Damos las gracias a James Perna para la ilustración gráfica. Este trabajo fue apoyado por subvenciones del Instituto Nacional de Salud Mental a YZ

Aprobación necesita ser obtenido de instituciones de origen antes del inicio de la cirugía y estudio de imágenes. Los experimentos descritos en este manuscrito se realizaron de acuerdo con las directrices y reglamentos de la Universidad de California, Santa Cruz Institucional Cuidado de Animales y el empleo Comisión. 1. Cirugía <ol><li> Autoclave todos los instrumentos quirúrgicos y esterilizar el área de trabajo con un 70% de alcohol a fondo antes de la cirugía. </li><li> Se anestesia el ratón por inyección intraperitoneal (IP) de solución anestésica KX (200 mg / kg de ketamina y 20 mg / kg de xilazina) de acuerdo con el peso corporal del ratón Nota:. Dosificación KX puede ser ajustado de acuerdo a la cepa, edad, y estado de salud de los ratones. También se recomienda la consulta veterinaria para determinar la dosis óptima. </li><li> Realice una prueba de toe-pinch periódicamente presionando los dedos del ratón y de la comprobación de una respuesta refleja para supervisar el estado de anestesia.Asegúrese de que el ratón esté totalmente anestesiado antes de iniciar la cirugía Nota:. Durante las sesiones de formación de imágenes prolongados, comprobar el estado de la anestesia del ratón periódicamente, administrar KX adicional si es necesario. </li><li> Coloque el ratón sobre una almohadilla de calentamiento para mantener la temperatura del cuerpo durante la cirugía. </li><li> Afeitarse la cabeza del ratón utilizando una hoja de afeitar para exponer el cuero cabelludo. </li><li> Esterilizar el área afeitada limpiando la piel con la alternancia de algodón con alcohol y betadine. Retire todos los recortes de pelo residuales. </li><li> Aplique suavemente ungüento para los ojos para lubricar los dos ojos, por lo tanto, la prevención del daño permanente causado por la deshidratación durante el experimento. </li><li> Hacer una incisión recta a lo largo de la línea media del cuero cabelludo, y mover la piel lateralmente hacia los bordes del cráneo. </li><li> Retire el tejido conectivo unido al cráneo. </li></ol> 2. Diluido-cráneo Preparación <ol><li> Identificar la región de imagen basada en ster. coordenadas eotaxic Nota: Trate de evitar los grandes vasos sanguíneos, lo que la penetración de luz bloque y difuminar las estructuras proyectadas. Se observa mejor vasculatura cuando el cráneo es húmeda con solución salina estéril. </li><li> Utilice el micro taladro de alta velocidad para diluir una región circular de cráneo (0,5-1,0 mm de diámetro). Mueva el taladro paralelo a la superficie del cráneo, en vez de sostener contra el cráneo y presionando hacia abajo. Taladro hasta tanto la capa de hueso compacto exterior y la capa esponjosa media se eliminan Nota:. Para evitar daños causados ​​por el sobrecalentamiento, evitar el contacto prolongado entre la broca y el cráneo. </li><li> Continuar el adelgazamiento de la capa de hueso compacto interior con una cuchilla microquirúrgica en el centro de la región de perforación-adelgazado. Mantenga la cuchilla microquirúrgica en un ángulo de aproximadamente 45 ° para raspar el cráneo sin presionar hacia abajo contra el cráneo hasta que un afinado de manera uniforme pequeña región (200-300 micras de diámetro) con un espesor de aproximadase obtiene LY 20-30 micras. Debido a la auto-fluorescencia del cráneo, el espesor se puede medir mediante el escaneo de la distancia entre la superficie superior e inferior del cráneo bajo el microscopio de dos fotones Nota:. Aunque un espesor cráneo de menos de 20 micras proporciona una buena calidad de imagen, no es recomendable porque hace que el proceso de adelgazamiento en posteriores sesiones de re-proyección de imagen difícil. Para evitar el exceso de adelgazamiento, el espesor del cráneo necesita ser comprobada periódicamente durante la cirugía (ver Discusión). </li></ol> 3. Inmovilización <ol><li> Retire con cuidado los restos de los huesos. </li><li> Coloque una pequeña gota de pegamento de cianoacrilato en cada borde de la abertura central de la placa de cabeza estéril, que ha sido tratado en autoclave (véase la Figura 1). Mantenga la placa de cabeza firmemente contra el cráneo con la región adelgazada situado en el centro de la abertura Nota:. Si el pegamento contamina el R adelgazadaegión por accidente, retire con cuidado con la cuchilla microquirúrgica. </li><li> Tire suavemente de la piel de los lados del cráneo hasta los bordes de la abertura central de la placa de cabeza. </li><li> Espere aproximadamente 10 minutos hasta que la placa de cabeza está bien adherida al cráneo. </li><li> Coloque la placa de cabeza en dos bloques laterales de la placa de sujeción y apriete los tornillos en los bordes de la placa de cabeza para inmovilizar el ratón sobre la placa de sujeción (véase la Figura 1) Nota:. Asegúrese de que no hay piel o los bigotes entre la placa de cabeza y los bloques antes de apretar los tornillos. Una buena preparación inmovilizada no debe mostrar ningún movimiento observable del cráneo bajo el microscopio de disección cuando la parte posterior del animal se dio unos golpecitos suavemente. </li><li> . Enjuagar el cráneo expuesto con solución salina para eliminar el pegamento no polimerizado Nota: Es importante eliminar cualquier pegamento restante, como pegamento no polimerizado difumina las imágenes y los daños objetivos de microscopio &lt;./ Li&gt; </ol> 4. Imaging <ol><li> Tome una foto de la vasculatura del cráneo expuesta con la región adelgazada como mapa 1, que se utiliza para la ubicación de la zona visualizada en las sesiones de formación de imágenes posteriores (véase la Figura 2). </li><li> Coloque el ratón bajo el microscopio de formación de imágenes. Ubicar la zona adelgazada bajo un objetivo de aire 10 veces usando epifluorescencia y mover el área más delgada para el centro de la vista. </li><li> Añadir una gota de solución salina en la parte superior del cráneo y cambiar a la objetivo 60X, que ha sido enjuagado con agua estéril. Seleccione una región donde las espinas dendríticas individuales están claramente visualizadas junto dendritas. Identificar y etiquetar la región correspondiente en el mapa 1 comparando vasculatura entre la vista de epifluorescencia y la foto vasculatura. </li><li> Sintonice la longitud de onda del láser de dos fotones de acuerdo con los fluoróforos. Por ejemplo, 920 nm para YFP; 890 nm para GFP; 1000 nm para DsRed y tdTomato 24. </li><li> Adquirir pilas de imágenes con 2pasos micras a lo largo del eje z usando el objetivo 60X. Esta pila de imagen cubre un área de 200 x micras aproximadamente 200 micras (512 x 512 píxeles) y se utiliza como mapa 2 para el traslado durante las sesiones de formación de imágenes posteriores (véase la Figura 2). </li><li> Adquirir nueve pilas de imágenes en el mapa 2 se utiliza el zoom digital de 3X. Cada pila de imagen cubre un área aproximada de 70 m x 70 m (512 x 512 píxeles), con 0,7 m pasos a lo largo del eje z Nota:. La intensidad del láser debe estar por debajo de 40 mW cuando se mide en muestras para reducir al mínimo la fototoxicidad. </li></ol> 5. Recuperación <ol><li> Tras las imágenes, separe con cuidado la placa de cabeza del cráneo. </li><li> Limpiar a fondo el cráneo y la piel para eliminar todo el pegamento restante Nota:. Cualquier pegamento que queda en la piel causará irritaciones y retrasar la cicatrización de la piel, mientras que el pegamento que queda en el cráneo provocará la erosión del cráneo y unngiogenesis en la capa de hueso recién crecido, por lo que la posterior reubicación y reconstrucción de imagen difícil. </li><li> Enjuague el cráneo y la piel con solución salina varias veces. </li><li> Suturar el cuero cabelludo con sutura quirúrgica estéril. </li><li> Mantenga al animal en una almohadilla térmica en una jaula separada. Administrar analgésico buprenorfina (0,1 mg / kg) por vía subcutánea para mitigar el dolor post-operatorio. Volver al animal a la jaula después de la recuperación completa. Vigilar estrechamente el animal (marque al menos una vez al día) hasta que la incisión haya sanado y se retiran las suturas. Administrar las inyecciones posteriores de buprenorfina analgésica si es necesario. </li></ol> 6. Creación de imágenes <ol><li> Repita los pasos 1.1 a 1.8. </li><li> Repita los pasos 3.1 a 3.6 </li><li> Localice región previamente fotografiado al comparar el patrón de vascularización a Mapa 1 y el patrón de ramificación dendrítica a Mapa 2. </li><li> Si se realiza la creación de las imágenes dentro de 1 semana, repita el paso 2.3 para quitar la fina capa de hueso recién crecido en la parte superior de la re adelgazadaregión utilizando la cuchilla microquirúrgica. Si reimaging se realiza después de más de 1 semana, repita los pasos tanto de 2.2 y 2.3 Nota:. La capa de hueso recién crecido consiste en una estructura menos condensada en comparación con la capa de hueso compacto original, que conduce a una menor calidad de imagen. Por lo tanto, para adquirir la misma calidad, es necesario diluir el cráneo ligeramente más de la sesión de formación de imágenes anterior. </li><li> Ajuste la posición y la orientación para obtener pilas de imágenes que coinciden previamente tomadas pilas de imágenes en el microscopio de dos fotones. </li><li> Adquirir imágenes como en el paso 4.6. </li><li> Repita los pasos 5.1 a 5.5 después de la impresión. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Para obtener una exitosa preparación de calavera adelgazado, varios pasos de este protocolo son cruciales. 1) El espesor del cráneo. El hueso craneal tiene una estructura de sándwich, con dos capas de hueso compacto de alta densidad y una capa intermedia de hueso esponjoso de baja densidad. Mientras que el micro taladro de alta velocidad es adecuado para la eliminación de las capas externas de hueso compacto y el hueso esponjoso, la cuchilla microquirúrgica es ideal para el adelgazamiento de la capa interior de hue...

La corteza de los mamíferos participa en muchas funciones cerebrales, de la percepción sensorial y el movimiento de control de abstraer el procesamiento de información y la cognición. Varias funciones corticales se basan en diferentes circuitos neuronales, que se componen de diferentes tipos de neuronas que se comunican e intercambian información en las sinapsis individuales. La estructura y la función de las sinapsis están siendo modificados constantemente en respuesta a las experiencias y patologías. En el cerebro maduro, la plasticidad sináptica toma la forma de ambos cambios de resistencia y la adición / eliminación de sinapsis, que juega un papel importante en la formación y mantenimiento de un circuito neuronal funcional. Las espinas dendríticas son los componentes postsinápticos de la mayoría de las sinapsis excitadoras en el cerebro de los mamíferos. La constante rotación y los cambios morfológicos de las espinas se cree que servirá como un buen indicador de las modificaciones en las conexiones sinápticas 1-7. Dos fotones de escaneo láser microscopy ofrece una penetración profunda a través, preparaciones opacas gruesas y baja fototoxicidad, que lo hace adecuado para las imágenes en directo en el cerebro intacto 8. En combinación con marcaje fluorescente, dos fotones de imágenes proporciona una poderosa herramienta para mirar en el cerebro vivo y siga reorganización estructural en las sinapsis individuales con alta resolución espacial y temporal. Varios métodos han sido utilizados para preparar los ratones para formación de imágenes en vivo 9-13. Aquí se describe una preparación de cráneo adelgazado de imágenes in vivo de dos fotones para investigar la plasticidad estructural de las espinas dendríticas postsinápticos en la corteza del ratón. Con este enfoque, los estudios recientes han representado una imagen dinámica de los cambios de la espina dendrítica en respuesta a las habilidades motoras de aprendizaje Con el aumento de la disponibilidad de animales transgénicos con subconjuntos neuronales marcados con fluorescencia y el rápido desarrollo de las técnicas in vivo de etiquetado, los procedimientos similares a los descritos aquí se pueden aplicar también de investigaciónTE otros tipos de células y regiones corticales, en combinación con otras manipulaciones, así como se utiliza en modelos de enfermedad 16-23.

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			<td height="21" style="height:21px;width:189px;">Ketamine</td>
			<td style="width:181px;">Bioniche Pharma</td>
			<td style="width:108px;">67457-034-10</td>
			<td style="width:775px;">Mixed with xylazine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Xylazine</td>
			<td style="width:181px;">Lloyd laboratories</td>
			<td style="width:108px;">139-236</td>
			<td>Mixed with ketamine for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saline</td>
			<td style="width:181px;">Hospira</td>
			<td style="width:108px;">0409-7983-09</td>
			<td>0.9% NaCl for injection and imaging</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Razor blades</td>
			<td style="width:181px;">Electron microscopy sciences</td>
			<td style="width:108px;">72000</td>
			<td>Double-edge stainless steel razor blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Alcohol pads</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">06-669-62</td>
			<td>Sterile alcohol prep pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Eye ointment</td>
			<td style="width:181px;">Henry Schein</td>
			<td style="width:108px;">102-9470</td>
			<td>Petrolatum ophthalmic ointment sterile ocular lubricant</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:189px;">High-speed micro drill</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">18000-17</td>
			<td>The high-speed micro drill is suitable for thinning the outer layer of compact bone and targeting a small area</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Micro drill steel burrs</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:108px;">19007-14</td>
			<td>1.4 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Microsurgical blade</td>
			<td style="width:181px;">Surgistar</td>
			<td style="width:108px;">6961</td>
			<td>The microsurgical blade is suitable for thinning the inner layer of compact bone and middler layer of spongy bone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Cyanoacrylate glue</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:108px;">NC9062131</td>
			<td>Fix the head plate onto the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Suture</td>
			<td style="width:181px;">Havard Apparatus</td>
			<td style="width:108px;">510461</td>
			<td>Non-absorbale, sterile silk suture, 6-0 monofilament</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Dissecting microscope</td>
			<td>Olympus</td>
			<td style="width:108px;"></td>
			<td>SZ61</td>
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			<td height="21" style="height:21px;width:189px;">CCD camera</td>
			<td>Infinity</td>
			<td style="width:108px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Two-photon microscope</td>
			<td style="width:181px;">Prairie Technologies</td>
			<td style="width:108px;"></td>
			<td style="width:775px;">Ultima IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">10X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 0.30, air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">60X objective</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:108px;"></td>
			<td>NA 1.1, IR permeable, water immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Ti-sapphire laser</td>
			<td style="width:181px;">Spectra-Physics</td>
			<td style="width:108px;"></td>
			<td>Mai Tai HP</td>
		</tr>
	</tbody>
</table>

two-photon in vivo imaging of dendritic spines in the mouse cortex using a thinned-skull preparation

En la corteza de los mamíferos, las neuronas forman redes muy complicadas y el intercambio de información en las sinapsis. Los cambios en la fuerza sináptica, así como la adición / eliminación de sinapsis, se producen de una manera dependiente de la experiencia, que proporciona la base estructural de la plasticidad neuronal. Como componentes postsinápticos de las sinapsis excitatorias más en la corteza, las espinas dendríticas se considera que son un buen indicador de las sinapsis. Tomando ventajas de la genética del ratón y técnicas de marcaje fluorescente, neuronas individuales y de sus estructuras sinápticas pueden ser etiquetados en el cerebro intacto. Aquí presentamos un protocolo de imágenes transcraneal utilizando dos fotones microscopía de escaneo láser para seguir la etiqueta fluorescente espinas dendríticas postsináptica a través del tiempo en vivo. Este protocolo utiliza una preparación de cráneo adelgazado, que mantiene el cráneo intacto y evita efectos inflamatorios causados ​​por la exposición de las meninges y la corteza. Por lo tanto, las imágenes pueden ser adquiridas inmediatamente después dose realiza rgery. El procedimiento experimental se puede realizar de forma repetitiva durante diversos intervalos de tiempo que van desde horas hasta años. La aplicación de esta preparación también se puede ampliar para investigar diferentes regiones y capas corticales, así como otros tipos de células, en condiciones fisiológicas y patológicas.

En YFP-H línea de ratones 25, proteína fluorescente amarilla expresa en un subconjunto de neuronas piramidales capa de V, que se proyectan sus dendritas apicales de las capas superficiales de la corteza. A través de la preparación de calavera adelgazado, los segmentos dendríticas marcados con fluorescencia pueden ser repetitivamente reflejados bajo el microscopio de dos fotones a través de varios intervalos de formación de imágenes, que van desde horas a meses. Aquí se muestra un ejemplo de una image...

Watch this Scientific Journal Video about Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation at JoVE.com

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-vivo-imaging-dendritic-spines-mouse-cortex-using-thinned

Research

JoVE Journal

Neuroscience

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

17.8K vistas.  University of California, Santa Cruz. 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.

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

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

Imagen crónica de la corteza visual del ratón usando una preparación de Diluido-cráneo

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Investigación

Neurociencias

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 ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

La detección de la hipoxia Microrregional en corteza cerebral de ratón por la imagen de dos fotones de fluorescencia endógena NADH

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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 ...

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 ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Diluido-skull técnica de ventana cortical para<em&gt; En Vivo</em&gt; Tomografía de Coherencia Óptica de imagen

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

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 ...

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 ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultánea de dos fotones<em&gt; En Vivo</em&gt; Imágenes de entradas sinápticas y Objetivos postsinápticos en la corteza del ratón retroesplenial

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

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 ...