This work was partially supported by National Institute of Deafness and Other Communications Disorders Awards R03-DC-012125 to D. A. Llano and F31-DC-013501 to B. J. Slater as well as the Carver Foundation.
The authors would like to thank Jason MacLean and Matthew Banks for technical advice with calcium imaging.

Todos los procedimientos fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Illinois. Todos los animales fueron alojados en las instalaciones de cuidado de los animales aprobados por la Asociación Americana para la Acreditación del Laboratorio Animal Care. Cada intento se hizo para reducir al mínimo el número de animales utilizados y reducir el sufrimiento en todas las etapas del estudio. 1. Preparación y Extracción de cerebro de ratón para rebanar <ol><li> Prepárese para la perfusión y la incubación rebanada. <ol><li> Aproximadamente 30 minutos antes de cortar, preparar alta solución de corte sacarosa y baja LCRa calcio para la incubación de la rebanada antes de la grabación o imágenes. </li><li> Coloque todas las herramientas necesarias (grandes tijeras romas finales, pequeñas tijeras de primavera, pinzas anatómicas, grandes tijeras o guillotina, iris tijeras, joyeros fórceps, 10 jeringas ml con aguja 27G x 1/2 pulgadas, pequeña pieza de 1 cm x 2 cm de filtro papel, y una punta de pipeta de Pasteur roto por sltransferencia de hielo) para la perfusión y corte en rodajas, y preparar la bandeja de perfusión. </li><li> Configurar la incubación de cámara con oxigenada LCRa en un baño de agua C ​​32 o y una placa de cultivo lleno de solución de corte. </li></ol></li><li> Preparar corte escenario para rebanar. <ol><li> Corte un pedazo pequeño de 3% de agar de aproximadamente 1 cm 3 para usar como respaldo para el cerebro y 1,5 cm x 1,5 mm x 3 mm para un bulto que se utilizará para apuntalar la IC. </li><li> Pegue el tope en el escenario con el adhesivo de cianoacrilato, así como la protuberancia en el lado derecho del tope de tal manera que forman un ángulo de 80 °. </li></ol></li><li> Retire el cerebro. <ol><li> Profundamente anestesiar a-p20 p12 (idealmente p14-18) ratón con ketamina 100 mg / kg y xylaxine 3 mg / kg (o comité de ética comparables procedimiento aprobado). Nota: Confirme el nivel de anestesia adecuado a través de la falta de respuesta a la pizca dedo del pie. Como el animal está bajo anestesia brevemente, pomada oftálmica es innecesaria. </li><li> Usando romoterminar tijeras, exponer la caja torácica de la apófisis xifoides hasta el cuello. <ol><li> Encuentra el proceso xifoides, y hacer un corte horizontal de aproximadamente 1,5 cm. </li><li> Hacer dos cortes verticales de los extremos del corte horizontal anterior a los hombros, aproximadamente 1,5 cm cada uno. </li></ol></li><li> Cortar a través del diafragma y las uniones costocondrales para exponer el corazón. </li><li> Con pequeñas tijeras de primavera, hacer un pequeño, 2-5 cortar en la aurícula derecha mm, desde el ventral a dorsal lado del corazón. </li><li> Usando una jeringa de 10 ml con una aguja 27G x 1/2 pulgadas, inyectar el ventrículo izquierdo y perfundir rápidamente el animal con alta solución de corte sacarosa. </li><li> Una vez que la sangre salga clara, utilizar tijeras grandes para quitar de la cabeza. Nota: No hemos evaluado sistemáticamente la utilidad de la perfusión. Sin embargo, en nuestra experiencia, la perfusión transcardíaca en ratones jóvenes esta se puede hacer con casi el 100% de éxito, y no tienen el beneficio de eliminar bl rojaood células, que pueden emitir fluorescencia e interferir con las imágenes. </li><li> Cortar la piel hacia abajo de la línea media para exponer el cráneo. </li><li> Usando iris dobladas tijeras, cortar el cráneo entre los ojos, y luego a partir de la corte entre los ojos, cuidadosamente cortado de anterior a posterior a lo largo de la línea media de la sutura con cuidado de no dañar el cerebro debajo. Nota: La dura por lo general, se corre con el cráneo. Si es necesario, retire la duramadre antes de retirar cuidadosamente el cerebro. </li><li> Usando joyeros fórceps, palanca abrir el cráneo y retire con cuidado el cerebro, a continuación, coloque el cerebro en una solución de corte. Tenga cuidado de no dañar las cortezas. </li></ol></li></ol> 2. Preparación del cerebro para rebanar <ol><li> Preparación del cerebro para el montaje en vibratome etapa. <ol><li> Usando una diapositiva marcado con dos líneas a 90 o y una línea diagonal a los 17 o desde la parte superior izquierda a la inferior derecha, intersección donde las dos líneas se encuentran, coloque la cara dorsal del cerebro hasta, utilizandouna hoja de afeitar, retire 2-4 mm del cerebro extremo rostral la creación de una superficie plana. </li><li> Coloque el lado caudal del cerebro en la superficie plana de nueva creación, y alinear la superficie dorsal del cerebro con la horizontal y la línea media del cerebro con la línea vertical. </li><li> Alinear la cuchilla de afeitar con la línea 17 o, incline la navaja en un ángulo de 30 ° y quite aproximadamente 3 mm de la corteza justo en un corte diagonal doble (17 o desde el plano horizontal y 60 ° de la corona). </li></ol></li><li> Montaje cerebro en vibratome etapa. <ol><li> Coloque un pequeño trozo de papel de filtro de 1 cm x 2 cm en la parte ventral del cerebro para que la dimensión larga es perpendicular a la línea media. </li><li> Con cuidado aplique una pequeña cantidad de adhesivo de cianoacrilato a la zona delante del antirretorno (y a la izquierda de la protuberancia). </li><li> Coloque el cerebro en diagonal de corte doble en el pegamento de manera que la parte caudal del cerebro y el rombencéfalo son prel op a la protuberancia y el lado derecho del cerebro está en contra del tope trasero. </li><li> Delicadamente presione hacia abajo en el cerebro, lo que garantiza que toda la superficie está en contacto con la cámara de corte. </li></ol></li></ol> 3. La obtención de la Colliculo-tálamo-cortical Slice <ol><li> Tomar rápidamente la etapa de corte y el lugar en el vibratome, llenar el escenario con una solución de corte. </li><li> Alinear la cuchilla con la parte superior (lado ventral) del cerebro. </li><li> Retire 1-1.5 mm de la parte superior del cerebro, retire 300-500 micras rodajas y evaluar a fondo después de la eliminación. Nota: Cuando la IC, el MGB, y el núcleo geniculado lateral (LGN) son visibles, y el giro dentado forma una forma de &#39;C&#39; tomar de una a dos 600 micras rodajas. Véase la Figura 3A. </li><li> Coloque las rebanadas en caliente (32 ° C) que sostiene la cámara. </li></ol> 4. Obtención de imágenes de la rebanada <ol><li> Preparación para la formación de imágenes autofluorescencia flavoproteína. <ol> &lt;li&gt; Prepare LCRa para la perfusión de la rebanada en una cámara estándar de registro electrofisiológico, LCRa burbuja con 95% de O2 / 5% de CO 2. </li><li> Tire electrodo de vidrio para la estimulación. Tenga en cuenta que varios diferentes electrodos estimulantes y configuraciones (vidrio, tungsteno, fibra de carbono, monopolar, bipolar) todo el trabajo muy bien en conjunto con imágenes autofluorescencia flavoproteína. </li><li> Encienda el ordenador, cámara, micromanipulador, fuente de luz, el software de estimulación, el software de captura de imágenes. </li><li> Perfundir la cámara de grabación con oxigenada LCRa, coloque el puente personalizado (diseño disponible en Materiales Suplementarios) para elevar la rebanada en la cámara. Nota: tasa de perfusión puede variar con cualquier individuo establecido; 5-15 ml / min se utiliza aquí. </li><li> Coloque la rebanada en la cámara, bajar el nivel de líquido en la cámara para evitar el corte de la elevación del puente mientras se coloca arpa especialmente construido sobre rebanada, teniendo cuidado de evitar la colocación de cuerdas del arpa sobre vías de interest (Figura 1A). Aumentar el nivel de líquido de aproximadamente 1-2 mm por encima de la rebanada para mantener el flujo LCRa encima y debajo de la rebanada. Nota: Si el reposicionamiento es necesario, utilice el final pipeta Pasteur roto o permita que el nivel LCRa a subir y utilizar fórceps con extremo cuidado. </li><li> Inserte electrodo de cloruro de plata en el electrodo de vidrio lleno de LCRa, inserte el electrodo de vidrio en micromanipulador y conectarse a los estímulos del aislador, y coloque cuidadosamente electrodo de vidrio en IC / sustancia blanca que lleva a tálamo (ver Figura 1). </li></ol></li><li> Flavoproteína adquisición de imágenes de autofluorescencia. <ol><li> Recoge imágenes a 4 Hz (utilizando un 2X macro objetivo infinito corregido (NA 0.13)) y una cámara de 105 seg, mientras que la estimulación eléctrica (cada estimulación consiste en 1 seg de 40 Hz 2 ms pulsos tejido en 0,05 Hz cinco veces), mientras que ilumina el tejido con azul claro desde 470 hasta 490 nm y por encima de la captura de 515 nm. Asegúrese de que la imagen no es ni dever o oscuro, ni apagada Figura 3 dejó la columna de referencia. Nota: El tiempo de recolección, la frecuencia de recogida, y la frecuencia de estimulación se pueden cambiar para adaptarse a la experiencia, el programa personalizado incluido en materiales complementarios se pueden modificar como tal. </li><li> Exportar imágenes a Matlab, y utilizando el programa escrito de encargo disponible en materiales complementarios para analizar la energía espectral de las imágenes a 0,05 Hz. La imagen resultante mostrará las vías conectados. Nota: El programa personalizado utiliza una transformada rápida de Fourier de los valores de los píxeles de la serie temporal para producir la imagen. </li></ol></li><li> Preparación para la imagen de calcio. <ol><li> Preparar pequeña cámara de incubación utilizando una placa de cultivo de 3 cm y la membrana cultura elevada para permitir LCRa para alcanzar la parte superior e inferior de la rebanada, y colóquelo sobre una almohadilla térmica para mantener la temperatura a aproximadamente 32 ° C </li><li> Rellene pequeña cámara de incubación con LCRa cálido y poco a poco la burbuja con un 95% de O2/5% de CO 2. </li><li> Mezclar 2 l de Pluronic F-127 ácido y 50 g de Fura-2AM disuelto en 48 l de DMSO. </li><li> Lugar rebanada en la pequeña cámara de incubación de la membrana elevada y con cuidado utilizando una micropipeta añadir mezcla de tinción directamente encima del MGB (u otra estructura de interés). </li><li> Cubra las rebanadas para prevenir la decoloración antes de la proyección de imagen, y que permiten rebanadas incubar durante 45 min - 1 hora. </li><li> Retire rebanadas a cámara de retención calentado durante 10-15 minutos para lavar el exceso de material. </li><li> Siga los pasos 4.1.1 al 4.1.6 para la preparación de estimular y la imagen de la rebanada-colliculo tálamo-cortical. </li></ol></li><li> Adquisición de imágenes de calcio. <ol><li> Recoge imágenes a 10 Hz (con un objetivo de inmersión 20X agua) durante 25 segundos, mientras que la estimulación eléctrica (cada estimulación consiste en un impulso de 2 ms) tejido en 0,2 Hz cinco veces mientras que ilumina el tejido con 365 nm de luz y la captura de fluorescencia por encima de 510 nm. </li&gt; <li> Exportar imágenes a Matlab y utilizando el programa escrito de encargo disponible en materiales complementarios para analizar la energía espectral de las imágenes a 0,2 Hz. La imagen resultante mostrará células activas. </li></ol></li></ol>

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The authors have nothing to disclose.

This protocol describes improvements upon a previously described colliculo-thalamocortical brain slice in p12-20 mouse to study information flow in the auditory system1. This method has a number of advantages over other, similar, brain slice preparations by retaining connections between more brain areas in a single slice, which gives investigators new tools to understand the interaction and interplay between auditory nuclei in the forebrain. There have been a few key modifications in this protocol, compared to...

En el sistema auditivo, aunque hay una transformación sustancial de información entre la periferia sensorial y el colículo inferior, hay una considerable procesamiento adicional antes de que alcance la corteza auditiva. Sabemos muy poco acerca de cómo ese proceso se hace y por lo tanto poco acerca de cómo esa transformación permite que el cerebro interpreta la información sensorial entrante. Con la excepción del olfato, cada uno de los sentidos tiene una organización muy similar con señales periféricas inicialmente siendo retransmitido con alta fidelidad que disminuye a medida que la señal asciende a la corteza. La corteza envía proyecciones a las estructuras inferiores para modular aún más la información entrante. Este complejo sistema ha sido estudiado en una variedad de maneras in vivo, así como en un número de preparaciones in vitro. En el primero, todas las conexiones están intactas, lo que permite al investigador para investigar cualquier conjunto de conexiones, mientras que el control de la entrada sensorial y la medición desalida en un área determinada. Con este enfoque, hay poco o ningún control de la gran variedad de otros insumos, incluyendo otros estímulos sensoriales, la excitación y la atención, dando lugar a una salida intensamente compleja. In vitro, rodajas de cerebro han sido cortadas para capturar ya sea un único conjunto de las proyecciones, o dos áreas cerebrales conectados, que permiten a los investigadores para estimular y evaluar los aferentes o áreas cerebrales diferentes. Estos son a menudo ya sea rebanadas thalamocortical o tectothalamic donde ya sea la entrada al tálamo o el tálamo y su salida a la corteza se conservan 2-5. Estas preparaciones permiten una amplia variedad de manipulaciones farmacológicas, eléctricas y optogenética. Sin embargo, con sólo dos regiones del cerebro, que evalúan principalmente la transferencia de información y carecen de la capacidad de evaluar la transformación de la información a medida que pasa a través del tálamo. También la proyección retículo-talámico, que puede desempeñar un papel en la modulación de la atención 6-9 es prESENT en este segmento. Aquí se demuestra mejoras sobre nuestra preparación previa 1, que permite el control investigador de varias entradas al tálamo para dar una perspectiva única de cómo las puertas tálamo y filtros de información. Nos pareja esta nueva preparación rebanada con imágenes de autofluorescencia flavoproteína para evaluar la conectividad rebanada y el análisis de activación a gran escala, imágenes de calcio en el tálamo para el análisis de la población neuronal, y la grabación de una sola célula para medir el impacto de las diversas entradas en un solo nivel celular. Para ayudar en el mantenimiento de estas conexiones generalizadas también hemos desarrollado una serie de modificaciones del ancla rebanada normal (también conocido como &quot;arpa&quot;) para la celebración de la corte de cerebro en su lugar y un puente para elevar la rebanada para mejorar la perfusión. El arpa está diseñado en forma de herradura modificada para rodear el corte y permitir puntos de fijación personalizables para las cuerdas del arpa. Tres cadenas sonadjunta de tal manera que i) se tiende horizontalmente a lo largo del borde medial de la rebanada, ii) Se extiende desde el extremo caudal del IC hasta el extremo caudal de la AC y iii) un extiende diagonalmente desde el borde medial de la rebanada a un área rostral a la AC (ver Figura 1 A). Muescas pequeñas en el marco de pegar (con pegamento de cianoacrilato) del arpa cadenas permiten una disminución en la cantidad de presión sobre el sector para ayudar a mantener la integridad de la rebanada (véase la Figura 1B). Mediante el uso de la impresión tridimensional, somos capaces de arpas de diseño personalizado a nuestras especificaciones únicas, así como los puentes que permiten el flujo ideal de líquido cefalorraquídeo artificial (LCRa) encima y debajo del tejido. Esto también mantiene grandes áreas de luz para penetrar en el tejido de la electrofisiología de pinzamiento zonal.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>High sucrose cutting solution</td>
			<td></td>
			<td></td>
			<td>in mM: 206 sucrose, 10.0 MgCl2, 11.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>Low calcium aCSF</td>
			<td></td>
			<td></td>
			<td>in mM: 126 NaCl, 3.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 1.0 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>aCSF</td>
			<td></td>
			<td></td>
			<td>in mM: 126 NaCl, 2.0 MgCl2, 10.0 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2.0 CaCl2, 2.5 KCl, pH 7.4</td>
		</tr>
		<tr>
			<td>Stimulus Isolator</td>
			<td>World Precision Instruments</td>
			<td>A360</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Life Technologies</td>
			<td>D12345</td>
			<td>Lot: 1572C502</td>
		</tr>
		<tr>
			<td>Fura-2AM</td>
			<td>Life Technologies</td>
			<td>F1201</td>
			<td>Lot: 144912</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Life Technologies</td>
			<td>P3000MP</td>
			<td>Lot: 1499369</td>
		</tr>
		<tr>
			<td>Large culture dish</td>
			<td>Fisherbrand</td>
			<td>08-757-13</td>
			<td>100x15mm culture dish</td>
		</tr>
		<tr>
			<td>Small culture dish</td>
			<td>Falcon</td>
			<td align="right">353001</td>
			<td>35x10mm culture dish</td>
		</tr>
		<tr>
			<td>Raised culture membrane</td>
			<td>Millicell</td>
			<td>PICMORG50</td>
			<td>Used to maintain oxygenated fluid perfusion on both sides of slice.</td>
		</tr>
		<tr>
			<td>Flavoprotein imaging fluorescence cube</td>
			<td>Olympus</td>
			<td>UMNIB</td>
			<td>470&#8211;490 nm excitation, 505 nm dichroic, 515 nm emission long pass.&#160; We have found that virtually any green fluorescence protein filter cube will work here.</td>
		</tr>
		<tr>
			<td>Calcium imaging fluorescence cube</td>
			<td>Omega Optical</td>
			<td>BX-18</td>
			<td>XF1005 365nm exitation, XF2001 400nm dichroic, XF3080 510nm emission</td>
		</tr>
		<tr>
			<td>Agar for blocking brain</td>
			<td></td>
			<td></td>
			<td>3% by weight in water</td>
		</tr>
		<tr>
			<td>Viper si Stereo Lithography Apparatus</td>
			<td>3D Systems</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

modification of a colliculo-thalamocortical mouse brain slice, incorporating 3-d printing of chamber components and multi-scale optical imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Un ejemplo de cortes de cerebro de ratón colliculo talamocortical obtenido en P15 ratón se muestra en la figura 2. El corte ideal deberá contener los cuatro grandes estructuras auditivas del cerebro medio y cerebro anterior IC, MGB, TRN, y CA, los cuales están activados cuando el IC es estimulada (Figura 2A). Utilizando el análisis de Fourier, la potencia espectral se mide en la frecuencia de estimulación eléctrica, con las regiones del cerebro que muestran conec...

Watch this Scientific Journal Video about Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging at JoVE.com

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

El uso de múltiples ángulos para cortar el cerebro pup ratón, mejoramos sobre una rebanada cerebral agudo descrito previamente-que captura las conexiones entre la mayoría de las principales estructuras del mesencéfalo y el prosencéfalo auditivas.

Modificación de un Colliculo-tálamo-cortical del ratón Cerebro Loncha, incorporación de la impresión 3-D de la Cámara Componentes e Imagen óptica multi-escala

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

modification-colliculo-thalamocortical-mouse-brain-slice

Research

JoVE Journal

Neuroscience

8.2K vistas.  University of Illinois Urbana-Champaign.  El uso de múltiples ángulos para cortar el cerebro pup ratón, mejoramos sobre una rebanada cerebral agudo descrito previamente-que captura las conexiones entre la mayoría de las principales estructuras del mesencéfalo y el prosencéfalo auditivas. 

Video: Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Escalable neumático matrices inyector para Viral focalización de los circuitos cerebrales intactas en 3-D

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Investigación

Neurociencias

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiples imágenes del ratón neuroanatómicos de Resonancia Magnética

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imágenes de detección de feromonas en una preparación de ratón Vomeronasal aguda lámina de tejido

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Un procedimiento para el implante Cámara Espinal para Longitudinal<em&gt; En Vivo</em&gt; Imágenes de la médula espinal de ratón

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Todo el montaje de imágenes de embriones de ratón Proyecciones sensorial Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

Imaging 3-D y Análisis de neuronas infectadas<em&gt; En Vivo</em&gt; Con<em&gt; Toxoplasma gondii</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, typically 3 mm x 3 mm. This protocol introduces a method for creating a substantially larger 7 mm x 6 mm cranial window exposing most of a cerebral hemisphere over the mouse temporal and parietal cortices (e.g., bregma 2.5 - 4.5 mm, lateral 0 - 6 mm). To perform this surgery, the head must be tilted approximately 30&#176; and much of the temporal muscle must be retracted. Due to the large amount of bone removal, this procedure is intended only for acute experiments with the animal anesthetized throughout the surgery and experiment.
The main advantage of this innovative large lateral cranial window is to provide simultaneous access to both medial and lateral areas of the cortex. This large unilateral cranial window can be used to study the neural dynamics between cells, as well as between different cortical areas by combining multi-electrode electrophysiological recordings, imaging of neuronal activity (e.g., intrinsic or extrinsic imaging), and optogenetic stimulation. Additionally, this large craniotomy also exposes a large area of cortical blood vessels, allowing for direct manipulation of the lateral cortical vasculature.

This protocol presents a method for creating a large unilateral craniotomy over the temporal and parietal regions of the mouse cerebral cortex. This is especially useful for real time imaging over an expansive area of a cortical hemisphere.

Un procedimiento de craneotomía lateral grande para el campo amplio de mesoescala imágenes ópticas de la actividad cerebral

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

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

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

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 ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Time-lapse Imágenes confocales de las neuronas migratorias en la cultura organotípica en rebanadas de cerebro de ratón embrionario<em&gt; En Utero</em&gt; Electroporación

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

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 ...