Este trabajo fue financiado por el Ministerio de ciencia y tecnología concede MOST104-2311-B-010-010-MY3, conceder MOST106-2321-B-010-012, los institutos nacionales de investigación de salud INDH-EX106-10429NI y las áreas de investigación centro Programa Beca de el Ministerio de educación a través del centro de investigación del cerebro, Universidad Nacional de Yang Ming en Taiwán, y Beca Postdoctoral becas MOST106-2811-B-010-031 (S.-Y.C.) y MOST105-2811-B-010-036 MOST106-2811-B-010-030 (H.-Y.K.).

Los protocolos animal aquí descritos han sido aprobados por el cuidado Animal y uso comités Universidad Nacional de Yang Ming.
1. preparación del titular de los cachorros neonatales en el aparato estereotáctica
<ol><li>Hacer la bandeja principal: cortar la parte inferior de un tubo de centrífuga de 1.5 mL (15 mm de largo) en la forma que se adapta a la cabeza de los cachorros neonatales quitando 1/5 de la pared del tubo.</li>
	<li>Tomar una caja de punta de pipeta con el tamaño correcto que encaja con el pedestal del aparato estereotáxicas y quite la cubierta superior. Pegue la cabeza bandeja preparada en el paso 1.1 y un cassette inclusión de tejido sobre la base de punta de bandeja con el pegamento de fusión en caliente. La altura del cassette inclusión de tejido es 7 m m, que se utiliza para apoyar el cuello y el cuerpo del cachorro en la posición fija de la cabeza.</li>
	<li>Lugar la configuración todo en un aparato estándar estereotáxicas.</li>
</ol>2. preparación de agujas de acero inoxidable de inyección de 30G
<ol><li>Limpieza previa de agujas de jeringa de 30 G por sumergiéndolas en cloroformo durante 3 días.</li>
	<li>Utilice pinzas para lenta y cuidadosamente saque la aguja desde el hub de polipropileno en una campana química.</li>
	<li>Lavar las agujas con etanol absoluto por 20 min seguido por enjuagues de etanol 70% por 3 × 10 min en un agitador a 50 rpm. Airee el agujas secas y almacenar las agujas limpias en una caja limpia a temperatura ambiente (RT) hasta su uso.</li>
</ol>3. prepare un adaptador para tubos de microinyección
<ol><li>Conecte la jeringa microliter una aguja 30G con un PE10 polietileno tubo (PE10). Preparar un adaptador de tubo (tubo PE20) PE20 polietileno para tender un puente sobre la aguja y la jeringa microliter. El uso del adaptador es necesario, porque el diámetro del tubo PE10 es mucho más pequeño que el de la aguja de la jeringa microliter de 26G.</li>
	<li>Preparar el tubo de la jeringa microliter: Conecte la aguja 30 G previamente limpiado con 5 cm de tubo PE20 y sellar la Unión con pegamento instantáneo. Este adaptador de tubería es reutilizable. Nota: Si la aguja de la jeringa de microinyección es 30G, la preparación (paso 3.1) y (paso 3.3) el uso de este adaptador no es necesario.</li>
	<li>Conecte el adaptador de tubo (preparado en el paso 3.1) con una jeringa microliter (10 μl).</li>
	<li>Conecte un extremo del PE10 el tubo (no más de 60 cm) en la aguja de 30G del adaptador de tubo PE20. Monte una nueva aguja 30G en el otro extremo del tubo PE10.</li>
</ol>4. Cargue el tubo de la microinyección con virus, colorante y agua destilada esterilizada
<ol><li>Retire el émbolo de la jeringa microliter. Utilizar una jeringa de 25G para cargar la jeringa microliter y su tubo de PE conectado con agua destilada esterilizada para remover el aire de la tubería.</li>
	<li>Coloque el émbolo de la jeringa microliter y empuje el émbolo hasta 2 μl de agua destilada se queda en el barril. No deje que el volumen de ser inferior a 2 μl de agua destilada.</li>
	<li>Montar con cuidado la jeringa microliter en la bomba de jeringa de tasa de flujo micro.</li>
	<li>Pipeta de pequeñas cantidades de tinte de 0,1% de verde rápido (preparado en solución salina 0.9% y filtrar con filtro de 0.22 μm) y los líquidos de virus a un trozo de parafilm.</li>
	<li>Retirar una pequeña cantidad de aire para hacer una burbuja de aire visible en el cruce entre la aguja 30G y tubo PE10 seguido de carga 0,7 μl del filtrado verde rápido en el tubo para probar el flujo de fluidos en la tubería de microinyección.</li>
	<li>Retirar otra pequeña cantidad de aire para hacer una segunda burbuja de aire seguida por la carga de los líquidos de virus en el tubo de microinyección.</li>
	<li>Coloque y fije la aguja de 30G microinyección en el brazo del aparato estereotáxicas.</li>
</ol>5. anestesia de ratones neonatales por hipotermia
<ol><li>Colocar el cachorro en una manga de guante de látex y sumergirlo en hielo hasta el cuello durante 5 minutos.</li>
	<li>El sujetador de pies de cría con pinzas para asegurarse de que no hay respuesta de retirada de sus pies.</li>
	<li>Pon el cachorro con la manga del guante de látex en la bandeja principal y algunos hielo picado alrededor de la manga de látex para mantener fría para la anestesia de la hipotermia.</li>
	<li>Aplique ungüento veterinario en los ojos del cachorro para prevenir sequedad mientras que bajo anestesia si es posible.</li>
</ol>6. microinyección
<ol><li>Preparan cirugía estéril frotando el instrumento estereotáxicas con etanol al 70%. Esterilizar el instrumental quirúrgico por inmersión en etanol al 70%.</li>
	<li>Frote la cabeza del cachorro con etanol al 70%. Ubicar el lambda de la señal en el cráneo y la lambda de la marca con un rotulador. Apunte la punta de la aguja a la lambda y fijar el antero-posterior (AP) y medial-lateral (ML) coordenadas como cero.</li>
	<li>Mueva el brazo de inyección al sitio de destino según las coordenadas X e Y del sitio de destino. Para el estriado de día postnatal (P) 2 crías, las coordenadas son: AP, +2,4 mm anterior a la lambda; ML, ±1. 0 mm lateral de la línea media; dorsal-ventral (DV), mm −1.7 del cráneo. Marque la posición de tinte verde rápido en PE10 tubo con una pluma.</li>
	<li>Bajar la aguja 30G lentamente a penetrar a través de la piel y el cráneo y luego traer hasta la punta de la aguja hasta que se detenga en la superficie del cráneo. Establecer la coordenada DV como cero.</li>
	<li>Haga descender la aguja 30G lentamente hasta llegar a la coordenada de la DV del sitio de destino. Espere 1 minuto permitir que el parénquima reasumir su forma normal. Ejecute el programa de la microinyección (100 nL/min).</li>
	<li>Asegúrese de que la marca de tinte verde rápido se está moviendo en el tubo de PE para que el líquido del virus es inyectado en el cerebro.</li>
	<li>Esperar 1 minuto después de la terminación de la microinyección y luego lenta y progresivamente lleve la aguja a la altura del 1/2 de profundidad DV dentro de los 30 s. Después de 30 s, retirar lentamente la aguja de la cabeza del cachorro.</li>
	<li>Repita el paso 6.2 a 6.7 hasta que se completen las microinyecciones de todos los sitios específicos.</li>
</ol>7. post quirúrgico recuperación del cachorro
<ol><li>El cachorro por 20 min en una incubadora de 33 ° C de calentamiento. Comprobar la recuperación de la cría de la hipotermia anestesia cada 5 min hasta que el cachorro ha recuperado la conciencia suficiente para mantener el recumbency esternal.</li>
	<li>Regresar el cachorro a la presa después de que el cachorro se recupera completamente.</li>
</ol>

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Los autores no tienen nada que revelar.

En el presente estudio, se demuestra un método simple y confiable estereotáctica para inyectar virus AAV en el cuerpo estriado del cerebro de ratón neonatal. Microinyectados virus AAV-eGFP-Cre en el estriado de los ratones de reportero Ai14 en P2 y luego analiza la expresión del gen reportero en P14. Encontramos que AAV transduced células GFP-positivas en el cuerpo estriado a nivel rostrocaudal. Por otra parte, casi todas las células GFP-positivas Co expresan el gen reportero tdTomato en células estriatales, lo qu...

Los estudios modernos de la estructura y función del cerebro requieren manipulación genética de genes específicos en células neuronales. Para probar las funciones de diferentes genes, ratones transgénicos con alelos del mutante, incluyendo nocaut y knock-in alelos se han generado habitualmente. Neurocirugía estereotáctica para roedores adultos es un método estándar para entregar localmente los medicamentos, virus, marcadores y otros reactivos a regiones específicas del cerebro de roedores1,2. Aplicación de la cirugía estereotáctica cerebral en ratones transgénicos permite manipular genéticamente las funciones de los genes y la actividad neuronal en poblaciones neuronales específicas del cerebro del ratón. La manipulación de tipo específico de célula proporciona un enfoque poderoso para descifrar las funciones neuronales en complejos circuitos neuronales del cerebro3,4,5.
Desarrollo neuronal del sistema nervioso comienza en las primeras etapas embrionarias, y continúan con los procesos de desarrollo después del nacimiento hasta el periodo juvenil. Maduración postnatal del sistema nervioso incluye el cableado sináptico precisa de circuitos neurales, que es esencial para las funciones fisiológicas y cognitivas del cerebro6. Por lo tanto, estudiar eventos del desarrollo que ocurren en tiempo neonatal es importante no sólo para comprender el desarrollo neural normal, pero también puede proporcionar penetraciones en la patogenesia de Neurodesarrollo y trastornos neuropsiquiátricos7 ,8. Aunque los métodos de cirugía estereotáctica cerebral de roedores adultos son fácilmente disponibles2,9, algunos protocolos están disponibles en internet para cirugía estereotáctica cerebral en ratones neonatales10,11. De hecho, microinyecciones estereotáxicas de reactivos en el cerebro de crías de ratón neonatal son difíciles, porque la cabeza del cachorro neonatal es demasiado frágil para ser fijado en el aparato estándar estereotáxicas. Sin embargo, la aplicación de cirugía estereotáctica cerebral en ratones transgénicos es factible para ratones neonatales12. Aquí, describimos un método simple con una instalación casera para realizar cirugía estereotáctica cerebral en crías de ratón recién nacido. Nos demuestran que esta técnica permite eliminar condicional floxed genes microinjecting recombinase de AAV-expresión Cre ADN en el cuerpo estriado de reportero gene ratones y condicional de ratones transgénicos floxed. Esta técnica también es aplicable para entregar los reactivos en el neonatal estriado de los ratones de tipo salvaje.

<table border="0" cellpadding="0" cellspacing="0" width="1134">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">30G PrecisionGlide Needle</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 305106</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Chloroform</td>
			<td style="width:273px;">JT Baker</td>
			<td style="width:199px;">9180-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Hamilton MICROLITER Syringe</td>
			<td style="width:273px;">Hamilton&#160;</td>
			<td style="width:199px;">80300</td>
			<td style="width:395px;">30G needle fit for PE10 tube; 26G needle needs a PE20 adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE20</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Polyethylene tubing PE10</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">427401</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Micro Flow Rate Syringe Pump</td>
			<td style="width:273px;">Longer Precision Pump Co.</td>
			<td style="width:199px;">TJ-2A (Controller) and L0107-2A (Drive Unit)</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">25G syringe</td>
			<td style="width:273px;">Becton Dickinson</td>
			<td style="width:199px;">REF 302105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Fast green</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:199px;">F-7252</td>
			<td style="width:395px;">0.1%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Standard Stereotaxic Instruments</td>
			<td style="width:273px;">RWD Life Science</td>
			<td style="width:199px;">68037</td>
			<td style="width:395px;">Without using 68030 Mouse/Neonatal Rat Adaptor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-FOXP2 antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab16046</td>
			<td style="width:395px;">Rabbit polyclonal to FOXP2, 1:4K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Anti-RFP antibody</td>
			<td style="width:273px;">Abcam</td>
			<td style="width:199px;">ab65856</td>
			<td style="width:395px;">Mouse monoclonal to RFP, 1:1K</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">BX63 microscope</td>
			<td style="width:273px;">Olympus</td>
			<td style="width:199px;">BX63</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">LSM 880 confocal microscope</td>
			<td style="width:273px;">Zeiss</td>
			<td style="width:199px;">LSM 880</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Goat anti-rabbit conjugated Alexa fluor594</td>
			<td style="width:273px;">Jackson lmmunoReserch Laboratories Inc.</td>
			<td style="width:199px;">111-585-003</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40</td>
			<td style="width:273px;">Penn Vector Core</td>
			<td style="width:199px;">AV-9-PV1848</td>
			<td style="width:395px;">Lot # CS0987, 5.506x1013 (GC/mL)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">AAV9.chicken actin-eGFP</td>
			<td style="width:273px;">AAV core, Institute of Biomedical Sciences, Academia Sinica, Taiwan</td>
			<td style="width:199px;">N/A</td>
			<td style="width:395px;">1x1014 (GC/ml)</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:269px;">B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">007914</td>
			<td>Ai14</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">B6(Cg)-Foxp2tm1.1Sfis/CfreJ</td>
			<td style="width:273px;">The Jackson Labtorary&#160;</td>
			<td style="width:199px;">026259</td>
			<td style="width:395px;">Foxp2fl/fl</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Dulbecco&#8217;s phosphate buffered saline</td>
			<td style="width:273px;">Corning&#160;cellgro</td>
			<td style="width:199px;">21-030-CVR</td>
			<td></td>
		</tr>
	</tbody>
</table>

stereotaxic surgery for genetic manipulation in striatal cells of neonatal mouse brains

Muchos genes se expresan en el cerebro embrionario y algunos de ellos se expresan continuamente en el cerebro después del nacimiento. Para tales genes persistentemente expresadas, pueden funcionan para regular el proceso de desarrollo o función fisiológica en el cerebro neonatal. Para investigar las funciones neurobiológicas de genes específicos en el cerebro, es esencial para desactivar genes en el cerebro. Aquí, describimos un método simple de estereotáctica para inactivar genes en el cuerpo estriado de los ratones transgénicos en ventanas de tiempo neonatal. AAV-eGFP-Cre virus fueron microinyectados en el estriado de los ratones de gen reportero Ai14 en día postnatal (P) 2 de cirugía estereotáctica cerebral. La expresión del gen reportero tdTomato fue detectada en P14 estriado, sugiriendo un éxito Cre-loxP mediada por recombinación de ADN en las células AAV-transduced estriada. Más validamos esta técnica por microinjecting virus AAV-eGFP-Cre en ratones P2Foxp2fl/fl . Etiquetado doble de GFP y Foxp2 demostró que las células GFP-positivas carecían de Foxp2 immunoreactivity en P9 estriado, lo que sugiere la pérdida de la proteína Foxp2 en AAV-eGFP-Cre transduced estriado células. Tomados en conjunto, estos resultados demuestran una canceladura genética efectiva por los virus de AAV-eGFP-Cre stereotaxically microinyectados en poblaciones neuronales específicas en el cerebro neonatal de ratones transgénicos floxed. En conclusión, nuestra técnica estereotáctica proporciona una plataforma fácil y simple para la manipulación genética en el cerebro de ratón neonatal. La técnica no sólo permite eliminar genes en regiones específicas del cerebro neonatal, pero también puede ser utilizado para inyectar drogas farmacológicas, trazadores neuronales, optogenetics genéticamente modificados y chemogenetics proteínas, indicadores de la actividad neuronal y otros reactivos en el cuerpo estriado del cerebro de ratón neonatal.

Para el primer conjunto del experimento, nos microinyectados 200 nL de AAV9.hSynapsin.HI.eGFP-Cre.WPRE.SV40 virus (AAV-eGFP-Cre, dilución 1/10 en tampón fosfato salino de Dulbecco) que expresan el recombinase de Cre DNA fundido con GFP en P2 estriado de los ratones Ai14. Los ratones Ai14 expresan el gen reportero de tdTomato sobre eliminación Cre-mediada del loxP flanqueado (floxed) cassette STOP (figura 2F). Los cerebros fueron cosechados en P14 de immuno...

Watch this Scientific Journal Video about Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains at JoVE.com

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Cirugía estereotáctica para manipulación genética en las células estriatales de cerebro de ratón Neonatal

Se describe un protocolo de cirugía estereotáctica con un dispositivo casero hecho de cabeza fija de reactivos microinjecting en el cuerpo estriado del cerebro de ratón neonatal. Esta técnica permite la manipulación genética en las células neuronales de regiones específicas del cerebro de ratón neonatal.

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

stereotaxic-surgery-for-genetic-manipulation-striatal-cells-neonatal

Research

JoVE Journal

Neuroscience

14.8K vistas.  National Yang-Ming University. Se describe un protocolo de cirugía estereotáctica con un dispositivo casero hecho de cabeza fija de reactivos microinjecting en el cuerpo estriado del cerebro de ratón neonatal. Esta técnica permite la manipulación genética en las células neuronales de regiones específicas del cerebro de ratón neonatal.

Video: Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

Cirugía estereotáctica de roedores y mejoras del bienestar de los animales de resultados para la revista Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

Investigación

Neurociencias

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Disección y Cultura de ratón dopaminérgico estriatal en explantes y tridimensionales ensayos matriz de colágeno

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

La cirugía estereotáxica para la lesión excitotóxica de áreas específicas del cerebro en la rata adulta

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

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 ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Inyección estereotáxica de un vector viral para la manipulación genética condicional en la médula espinal de ratón

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

La manipulación genética del ratón en desarrollo a través de Hipotálamo<em&gt; En el útero</em&gt; La electroporación

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Todo el montaje de la tinción de inmunofluorescencia del Neonatal ratón Retina de investigar Angiogénesis<em&gt; En vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

La manipulación genética de las neuronas cerebelosa gránulo<em&gt; In Vitro</em&gt; Y<em&gt; En Vivo</em&gt; Estudiar Neuronal Morfología y Migración

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Inyección Viral Intracerebroventricular de Neonatología del ratón Cerebro de transducción neuronal persistente y generalizada

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Tomografía de coherencia óptica: Proyección de imagen de ratón ganglionares de la retina las células In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Cirugía estereotáctica para manipulación genética en las células estriatales de cerebro de ratón Neonatal

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Capítulos en este video

Cirugía estereotáctica de roedores y mejoras del bienestar de los animales de resultados para la revista Behavioral Neuroscience

Disección y Cultura de ratón dopaminérgico estriatal en explantes y tridimensionales ensayos matriz de colágeno

La cirugía estereotáxica para la lesión excitotóxica de áreas específicas del cerebro en la rata adulta

La visualización y la manipulación genética de las dendritas y espinas en la corteza cerebral y el hipocampo del ratón usando

Inyección estereotáxica de un vector viral para la manipulación genética condicional en la médula espinal de ratón

La manipulación genética del ratón en desarrollo a través de Hipotálamo

Todo el montaje de la tinción de inmunofluorescencia del Neonatal ratón Retina de investigar Angiogénesis

La manipulación genética de las neuronas cerebelosa gránulo

Inyección Viral Intracerebroventricular de Neonatología del ratón Cerebro de transducción neuronal persistente y generalizada

Tomografía de coherencia óptica: Proyección de imagen de ratón ganglionares de la retina las células In Vivo