La mayor parte del trabajo y el personal involucrado fueron apoyados por el NIH subvención NS060677 y en parte por el NIH otorga MH099559 y MH104069 (a BSK). Parte del trabajo también fue apoyado por la Fundación CHDI.

Todos los animales protocolos fueron de acuerdo con los Institutos Nacionales de Salud de Guía para el Cuidado y Uso de Animales de Laboratorio y fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la UCLA. 1.1) Preparar Micropipeta y AAV2 / 5 Virus Cargando <ol><li> Utilice micropipetas de vidrio de borosilicato finas para la inyección del virus. Tire de la micropipeta utilizando un programa tirando de dos pasos con un extractor vertical. Bevel la pipeta en un ángulo de 40 ° utilizando un molinillo de pipeta. La pipeta que es ideal para su uso tendrá un diámetro de punta de 20 - 40 micras y una longitud de vástago de 6-7 mm. Autoclave la pipeta y los instrumentos quirúrgicos. El taladro se esteriliza por frotándolos con etanol al 70%. </li><li> Guarde el virus AAV2 / 5-GfaABC 1 D.cyto-GCaMP3.SV40 (1,5 x 10 13 gc / ml), a -80 ° C en 10 ml de alícuotas. Justo antes de la cirugía, sacar las alícuotas del congelador y almacenar en hielo hasta que se utilizaron para llenar ªe micropipeta. </li><li> Llene la micropipeta con virus usando una bomba de jeringa. Conectar firmemente un extremo de un trozo de tubo, a través de un tubo de tamaño apropiado de compresión apropiado, a una jeringa de vidrio con un émbolo reforzada. Coloque la micropipeta autoclave hasta el otro extremo de la pieza de tubería a través de una compresión de la aguja extraíble de tamaño apropiado apropiado. </li><li> Antes de cargar el vector viral, eliminar las burbujas de aire del sistema de bombeo incluyendo tubo, jeringa y micropipeta por llenar el sistema con aceite mineral coloreado con rojo Sudán IV (~ 1 mg / 50 ml) en una jeringa de 1 ml con una aguja (27 G ). </li><li> Coloque un trozo limpio de Parafilm bajo la micropipeta de vidrio, y dispensar de 5 - 10 l de la vector viral en el Parafilm. </li><li> Aspirar el vector viral moviendo el émbolo de la jeringa hacia atrás a 0,5 l / min, y marcar el límite entre el vector y el aceite en la pipeta de vidrio. </li></ol> 1.2) La anestesia, corte de pelo, Head Fijación y preparación por una craneotomía <ol><li> Poner un ratón (P49-63, C57BL / 6) en la cámara llena de N 2 O y O 2, y 5% de isoflurano. Evaluar la profundidad de la anestesia por pérdida de movimiento intencional y un ritmo respiratorio más lento (~ 60-90 / min). Cortar el pelo en la cabeza cuando se anestesia el ratón. </li><li> Coloque el ratón en el marco estereotáxico, con la cabeza protegida por barras de oído contundentes y su nariz colocados en un sistema de anestesia y ventilación. Mantener isoflurano continua a 2 - 3%. Aplicar las lágrimas artificiales pomada para ambos ojos ya que los ratones pierden su reflejo de parpadeo bajo anestesia. </li><li> Administrar 0,05 ml de buprenorfina (0,1 mg / ml) mediante inyección subcutánea para el alivio del dolor. </li><li> Área quirúrgica limpia con 10% de povidona yodada y alcohol al 70% tres veces, alternando entre las dos soluciones y comenzando con povidona yodada. Comience desde la parte frontal y limpie hacia la parte posterior para eliminar los pelos ya cortadas. </li><li> Hacer una incisión en la piel en la parte superior of la cabeza que comienza entre los ojos y termina entre las orejas. </li></ol> 1.3) craneotomía y microinyecciones <ol><li> Retire el periostio al golpear con un hisopo de algodón y luego seque la superficie del cráneo con bolas de algodón dentales. Hacer una marca alrededor del sitio de inyección, y perforar alrededor del borde de la marca usando una pequeña rebaba de acero accionado por un taladro de alta velocidad. </li><li> Retire el hueso y limpiar la superficie con solución salina. </li><li> Coloque la pipeta en el dorsal izquierdo lateral estriado con las siguientes coordenadas (mm): 0.8 anterior-posterior, medial-lateral: 2.0, dorsal-ventral de la superficie pial: -2.4 13. Tenga en cuenta que estas coordenadas significan que la punta de la aguja de microinyección se encuentra justo por encima del cuerpo estriado dorsolateral (Figura 1A, B), lo que significa que penetra sólo ligeramente dentro de este núcleo. Evitar los vasos sanguíneos perjudiciales. Si la pipeta pasa a ser justo en un vaso sanguíneo, ajustar ligeramente elcoordina por ~ 0,1 a 0,2 mm. </li><li> Iniciar la inyección con velocidad de inyección fijada en 0,2 l / min, y típicamente inyectar de 1 a 1,5 l de virus. </li><li> Deja la pipeta en el lugar después de la inyección durante 10 min, y luego retirar la pipeta lentamente. </li><li> Termine por el cierre de la herida quirúrgica con una sutura continua de sutura de nylon externa. </li></ol> 1.4) Recuperación <ol><li> Ponga el ratón en una jaula limpia sobre una almohadilla caliente durante 24 horas. No devuelva un ratón que ha sido sometido a cirugía para la compañía de otros animales hasta que se recupere completamente. No deje el ratón sin atención hasta que se haya recuperado el conocimiento suficiente para mantener el decúbito esternal. </li><li> Añadir Trimetoprim Sulfamethoxiazole (5 ml por 500 ml de agua, que contienen 40 mg de trimetoprim y 200 mg sulfamethoxiazole) en el agua durante una semana. Administrar buprenorfina dos veces al día durante un máximo de 3 días después de la cirugía. </li><li> Mantener el ratón en un ciclo de luz-oscuridad de 12 horas, alimentado y regado diario, unnd se pondera una vez por día hasta 3 días después de la cirugía. En estos 3 días, cualquier ratón con una pérdida de más del 10% del peso corporal se considera como comprometida, y se practicó la eutanasia en lugar de ser sometida a experimentación. </li><li> Por separado, cambiar la jaula del ratón dos veces por semana, y vigilar para la salud general al menos una vez por día. Por lo general, el ratón se utiliza dentro de 2 - 3 semanas después de las microinyecciones estereotáxica. </li></ol> 2. aguda del cerebro rebanada preparación para confocal Ca 2+ Imaging 2.1) Preparación de soluciones de grabación y corte. <ol><li> Preparar la solución que comprende (mM) de corte: 194 sacarosa, 30 NaCl, 4,5 KCl, 10 D-glucosa, 1 MgCl 2, 1,2 NaH 2 PO 4, 26 y NaHCO 3, saturado con 95% O 2 y 5% de CO 2. </li><li> Preparar la solución de grabación que comprende (mM): 124 NaCl, 4,5 KCl, 1 MgCl 2, 10 D-glucosa, 2 CaCl 2, 1,2 NaH 2 PO 4 </sub&gt;, y el 26 de NaHCO3; pH 7.3 a 7.4, 290 a 295 mOsm, saturada con 95% de O2 y 5% de CO 2. Llene el vaso de precipitados de cortes de cerebro con la celebración de solución de grabación, y la mantendrá a 34 ° C. </li></ol> 2.2) El rebanar <ol><li> De dos a tres semanas después de la microinyección estereotáxica, profunda y terminal anestesiar el ratón, y luego decapitar a ella. </li><li> Extraer el cerebro, y el uso de una cuchilla para eliminar el hemisferio derecho no inyectado, y montar el uno a la izquierda en la bandeja vibratome utilizando pegamento. Llenar la bandeja vibratome con tampón de hielo de corte en frío, y mantener la saturación con 95% de O2 y 5% de CO 2. </li><li> Cortar rodajas de cerebro del cuerpo estriado coronal o sagital a 300 micras de espesor. Por lo general, 4 - 5 coronal, o 6 - 7 rebanadas estriatales sagitales se pueden recoger. </li><li> Transferir las rebanadas al vaso de precipitados con rodaja calentado a 34 ° C, y mantenerlos allí durante 30 minutos antes de guardarlos a temperatura ambiente para su posterior recording. </li><li> Se incuban las rodajas de cerebro en memoria intermedia de grabación oxigenada a temperatura ambiente durante al menos 30 minutos antes de la [Ca 2 +] i de imágenes. </li></ol> 3. La inmunohistoquímica (IHC) <ol><li> Dos semanas después de la inyección de virus, perfundir el ratón transcardially con PBS seguido por 10% de formalina en PBS. </li><li> Retire, posterior a fijar durante la noche, y cryoprotect el cerebro en tampón sacarosa al 30% por lo menos durante 2 días. </li><li> Preparar 40 micras secciones usando un microtomo criostato. </li><li> Enjuague las secciones 3 veces con PBS a 10 minutos de intervalo. </li><li> Incubar las secciones durante 1 hora en PBS con 10% de suero normal de cabra y con 0,5% de Triton X-100. </li><li> Incubar las secciones durante la noche a 4 ° C en anticuerpo primario preparado en PBS con 0,5% de Triton X-100. Los anticuerpos primarios utilizados fueron los siguientes: pollo anti-GFP 1: 1000, de ratón anti-S100β 1: 400, de ratón anti-NeuN 1: 600, ratón anti-sintetasa glutamina 1: 300. </li><li> Enjuague las secciones 3 veces con PBS a 10 men intervalos. </li><li> Incubar las secciones con anticuerpos secundarios preparados en PBS con 10% de suero normal de cabra durante 2 hr a temperatura ambiente. Los segundos anticuerpos fueron los siguientes: de cabra anti-ratón Alexa546 1: 500, de cabra anti-pollo Alexa488 1: 500. </li><li> Monte las secciones sobre portaobjetos de vidrio, secos y aplicar varias gotas de Antifade medio de montaje, a continuación, cubrir lentamente las secciones con un cubreobjetos de vidrio. Guarde las diapositivas a 4 ° C. </li><li> Tomar imágenes en un microscopio confocal con una lente de inmersión en aceite 40X con una apertura numérica de 1,3. </li></ol> 4. confocal [Ca 2 +] i Imaging NOTA: [Ca2 +] i la formación de imágenes se realizó utilizando un microscopio confocal con una lente objetivo de 40X de inmersión en agua con una apertura numérica de 0,8. <ol><li> Entre 1 y 5 horas después de cortar, lugar rebanada en la cámara de grabación y superfuse con tampón de grabación oxigenada a temperatura ambiente con velocidad de flujo de 1 - 2 ml pormin. A continuación, coloque un arpa de platino con cuerdas de nylon en la parte superior de la rebanada para minimizar el movimiento durante el experimento. </li><li> Visualizar y localizar el cuerpo estriado dorsal con brillante luminiscencia campo bajo un objetivo de inmersión en agua 10 veces. </li><li> Cambie a la lente objetivo de 40X de inmersión en agua, y encienda la línea de 488 nm del láser de argón. Al cambiar el plano focal, encontrar células situadas a unos 20 m por debajo de la superficie de corte, presentan fluorescencia basal en Soma, ramas y ramillas. Es de destacar que las células comprometidas suelen mostrar nivel de fluorescencia por encima del promedio, lo que debe evitarse. </li><li> Utilice el modo &#39;scan marco &quot;para iniciar la exploración. Por lo general, la intensidad del láser ajustado a 0,5 - 5% de de la salida máxima es suficiente para imagen [Ca2 +] i con cito-GCaMP3. Para el seguimiento de [Ca 2 +] i dinámica en todo el territorio de los astrocitos, la &#39;exploración de cuadros&#39; con una velocidad de barrido de 1 segundo por fotograma o más rápido es RECOMEnded, pero esto tiene que ser decidido en función del experimento en cuestión. </li><li> Si es necesario, a la imagen [Ca 2 +] i en los procesos, utilice ampliación digital de 2 a 3 veces para supervisar 1 - 2 astrocitos en todo el campo de visión. </li><li> Para evitar píxeles saturados durante la grabación, utilice el modo de búsqueda &#39;HiLo&#39; a Pseudocolor las células. Comience siempre con una &quot;grabación piloto&quot; durante unos 5 minutos, para probar si el color rojo aparece como indicación de saturación. Si es así, menor será la potencia de salida del láser, o bajar la PMT / Ganancia / valores de desplazamiento y realizar otra grabación piloto para probar los valores de píxeles están en el rango no saturada. Por lo general, los parámetros combinados utilizan para astrocito de imágenes [Ca 2 +] i en el cuerpo estriado son las siguientes: la potencia del láser de salida 0,5 a 5% (de 10 mW), PMT 550-650 Voltios, Ganancia 2,0 - 3,5 veces, y el offset 0 - 5%. </li></ol>

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Los autores no tienen nada que revelar.

Los métodos descritos en el presente documento nos han permitido expresar cito-GCaMP3 en astrocitos del cuerpo estriado en vivo para su posterior [Ca 2 +] i de imágenes in situ. Este método tiene ventajas sobre el uso transgénica o ratones knock-in, incluyendo la expresión robusta de la proteína objetivo, rapidez y flexibilidad de aplicación experimental y especificidad anatómica. La expresión de GCaMP3 usando AAV2 / 5 se encontró que era específica y robusta. La combin...

Los astrocitos son células gliales ubicuas y abundantes del cerebro. Está bien establecido que los astrocitos sirven de soporte vital y las funciones homeostáticas que incluyen el almacenamiento en búfer de concentración de K + en el espacio extracelular, la captación de neurotransmisores, así como proporcionar nutrientes. Sin embargo, estudios recientes muestran que también muestran [Ca 2 +] i señales, que se producen de forma espontánea y se incrementan por la actividad neuronal 1. La existencia de los astrocitos [Ca 2 +] i de señalización se ha pensado cada vez más para activar su comunicación con las neuronas, y como tal se ha interpretado como una forma de &quot;Ca 2+ excitabilidad&quot; dentro de los astrocitos. Los datos disponibles en las últimas dos décadas sugieren dos escenarios en los que los astrocitos y las neuronas pueden comunicarse, tal vez de una manera bidireccional. En primer lugar, los astrocitos a menudo responden con un aumento de la [Ca 2 +] i cuando se activa mediante neurotransmisores yneuromoduladores liberados por las neuronas 2. En segundo lugar, la [Ca 2 +] i se incrementa dentro de los astrocitos provoca la liberación de moléculas de señalización de los astrocitos que a su vez puede afectar a las neuronas y los vasos sanguíneos. La evidencia sugiere que las moléculas liberadas a partir de astrocitos conducen a cambios en las funciones de las sinapsis, circuitos y en última instancia el comportamiento de 3-5 a través de la señalización de astrocitos a neurona. Sin embargo, esto sigue siendo un área de investigación en rápido desarrollo, y se ha argumentado que una comprensión mejor y más detallada de los astrocitos [Ca 2 +] i es necesaria para resolver algunas de las incertidumbres actuales 6. En trabajos anteriores, se demostró que la carga a granel de orgánicos de Ca 2+ colorantes indicadores en los astrocitos no puede detectar con fiabilidad [Ca 2 +] i señales dentro de los astrocitos en la totalidad de la cultura e in situ 7-10. Estos resultados han sido discutidos por nosotros y otros 6,11,12. El Emerging imagen es que el [Ca 2 +] i señales dentro de los procesos de astrocitos (por ejemplo, ramas y ramillas), que son los sitios principales para la interacción con las neuronas y los vasos sanguíneos, rara vez han sido exploradas en detalle. Recientemente, el uso de indicadores de calcio codificados genéticamente (Gecis) tales como GCaMP3 citosólica, GCaMP5G y GCaMP6 y membrana plasmática versiones atados (por ejemplo, Lck-GCaMP3) ha permitido el estudio de la [Ca2 +] i en las señales de pequeños compartimentos de astrocitos tales procesos como delgadas, cerca de la membrana plasmática y dentro de territorios enteros 7,8. Sin embargo, tienen una desventaja Gecis sobre orgánicos Ca 2+ colorantes indicadores y que es el requisito de métodos genéticos para entregar los genes que codifican selectivamente a los astrocitos in vivo durante períodos de semanas para el Gecis sea apropiadamente expresado. Expresión in vivo se logra típicamente usando ratones transgénicos, knock-en ratones o con la entrega de aplicaciones a base de viruscucarachas. En el presente artículo JoVE informamos métodos y procedimientos empleados para entregar Gecis a los astrocitos del cuerpo estriado utilizando virus adeno-asociado. Nos centramos en cito-GCaMP3 como un ejemplo, pero el mismo procedimiento básico funciona para cualquier otra GECI o fluorescente reportero base de proteínas.

<table><tbody><tr><td>Syringe Pump</td><td>Harvard Apparatus</td><td>704506</td><td></td></tr><tr><td>Glass Capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td></td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>Micropipette grinder</td><td>Narishige</td><td>EG-40</td><td></td></tr><tr><td>pZac2.1 GfaABC&lt;sub&gt;1&lt;/sub&gt;D.cyto-GCaMP3</td><td>Addgene</td><td>44331</td><td>a plasmid sent to UPenn Vector Core for virus packaging</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309628</td><td></td></tr><tr><td>syringe needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>AAV2/5 virus</td><td>UPenn vector core</td><td>NA</td><td></td></tr><tr><td>Sudan red IV</td><td>Sigma-Aldrich</td><td>67386</td><td></td></tr><tr><td>Mineral oil</td><td>CVS Pharmacy</td><td>152355</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050 S</td><td></td></tr><tr><td>Stereotaxic instrument</td><td>David Kopf Instruments</td><td>900LS</td><td></td></tr><tr><td>High Speed Rotary Micromotor Kit</td><td>FOREDOM</td><td>K.1070</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Santa cruz biotechnology</td><td>sc-281692</td><td></td></tr><tr><td>Super Glue</td><td>Krazy&amp;reg;Glue</td><td>KG925</td><td></td></tr><tr><td>Microslicer</td><td>Ted Pella</td><td>DTK-Zero 1</td><td></td></tr><tr><td>Confocal microscopes</td><td>Olympus</td><td>FV300 and FV1000</td><td></td></tr><tr><td>Normal goat serum</td><td>Vector</td><td>S-1000</td><td></td></tr><tr><td>chicken anti-GFP</td><td>Abcam</td><td>ab13970</td><td></td></tr><tr><td>mouse anti-s100&amp;beta;</td><td>Sigma-Aldrich</td><td>S2532</td><td></td></tr><tr><td>mouse anti-NeuN</td><td>Millipore</td><td>MAB377</td><td></td></tr><tr><td>mouse anti-glutamine synthetase</td><td>Millipore</td><td>MAB302</td><td></td></tr><tr><td>goat anti-mouse-Alexa546</td><td>Invitrogen</td><td>A11003</td><td></td></tr><tr><td>goat anti-chicken-Alexa488</td><td>Invitrogen</td><td>A11039</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Cover Glass</td><td>Fisher Scientific</td><td>12-548-5J</td><td></td></tr><tr><td>Mounting Medium</td><td>Vector</td><td>H-1000</td><td></td></tr></tbody></table>

imaging intracellular ca2+ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Para la expresión específica de astrocitos de cito-GCaMP3 en el cuerpo estriado, se utilizó virus adeno-asociado (AAV) del serotipo 5, y el promotor de GFAP GfaABC 1 D (Figura 1A), que se ha demostrado previamente para conducir GCaMP3 robusto y gen reportero expresión en astrocitos del hipocampo y corticales 8,14. Dos semanas después de la microinyección virus en el cuerpo estriado de ratón, el ratón (~ 10 semanas de edad) y se perfundió IHC se realizó en secciones de cer...

Watch this Scientific Journal Video about Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators at JoVE.com

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Imaging intracelular de Ca<sup&gt; 2 +</sup&gt; Las señales en estriatal astrocitos de ratones adultos a través de indicadores de calcio codificados genéticamente-

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

14.7K vistas.  University of California Los Angeles. The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ. 

Video: Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

Imagen de calcio funcionales en el desarrollo de redes corticales

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Investigación

Neurociencias

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Cerca de la medición de la membrana plasmática y Global Dynamics calcio intracelular en los astrocitos

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biología

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

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

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

Dos registros electrofisiológicos de sinápticamente respuestas evocadas neuronal y astroglial en agudas rodajas de hipocampo

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

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

La inducción de la plasticidad de astrocítico Receptores por manipulación de Neuronales cocción Precios

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Cultivo In Vivo-como astrocitos murinos mediante el protocolo AWESAM rápido, sencillo y barato

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

Ex Vivo Proyección de imagen del calcio celular específicos de señalización en la sinapsis tripartita del diafragma del ratón

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Disección de Local Ca2 + señales en las células cultivadas por membrana dirigidos Ca2 + indicadores

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Obtención de imágenes de ca2+ mitocondrial en astrocitos y neuronas utilizando indicadores de Ca2+ codificados genéticamente (GECI)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

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 ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagen Ca Local<sup&gt; 2 +</sup&gt; Las señales en las células de mamífero en cultivo

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Imaging intracelular de Ca

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