Nos gustaría dar las gracias a Britta Kaltwasser por su excelente asistencia técnica y Mahesh Kumar Teli para organizar la preparación de la filmación de video.

1. Animales <ol><li> Los experimentos se llevaron a cabo de acuerdo con las directrices del NIH para el cuidado y uso de animales de laboratorio y aprobados por las autoridades locales. Para todos los experimentos, C57BL6 / J ratones de tipo salvaje, ApolipoproteinE - / - (ApoE KO) y ratones SV129 (12-16 semanas de edad, 26-30 g de peso corporal, 5-6 animales por grupo experimental) fueron utilizados. </li></ol> 2. La tinción de los vasos cerebrales con látex de color <ol><li> Preparar una mezcla de 25 l tinta de carbón negro (Herlitz, Alemania) con 0,5 ml de compuesto de látex (Pébéo, Francia) en una proporción 1:20 en un tubo de PE y calentar la mezcla a 37 ° C en un baño de agua. Recoger la mezcla en una jeringa de 2 ml con una aguja de 28-30G antes de anestesiar al animal. </li><li> Disolver 50 mg de polvo de hidrocloruro de papaverina en 1 ml de solución salina normal estéril. Recoger la solución en una jeringa de insulina. Rompa la aguja de otra jeringa de insulina estéril. Colocar esta aguja en el extremo de un c 20m de largo PE10 tubo. Ahora fije este tubo PE10 en la aguja de la jeringa de insulina que contiene solución de hidrocloruro de papaverina para ser inyectado a través de la vena femoral para asegurar la vasodilatación y el llenado adecuado de los vasos. </li><li> Preparar otros dos jeringas de insulina que contienen cada uno 1 ml de solución salina. Las jeringas de insulina permitir la entrega suave del fluido inyectado en una ubicación precisa. Sin embargo, debido a la alta viscosidad del látex, alternativa jeringas de 1 ml con agujas extraíbles más amplios deben ser utilizados. </li><li> Tome todas las jeringas esterilizadas y los instrumentos de disección cercanas a la etapa de operación. Extender una hoja draper quirúrgico alrededor de la etapa de operación. La etapa de operación debe estar suficientemente iluminada y un microscopio de operación puede ser utilizado si es necesario. </li><li> Ahora se mide el peso corporal de un ratón C57BL6 / J y luego inducir la anestesia con isoflurano 5% vaporizado en 70% de N 2 O y 30% O 2. Continuar la anestesia con isoflurano 1% de vaporizado. Después POSICIONAM adecuadag del ratón sobre su espalda y la fijación de las extremidades, evaluar la profundidad de la anestesia. A continuación, cortar la piel sobre la vena femoral izquierda y localizar la vena. Insertar la punta afilada de la aguja que se coloca en PE10 tubo e inyecte lentamente solución de hidrocloruro de papaverina a una velocidad de 100 l por min (50 mg / kg de peso corporal). </li><li> Después de la inyección, corte de la cavidad abdominal y el diafragma perforar el. Cortar las costillas para exponer el corazón sin dañar o hematomas ella. Clip de la aorta torácica descendente con un fórceps arteriales. Haga un corte brusco en el atrio derecho para permitir que la sangre venosa drene hacia fuera. Inyectar 2 ml de solución salina en el ventrículo izquierdo, seguido por la inyección del látex coloreado manualmente con una ligera presión sobre 18-20 seg. Use las jeringas precargadas uno tras otro. Evitar la inyección de burbujas. </li><li> Después de la inyección de látex, dejar al animal durante 5 min. Decapita el animal y retire con cuidado todo el cerebro. Tome el buen cuidado de no dañar la arquitectura recipiente y cortex mientras se quita el hueso del cráneo y las meninges. Coloque el cerebro en un plato de cultivo celular de 60 mm que contiene 8.6 ml de 4% PFA para tomar una foto. Coloque una regla de medición en el plato transparente. Tomar fotos con un aumento de 25x de la dorsal y la superficie ventral del cerebro con una cámara conectada al microscopio. Las fotos se deben tomar en un ángulo de 90 ° entre la placa de Petri y el objetivo del microscopio con el fin de cuantificar la distancia original. </li><li> Repita el procedimiento en el otro 4-5 animales. </li></ol> 3. La tinción de los vasos cerebrales con mezcla de tintas de Carbono Negro <ol><li> Para comparar los resultados de base de látex tinción vascular cerebral con nuestro protocolo, prepara una mezcla de 100 l de Herlitz Stempelfarbe tinta (CB1) con 900 l de Pelican Scribtol Schwarz tinta (CB2) en un tubo de 1,5 ml EP. Preparar dos tubos de PE para hacer un volumen total de 2 ml de mezcla de CB1 y CB2 en una relación 1:9. Recoger la mezcla en dos jeringas de insulina separadas (1ml cada uno). Tapar las jeringas y colocarlos en un baño de agua caliente a 37 ° C. Recoger 2 ml de solución salina en otros dos jeringas de insulina, y colocarlos en un baño de agua también. </li><li> Se anestesia el animal y repetir el mismo procedimiento descrito anteriormente, excepto para la inyección de hidrocloruro de papaverina. Después de la inyección de 2 ml salaine en el ventrículo izquierdo, inyectar una mezcla de 2 ml de tintas de negro de carbono en lugar del látex de color. Realice las inyecciones a mano con una ligera presión sobre 18-20 segundos por ml. Clip de la aorta torácica descendente antes de las inyecciones. </li><li> El sacrificio del animal, retire el cerebro y tomar fotos. </li><li> Para conservación a largo plazo del cerebro, poner el cerebro en 4% de PFA durante la noche seguido por incubación con sacarosa con una concentración gradualmente creciente hasta que el cerebro flotante sumerge totalmente (5% seguido de 15% y luego% 30). Los cerebros de los animales perfundidos con el látex de color también puede ser conservada de la misma manera. </li><li> Lleve a cabo elprocedimiento en otros animales de 4-5. Para comparar la diferencia en la anatomía cerebral vascular debido a diferentes antecedentes genéticos o cepas, repita el protocolo en igual número de ratones ApoE KO y SV129, respectivamente. </li><li> Para estudiar la anatomía vascular en condiciones isquémicas, inducir transitorio de isquemia cerebral focal por 45 min o 90 min de acuerdo con un protocolo estándar de la oclusión intraluminal de la MCA bajo anestesia profunda (una descripción detallada de la cirugía está más allá del alcance de este artículo, la lector es referido a Doeppner et al., 2010). Al final del período de reperfusión planeado (1 día, o 5 días), lleve a cabo la tinción vascular con CB1 + CB2 únicas tintas y manchar todo el cerebro con 2% TTC solución a 37 ° C durante 5-10 minutos para identificar el volumen de infarto . TTC se reduce a formazán coloreado rojo por las enzimas mitocondriales (particularmente succinato deshidrogenasa). Después de la tinción TTC, las manchas de los tejidos metabólicamente activas de color rojo oscuro, mientras que el tejido infartado se mantiene unstained y parece blanco debido a la enzima mitocondrial disfuncional y desnaturalizado. Recoger imágenes de la misma manera como se describió anteriormente. </li></ol> 4. Estudio de los territorios vasculares cerebrales <ol><li> Para analizar la anatomía de los vasos cerebrales, las imágenes de la superficie dorsal del cerebro o bien teñidas con colores látex o CB1 CB2 + tintas pueden ser analizados con el software ImageJ (a Java basada en software de dominio público utilizado para el procesamiento de imágenes y análisis). Los cerebros perfundidos con látex de color puede mostrar grados variables de tinción recipiente situado en la superficie dorsal, mientras CB1 CB2 + perfusión se mancha todos los vasos en ambas superficies ventral y dorsal. </li><li> Abra un archivo de imagen con el software ImageJ. Identificar todos los puntos de anastomosis entre la ACA y MCA. Un punto de la anastomosis se define como el lugar donde el diámetro de los vasos es el más restringido o la distancia media entre los puntos más próximos de ramificación de la ACA y las ramas de MCA, respectivamente 2. <sarriba&gt; Trace una línea imaginaria (línea anastomótica), marcando los puntos de anastomosis con la ayuda de la herramienta de dibujo de línea segmentada y el plugin &quot;línea punteada&quot;. </li><li> Establecer la escala en mm. Mida la distancia entre la línea anastomótica y la línea media a 4 mm caudal al polo frontal del cerebro utilizando la herramienta de dibujo de línea recta y una herramienta de &quot;medida&quot;. Hacer lo mismo a 6 mm en sentido caudal desde el polo frontal. Analizar imágenes 3-4 por animal y calcular el valor medio. Compare los valores de los diferentes grupos de animales para identificar la variación de la anatomía vascular cerebral. En C57BL6 / J animales, la línea anastomótica entre la ACA y MCA se puede remontar más cerca de la línea media a 4 mm de menos 6 mm caudal del polo frontal. Ratones KO ApoE presentará ninguna diferencia significativa en este sentido. Por el contrario, en ratones SV129 la línea anastomótica se encuentran mm más allá y paralelamente a la línea tanto a 4 mm y 6 caudalmente desde el polo frontal. </li><li> Para el análisis de la anatomía vascular in condiciones isquémicas, realizar los mismos cálculos antes mencionados. Identifique el borde del infarto como el margen de tejido de color rojo y negro que representa el tejido metabólicamente activo y el tejido necrótico sin teñir, respectivamente. Medir la distancia de la frontera infarto de la línea media a 4 mm y 6 mm caudal a partir del polo frontal del cerebro. Se recoge el valor medio de las imágenes 3-4 por animal. Comparar los resultados entre diferentes isquemia y reperfusión períodos. </li></ol>

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	<li>Meng, H., Peng, Y., 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=Nuclear+contrast+angiography:+A+simple+method+for+morphological+characterization+of+cerebral+arteries.">Nuclear contrast angiography: A simple method for morphological characterization of cerebral arteries.</a> Brain Research. 1261, 75-81 (2009).</li><li>Maeda, K., Hata, R., 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=Differences+in+the+cerebrovascular+anatomy+of+C57black/6+and+SV129+mice.">Differences in the cerebrovascular anatomy of C57black/6 and SV129 mice.</a> Neuroreport. 9 (7), 1317-1319 (1998).</li><li>Coyle, P., Jokelainen, P. 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R., Nagel, F., 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=TAT-Hsp70-mediated+neuroprotection+and+increased+survival+of+neuronal+precursor+cells+after+focal+cerebral+ischemia+in+mice.">TAT-Hsp70-mediated neuroprotection and increased survival of neuronal precursor cells after focal cerebral ischemia in mice.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 29, 1187-1196 (2009).</li><li>Todo, K., Kitagawa, K., 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=Granulocyte-macrophage+colony-stimulating+factor+enhances+leptomeningeal+collateral+growth+induced+by+common+carotid+artery+occlusion.">Granulocyte-macrophage colony-stimulating factor enhances leptomeningeal collateral growth induced by common carotid artery occlusion.</a> Stroke. 39 (6), 1875-1882 (2008).</li><li>Sugiyama, Y., Yagita, Y., 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=Granulocyte+colony-stimulating+factor+enhances+arteriogenesis+and+ameliorates+cerebral+damage+in+a+mouse+model+of+ischemic+stroke.">Granulocyte colony-stimulating factor enhances arteriogenesis and ameliorates cerebral damage in a mouse model of ischemic stroke.</a> Stroke. 42 (3), 770-775 (2011).</li><li>Busch, H. 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R., 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=Increased+blood-brain+barrier+permeability+and+brain+edema+after+focal+cerebral+ischemia+induced+by+hyperlipidemia:+Role+of+lipid+peroxidation+and+calpain+1/2,+matrix+metalloproteinase-2/9,+and+RhoA+overoxidation.">Increased blood-brain barrier permeability and brain edema after focal cerebral ischemia induced by hyperlipidemia: Role of lipid peroxidation and calpain 1/2, matrix metalloproteinase-2/9, and RhoA overoxidation.</a> Stroke. 42, (2011).</li><li>Coyle, P., Jokelainen, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+outcome+to+middle+cerebral+artery+occlusion+in+spontaneously+hypertensive+stroke-prone+rats+(SHRSP)+and+Wistar+Kyoto+(WKY)+rats.">Differential outcome to middle cerebral artery occlusion in spontaneously hypertensive stroke-prone rats (SHRSP) and Wistar Kyoto (WKY) rats.</a> Stroke. 14, 605-611 (1983).</li><li>Coyle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diameter+and+length+changes+in+cerebral+collaterals+after+middle+cerebral+artery+occlusion+in+the+young+rat.">Diameter and length changes in cerebral collaterals after middle cerebral artery occlusion in the young rat.</a> The Anatomical Record. 210 (2), 357-364 (1984).</li></ol>

No hay conflictos de interés declarado.

La perfusión de CB1 + CB2 por inyección manual puede llevarse a cabo con éxito por sin entrenamiento intensivo, ya que no implica ningún dispositivo específico para implicar cierta presión 2,3. La heterogeneidad de los resultados de perfusión en nuestro protocolo es también insignificante. Sólo 1 de cada 16 animales no isquémicas animales y 3 de cada 20 animales isquémicos han mostrado perfusión incompleta. En estos casos, la incorporación de burbujas durante la perfusión de solución salina que...

<table border="1"><tbody><tr><td> Nombre de reactivo </td><td> Nombre de la compañía </td><td> N º de catálogo </td></tr><tr><td> Scribtol Schwarz (CB2) </td><td> Pelican, Alemania </td><td> 221 135 </td></tr><tr><td> Stempelfarbe (CB1) </td><td> Herlitz PBS AG, Alemania </td><td> 10417202 </td></tr><tr><td> Gedeo Latex </td><td> Pébéo, Francia </td><td> 13042B </td></tr></tbody></table>

intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels

La estructura anatómica de los vasos cerebrales es un factor determinante para la hemodinámica cerebral, así como la gravedad de la lesión después de lesiones isquémicas. La vasculatura cerebral dinámicamente responde a varios estados fisiopatológicos y presenta considerables diferencias entre cepas y bajo condiciones de manipulaciones genéticas. Esencialmente, una técnica fiable para la tinción de vasos intracraneales es esencial con el fin de estudiar la patogénesis de la apoplejía isquémica. Hasta hace poco, un conjunto de diferentes técnicas se ha empleado para visualizar la vasculatura cerebral incluyendo la inyección de resina de baja viscosidad, araldite F, gelatina mezclada con diversos colorantes 1 (es decir, rojo carmín, India tinta) o látex con 2 o 3 sin negro de carbón. La perfusión de blanco compuesto de látex a través de la aorta ascendente se ha reportado por primera Coyle y Jokelainen 3. Maeda et al. 2 han modificado el protocolo mediante la adición de carbon negro de tinta para el compuesto de látex para la visualización mejorada de contraste de los vasos después de la perfusión de solución salina del cerebro. Sin embargo, la perfusión ineficiente y llenado inadecuado de los recipientes se experimentan con frecuencia debido a la alta viscosidad del compuesto de látex 4. Por lo tanto, se ha descrito una técnica simple y rentable utilizando una mezcla de dos tintas de carbono comercialmente disponibles negros (CB1 y CB2) para visualizar la vasculatura cerebral de una manera reproducible 5. Hemos mostrado que la perfusión con CB1 CB2 + en ratones resulta en la tinción de los vasos cerebrales significativamente más pequeños en una densidad más alta en comparación con la perfusión de látex 5. A continuación, describimos nuestro protocolo para identificar los puntos de anastomosis entre la parte anterior (ACA) y las arterias cerebrales medias (MCA) para el estudio de las variaciones de los vasos en ratones con diferentes antecedentes genéticos. Por último, demostrar la viabilidad de nuestra técnica en un modelo transitorio de isquemia cerebral focal en ratones mediante la combinación de CB1 +CB2 mediada recipiente de tinción con tinción con TTC en diversos grados de lesiones isquémicas.

El protocolo descrito aquí supera las limitaciones técnicas de visualización convencional a base de látex de la vasculatura cerebral de roedor. Figura 1A muestra que la perfusión de la siguiente látex coloreado, sólo los vasos grandes en la superficie ventral están manchadas, dejando la superficie dorsal sin teñir. El resultado es también muy variable. Sólo un animal de cada seis muestra la tinción parcial de la ACA y MCA el visible en la superficie dorsal del cerebro (datos no mostrados). P...

Watch this Scientific Journal Video about Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels at JoVE.com

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Análisis de la anatomía cerebrovascular roedor juega un papel importante en la investigación de accidente cerebrovascular experimental. En este contexto, la perfusión intravascular con látex de color ha sido considerado como una herramienta estándar para varios años. Sin embargo, esta técnica implica distintas limitaciones técnicas, que socavan su reproducibilidad. Aquí se describe un método simple para visualizar los vasos cerebrales de una manera reproducible. La inyección de una mezcla de dos tintas negras de carbono comercialmente disponibles a través de los resultados de miocardio en el ventrículo izquierdo llenado adecuado de los vasos cerebrales, con visualización de alto contraste. Hemos aplicado con éxito esta técnica para identificar los puntos de anastomosis entre territorios vasculares cerebrales de ratones con diferentes antecedentes genéticos. Finalmente dan evidencia de que este método novedoso y sencillo para la tinción recipiente se pueden combinar con cloruro de trifeniltetrazolio (TTC) Tinción - una herramienta ampliamente utilizada para observar y analizar los volúmenes de infarto en ratones.

La perfusión intravascular de Carbono Negro Tinta fiable permite la visualización de los vasos cerebrales

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

intravascular-perfusion-carbon-black-ink-allows-reliable

Research

JoVE Journal

Neuroscience

15.3K vistas.  University of Duisburg-Essen Medical School. Análisis de la anatomía cerebrovascular roedor juega un papel importante en la investigación de accidente cerebrovascular experimental. En este contexto, la perfusión intravascular con látex de color ha sido considerado como una herramienta estándar para varios años. Sin embargo, esta técnica implica distintas limitaciones técnicas, que socavan su reproducibilidad. Aquí se describe un método simple para visualizar los vasos cerebrales de una manera reproducible...

Video: Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Modelo de isquemia cerebral focal por oclusión endovascular de sutura de la arteria cerebral media en la rata

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Investigación

Neurociencias

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Reproducción automática de vídeo interactivo de Estudios de la Comunicación Animal

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Marcaje fluorescente retrógrada Permite dirigida extracelular sola unidad de grabación de neuronas identificadas<em&gt; En vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando<em&gt; En el útero</em&gt; La electroporación

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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

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

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

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

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

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Electrofisiología en aislados Preparativos Cord-tronco cerebral espinal de roedores recién nacidos permite la grabación de salida de red neuronal Respiratorio

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

La purificación de los buques de cerebro de ratón

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

El aislamiento y la canulación del parénquima cerebral arteriolas

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Intracerebroventricular y por inyección intravascular de partículas víricas y fluorescentes microperlas en el cerebro neonatal

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Un modelo de rata de hipoperfusión leve intrauterina con microcoil de transcatheter estenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...