La financiación de este trabajo fue proporcionado por un Passe Garnett y Williams subvención Proyecto Memorial Foundation Rodney R. Lim y AJ Camp.

1. Animales <ol><li> Los ratones se obtuvieron del Centro de roedores Australiano (ARC, Perth, Australia) y se mantuvieron en la Universidad de Sydney Bosch Animalario en un ciclo de luz / oscuridad de 12 horas normal con enriquecimiento ambiental. Todos los experimentos descritos fueron aprobados por la Universidad de Sydney Comité de Ética Animal. </li><li> Ratones machos y hembras (C57/Bl6) se utilizaron para todos los experimentos ya que esta cepa se utiliza comúnmente como el fondo para la manipulación genética, y pueden considerarse equivalentes a wildtype 8-9. </li></ol> 2. Preparación del tejido <ol><li> Preparar 300 ml de un fluido artificial basado en glicerol cefalorraquídeo (ACSF) formado por (en mM): 26 NaHCO3, 11 de glucosa, 250 glicerol, 2,5 KCl, 1,2 NaH 2 PO 4, 1,2 MgCl2, CaCl2 y 2.5. Antes de la adición de CaCl2, gas de la solución con carbógeno (95% de O2 y 5% de CO 2) para establecer APH de 7,4 y evitar la precipitación de calcio (nubosidad). Congelar la solución en un congelador a -80 ° C durante 45 min de manera que se forma una suspensión de hielo. </li><li> Profundamente anestesiar a los ratones con ketamina (100 mg / kg) (Parnell, Alejandría, Australia) a través de una inyección intra-peritoneal. </li><li> Una vez reflejos pizca extremidades posteriores son ratones decapitar ausentes utilizando tijeras afiladas de acero inoxidable y hacer una incisión sagital con una hoja de afeitar (redondeado # 22) para exponer el cráneo. En este punto ya lo largo de los pasos 02.03 a 02.08 de la bóveda craneal, cerebro y aparato vestibular subyacente debe mantenerse lo más fresco posible por la aplicación regular de ACSF helada sobre el tejido. </li><li> Usando el brazo puntas de las tijeras del dibujo estándar (FST, North Vancouver, Canadá) hacer una pequeña incisión en el cráneo en Lambda y se corta a lo largo de la sutura sagital. Asegúrese de que el cerebro no se &quot;arrastrado&quot; por la cuchilla de cizalla durante este paso. </li><li> Retire con cuidado los huesos parietales de distancia lateral y el hueso occipital posteriorly con baja curva pearson gubia (FST). </li><li> Una pequeña espátula de acero inoxidable se utiliza a continuación para levantar suavemente el cerebro de la superficie de la fosa craneal media y posterior, y el nervio vestibulocochlea expuesta (CNVIII) cortada a medio camino entre el oído interno y el tronco cerebral con un par de tijeras iris finas. Cortar este nervio minimiza la tensión indebida en los axones de las fibras aferentes primarias y sus conexiones con las células ciliadas. </li><li> Después de la sección transversal del CN VIII del cerebro se elimina en su totalidad. </li><li> El laberinto vestibular es ahora claramente visible en la fosa craneal media, con la cóclea apunta anteromedialmente. Pinzas gubias de cada lado de la laberinto vestibular antes de la escisión suavemente agarrando el canal semicircular anterior y tirando lateralmente. </li><li> Sumerja los laberintos extirpados (Figura 1) en un plato de disección que contiene la ACSF helada, gaseado continuo descrito en la sección 2.1. Bajo un microscopio estereoscópico, mantenga el laberinto dela base del plato de agarre por la cóclea. Utilice unas pinzas finas para rascarse lejos en el hueso que cubre la ampolla del canal semicircular anterior. Una vez que se consigue un pequeño agujero en el hueso comenzar a chasquear el hueso fuera de la ampolla. Se debe tener cuidado de no empujar la pinza a través del hueso ya que esto puede causar daños en el laberinto membranoso subyacente y el epitelio sensorial. Continúe con este procedimiento hasta tanto el anterior y los conductos semicirculares membranosos horizontales adyacentes y ampollas están expuestos (Figura 2). </li></ol><img alt="Figura 1" src="/files/ftp_upload/50471/50471fig1.jpg" /> Figura 1. El aislado del ratón laberinto vestibular. A. El panel izquierdo) Representación esquemática de los aislados del ratón laberinto vestibular. Los puntos importantes de referencia para acceder al epitelio sensorial vestibular, la cóclea, anterior y horizontal canales semicirculares son de laboratorioeLED. Los asteriscos indican la ampolla del canal semicircular que contiene el epitelio sensorial vestibular. B. Panel de la derecha) Fotomicrografía de la laberinto vestibular aislado de un ratón de 1 mes de edad. <img alt="La figura 2" src="/files/ftp_upload/50471/50471fig2.jpg" /> Figura 2. Exposición del laberinto vestibular membranoso. El hueso que recubre la ampolla del canal semicircular anterior y horizontal han sido rayado para revelar las ampollas membranosas negro / marrón con manchas y los nervios ampulares asociados (CNVIII). El esquemática en el panel inferior representa las estructuras en la región resaltada de la microfotografía y muestra la relación de los conductos del conducto semicircular a las ampollas y CNVIII. <ol start="10"><li> Con unas pinzas finas, levante suavemente las ampollas y utrículo asociado lejos del laberinto óseo, asegurando que la región central de las ampollas (contanando el epitelio sensorial) no está dañado. En algunos casos, la parte proximal del conducto semicircular membranosa puede ser necesario cortar con unas tijeras iris para liberar las ampollas de la médula. </li><li> Transferir la tríada que contiene los dos ampollas y utrículo en una placa de Petri llena de L-15 de Leibovitz medio de cultivo (Sigma-Aldrich, St Louis, MO). Use una fibra óptica para &quot;backlight&quot; el tejido. Esto permite una visualización clara de la cresta que contiene el epitelio sensorial dentro de las ampollas. </li><li> Con cuidado de una incisión en el &quot;techo&quot; moteado del utrículo con las finas tijeras iris. Continuar esta incisión a través del techo de la anterior y a continuación, las ampollas horizontal. Hacer la incisión lo más cerca posible del borde del epitelio sensorial como sea posible sin contacto con él (Figura 3A). Asegúrese de que no queden trozos de membrana que recubre el epitelio sensorial (Figura 3B) </li><li> Transferir la preparación intacta semi aislado de una pequeña glascámara de registro s de fondo lleno de L-15 medios de comunicación. Pesar abajo de la preparación utilizando una rejilla de fibras de nylon finas fijadas a un alambre de platino en forma de U aplanada (Figura 3B). Idealmente, las fibras de la red no deben superponerse el epitelio sensorial, sin embargo, en algunos casos donde se requiere una mayor estabilidad, una sola fibra transección del epitelio sensorial tal vez preferido. </li></ol><img alt="Figura 3" src="/files/ftp_upload/50471/50471fig3.jpg" /> Figura 3. El aislado preparación semi-intacta del epitelio. A. Representación esquemática sensorial vestibular de la preparación semi-intacta y configuración de los electrodos. La ampolla que cubre la cresta ha sido &quot;de-techo&quot; para exponer la superficie del epitelio sensorial (verde). B. Microfotografía de la semi-intacta preparación &quot;tríada&quot; que muestra el anterior (ac) y horizontal (hc) crista (utrículo oscurecido serhind ac). Tenga en cuenta la fibra de nylon utilizado para asegurar la preparación de la base de la cámara de registro. Barra de escala:. 100 micras C. Un electrodo de registro colocado en una celda de pelo vestibular individual. Barra de escala: 15 m. 3. Electrofisiología <ol><li> Perfundir la preparación semi-intacta con oxigenada continuamente medio L-15. Los medios de comunicación contiene un indicador de pH (rojo de fenol) y el color del medio deben ser controlados a través de las grabaciones. El pH con el oxígeno debe ser 7.3 a 7.4 y corresponden a un color rojo. </li><li> Preparar las pipetas de registro de 1,5 mm (1,19 mm ID) de vidrio de borosilicato usando un protocolo de dos pasos (paso de calentamiento 1: 72, y el paso de calor 2: 50) en un extractor de micropipeta (Narishige, Japón, modelo PP-830) para lograr una impedancia de final 3-4 mW. </li><li> Llenar el vástago de la pipeta con 3-4 mm de potasio solución interna con base de fluoruro que contiene (en mM) 110 KF, KCl 12, 27 KOH, 1 NaCl, 10 HEPES, 10 EGTA, 1,8 MgCl 2,3 D-glucosa, 2 y Na-ATP, pH 7,4 con KOH. </li><li> Envolver el vástago de la pipeta 2-3 veces y lo más abajo de la punta como sea posible con una tira delgada de parafina para aislar el electrodo y reducir la capacitancia pipeta. </li><li> Coloque la pipeta en el epitelio sensorial bajo aumento de potencia (5X) en un microscopio vertical (Olympus BX51). Cambie a alta potencia (40X) y visualizar las células ciliadas vestibulares individuales con una cámara CCD adjunta. </li><li> Posición pipeta sobre la membrana de una célula de pelo visualizaron (Figura 3C) usando un micromanipulador (Sutter Instruments, California, EE.UU.). Una vez que se logra un sellado gigaohmio, la ruptura de la membrana celular con una pequeña cantidad de presión negativa aplicada a través de un puerto de succión en el soporte de pipeta. </li><li> Realiza grabaciones de fijación de voltaje de células enteras utilizando técnicas estándar 8-9. </li></ol> 4. Dos fotones Microscopía <ol><li> Preparar una solución salina al 0,9% que contiene 5 mM de Oregon Green488 BAPTA-1 (OGB-1; sal hexapotassium; Invitrogen, Alemania). </li><li> Aunque todavía sumergido, coloque el &quot;de-techo&quot; preparación tríada semi-intacta en un pequeño trozo de papel de filtro (4x4 mm, 0,8 m de espesor; Millipore, Alemania) asegurarse de que el epitelio sensorial está obstruida desde arriba. </li><li> Transferir el papel de filtro y la preparación sobre el electrodo de platino base de solución salina cubierta (7 l) de un electroporador que consiste en la combinación de un generador de impulsos y un amplificador de banda ancha, y la cubierta con un 5 l de solución 5 mM de calcio sintético colorante indicador de Oregon Green 488 BAPTA-1 (Figura 4). </li></ol><img alt="Figura 4" src="/files/ftp_upload/50471/50471fig4.jpg" /> La Figura 4. Electroporación granel. A. esquemática en sección transversal de configuración que muestra la electroporación. La preparación semi-intacta (*) se coloca en un trozo de papel de filtro sumergido en dy calcioe (Oregon Green 488 BAPTA-1) y la corriente aplicada entre 2 electrodos de platino. Adaptado de Briggman y Euler, 2011 10. <ol start="4"><li> Traer la parte superior paralelo electrodo de platino con el electrodo de base a una distancia de aproximadamente 2 mm, teniendo cuidado de no perturbar la orientación de la preparación, cuando el contacto con el verde 488 BAPTA-1 solución de Oregon recibe. </li><li> Pasar un breve impulso de corriente a través de la preparación (Parámetros para la corriente; 13 V, 10 mseg de anchura de pulso, frecuencia 1 Hz, 10 pulsos de onda cuadrada) 10-11. </li><li> En la parte inferior de una cámara de registro con fondo de vidrio lleno de medio L-15, colocar una pequeña gota de aceite de silicona y la posición de la preparación semi-intacta en él, de nuevo asegurándose de que el epitelio sensorial es sin obstrucciones desde arriba. Para garantizar la estabilidad en toda la imagen, vuelva a colocar la rejilla de nylon en la preparación como se describe anteriormente (véase el punto 2.13). </li><li> El uso del microscopio de dos fotones realizar grabaciones ópticas de esponla actividad del calcio nea en el epitelio sensorial (Figura 7), utilizando protocolos estándar de imaginología, 10-11. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Los mecanismos que subyacen a nuestro sentido del equilibrio han recibido poca atención en comparación con otros sistemas sensoriales, por ejemplo, los sistemas visual y auditivo. De los estudios que han investigado los cambios en la función vestibular o el balance, la mayoría se han centrado en medidas conductuales como el reflejo vestíbulo-ocular, con un conocimiento incompleto de los pilares fundamentales de la balanza de las propias células ciliadas vestibulares de construcción. Los estudios que se h...

A pesar de la importante contribución del sistema vestibular en nuestra vida diaria, una clara comprensión de los procesos responsables de la disminución observada en la función vestibular con la edad, siendo difícil de alcanzar. Una razón para esta falta de conocimiento es que la disminución de la función vestibular ha sido casi exclusivamente explorado el uso de ensayos de comportamiento, incluyendo el reflejo vestíbulo-ocular (VOR), un indicador preciso de la función vestibular extrínseca, pero proporciona información limitada sobre los cambios de los componentes intrínsecos . Esto es un obstáculo importante para nuestra comprensión de la función de las células ciliadas vestibulares en la salud, la enfermedad o el envejecimiento. Aunque ha habido muchos estudios sobre las células ciliadas vestibulares individuales, una deficiencia importante ha sido la dependencia de las preparaciones de células de cabello agudos, en los que las células capilares y terminales aferentes incluso cáliz son retirados de su ambiente normal mediante tratamiento mecánico y / o enzimática. Tal enfoques inevitablea perturbar el delicado microarquitectura entre las células ciliadas y del cáliz, y el pelo de células y células de apoyo. Con el desarrollo de preparaciones semi-intacta 1-5, y un ratón aislado preparación laberinto 6, ahora existe la oportunidad de estudiar las distintas formas de comunicación sináptica en condiciones que se parecen más a los de vivo. En efecto, Lim et al. (2011) mostró marcadas diferencias en las corrientes de células enteras grabadas de tipo agudo aislado células ciliadas vestibulares I en comparación con aquellos que permanecieron incrustados dentro del neuroepitelio. Específicamente, se cree potasio a acumularse en el espacio intercelular, entre la célula de pelo y cáliz aferente, y altera significativamente la respuesta de la célula de pelo 7. Este tipo de información sería imposible de obtener sin la preparación semi-intacta del epitelio sensorial vestibular se describe aquí. Se demuestra la preparación semi-intacta del ratón crista 3, Y muestran resultados representativos obtenidos de parche de células enteras de electrofisiología y de imágenes de calcio de dos fotones.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Leibovitz medium L-15</td>
 <td>Sigma Aldrich</td>
 <td>L4386-10X1L</td>
 <td></td>
 </tr>
 <tr>
 <td>BAPTA-1-oregon green</td>
 <td>Invitrogen</td>
 <td>O6806 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT </td>
 </tr>
 <tr>
 <td>Stereo microscope</td>
 <td>Leica Microsystems</td>
 <td>A60S</td>
 <td></td>
 </tr>
 <tr>
 <td>Upright microscope</td>
 <td>Olympus</td>
 <td>BX51WI</td>
 <td></td>
 </tr>
 <tr>
 <td>Two-photon microscope</td>
 <td>Olympus/La Vision</td>
 <td>BX51WI/ TriMScope II</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont #5 SF Forceps</td>
 <td>FST</td>
 <td>11252-00</td>
 <td></td>
 </tr>
 <tr>
 <td>Friedman-Pearson Rongeurs</td>
 <td>FST</td>
 <td>16221-14</td>
 <td></td>
 </tr>
 <tr>
 <td>Standard Pattern Scissors</td>
 <td>FST</td>
 <td>14001-12</td>
 <td></td>
 </tr>
 <tr>
 <td>InstraTECH A-D converter</td>
 <td>HEKA</td>
 <td>ITC-18</td>
 <td></td>
 </tr>
 <tr>
 <td>Sutter Micromanipulator</td>
 <td>Sutter</td>
 <td>MP-225/M</td>
 <td></td>
 </tr>
 <tr>
 <td>multiclamp amplifier</td>
 <td>Axon Instruments</td>
 <td>700B</td>
 <td></td>
 </tr>
 <tr>
 <td>Data acquisition software (electrophysiology)</td>
 <td>Axograph</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Imspector Data acquisition software (two-photon)</td>
 <td>Max Planck innovation</td>
 <td>N/A</td>
 <td></td>
 </tr>
 </tbody>
</table>

an isolated semi-intact preparation of the mouse vestibular sensory epithelium for electrophysiology and high-resolution two-photon microscopy

Comprensión de las células ciliadas vestibulares funcionan en condiciones normales, o cómo el trauma, la enfermedad y el envejecimiento interrumpir esta función es un paso vital en el desarrollo de enfoques preventivos y / o nuevas estrategias terapéuticas. Sin embargo, la mayoría de los estudios en busca de la función vestibular anormal no han sido a nivel celular pero centrado principalmente en ensayos de comportamiento de la disfunción vestibular, tales como los análisis de la marcha y el rendimiento reflejo vestíbulo-ocular. Si bien este trabajo ha dado información valiosa sobre lo que sucede cuando las cosas van mal, hay poca información obtenida sobre las causas subyacentes de la disfunción. De los estudios que se centran en los procesos celulares y subcelulares que subyacen a la función vestibular, la mayoría han confiado en forma aguda las células ciliadas aislados, desprovistos de sus conexiones sinápticas y el entorno celular de soporte. Por lo tanto, un importante reto técnico ha sido el acceso a las células ciliadas vestibulares exquisitamente sensibles en una preparaciónración que está menos perturbado, fisiológicamente. Aquí se demuestra una preparación semi-intacta del ratón epitelio sensorial vestibular que conserva el micro-entorno local, incluyendo las células ciliadas / complejos aferentes primarias.

Las propiedades electrofisiológicas de las células ciliadas vestibulares dependen de la microarquitectura complejo en el que están incrustados 7. Figura 5 muestra que la preparación epitelio vestibular semi-intacta se puede utilizar para diferenciar entre el tipo de células ciliadas (figura 5A), células de pelo de tipo II que (Figura 5B), y el cáliz del aferente primario (Figura 5C) sobre la base de características conductancias de c?...

Watch this Scientific Journal Video about An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy at JoVE.com

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Análisis de la función de las células ciliadas vestibulares se complica por su ubicación en lo profundo de la parte más dura del cráneo, hueso temporal pétreo. La mayoría de los estudios de células pilosas funcionales han utilizado intensamente células ciliadas aislados. A continuación se describe una preparación semi-intacta de ratón epitelio vestibular para estudios electrofisiológicos y de microscopía de dos fotones.

Una preparación semi-intacta aislada del ratón epitelio sensorial vestibular de Electrofisiología y de alta resolución de la microscopía de dos fotones

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

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Research

JoVE Journal

Neuroscience

11.8K vistas.  University of Sydney. Análisis de la función de las células ciliadas vestibulares se complica por su ubicación en lo profundo de la parte más dura del cráneo, hueso temporal pétreo. La mayoría de los estudios de células pilosas funcionales han utilizado intensamente células ciliadas aislados. A continuación se describe una preparación semi-intacta de ratón epitelio vestibular para estudios electrofisiológicos y de microscopía de dos fotones.

Video: An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Imágenes in vivo de la médula espinal de ratón Utilizando microscopía de dos fotones

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Investigación

Neurociencias

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Preparación y tejido de fibras inmunotinción ratón nerviosas sensoriales que inervan la piel y los huesos de extremidades

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In vivo de dos fotones de imágenes de dependientes Experience-Los cambios moleculares en las neuronas corticales

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Un<em&gt; In-vitro</em&gt; Preparación de las neuronas entéricas aisladas y células gliales del plexo mientérico del ratón adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

En Vivo De dos fotones microscopía de terminaciones nerviosas individuales en la piel

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

De dos fotones<em&gt; In vivo</em&gt; Imágenes de las espinas dendríticas en la corteza del ratón Usando un Diluido-cráneo Preparación

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

De dos fotones de imágenes de Dinámica Celular en la médula espinal de ratón

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Cuantificación de Cerebral Vascular Arquitectura utilizando microscopía de dos fotones en un modelo de ratón de la neuroinflamación inducida por el VIH

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

Preparación fisiológica de las células ciliadas del sáculo de la rana toro americana (<em&gt; Catesbeiana</em&gt;)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

En Vivo Proyección de imagen de dos fotones de neuronas corticales en ratones neonatales

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...