This work was supported by a Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (T.I.) and Grants-in-Aid for Scientific Research (C) from MEXT (S.T. and K.N).

Los protocolos y uso de animales experimentales fueron aprobados por los comités de cuidado animal y el uso institucional de la Universidad de Yamanashi y la Universidad de Waseda. Cuidado de los animales se realizó de acuerdo con las directrices institucionales. 1. Preparación CPEC <ol><li> Preparar los siguientes aparatos y materiales: un microscopio estéreo, preferiblemente capaz de transmitir la iluminación desde la parte inferior; un par de pinzas de relojero (Dumont # 3 o # 4), llama-esterilizado, tijeras de funcionamiento rectas, llama-esterilizado; dos platos de 10 cm de plástico estériles que contienen 20 ml de hielo frío medio Leibovitz L-15; Platos con fondo de cristal de 35 mm que contienen 2 ml RT Leibovitz medio L-15; un vaso de precipitados de 100 ml que contiene 70% de etanol; cachorros neonatales ratón. </li><li> Brevemente sumerja un ratón neonatal en etanol al 70% y la eutanasia de forma rápida por decapitación con las tijeras que operan. </li><li> Coloque la cabeza inmediatamente helada Leibovitz L-15 en el medio estéril 10 cm dish. </li><li> Retire la piel de los bóveda craneal utilizando el par de pinzas relojero, cortar el cráneo para exponer el cerebro, y luego cortar los nervios craneales para aislar todo el cerebro. </li><li> Transferir el cerebro a un nuevo plato que contiene helado Leibovitz L-15 medio y observar bajo el microscopio estereoscópico, asegurándose de que el cerebro está completamente inmerso en el medio. </li><li> Establezca la cara dorsal del cerebro hacia arriba, y orientar el cerebro para que los bulbos olfatorios residen en la posición tres (para personas diestras). Mantenga el cerebro suavemente con las pinzas en la mano izquierda. </li><li> Usando las pinzas de disección finas (Dumont # 3 o # 4) en la mano derecha, cortar el cuerpo calloso y debajo del parénquima que conecta los hemisferios cerebrales, a lo largo de la fisura longitudinal del cerebro. </li><li> Presione suavemente los hemisferios cerebrales de distancia a los lados laterales y exponer la fisura cerebral transversal. </li><li> Separar los hemisferios pellizcando a cabo ªe parénquima entre el hemisferio y el tálamo. </li><li> Retire con cuidado el plexo coroideo del ventrículo lateral que se adjuntan a la parte lateral del hipocampo por el Affixa lámina. </li><li> Transferir el plexo coroideo aisladas para el plato de fondo de vidrio de 35 mm que contiene frescos medio Leibovitz L-15, y las cubrirás de un peso (Lista de Materiales) suavemente para mantener el tejido en su lugar. </li></ol> 2. imágenes en vivo de CPEC cilios <ol><li> Confirmar ultravioleta adecuada (UV) y el filtro (s) deducido (IR) de corte para bloquear la luz corta que 400 nm y más de 700 nm, y que de densidad neutra (ND) Filtros (25% y 6%) se inserta en la trayectoria de la luz del microscopio invertido. </li><li> Ajuste el enfoque de la lente objetivo más o menos a ojo, y luego ajustar el condensador de modo que el centro y el enfoque para ajustarse a la iluminación Köhler. Insertar un contraste de interferencia diferencial apropiado (DIC) prisma, un elemento de DIC, así como elementos del analizador y el polarizador en el liruta luchar para ajustarse a la óptica DIC. </li><li> Ajustar el contraste de la vista por la posición del prisma DIC modo que la estructura de la superficie del tejido es más reconocible. Si todos los cilios de las células diana son móviles, una visión clara de cilios móviles no se puede obtener por el ojo a causa de su movimiento. </li><li> Cambie la trayectoria de la luz a la cámara de video, y quitar los filtros ND para aumentar la potencia de la luz. </li><li> Utilice la cámara en el modo de enfoque para ajustar el campo de visión y el enfoque. Durante el enfoque, en el que las imágenes de vídeo se visualizan en tiempo real en el monitor, una visión clara de cilios móviles no está disponible. </li><li> Utilice la cámara a 200 Hz con un tiempo de exposición de 0,1 ms para el período (segundos a minutos) deseado. Después de la adquisición de la pila de imágenes, cuadros individuales se mostrarán las estructuras ciliares claras. Si bordes ciliares están borrosas, aumentar la velocidad de fotogramas o utilizar un tiempo de exposición corto. </li><li> Registrar el movimiento de los cilios CPEC dentro 25-60 minutos después de la eutanasia en LeibovitzL-15 medio. </li></ol> 3. Análisis de movimiento ciliar <ol><li> Rastrear manualmente superando patrones de cada cilio en el monitor de la computadora. Marcar las posiciones de punta ciliar en cada cuadro con el puntero del ratón, que se ensamblan para obtener información trayectoria de cada cilio. Cualquiera de analizar la información de trayectoria utilizando el mismo software o exportar a otras aplicaciones más generales para su posterior análisis. La eficiencia de este paso de análisis se describe en la discusión. </li><li> Clasificar las trayectorias en dos modos de movimiento, de ida y vuelta o de rotación, por ojo. </li><li> Calcular la frecuencia de latido ciliar (CBF) utilizando la siguiente fórmula: [CBF = (número de fotogramas por segundo) / (número promedio de marcos para un solo latido)] 14, que se puede obtener a partir de un diagrama de movimiento punta ciliar (Figura 3 ). Repetir este cálculo para múltiples ciclos de batido ciliar, porque otra cilios en la misma célula puede interferir con el movimiento de cada cilium, resultando en irregularidad. </li><li> Para analizar la uniformidad angular de la ciliar superando ejes dentro de una sola célula, definir el ángulo θ golpes para cada trayectoria (Figura 4). Para trayectorias de nuevo adelante y hacia atrás, ajuste la posición de la punta cilios a una línea recta, y definir θ como el ángulo de la línea hace con el eje x. Para las trayectorias de rotación, encajar las posiciones a una elipse, y definir θ como el ángulo del eje mayor de la elipse hace con eje x. Los detalles de la instalación se describen en la sección de Resultados Representante. </li><li> Para obtener una descripción cuantitativa de cada trayectoria, calcular generalizada relación de aspecto AR. Brevemente, rotar la trayectoria por - θ y definir AR como la relación entre las anchuras de la distribución a lo largo de x - y -axes Y (Figura 4B). Los detalles se muestran en la sección Resultados Representante, y la interpretación, El significado y la limitación de los parámetros se describen en la Discusión. </li></ol> 4. Preparación de muestras para SEM Nota: SEM es un método importante para evaluar el estado de los cilios en CPECs de una manera integral. Para preparar muestras para SEM, un procedimiento estándar se informó anteriormente 15 se emplea con ligeras modificaciones. <ol><li> Antes de la disección de los tejidos del cerebro, preparar el fijador en un vial de vidrio de 5 ml con un tapón de polietileno. El fijador se compone de 2% de paraformaldehído, 2.5% de glutaraldehído (solución media de Karnovsky 16) en tampón fosfato 0,1 M, pH 7,4. </li><li> Diseccionar el tejido del cerebro como se describe en el paso 1. </li><li> Brevemente enjuagar el tejido aislado con solución salina equilibrada de Hank (HBSS) en un nuevo plato y luego fijar el tejido en el fijador en el vial de vidrio durante 1 hora a temperatura ambiente. Utilice pipetas de transferencia desechables y manipular las muestras gently. Después de enjuagar en HBSS, el tejido se vuelve pegajosa. <ol><li> Para transferir los especímenes en la solución de fijación, expulsar lentamente una pequeña cantidad de solución que contiene el tejido de la pipeta de transferencia como una gotita y añadir a la fijador. </li></ol></li><li> Después de la fijación, deseche el fijador y lavar el tejido con tampón fosfato tres veces. </li><li> Sumergir el tejido en una solución de sacarosa al 10% para lavar los aldehídos restantes. Para garantizar la eliminación completa de los aldehídos, sumergir las muestras en la solución durante 10 min y luego repetir dos veces con 10% de sacarosa fresco. Este paso es importante para lograr la adecuada post-fijación en los pasos posteriores. </li><li> Sumergir el tejido en una solución de 1% de tetróxido de osmio en tampón fosfato durante 30 min y luego se coloca en hielo durante post-fijación. Juzgar el grado de osmification por el color de la muestra: cuando aldehídos se eliminan por completo, la muestra es de color negro. </li><li> Lavar las muestras de tejidos después de la fijada extensamente con doble destiladad agua varias veces. </li><li> Deshidratar las muestras por inmersión en concentraciones graduadas de etanol, por lo general 65%, 75%, 85%, 95%, 99%, y 100%, durante 10 min cada uno. Obtener etanol anhidro mediante la colocación de tamices moleculares en etanol 99,5% de una botella que acaba de adquirir. Repita deshidratación con etanol anhidro tres veces. </li><li> Colocar las muestras deshidratadas en acetato de isoamilo, un reactivo de sustitución de secado de punto crítico, para 10 min. Repita este paso dos veces. Este reactivo se evapora rápidamente y la muestra puede convertirse en seco, lo que resulta en la destrucción por la tensión superficial. Por lo tanto, no secar la muestra por completo. </li><li> Después de que el intercambio final de acetato de isoamilo, eliminar la mayor parte del disolvente, inmediatamente envolver el vial de vidrio abierto con papel de aluminio, y colocar el vial en hielo seco. Usando una aguja o unas pinzas finas, hacer varios agujeros en la lámina que cubre la boca del vial, de modo que el dióxido de carbono líquido fluye fácilmente en el vial en el secador de punto crítico. Continúe con el siguiente pasolo más rápido posible. </li><li> En este paso, minimizar el arrastre de acetato de isoamilo en la cámara de la secadora, pero no dejes que la muestra se seque completamente antes de secado de punto crítico. Además, no deje la muestra en hielo seco durante un tiempo innecesariamente largo para evitar la formación de escarcha en el vial. </li><li> La transferencia de los viales de vidrio envueltas en papel aluminio que contienen las muestras de tejido en el secador de punto crítico que asegura la estructura de la superficie del tejido permanece intacta, mientras que la eliminación de agua contenida en el tejido. La información detallada sobre el funcionamiento del secador de punto crítico puede obtenerse a partir de las instrucciones del fabricante. </li><li> Maneje las muestras cuidadosamente con un palillo de dientes para minimizar el daño mecánico. Las muestras de tejido secas resultantes son frágiles. Montar las muestras en trozos de metal y escudo de oro-paladio usando una pulverización catódica de iones. </li></ol> 5. Observación por SEM <ol><li> Observe por SEM y grabar imágenes con una cámara digitalequipado para microscopio electrónico de barrido. </li><li> Transferir datos de imagen digital a un PC para su análisis. </li></ol>

<ol>
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S., Ben-Shahar, Y., Moninger, T. O., Kline, J. N., Welsh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motile+cilia+of+human+airway+epithelia+are+chemosensory.">Motile cilia of human airway epithelia are chemosensory.</a> Science(New York, N.Y). 325 (5944), 1131-1134 (2009).</li><li>Kiprilov, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells+in+culture+possess+primary+cilia+with+hedgehog+signaling+machinery).">Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery).</a> The Journal of Cell Biology. 180 (5), 897-904 (2008).</li><li>Hirokawa, N., Tanaka, Y., Okada, Y., Takeda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+flow+and+the+generation+of+left-right+asymmetry.">Nodal flow and the generation of left-right asymmetry.</a> Cell. 125 (1), 33-45 (2006).</li><li>Narita, K., Kozuka-Hata, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+multiple+primary+cilia+reveals+a+novel+mode+of+ciliary+development+in+mammals.">Proteomic analysis of multiple primary cilia reveals a novel mode of ciliary development in mammals.</a> Biology Open. 1 (8), 815-825 (2012).</li><li>Takeda, S., Narita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+vertebrate+cilia,+towards+a+new+taxonomy.">Structure and function of vertebrate cilia, towards a new taxonomy.</a> Differentiation; Research in Biological Diversity. 83 (2), S4-S11 (2012).</li><li>Ikegami, K., Sato, S., Nakamura, K., Ostrowski, L. E., Setou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin+polyglutamylation+is+essential+for+airway+ciliary+function+through+the+regulation+of+beating+asymmetry.">Tubulin polyglutamylation is essential for airway ciliary function through the regulation of beating asymmetry.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (23), 10490-10495 (2010).</li><li>Foster, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+ciliary/flagellar+beating+of+Chlamydomonas.">Analysis of the ciliary/flagellar beating of Chlamydomonas.</a> Methods in Cell Biology. 91, 173-239 (2009).</li><li>Lechtreck, K. -. F., Sanderson, M. J., Witman, G. 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A., Rutman, A., O&#8217;Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+beat+pattern+is+associated+with+specific+ultrastructural+defects+in+primary+ciliary+dyskinesia.">Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia.</a> The Journal of Allergy and Clinical Immunology. 112 (3), 518-524 (2003).</li><li>Takeda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left-right+asymmetry+and+kinesin+superfamily+protein+KIF3A:+new+insights+in+determination+of+laterality+and+mesoderm+induction+by+kif3A-/-+mice+analysis.">Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/- mice analysis.</a> The Journal of Cell Biology. 145 (4), 825-836 (1999).</li><li>Karnovsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+formaldehyde-glutaraldehyde+fixative+of+high+osmolarity+for+use+in+electron+microscopy.">A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy.</a> The Journal of Cell Biology. 27, 137-138A (1965).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia. Este documento tiene por objeto informar metodología detallada para observar la motilidad de los cilios en los tejidos del plexo coroideo aisladas. Novedades científicas se han reportado en estudios anteriores 1,8.

Perspectivas de este método Aunque la técnica descrita aquí no proporciona un análisis más detallado de los cilios que los métodos publicados anteriormente, la importancia de esta técnica reside en la simplicidad del sistema y la eficacia de costes, que se puede aplicar fácilmente a cualquier tipo de detección de la motilidad ciliar ex vivo. En particular, TI Workbench proporciona una interfaz simple y fácil de usar que permite a los investigadores observar y analizar la m...

Cilia are hair-like projections on the surface of most vertebrate cells, which have attracted attention by medical researchers because of a class of diseases termed ciliopathies2&#8211;4. Despite the ubiquitous expression of the organelle, a wide variety of ciliary functions have been reported, including motility and biosensing. For example, motile cilia on the mucoepithelial surface transport mucus5 and epithelial debris to the outlet of tracts, thereby preventing disease by clearing the surface of epithelia. Moreover, during early developmental periods and embryonic stages, cilia regulate the proliferation of stem cells6, and are involved in the determination of left&#8211;right asymmetry of the vertebrate body7. 
Choroid plexus epithelial cells (CPECs) are derivatives of neuroepithelial cells that cover the surface of the choroid plexus tissue in the brain, which play important roles in maintaining homeostasis of the intracranial environment by production of cerebrospinal fluid (CSF). It has been previously demonstrated that CPECs have multiple non-motile cilia that regulate the production of CSF through G-protein-coupled receptors that are specifically concentrated on the cilia8. Although these cilia had been regarded as quiescent non-motile cilia, it was discovered that some CPEC cilia exhibit transient motility during the neonatal period1. This finding was quite important because it revealed that so-called non-motile cilia are not necessarily immotile from the beginning of development and might display transient motility during specific time windows, possibly in response to specific physiological demands and functions9. To precisely describe the motile nature of CPEC cilia, it is necessary to develop an ex vivo observation system that encompasses analysis of the kinetic profiles unique to CPEC cilia. 
With respect to motility, although several technical reports have described observations of the motile cilia of the tracheal epithelium5,10, motile single-cell flagella11, so-called conventional motile cilia12, and nodal cilia13, detailed analytical methods applicable to relatively undulated structures such as the choroid plexus have not been well documented so far. Moreover, a high time resolution is required to analyze the ciliary movement of CPECs, in which expensive high-speed cameras are indispensable. To circumvent this necessity and simplify monitoring the ciliary motility of various cell types, a low cost, high-speed camera has been introduced, and an easily accessible and convenient method to record the motility of motile cilia, especially to describe the speed and pattern of motion of each cilium, has been developed1. Moreover, original image analysis software &#8220;TI Workbench&#8221; has been used here to facilitate detailed analysis of motility. Collectively, this method provides a new concise strategy to analyze ciliary motion together with correlative scanning electron microscopy (SEM), which can be adopted in a wide range of cilium research.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>for both live imaging and SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stereo microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr>
			<td>flat paper towel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#934;10-cm plastic dish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-mL beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>straight operating scissors</td>
			<td>Sansyo</td>
			<td>S-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>watchmaker forceps</td>
			<td>Dumont</td>
			<td>No.DU-3 or -4, INOX</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bottom dish</td>
			<td>Matsunami Glass</td>
			<td>D110300</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-mL glass vial with a polyethylene cap</td>
			<td>Nichiden Rika-Glass</td>
			<td>PS-5A</td>
			<td></td>
		</tr>
		<tr>
			<td>transfer pipette</td>
			<td>Samco Scientific</td>
			<td>SM251-1S</td>
			<td>for specimen tranfer</td>
		</tr>
		<tr>
			<td>toothpick</td>
			<td></td>
			<td></td>
			<td>for specimen transfer</td>
		</tr>
		<tr>
			<td>ion sputter with gold-palladium</td>
			<td>Hitachi</td>
			<td>E-1030</td>
			<td></td>
		</tr>
		<tr>
			<td>critical point dryer</td>
			<td>Hitachi</td>
			<td>HCP-2</td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>100 W mercury lump housing and power supply</td>
			<td>Olympus</td>
			<td>U-ULH and BH2-RFL-T3</td>
			<td></td>
		</tr>
		<tr>
			<td>100 W mercury lamp</td>
			<td>Ushio</td>
			<td>USH103D</td>
			<td></td>
		</tr>
		<tr>
			<td>DIC condenser, n.a. 0.55</td>
			<td>Olympus</td>
			<td>IX-LWUCD</td>
			<td></td>
		</tr>
		<tr>
			<td>electrrical shutter</td>
			<td>Vincent Associates</td>
			<td>VS35S22M1R3-24 and VMM-D1</td>
			<td>manual shutter can be used.</td>
		</tr>
		<tr>
			<td>band-pass filter (400-700 nm, &#934;45 mm)</td>
			<td>Koshin Kagaku</td>
			<td>C10-110621-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ND filter (&#934;45 mm)</td>
			<td>Olympus</td>
			<td>45ND6, 45ND25</td>
			<td>combination of 25% and 6% ND filters are used</td>
		</tr>
		<tr>
			<td>objective lens (water immersion) with DIC element</td>
			<td>Olympus</td>
			<td>UApo 40XW/340, n.a., 1.15 with IX-DPAO40</td>
			<td></td>
		</tr>
		<tr>
			<td>high-speed video camera</td>
			<td>Allied Vision Technologies</td>
			<td>GE680</td>
			<td>&#62;= 200 Hz frame rate and 1 msec expose time</td>
		</tr>
		<tr>
			<td>image acquisition / analysis software</td>
			<td>in-hous software</td>
			<td>TI Workbench</td>
			<td>capable of acquisition at high frame rates.</td>
		</tr>
		<tr>
			<td>PC for camera control / analysis</td>
			<td>Apple</td>
			<td>Mac Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>vibration isolation table</td>
			<td>Meiritsu Seiki</td>
			<td>AD0806</td>
			<td></td>
		</tr>
		<tr>
			<td>weight for tissue</td>
			<td>Warner Instruments</td>
			<td>slice anchor kits</td>
			<td>It can be made with nylon mesh glued to a U-shape squashed &#934;0.5mm platinum wire.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>inverted microscope</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>scanning electron microscope</td>
			<td>JEOL</td>
			<td>JSM-6510</td>
			<td></td>
		</tr>
		<tr>
			<td>for live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol&#160;</td>
			<td>Wako Chemicals</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz L-15 medium</td>
			<td>Life Technologies</td>
			<td>11415-064</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>for SEM preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol&#160;</td>
			<td>Wako Chemicals</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Life Technologies</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde&#160;</td>
			<td>Merck</td>
			<td>1040051000</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaraldehyde&#160;</td>
			<td>Nacalai tesque</td>
			<td>17003-05</td>
			<td></td>
		</tr>
		<tr>
			<td>isoamyl acetate</td>
			<td>Nacalai tesque</td>
			<td>02710-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Sieves 4A 1/8&#160;</td>
			<td>Wako Chemicals</td>
			<td>130-08655</td>
			<td>for preparation of anhydrous ethanol</td>
		</tr>
		<tr>
			<td>phosphate buffer saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffer, 0.1 M</td>
			<td></td>
			<td></td>
			<td>To make 100 ml, mix 19.0 ml of 0.1 M NaH2PO4 and 81.0 ml of 0.1 M Na2HPO4&#160;</td>
		</tr>
		<tr>
			<td>monosodium phosphate (dihydrate)</td>
			<td>Nacalai tesque</td>
			<td>31718-15</td>
			<td></td>
		</tr>
		<tr>
			<td>disodium phosphate (anhydrous)</td>
			<td>Nacalai tesque</td>
			<td>31801-05</td>
			<td></td>
		</tr>
		<tr>
			<td>suclose</td>
			<td>Nacalai tesque</td>
			<td>30406-25</td>
			<td></td>
		</tr>
		<tr>
			<td>osmium tetroxide</td>
			<td>Nisshin EM</td>
			<td align="right">300</td>
			<td></td>
		</tr>
		<tr>
			<td>dry ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

observation of the ciliary movement of choroid plexus epithelial cells ex vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Una visión general del flujo de trabajo se muestra en la Figura 1, incluyendo imágenes de los dispositivos. Observaciones de movimiento vivo de CPECs Película 1 muestra una película de CPECs aisladas a partir de un ratón perinatal, y la película 2 muestra una vista ampliada de las imágenes en la película 1. Debe tenerse en cuenta que las puntas ciliares individuales son menos claros en im...

Watch this Scientific Journal Video about Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo at JoVE.com

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Observación del Movimiento ciliar de las células epiteliales del plexo coroideo<em&gt; Ex Vivo</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

observation-ciliary-movement-choroid-plexus-epithelial-cells-ex

Research

JoVE Journal

Neuroscience

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

12.1K vistas.  Waseda University. In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Video: Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

Ex Vivo organotípicos Modelo corneal aguda de herpes simplex virus tipo epitelial que la infección

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

Investigación

Neurociencias

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Determinación rápida del umbral nociceptivo térmico en ratas diabéticas

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

Ex cultivo in vivo de la Plenaria, los cerebros en desarrollo de Drosophila

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

Una descripción visual de la disección de la vasculatura cerebral superficial y las meninges asociada y el plexo coroideo del cerebro de rata

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex vivo la Producción de la Bombilla órgano vomeronasal y accesorios olfativa Intacto

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Agudo<em&gt; In Vivo</em&gt; Grabaciones electrofisiológicas de potenciales de campo locales y actividad de múltiples unidades de la vía hiperdirecta en ratas anestesiadas

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

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

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

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

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

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

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

In vivo Calcium Imaging in Mouse Inferior Olive

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

In vivo Calcium Imaging in Mouse Inferior Olive

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

In vivo Imágenes de calcio en la aceituna inferior de ratón

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

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

Ex Vivo Liberación del péptido relacionado con el gen de la calcitonina del sistema trigémino vascular en roedores

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

Preparación del nervio ciático de rata para neurofisiología ex vivo

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

Observación del Movimiento ciliar de las células epiteliales del plexo coroideo

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