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 border="0" cellpadding="0" cellspacing="0" >
	<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 GfaABC1D.cyto-GCaMP3</td>
			<td >Addgene</td>
			<td>44331</td>
			<td >a plasmid sent to UPenn Vector Core for virus packaging</td>
		</tr>
		<tr >
			<td >I 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&#174;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&#946;</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

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

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Las respuestas de imagen neuronal en los preparativos de la rebanada del órgano vomeronasal Expresando un sensor de calcio genéticamente codificados

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Investigación

Neurociencias

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

La grabación simultánea de señales de calcio en neuronas identificadas y comportamiento de alimentación de Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modelado astrocitoma Patogénesis<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Uso cortical astrocitos o células troncales nerviosas de ratones condicionales, Genéticamente Manipulados

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Membrana de imagen potencial con dos tipos de sensores de tensión fluorescentes genéticamente codificados

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Control óptico de una proteína neuronal utilizando un ácido Unnatural Amino genéticamente codificados en las neuronas

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

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 ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Indicadores de sinapsis únicas de liberación y aceptación de glutamato en rebanadas cerebrales agudas de ratones normales y Huntington

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Visualización y análisis del transporte intracelular de organelos y otros Cargos en astrocitos

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Minimizar la hipoxia en las rebanadas de hipocampo de ratones adultos y envejecidos

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

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.