This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

NOTA: Todos los procedimientos y manejo de los animales cumplido con las pautas de ética NIH y los requisitos del protocolo aprobado del Comité de Cuidado y Uso de Animales Institucional de la Universidad de California en San Francisco. 1. Preparación del Animal de Cirugía <ol><li> Llevar a cabo procedimientos quirúrgicos en un espacio dedicado limpio. Autoclave todos los instrumentos quirúrgicos, esterilizar con un esterilizador de vidrio de grano antes de la cirugía. NOTA: En este protocolo, utilice el día postnatal 10-12 (P10-12) ratones FVB. Diferentes edades y razas de ratones se pueden utilizar para satisfacer las necesidades de un proyecto específico. Ratones mayores de P40 puede ser un reto porque el hueso bulla se hace más difícil. </li><li> Anestesiar a los ratones con inyección intraperitoneal de una mezcla de clorhidrato de ketamina (100 mg / kg), clorhidrato de xilazina (10 mg / kg) y acepromacina (2 mg / kg). Comienza la preparación quirúrgica sólo después de que el animal ya no responde a los estímulos dolorosos, como la pizca dedo del pie. Si necessary, administrar una dosis de refuerzo (un quinto de la dosis original) del cóctel anestésico para restaurar el plano original de anestésico. NOTA: Tenga cuidado en este paso porque la mayor parte de la mortalidad observada en esta edad es causada por sobredosis de anestésico. </li><li> Cubrir los ojos del animal con una pomada oftálmica de protección para mantener los ojos húmedos durante la anestesia, la supresión de parpadeo reflejo del animal. </li><li> Coloque el ratón con cuello extendido sobre una almohadilla caliente durante todo el procedimiento hasta que el ratón está totalmente despierto, para prevenir la hipotermia post-anestesia. </li><li> Afeitarse la región post-auricular izquierda con un clipper y desinfectar con etanol al 70% y la povidona yodada antes de la manipulación quirúrgica. </li></ol> 2. El procedimiento quirúrgico y la inyección del vector <ol><li> Utilice un enfoque post-auricular para exponer la bulla timpánica. Incisión en el tejido subcutáneo con unas tijeras pequeñas para exponer el músculo post-auricular. Después de la retracción del tejido adiposo a Mlado posterior e de la incisión, separar los músculos a la derecha y el lado izquierdo perpendicular a la incisión para exponer el hueso temporal. Asegúrese de que esta incisión es el tiempo suficiente para trabajar cómodamente con una visualización adecuada. </li><li> Perforar la bulla timpánica con una aguja 25 G y ampliar el agujero como sea necesario con unas pinzas por el pelado de la columna vertebral para permitir el acceso a la espira basal de la cóclea, y luego ampliar suficientemente para visualizar la arteria estapedial y la membrana de la ventana redonda (RWM) . </li><li> Perforar el RWM suavemente en el centro con una pipeta capilar de borosilicato. Observe flujo de salida de líquido a través de la RWM en este punto; esto es normal. Esperar hasta que el flujo de salida se ha estabilizado (el líquido effluxed de la cóclea se seca con un papel de filtro estéril). NOTA: Tenga cuidado al sostener y hacer avanzar la aguja de vidrio para que la perforación RWM es lo más pequeño posible. Dependiendo del microscopio usado, tienen las pipetas usando un micromanipulador o a mano usando una pipetatitular. </li><li> Preparar las pipetas para inyección con una pipeta extractor se preparan de la misma manera pipetas de patch clamp. Asegúrese de que el diámetro de la punta es grande (unos 15 m de diámetro) para que dispensó el virus de entrada y salida se hace fácilmente. </li><li> Elaborar 1 a 2 l de AAV1-VGLUT3, AAV1-GFP o AAV2-GFP en una pipeta de inyección. Después se estabiliza el flujo de salida (5 a 10 minutos), microinyectar este volumen fijo en la rampa timpánica por el mismo agujero hecho previamente en RWM. </li><li> Sellar el nicho RW rápidamente después de sacar la pipeta con un pequeño tapón de músculo y seguro que con una pequeña gota de adhesivo tisular colocado en el músculo para evitar la fuga de la ventana redonda. NOTA: Selle este agujero muy bien después se retira la aguja con un pequeño tapón de músculo para evitar cualquier fuga perilinfa de la cóclea, este paso es fundamental para preservar la audición. El fracaso para sellar completamente el VM se traducirá en la pérdida de audición en el tiempo. </li><li> Después de la inyección, cubra elagujero en la ampolla auditiva con el tejido adiposo, los músculos de regreso post-auricular y el tejido adiposo a su posición normal y la sutura de la herida en capas con una sutura absorbible crómico 6-0 o más pequeño. </li><li> Desinfectar la herida con povidona yodada. </li></ol> 3. Cuidado posoperatorio <ol><li> Coloca los ratones en una jaula caliente y no los deje sin supervisión hasta que estén completamente recuperados luego pasar de nuevo con la madre. Se recomienda retirar el macho de la jaula antes de poner los cachorros de vuelta con la madre. </li><li> Administrar subcutánea carprofeno (2 mg / kg) para la analgesia después de la operación y cada 24 h durante 3 días a partir de entonces, para gestionar la inflamación y el dolor. Monitorear los animales diariamente para detectar signos de sufrimiento, pérdida anormal de peso, dolor o infección. Generalmente por el 3er día después de la cirugía todos los ratones deberían estar actuando normalmente. Si cualquier signo de sufrimiento o la enfermedad aparecen en un ratón después de los 3 días, considere la eutanasia de tél ratón según las directrices institucionales. </li></ol> 4. Evaluación de la función coclear después de un parto Viral Utilizando respuesta auditiva del tronco cerebral (ABR) Grabaciones NOTA: La respuesta auditiva del tronco cerebral (ABR) es una auditivo potencial extraído de la actividad eléctrica en curso en el cerebro y grabado a través de electrodos colocados bajo el cuero cabelludo evocado. El animal se estimula con el sonido. La grabación resultante consiste en cinco ondas que reflejan la actividad eléctrica de los puntos sucesivos en la vía auditiva en los primeros 10 mseg después de la aparición de un estímulo auditivo. <ol><li> Anestesiar ratones por inyección intraperitoneal de una mezcla de clorhidrato de ketamina y clorhidrato de xilazina como se describe en la sección 1.2 de este protocolo, sólo que sin la acepromacina. </li><li> Coloque el ratón sobre una almohadilla de calefacción en toda la prueba de audición hasta que el ratón está totalmente despierto, para prevenir la hipotermia post-anestesia. NOTA: El usolos siguientes estímulos sonoros en este experimento: haga clic (5 milisegundos de duración; 31 Hz tasa de presentación). </li><li> Coloque los electrodos de aguja subdérmicos en el vértice, por debajo del pabellón auricular del oído izquierdo (de referencia), y por debajo de la oreja contralateral (tierra) y empezar ABRs grabación como se describe anteriormente en una cámara insonorizada 27,28. Grabar formas de onda ABR en 5 intervalos de nivel de presión sonora dB por debajo del máximo de amplitud. </li><li> Determinar los umbrales ABR postoperatorio tan pronto como 4 días después del parto viral NOTA: Umbral se define como el nivel de estímulo más bajo al que de respuestas de los picos para las ondas I-V eran claramente y repetidamente presente en la inspección visual. </li><li> Mida Wave I para analizar la actividad del nervio coclear. El nivel de estímulo más bajo que produce una forma de onda de ABR detectable se define como el umbral. </li></ol> 5. Coclear Transgén expresión de la proteína mediante inmunofluorescencia <ol><li> Anestesiar ratones por una sobredosis de intrapinyección eritoneal de una mezcla de clorhidrato de ketamina y clorhidrato de xilazina como se describe en la sección 1.2 de este protocolo, sólo que sin la acepromacina. </li><li> Decapitar la cabeza solamente después que el animal ya no responde a los estímulos dolorosos, como la pizca dedo del pie. </li><li> Diseccionar las cócleas y proceso para todo el montaje de inmunofluorescencia como se describe 26. Un protocolo detallado de coclear inmunofluorescencia de montaje en toda utilizando anticuerpos anti-7a anti-miosina VGLUT3 y se describe en Akil et al., 2013 29. </li><li> Para el etiquetado GFP, incubar los cocleares enteros monta la noche a 4 ° C con un anticuerpo de conejo anti-GFP en dilución 1: 250. </li><li> Enjuague el cochleae dos veces durante 10 min con PBS y luego se incuba durante 2 horas en IgG de cabra anti-conejo conjugado con Cy2 diluido 1: 4000 en PBS. </li><li> Enjuague el cochleae dos veces con PBS durante 10 minutos y se incuba con DAPI durante 15 min. </li><li> Monte el cochleae en portaobjetos de vidrio y observar under un microscopio con inmunofluorescencia confocal. </li></ol>

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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=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. G., 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=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

En este trabajo, se describe en detalle una técnica que se puede utilizar para la terapia génica coclear, con el objetivo de restaurar o rescatar la función auditiva normal que se ve comprometida por un defecto genético. Como es típicamente atraumática, este enfoque es seguro para la transferencia de genes coclear u otras terapias moleculares potenciales 30. Se han descrito otros enfoques para la terapia coclear, incluyendo un enfoque ventral 24, cocleostomía 31,32 y el saco endol...

La terapia génica durante mucho tiempo ha sido sugerido como un posible tratamiento para la pérdida auditiva genética, pero el éxito en esta área ha sido difícil de alcanzar 1. Hasta la fecha, las metodologías mediadas por virus han predominado por la capacidad teórica para dirigirse a tipos específicos de células dentro de la cóclea relativamente inaccesible. Ambos adenovirus (AV) y el virus adeno-asociado (AAV) se han utilizado para la entrega de genes coclear. AAV son ventajosos en la cóclea por un número de razones. Ellos son virus deficientes en la replicación y pueden transferir de manera eficiente moléculas transgénicos para diferentes tipos de células incluyendo neuronas, un objetivo importante para un número de causas de la pérdida de audición. Entrada AAV en la célula está mediada por receptores específicos 2; Por lo tanto, la elección de un serotipo particular, debe ser compatible con los tipos de células a ser transducidas. AAV puede transfectar eficazmente células ciliadas 3 e incorporar en el genoma del huésped, lo que resulta en una expresión estable y a largo plazo de la traproteína nsgenic y cambio fenotípico en la celda 4. Aunque no necesariamente ventajoso para aplicaciones de corto plazo, tales como la regeneración de células de pelo, expresión a largo plazo es muy importante para el rescate estable de defectos genéticos. Debido AAV no están asociados con ninguna enfermedad humana o infección y demuestran no ototoxicidad 5,6,7, son un candidato ideal para su uso en la terapia génica para formas heredadas de la pérdida de audición 8. Transferencia de material genético exógeno en el oído interno de los mamíferos utilizando vectores virales se ha estudiado en la última década y se perfila como una técnica prometedora para el tratamiento de las formas tanto genéticas y adquiridas de la pérdida de 9 de la audición. La cóclea es potencialmente una diana ideal para la terapia génica por varias razones: 1) su volumen pequeño requiere una cantidad limitada de que el virus sea necesario; 2) su relativo aislamiento de otros efectos secundarios límites del sistema de órganos; y 3) sus cámaras llenas de líquido facilitan viralentrega en todo el laberinto 10, 11,12,13,14, 15. Los modelos de ratón de la sordera congénita permiten el uso de muchos métodos de estudio para monitorear el desarrollo del oído interno de una manera sistemática, replicable. Mientras que el pequeño tamaño de cochleae ratón no presenta cierta dificultad quirúrgica, el ratón sirve como un modelo extremadamente importante en el estudio de la pérdida auditiva genética, con varias ventajas sobre otras especies experimentales 16. Los modelos de ratón permiten la evaluación de una serie de características a través de análisis de ligamiento genético, colección de observaciones morfológicas detalladas, y la simulación de escenarios patógenos; Como tales, son buenos candidatos para la terapia génica mediada por virus. Extensos estudios genéticos en ratones combinados con los avances tecnológicos han hecho posible la generación de ratones modificados genéticamente de forma reproducible a través de laboratorios de 17,18, 19, 20,21. Furthermore, existen numerosos modelos para ambos adquiridas y heredadas de la audición fenotipos de pérdida en ratones, lo que permite a rigurosas pruebas en este modelo animal 22, 23,24. Por lo tanto, la corrección de la audición mediante la terapia génica mediada por virus en un modelo de ratón es un primer paso apropiado en la búsqueda de una cura para la enfermedad humana. Hemos demostrado previamente que los ratones transgénicos que carecen de glutamato vesicular transportador 3 (VGLUT3) nacen sordos debido a la falta de liberación de glutamato en la sinapsis cinta IHC 25. Debido a que esta mutación no conduce a una degeneración primaria de las células ciliadas sensoriales, estos ratones mutantes son potencialmente un excelente modelo en el que para probar la terapia génica coclear para la pérdida de audición congénita. Hasta la fecha, un número de técnicas de administración virales para la terapia génica coclear se han descrito, incluyendo la difusión membrana de la ventana redonda, la inyección de membrana de la ventana redonda, y la entrega a través de una cocleostomía. Hay potenteventajas ial y desventajas de cada uno de estos enfoques 9. Aquí mostramos un método quirúrgico para la entrega de genes mediada por virus en el oído interno VGLUT3 KO ratón a través de la membrana de la ventana redonda (RWM). El método de inyección de RWM post-auricular es mínimamente invasiva con la preservación audiencia excelente, y es relativamente rápido. Como hemos publicado anteriormente, en un esfuerzo para restaurar la audición en este modelo de ratón, un vector de AAV1 que lleva el gen VGLUT3 (AAV1-VGLUT3) se introdujo en la cóclea de estos ratones sordos en el día postnatal 12 (P! @), Resultando en la restauración de la audición 26. Audición en los ratones KO VGLUT3 se verificó por la respuesta del tronco encefálico (ABR), mientras que la expresión de proteínas transgén se verificó mediante inmunofluorescencia (IF). Por tanto, esta metodología demuestra que la terapia génica mediada por virus-puede corregir un defecto genético que de otro modo resulta en sordera.

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			<td height="20" style="height:20px;">TDT BioSig III System&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>&#160;Tucker-Davis Technologies</td>
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surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

La terapia génica, que se utiliza para lograr la recuperación funcional de la sordera neurosensorial, se compromete a conceder una mejor comprensión de los mecanismos moleculares y genéticos subyacentes que contribuyen a la pérdida de la audición. Introducción de vectores en el oído interno debe hacerse de una manera que distribuye ampliamente el agente a lo largo de la cóclea al tiempo que minimiza la lesión a las estructuras existentes. Este manuscrito describe un enfoque quirúrgico post-auricular que se puede utilizar para la terapia coclear ratón usando molecular, farmacológica, y la entrega viral a ratones día postnatal 10 y mayores a través de la membrana de la ventana redonda (RWM). Este abordaje quirúrgico permite una entrega rápida y directa en la rampa timpánica y reducir al mínimo la pérdida de sangre y evitar la mortalidad de los animales. Esta técnica implica un daño insignificante o no a las estructuras esenciales del oído medio e interno, así como los músculos del cuello, mientras su totalidad preservar la audición. Para demostrar la eficacia de esta técnica quirúrgica, el glutam vesicularcomió transportador 3 knockout (KO VGLUT3) los ratones se utilizará como un ejemplo de un modelo de ratón de la sordera congénita que recupera la audición después de la entrega de VGLUT3 al oído interno utilizando un virus adeno-asociado (AAV-1).

Para verificar las características técnicas y utilidad del enfoque post-auricular para la terapia molecular coclear, AAV1-VGLUT3, AAV1-GFP y AAV2-GFP se entregaron en ratones P10-12 oído interno a través de la RWM. Este enfoque demuestra con éxito la expresión del transgén dentro de las células ciliadas internas (IHC) (VGLUT3 Figura 1 y Figura 2 GFP y GFP Figura 3A), las células ciliadas externas (OHC) (GFP Figura 2) y células de soporte (GFP ...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Método quirúrgico para la entrega de genes mediada por virus de la oreja del ratón Interior a través de la ventana de membrana Ronda

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.1K vistas.  University of California, San Francisco. The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane. 

Video: Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Microscopia intravital de la microcirculación del cerebro del ratón con una ventana craneal cerrado

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Investigación

Neurociencias

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Médula Espinal Electrofisiología II: Fabricación de electrodos de aspiración extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Aislar las células que expresan LacZ de tejidos de ratón del oído interno mediante citometría de flujo

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Transferencia de genes a la oreja del ratón en desarrollo por el interior<em&gt; En Vivo</em&gt; La electroporación

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

Un pulido y reforzado Diluido cráneo-Ventana para a largo plazo de imágenes del cerebro de ratón

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Grabaciones de patch clamp en Inner células del oído pelo Aislado de pez cebra

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Formación de imágenes pHluorin-etiquetados Inserción del receptor a la membrana plasmática en neuronas primarias de ratón en cultivo

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologías combinadas para definir el papel de la membrana plasmática de entrega Durante Axon ramas y morfogénesis neuronal

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Técnica ratón Microcirugía de infusión para Targeted Sustancia de entrega en el SNC<i&gt; vía</i&gt; La arteria carótida interna

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Laminectomía e implantación de la ventana de la médula espinal en el ratón

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...