Authors would like to thank Charisse Montgomery for her editing service. A. Alomran&#8217;s work partially fulfills the requirements for a master&#8217;s degree in Pharmacology.This work is funded by The University of Toledo&#8217;s intramural startup fund for W.A.A and NIH grant (DK080640) for S.M.N.

Los procedimientos para el uso de los animales fueron aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC) de la Universidad de Toledo, de acuerdo con las directrices del Comité de Cuidado y Uso de Animales institucional de los Institutos Nacionales de Salud y la Guía para el Cuidado y Uso de animales de laboratorio. 1. Extracción del cerebro, Seccionamiento y Tejidos Preparación <ol><li> Sacrificio de tipo salvaje cepa de ratón C57BL / 6 por profundamente eutanasia con CO2 asfixia durante 5 min. Asegurar la muerte por dislocación cervical. </li><li> Limpie el cabezal de ratón con etanol al 70%. </li><li> Realizar craniectomía usando tijeras y pinzas estériles por primera tira de la piel de, a partir de la parte superior de la cabeza para exponer el cráneo. </li><li> Entonces, cuando se expone el cráneo, retire el cráneo por el pelado de la pieza por pieza de hueso, comenzando desde el lado posterior y en movimiento hacia el lado anterior. Tenga cuidado de no destruir los ventrículos cerebrales. </li><li>Recoge todo el cerebro. </li><li> Coloque el cerebro en un plato de 100 mm de Petri que contiene DMEM / de alta glucosa suplementado con 10% de suero bovino fetal (FBS) y solución de penicilina / estreptomicina 1% que contiene 10.000 unidades / ml de penicilina y 10.000 g / ml de estreptomicina y pre-calentado a 37 ° C. </li><li> Cortar el cerebro en el plano sagital mediano a mano con una cuchilla afilada, y obtener la primera sección de 100-200 mm de cada medio usando un vibratome. </li><li> Enjuague el tejido cerebral con C salina tamponada con fosfato (PBS 1x) solución de 37 ° pre-calentado. </li><li> Coloque inmediatamente la sección del cerebro en DMEM / media-alta de la glucosa pre-calentado a 37 ° C. </li></ol> 2. Vivo Configuración de imagen y configuración <ol><li> Coloque las secciones de tejidos cerebrales en 30 mm placas de cultivo con fondo de cristal que contienen 1 ml de DMEM / medios-altos de glucosa. Ajustar entorno de cámara cerrada del microscopio a 37 ° C, 95% / 5% O 2 / CO contenido de 2 (Figura 1 </strong&gt;). </li><li> El uso de un lente objetivo de inmersión en aceite 60X, recoger las células ependimarias / imágenes cilios colocando primero una gota de aceite en la lente objetivo de 60X y se centra en las células con DIC regularmente transmiten luz. </li><li> A continuación, siga la dirección del movimiento de la burbuja DMEM como una guía para la ubicación de los cilios ependymal móviles como golpes ciliares crear una especie de movimiento de la burbuja en la zona. Elija un área que contiene las células sanas con cilios móviles en el ventrículo lateral del cerebro utilizando el filtro DIC. Una vez que los cilios ependymal se encuentran, ajustar la luz y el enfoque para obtener una imagen satisfactoria. </li><li> Establezca los parámetros de imagen en vivo de acuerdo con un propósito específico utilizando el software de imágenes Metamorph. En la presente manifestación, adquirir veinte imágenes de cuatro bits con el conjunto de intervalos cámara a 1 x 1 en combinación con 60X objetiva y tiempo de exposición 5/10 ms. </li><li> Recoge las imágenes DIC abriendo la abertura microscopio para un nivel óptimo con el fin de tener un mínimo de exptiempo osure. Observar las imágenes en directo corriente a la cámara para proporcionar la adquisición de imágenes rápido e inmediato sin demora. Calcular la velocidad de latido cilios basado en el requisito de los tiempos de exposición mínimos para obtener suficiente contraste de la imagen. </li></ol> 3. Visualización y análisis de datos <ol><li> Calcular el número de golpes cilios contando el número de golpes en 1 min. Para ello, la disminución de la velocidad del vídeo y contando el número de pulsaciones utilizando un contador de células o una herramienta similar. </li><li> Para calcular la frecuencia de las palizas, multiplique el tiempo de exposición a la que el vídeo se graba por el número de los marcos o imágenes en tiempo lapso adquiridos para obtener el número de segundos. (Ejemplo: tiempo de exposición 5 ms 200 marcos = 1000 ms o 1 segundo). </li><li> Calcular el número de golpes en un segundo para obtener la frecuencia que se expresa como el número de golpes por uno sec. Para ello, dividiendo el número de golpes cilios más de un segundo entre el tiempoval (Ejemplo: cilios late 50 veces en un video de 200 marcos grabado en tiempo de exposición de 5 ms es decir 5 ms x 200 fotogramas = 1 seg; ahora dividir 50 latidos por 1 seg = 50 Hz). </li><li> Calcular el ángulo de golpeo ciliar mediante la evaluación del camino recorrido por los cilios ependymal durante tanto las carreras de trabajo y recuperación. Realice esto de acuerdo con un método descrito previamente, con modificaciones menores 11. Se observa el movimiento preciso de los cilios individuo durante el ciclo de latido completo. </li><li> En una hoja de acetato colocado sobre el monitor, dibujar una línea horizontal a lo largo del borde ependimarias y una línea vertical a través de la posición de la línea media de los cilios en el inicio de la carrera de potencia. </li><li> Trazar la posición precisa de la trama cilio por trama a medida que se mueve hacia adelante durante la carrera de potencia. De una manera similar, trazar el movimiento de los cilios durante la carrera de recuperación. </li><li> Calcular el ángulo de golpeo ciliar de la desviación máxima del cilio de la línea media durante tél carrera de potencia, así como la carrera de recuperación. </li></ol> 4. El calcio de grabación de la señal <ol><li> Después de cortar el cerebro, enjuagar brevemente el corte de cerebro con 1x PBS o PBS de Dulbecco (pH 7,0). Preparar fresco Fluo-2 para evitar la extinción de la fluorescencia y para obtener una buena relación señal-ruido. </li><li> Preparar 1 mM solución madre de Fluo-2 solución en dimetilsulfóxido (DMSO), mezclar y agitar la solución durante al menos 5 min para asegurar que Fluo-2 se disuelve homogéneamente en DMSO. </li><li> Diluir la solución madre de Fluo-2 en 500 ml de DMEM / de alta glucosa suplementado con 2% B27 pre-calentado a 37 ° C a una concentración final de 20 mg / ml. NOTA: B-27 es un suplemento libre de suero optimizado que contiene vitamina A, cóctel antioxidante y la insulina utilizada para apoyar la viabilidad a corto o largo plazo del hipocampo y otras neuronas del SNC. </li><li> Incubar inmediatamente el corte de cerebro con 20 mg / ml Fluo-2 durante 30 minutos a 37 ° C en un plato con fondo de cristal. </li><li> Para determinar la concentración de carga óptima de fluoróforo calcio Fluo-2 y para evitar la toxicidad celular tinte de calcio, las células de desafío con el ATP, y comprobar la viabilidad celular mediante la determinación de la evolución en el tiempo y la magnitud máxima de señales de calcio en respuesta a ATP. </li><li> Grabar el vídeo para la oscilación de calcio a una velocidad de captura de 5 mseg durante un mínimo de 1 seg (200 fotogramas por segundo), con longitudes de onda de excitación y emisión de 488 nm y 515 nm, respectivamente (Película 2). </li><li> Para distinguir la señal de calcio Fluo-2 de autofluorescencia o artefactos de movimiento, asegúrese de que las intensidades emitidas a 515 nm se controla por separado. NOTA: Fluo-2 no es el fluoremetric calcio adecuado para cuantificar el calcio intracelular. Sin embargo, es un excelente tinte de calcio dinámica para detectar cambios rápidos de calcio, tales como oscilaciones de calcio. Para la cuantificación de calcio más precisa, se recomienda Fura-2. Sin embargo, los cambios dinámicos de Fura-2 están limitados por su radiométricala naturaleza del colorante. </li><li> Siga las fórmulas proporcionadas por el fabricante para calcular los valores exactos de calcio intracelular libres. Ejemplo [Ca 2 +] = Kd × (R - R min) / (R max - R). Donde K d es la constante de disociación del colorante desde el calcio liberado, R es la fluorescencia medida a 488, y R y R min max son las relaciones de fluorescencia a concentración máxima de iones mínimo y 12. </li></ol> 5. Inmunofluorescencia Microscopía <ol><li> Fijar las secciones de cerebro con solución salina tamponada con fosfato que contiene 4% de paraformaldehído (PFA) y 2% de sacarosa durante 10 min. Alternativamente, arreglar todo el cerebro con 4% PFA sección y luego en 50 secciones mm utilizando un criostato. </li><li> Lavar el tejido tres veces con 1x PBS durante 5 min cada vez. </li><li> Incubar la rebanada cerebro con un solutien 0,1% de Triton-X en 1X PBS durante 5 min, luego enjuagar tres veces con 1x PBS durante 5 min cada vez. </li><li> Incubar los cortes de cerebro con el anticuerpo primario de ratón, antiacetylated a-tubulina, utilizado a una dilución de 1: 5000 en 10% de FBS en 1x PBS durante una hora a temperatura ambiente (RT) o durante la noche a 4 ° C. </li><li> Lavar el tejido tres veces con 1x PBS durante 5 min cada vez. </li><li> Incubar la rebanada del cerebro en anticuerpo secundario, IgG anti-ratón fluoresceína a una dilución de 1: 500 en 10% de FBS en PBS 1x solución durante 1 hora a RT. </li><li> Antes de la observación bajo un microscopio fluorescente, contratinción la sección con 4 &#39;, 6-diamidino-2-fenilindol (DAPI) durante 5 min para teñir el núcleo (o ADN) 13. Para minimizar fotoblanqueo, la imagen de las secciones de inmediato con el tiempo de exposición mínimo posible. </li></ol>

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No hay conflictos de interés declarado.

Describe aquí es un protocolo para la preparación de tejido cerebral de ratón tanto para formación de imágenes en vivo y microscopía de fluorescencia que proporciona una rápida y sensible estrecha observación de los cilios ependymal dentro de los ventrículos cerebrales. Esta técnica no se limita a sólo el ventrículo lateral; que podría ser utilizado para observar los cilios en otros ventrículos cerebrales. Esta técnica de imagen proporciona una transmisión en vivo que se asemeja el movimiento del LCR por...

Los cilios son orgánulos basado-microtúbulos sensoriales que se extienden desde la superficie de la célula con el medio ambiente extracelular. Dependiendo de su nivel de organización de los microtúbulos, los cilios se pueden clasificar en dos tipos - &quot;9 + 0&quot; o &quot;9 + 2&quot;. Funcionalmente, en base a su motilidad, éstos pueden ser clasificados como móviles o no móviles cilios 1. Cilios primaria es un término comúnmente utilizado para referirse a &quot;9 + 0&quot; cilios no móviles. Estos tienen nueve microtúbulos doblete paralelas (denotados por &#39;9&#39;) y un par central de microtúbulos está ausente dentro de la vaina central (denotado por &#39;0&#39;). Sin embargo, algunos &quot;9 + 0&quot; cilios, como cilios nodales, que regulan la lateralidad embrionaria son móviles 2. Por otro lado, cilios móviles se caracterizan, además de los nueve dobletes de microtúbulos paralelos, por un par central adicional de dobletes de microtúbulos y se asocia con proteínas motoras dineína para facilitar la motilidad. Además, algunos &quot;92 &quot;cilios como cilios olfativos son no móviles 3. Las células ependimarias que recubren los ventrículos cerebrales y el canal central de la médula espinal se caracterizan por cilios móviles que propulsar el líquido cefalorraquídeo (LCR) a lo largo de los ventrículos cerebrales 4. El objetivo general de este método es el de facilitar el estudio de la dinámica de los cilios móviles y anormalidades estructurales. La salud y el desarrollo del cerebro en gran medida dependen de la circulación eficiente de LCR dentro de los ventrículos cerebrales. Por ejemplo, el flujo de LCR normal y el equilibrio de líquidos requieren latido normal y cilios ependymal funcional 5,6, que a su vez juega un papel crítico en la regulación del movimiento direccional de las células neuronales y la migración de células madre 7. Como tal, la función de los cilios ependymal anormal o estructura puede conducir a flujo del LCR anormal, que se asocia con la hidrocefalia, una condición médica en la que hay una acumulación anormal de LCR en los ventrículos del blluvia. En consecuencia, esto puede causar un aumento de la presión intracraneal y la ampliación progresiva de la cabeza, convulsiones, visión de túnel, y la discapacidad mental 8. Las ventajas de esta técnica sobre los métodos existentes es que se permitió por primera vez a reportar tres distintos tipos de células ependimarias: I, II, y III, en función de su única frecuencia de golpeo ciliar y golpeando ángulo. Estas células ependimarias se localizan dentro de ciertas regiones de los ventrículos cerebrales. Además, los efectos de la edad y agentes farmacológicos tales como alcohol y cilostazol en la modificación de los tipos de células ependimarias o sus localizaciones se pueden demostrar, que no era posible antes de esta clasificación de células ependimarias. Cilostazol es un inhibidor de la fosfodiesterasa-3, una enzima que metaboliza cAMP a AMP y también regula el calcio intracelular 9. Utilizando el análisis de imágenes de fluorescencia de alta velocidad permite obtener imágenes y cuantificación de la intracelular únicopropiedades de oscilación de calcio de las células ependimarias. Por ejemplo, tanto el alcohol y el cilostazol alteraron significativamente la frecuencia de golpeo ciliar ependimarias, así como las oscilaciones de calcio intracelulares propiedades, que a su vez, podría conducir a un cambio en la reposición de volumen de líquido cefalorraquídeo por cilios ependymal 10. En resumen, esta técnica fue clave para proporcionar la primera evidencia de tres tipos distintos de células ependimarias con diferentes propiedades de oscilación de calcio. En la siguiente sección, se proporciona una visión detallada paso a paso del procedimiento, prestando especial atención a la preparación y manipulación de tejidos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/HIGH GLUCOSE</td>
			<td>Cellgro Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30088-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermo Scientific</td>
			<td>SH30256-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S-2395</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-2&#160;</td>
			<td>TEF Labs</td>
			<td>#0200</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD HardSet Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetylated a-tubulin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T7451&#160;</td>
			<td>clone 6-11B1</td>
		</tr>
		<tr>
			<td>FITC Anti-mouse antibody</td>
			<td>Vector Labs</td>
			<td>FI-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture plate</td>
			<td>VWR Vista Vision</td>
			<td>30-2041</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slip (18x18)</td>
			<td>VWR Vista Vision</td>
			<td>16004.326</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>Leica VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>Leica CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>Nikon&#160;</td>
			<td>Nikon TE2000</td>
			<td>60X oil&#160;</td>
		</tr>
		<tr>
			<td>Microscope cover glass 24x60mm</td>
			<td>VWR Vista Vision</td>
			<td>16004-312</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI filter cube</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of the ependymal cilia in the lateral ventricles of the mouse brain

Células ependimarias Multiciliated alinean los ventrículos en el cerebro adulto. Función o estructura de los cilios ependymal anormal se asocia con diversos déficits neurológicos. La corriente de imágenes ex vivo en directo de la técnica de los cilios ependymal móviles permite un estudio detallado de la dinámica ciliares siguiendo varios pasos. Estos pasos incluyen: ratones eutanasia con dióxido de carbono de acuerdo con los protocolos de la Universidad de Animal Care Institucional de Toledo y el empleo Comisión (IACUC); craniectomía seguido de la eliminación del cerebro y la disección del cerebro sagital con un vibratome o cuchilla afilada para obtener secciones muy finas a través de los ventrículos laterales del cerebro, donde los cilios ependymal puede ser visualizado. La incubación de las rebanadas del cerebro en un plato con fondo de cristal personalizado que contiene medio de Eagle modificado (DMEM) / alta glucosa de Dulbecco a 37 ° C en presencia de 95% / 5% O 2 / CO mezcla 2 es esencial para mantener vivo el tejido duranteel experimento. Un video de la paliza cilios se registra a continuación, utilizando una alta resolución de microscopio de contraste de interferencia diferencial. El vídeo se analiza a continuación, cuadro por cuadro para calcular la frecuencia de latido ciliar. Esto permite distinta clasificación de las células ependimarias en tres categorías o tipos en función de su frecuencia de latido ciliar y el ángulo. Además, esta técnica permite el uso de análisis de imágenes de fluorescencia de alta velocidad para caracterizar las propiedades únicas intracelulares de oscilación de calcio de células ependimarias, así como el efecto de agentes farmacológicos en las oscilaciones de calcio y la frecuencia de latido ciliar. Además, esta técnica es adecuada para formación de imágenes de inmunofluorescencia para la estructura ciliar y ciliar proteína de localización de estudios. Esto es particularmente importante en los estudios de diagnóstico de enfermedades y fenotipo. La principal limitación de la técnica se atribuye a la disminución en el movimiento cilios móviles en vivo como el tejido cerebral comienza a morir.

Medición de la función cilios ependymal en cerebro de ratón vivo El método descrito en este protocolo se utiliza para controlar la función cilios ependymal y estructura en el tejido fresco diseccionado desde el cerebro del ratón, así como para vigilar y estudiar los cilios frecuencia paliza. Los pasos seguidos para llevar a cabo un experimento completo se representan en un diagrama de flujo esquemático (Figura 1). Es muy recomendable que el experimento se lleva a cab...

Watch this Scientific Journal Video about Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain at JoVE.com

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

El uso de contraste de interferencia diferencial (DIC) de microscopía de alta resolución, una observación ex vivo de la paliza de los cilios ependymal móviles ubicada dentro de los ventrículos cerebrales de ratones se demostró por imágenes en vivo. La técnica permite una grabación de la frecuencia de latido ciliar único y ángulo superando así como sus propiedades de estimulación de oscilación de calcio intracelular.

Imágenes en vivo de la ependimarios cilios en los ventrículos laterales del cerebro del ratón

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

live-imaging-ependymal-cilia-lateral-ventricles-mouse

Research

JoVE Journal

Neuroscience

13.6K vistas.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences.  El uso de contraste de interferencia diferencial (DIC) de microscopía de alta resolución, una observación ex vivo de la paliza de los cilios ependymal móviles ubicada dentro de los ventrículos cerebrales de ratones se demostró por imágenes en vivo. La técnica permite una grabación de la frecuencia de latido ciliar único y ángulo superando así como sus propiedades de estimulación de oscilación de calcio intracelular. 

Video: Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

La neuromodulación y mitocondrial de transporte: Imágenes en vivo en las neuronas del hipocampo durante largos períodos de tiempo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Investigación

Neurociencias

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

Drenaje Manual de los ventrículos del cerebro embrionario de pez cebra

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Actividad telomerasa en las diversas regiones del cerebro de ratón: La telomerasa no radiactivo Repetir Protocolo de Amplificación (TRAP) Ensayo

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

Modelado 3D del lateral Ventrículos y histológico Caracterización de Tejidos periventricular en los seres humanos y de ratón

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Las fibras serotoninérgicos proyección de imagen en la médula espinal de ratón El uso de la claridad / Técnica CÚBICO

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Grabación de la plasticidad sináptica en rebanadas Hippocampal agudas mantenidas en un pequeño volumen reciclado - perfusión- y sistema de cámara de tipo inmersión

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

Proyección de imagen de las células de la corteza Cerebral primaria usando un sistema 2D de la cultura en vivo

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Explorar el espacio profundo - descubrimiento de la anatomía de las estructuras periventriculares para revelar los ventrículos laterales del cerebro humano

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.