medio de cultivo para la célula de recubrimiento <ul><li> Medio Neurobasal </li><li> 2% suplemento B27 Neurobasal </li><li> 2 mM de glutamato mediante la adición de 1% 100x GlutaMAX </li><li> 1% de penicilina-streptomysin </li><li> 5% de suero fetal bovino SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> Medio de cultivo de astrocitos <ul><li> Dulbecco modificado eagle glucosa medio-alto </li><li> 10% de suero fetal bovino F4135 </li><li> 1% de penicilina-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Materiales Tabla 1. Utilizados en el cultivo de microfluidos plataforma basada axón neuronal y glial sistema de co-cultivo. </td></tr></tbody></table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

Neurona adecuada a la interacción glia es fundamental para la función fisiológica del sistema nervioso central (SNC). Esta comunicación bidireccional está sofisticadamente mediada por vías de señalización específicas entre neuronas y glía 1,2. Identificación y caracterización de estas vías de señalización es esencial para la comprensión de cómo neurona a la interacción glia formas CNS fisiología. Anteriormente, los cultivos de neuronas y células gliales mixtos han sido ampliamente utilizados para la prueba de las vías de señalización entre las neuronas y la glía. Lo que hemos aprendido de estos preparativos y otras herramientas in vivo, sin embargo, se ha sugerido que la señalización mutuas entre neuronas y glía ocurrieron a menudo en compartimentos específicos dentro de las neuronas (es decir, axón, dendrita, o soma) 3. Esto hace que sea importante para desarrollar un nuevo sistema de cultivo que permite la separación de los compartimentos neuronales y, específicamente, examina la interacción entre células gliales y neuroNAL / axones dendritas. Además, el sistema de cultivo mixto convencional no es capaz de diferenciar los factores solubles y directos señales de contacto entre la membrana neuronal y glial. Además, la gran cantidad de neuronas y células gliales en el co-cultivo convencional sistema carece de la resolución necesaria para observar la interacción entre un solo axón y una célula glial. En este estudio, se describe un axón y un sistema nuevos glia co-cultivo con el uso de una plataforma de cultura microfluídico (MCP). En este sistema de co-cultivo, las neuronas y las células gliales se cultivó en dos cámaras separadas que están conectadas a través de múltiples canales centrales. En esta plataforma de microfluidos cultura, sólo los procesos neuronales (especialmente los axones) puede entrar en el lado glial a través de los canales centrales. En combinación con el etiquetado potente proteína fluorescente, este sistema permite el examen directo de las vías de señalización entre las interacciones axonal / dendrítico y glial, tals axón mediada por la regulación transcripcional en células gliales, el tráfico del receptor mediada por células gliales en terminales neuronales y gliales mediada por el crecimiento del axón. El diámetro estrecho de la cámara también significativamente prohíbe el flujo del medio de neurona enriquecido en la cámara de glial, facilitando el sondeo de la membrana directa-proteína interacción entre axones / dendritas y superficies gliales.

Time-lapse de imágenes de análisis axón inducida por la activación del promotor GLT1 en astrocitos La neurona de compartimentado y el sistema de co-cultivo de astrocitos permite sólo los procesos neuronales, especialmente los axones, para interactuar selectivamente con los astrocitos. Tras el establecimiento con éxito de axón y astrocitos (u otras células gliales) co-cultivo en el ensamblado MCP, diferentes tipos de interacciones gliales del axón puede ser estudiado como; axón inducid...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Este estudio describe los procedimientos de instalación de un axón neuronal y la novela (astro) glia plataforma de co-cultivo. En este sistema de co-cultivo, la manipulación de la interacción directa entre un solo axón (y células gliales único) se hace factible, permitiendo un análisis mecanicista de la neurona mutuo de señalización gliales.

Análisis de imágenes de Neuron a la interacción glía en la Plataforma Cultura Microfluidic (MCP)-basada en Axón Neuronal y Sistema Glia Co-cultura

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

imaging-analysis-neuron-to-glia-interaction-microfluidic-culture

Research

JoVE Journal

Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

14.9K vistas.  Tufts University. Este estudio describe los procedimientos de instalación de un axón neuronal y la novela (astro) glia plataforma de co-cultivo. En este sistema de co-cultivo, la manipulación de la interacción directa entre un solo axón (y células gliales único) se hace factible, permitiendo un análisis mecanicista de la neurona mutuo de señalización gliales.

Video: Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-cultivo de neuronas de rata primaria cortical y la glía

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Investigación

Neurociencias

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Análisis de Desarrollo del diente Germ Inervación Uso de dispositivos de microfluidos Co-cultura

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

La indirecta neurona-astrocito Coculture Ensayo: Un<em&gt; In Vitro</em&gt; Puesta en marcha de la investigación detallada de las interacciones neurona-glía

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Aislamiento y cultura de las neuronas primarias y Glia de la vejiga urinaria de rata adulta

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

Lesión neuronal dirigida para la desconexión no invasiva del circuito cerebral

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Disección y aislamiento de la glía murina de múltiples regiones del sistema nervioso central

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Un modelo de cultivo celular para estudiar el papel de las interacciones de neuronas y glia en isquemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imágenes de calcio en la aceituna inferior de ratón

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Visualización de cambios en el circuito Neuron-Glia con la técnica de imágenes de calcio

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Trasplante de microglía derivada de células madre pluripotentes inducidas por humanos en ratones cerebros inmunocompetentes a través de una ruta transnasal no invasiva

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...