Este trabajo fue apoyado por el Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares conceder NS081333 (a CMT), Asociación de Alzheimer subvención NIRG-10-171721 y el Instituto Nacional de Salud Mental de subvención MH096702 (a UH), y el Instituto Nacional sobre el Envejecimiento financiados Investigación de la Enfermedad de Alzheimer Centro de la Universidad de Columbia subvención piloto AG008702 (a YYJ y JB).

Nota: Para todos los experimentos con animales, institucional y directrices nacionales para el cuidado y uso de animales de laboratorio fueron seguidos. 1. Preparar instrumentos quirúrgicos y soluciones para la cirugía <ol><li> Autoclave todos los instrumentos quirúrgicos de acero inoxidable. </li><li> Preparar etanol al 70% por dilución de 200 a prueba de etanol absoluto con el grado molecular de agua desionizada destilada estéril. </li><li> Coloque la aguja G 29 a la jeringa Hamilton. Limpie el interior de Hamilton jeringa y la aguja mediante la elaboración y el agua nanopura expulsión repetidamente durante 1 min. Repita este procedimiento con etanol al 70%. <ol><li> Retire el émbolo y la aguja de la jeringa Hamilton. Secar las partes en una campana de flujo laminar S / N. </li><li> Irradiar la aguja y jeringa con luz ultravioleta durante 30 minutos antes de su uso. </li></ol></li><li> Preparar la solución salina disolviendo NaCl en agua de calidad molecular hasta un peso final a la concentración de volumen de 0,9%. Esterilize la solución por filtración a través de filtro de 0,2 micras de poro. </li></ol> 2. Prepare Oligomeric Aß 1-42 <ol><li> Monomerize y resuspender recombinante humana 1-42 en DMSO a 5 mM exactamente como se describe en la publicación de Fa &#39;y otras 13 personas. </li><li> Diluir 5 mM 1-42 con (1x) PBS estéril a 100 mM en el día antes de la cirugía. </li><li> Mezclar la solución bien por trituración. </li><li> Incubar Aß 1-42 solución durante 12 horas a 4ºC. Nota: Las soluciones de control, tales como la dilución de DMSO en PBS o revueltos / inversa 1-42 péptido debe ser preparado de la misma manera como Aß 1-42. </li></ol> 3. Determinar Coordenadas Injection <ol><li> Utilice los adultos atlas del cerebro del ratón para determinar la exacta anterior-posterior (AP), medio-lateral (ML) y dorsal-ventral (DV) coordenadas de la región del cerebro de interés 14. Note: Para ilustrar la técnica que elegimos el giro dentado del hipocampo como el objetivo con las siguientes coordenadas del bregma: AP, -2,00 mm; ML, ± 1,3 mm; DV, -2,2 mm. El signo negativo que precede al valor AP indica que es 2,00 mm posterior del bregma. El signo ± anterior el valor ML indica la dirección izquierda y derecha del centro. Por último, el signo negativo que precede a la DV indica que se está moviendo desde la superficie ventral del cerebro. </li></ol> 4. Instalación de marco estereotáxica <ol><li> Use una máscara quirúrgica cara, bata de laboratorio limpia y guantes quirúrgicos estériles. </li><li> Limpie instrumento estereotáxico y adaptador de ratón con etanol al 70%. </li><li> Establecer paño quirúrgico estéril sobre el mostrador. </li><li> Coloque el instrumento estereotáxica con el adaptador del ratón encima de la sábana quirúrgica estéril. </li><li> Limpie la almohadilla del ratón con etanol al 70%. Coloque la almohadilla térmica sobre la cama marco estereotáxico. Utilice labora propósito generalcinta de etiquetado tory en cinta por la almohadilla térmica sobre la cama adaptador de ratón. <ol><li> Coloque la almohadilla eléctrica a la bomba de recirculación de agua caliente de acuerdo a las instrucciones del fabricante. </li><li> Encienda la bomba de recirculación de agua caliente y ajustar la temperatura a 37 ° C, y luego esperar hasta que alcance la temperatura. </li></ol></li><li> Fije la 50 l Hamilton jeringa con una aguja de 29 G en el marco estereotáxico atornillándolo en el eje vertical de inyección motorizada de acuerdo con las instrucciones del fabricante. </li><li> Encienda el esterilizador de cuentas y espere hasta que alcance la temperatura preestablecida por el fabricante. Nota: Esto se utiliza para la esterilización de instrumentos de acero inoxidable entre los animales. Se necesita instrumentos 20 seg de contacto con los granos para la esterilización. </li><li> Encienda el plato caliente y ponerlo a 42 ° C y colocar una jaula vacía limpia cima. </li><li> Mientras que la jeringa Hamilton se fija en el marco estereotáxico utilizar el i estereotáxico motorizadonjector elaborar la solución Aß 1-42. Nota: Elaborar más de 4 l de la solución en la jeringa y luego usar el inyector estereotáxica para establecer el volumen de inyección. Operar el inyector de acuerdo con las instrucciones del fabricante. </li></ol> 5. Preparación de los animales <ol><li> Determinar el peso del ratón utilizando la escala de pesaje. </li><li> Anestesiar ratón con cóctel de ketamina / xilazina a 100 mg / kg de ketamina y 10 mg / kg de xilazina mediante la inyección por vía intraperitoneal (IP). Monitorear la profundidad de la anestesia por la pérdida del reflejo pizca dedo del pie. </li><li> Una vez sedado el ratón tome la cortadora de cabello y afeitarse la cabeza para exponer la piel sobre el cráneo. </li><li> Coloca el ratón encima de la almohadilla térmica en la parte superior de la cama estereotáxica. </li><li> Utilice la espátula para abrir la boca y colocar los dientes incisivos dentro de la guardia de los dientes. Asegure el revólver sobre la cara del ratón. Sujete suavemente hacia abajo y no demasiado apretado. </li><li> Posición los travesaños del oído bilaterales en meato auditivo para asegurar la cabeza. Mueva cada travesaño hasta que golpea el cráneo, y luego gire el tornillo para fijarlo. <ol><li> Para asegurarse de que la cabeza está asegurado utilice su dedo índice para empujar suavemente hacia abajo en la cabeza. Si la cabeza está bien asegurado no va a dar o mover cuando se presiona. </li></ol></li><li> Aplique una gota de crema para los ojos o colirio para los ojos para mantenerlos húmedos. Asegúrese de que los ojos están húmedos durante todo el procedimiento quirúrgico. </li><li> Para desinfectar el sitio quirúrgico usar hisopos de algodón estériles para aplicar solución de betadine a la piel sobre el cráneo, seguido por etanol al 70%. Realice el betadine alterna y etanol 70% limpieza 2 veces más. </li></ol> 6. La cirugía y la Infusión <ol><li> Utilice un bisturí para realizar una incisión de 2-3 mm de la línea media del cuero cabelludo, y luego usar una escalera tijeras finas para extender la línea de incisión para 1,0 a 1,5 cm para exponer la sutura sagital, bregma y lambda del cráneo, señals que las coordenadas estereotáxicas se basan en. Utilice micro abrazaderas para mantener la piel a pedazos. Nota: Las posiciones bregma y lambda se explican en el atlas del cerebro de ratones &quot;el cerebro del ratón en estereotáxica Coordenadas&quot; 14. </li><li> Tome un bastoncillo de algodón, sumergirlo en una solución salina estéril, y lo utilizan para limpiar el cráneo. A continuación, utilice un hisopo de algodón limpio y seco para secar el cráneo. </li><li> Utilice una punta fina pluma estéril para marcar un punto en el bregma. Utilice el micromanipulador estereotáxico para colocar la jeringa Hamilton de modo que la punta de la aguja apenas toca el punto en el bregma. <ol><li> Graba el DV coordenadas. </li><li> Para comprobar si el eje AP del cráneo es de nivel se mueve la jeringa Hamilton posteriormente para que la punta de la aguja toca el punto lambda. </li><li> Anote la posición DV. Asegúrese de que el DV coordenadas de bregma y lambda están a 0,5 mm. Nota: El objetivo es reducir al mínimo la diferencia entre DV bregma y lambda. </li></ol></li><li> Úsalos a ellosicromanipulator para volver la punta de la aguja de la jeringa Hamilton de nuevo al punto bregma. </li><li> Anote la posición inicial AP. </li><li> Calcular la posición final AP restando 2,00 mm desde la AP a partir de coordenadas. </li><li> Utilice el micromanipulador para reposicionar la aguja de la jeringa Hamilton a la final AP coordenadas. </li><li> Graba la corriente ML coordenadas. </li><li> Calcular la izquierda y derecha que termina ML coordina mediante la adición o sustracción de 1,3 mm a partir de la ML de coordenadas. </li><li> Utilice el micromanipulador para mover la aguja de la jeringa Hamilton ya sea a la ML terminando de coordenadas. <ol><li> Toque la punta de la aguja a la superficie del cráneo en ambos terminan coordenadas ML y registrar las coordenadas DV y asegurarse de que los valores están dentro de 0,5 mm. Nota: El objetivo es reducir al mínimo la diferencia entre los dos DV terminando ML coordenadas. </li><li> Mientras que en un final coordinar tirar de la aguja hasta 0,5-1 cm por encima del cráneo. Tome una punta fina pluma estéril para marcar un punto en el the superficie del cráneo. Repita este paso para el lado contralateral del cráneo. </li></ol></li><li> Gire la jeringa Hamilton clara fuera del camino. </li><li> Adjuntar 0,8 mm cabeza de perforación a la perforación. Tome el taladro con ambas manos, ajuste los codos sobre la superficie de la mesa para la estabilidad, y la posición de la cabeza de perforación ligeramente por encima del punto sharpie en la izquierda o la derecha ML coordinan. Activar el taladro, bajar la punta de la broca sobre la superficie del cráneo para introducir un agujero en el cráneo. Repita este paso para el lado contralateral. Nota: Algunos sangrado puede ocurrir a partir de los orificios de reciente introducción. Si hay sangrado luego usar un hisopo de algodón limpio y seco para secarse la sangre. </li><li> Mueva la jeringa Hamilton de nuevo en su posición sobre cualquiera de los agujeros de LD de reciente introducción. Baje la aguja Hamilton justo después del cráneo. En este punto, tenga cuidado de no perforar el cerebro. Para asegurarse de que la aguja ha pasado el cráneo tome su dedo índice y empuje suavemente la aguja contra el cráneo para hacer sure la aguja no salir del agujero. </li><li> Graba el DV a partir de coordenadas. </li><li> Calcular el DV terminando coordinar restando 2,2 mm de la DV comenzando coordenadas. </li><li> Baje la aguja hasta el DV terminando de coordenadas. </li><li> Activar la bomba de inyección estereotáxica para bombear 4 l de 1-42 en la circunvolución dentada a una velocidad de 0,5 l / min. </li><li> Cuando la infusión se ha completado dejar que la aguja permanezca en su lugar por un adicional de 1 min para minimizar el reflujo de la solución fuera de la zona de inyección. </li><li> Mover la aguja al otro lado del cerebro y repita los pasos 6.13 a 6.18. </li></ol> 7. La eliminación de animales y Cuidado Postoperatorio <ol><li> Desatornille las barras de oído y protección para la cara / nariz. Retire el ratón del aparato. </li><li> Utilice un estudiante fórceps patrón estándar para tirar de cerrar el cuero cabelludo y luego sellar la herida con sutura. </li><li> Inyectar 1 ml de solución salina estéril en el ratón a través de IP para la hidratación. </li><li> Enbuprenorfina yecto (0,1 mg / kg) por vía subcutánea a aliviar el dolor. </li><li> Mantener el ratón en la jaula vacía limpio situado en la parte superior de 42 ° C placa caliente hasta que se despierta, y luego devolverlo a su jaula de la vivienda con mucha comida y agua. </li><li> Administrar buprenorfina por vía subcutánea cada 12 horas durante 3 días para el alivio del dolor. </li></ol> Remoción 8. Sutura <ol><li> Anestesiar el ratón con cóctel de ketamina / xilazina a 100 mg / kg de ketamina y 10 mg / kg xlyazine por IP. Nota: Esto se hace 7-10 días después de la cirugía. </li><li> Utilice un estudiante fórceps patrón estándar y tijeras finas rectas para eliminar la sutura. </li><li> Inyectar 1 ml de solución salina estéril en el ratón a través de IP para la hidratación. </li><li> Mantener el ratón en una jaula vacía limpio situado en la parte superior de 42 ° C placa caliente hasta que se despierta, y luego devolverlo a su jaula de la vivienda con mucha comida y agua. </li></ol> Sacrificio y Tejidos Procesamiento 9. Animal <ol><li> Anestesiar el ratón y el perfuse con paraformaldehído al 4% 15, retire el cerebro, y procesarlo para cryosectioning 16. Nota: El momento del sacrificio de los animales depende del experimentador. Sin embargo, para nuestros estudios hemos elegido los 7 y 14 días después de la cirugía. </li></ol>

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R., Zaborszky, L., Heimer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+GABAergic+and+cholinergic+neurons+in+the+rat+diagonal+band.">Distribution of GABAergic and cholinergic neurons in the rat diagonal band.</a> Neuroscience. 17 (2), 439-451 (1986).</li><li>Clarke, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+innervation+of+the+rat+dentate+gyrus:+an+immunocytochemical+and+electron+microscopical+study.">Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study.</a> Brain research. 360 (1-2), 349-354 (1985).</li><li>Garcia-Osta, A., Alberini, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+mediates+memory+formation.">Amyloid beta mediates memory formation.</a> Learning &amp; memory. 16 (4), 267-272 (2009).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Para lograr un éxito Aß 1-42 inyección experimentador o cirujano debe: 1) utilizar una técnica aséptica; 2) identificar correctamente la región del cerebro de intereses con coordenadas precisas; 3) ser capaz de asegurar adecuadamente el ratón en el marco estereotáxico con el cerebro nivelado en el eje AP y ML; 4) tener la capacidad de operar el micromanipulador con precisión; 5) garantizar la atención postoperatoria adecuada. Si se siguen estos pasos clave del ratón debe sobrevivir a la cirugía si...

Depósitos de placa amiloide, que se componen de beta-amiloide (Aß 1-42), son una característica clave de la patología de la enfermedad de Alzheimer (EA). Numerosos estudios han demostrado que los niveles elevados o tóxicos de oligomérica recombinante Aß 1-42 suscitar la muerte neuronal, distrofia sináptica, la pérdida y disfunción; así como los déficit de aprendizaje y memoria 1-4. Las regiones del cerebro afectadas incluyen el hipocampo, la corteza y las estructuras subcorticales tales como el cerebro anterior basal y el 5,6 amígdala. Hasta la fecha, hay varios modelos de ratones transgénicos que intentan simular la Aß 1-42 patología de la EA. Dependiendo de la cepa estos animales demuestran ser útiles para examinar seleccione características patológicas de la AD. Desafortunadamente, con la excepción de 2 líneas transgénicas, APP23 y 5XFAD, estos ratones no replicar completamente la pérdida neuronal, un aspecto clave de la EA. Incluso con la pérdida neuronal observada en APP23 y 5XFAD, la obser muerte neuronalved era sutil, dependiente de la edad, y aislada a una pocas regiones selectas 7,8. La infusión directa de oligómeros Aß 1-42 en el cerebro del ratón de tipo salvaje proporciona un excelente modelo in vivo que reproduce el aspecto de la muerte neuronal en amyloidopathy 1,9,10. A diferencia de los modelos de ratones transgénicos utilizados comúnmente el modelo de infusión de Aß 1-42 oligomérica es ideal para la elevación aguda Aß 1-42 niveles de una manera espacial y temporal. La ventaja de usar los ratones de tipo salvaje para este modelo evita potenciales de compensación o los efectos secundarios de las mutaciones introducidas en líneas de ratones transgénicos. Estudios anteriores han demostrado que la infusión de niveles tóxicos de 1-42 en el hipocampo provoca la muerte de neuronas en la vecindad del sitio de inyección dentro de 1 semana 1. Además, en consonancia con la observación de que Aß 1-42 es tóxico para las neuronas colinérgicas del cerebro anterior basal 11 lacolinérgicos neurona (BFCN) población que se proyecta en el hipocampo se reduce un 20-50% dentro de 7 a 14 días después de beta-amiloide infusión de 1,10 en ratones, lo que permite de manera efectiva para los exámenes de circuitos neuronales aisladas en el cerebro. Desde proyecto BFCN ipsilateralmente para el giro dentado del hipocampo 12, para el control de la parte / vehículo y oligómeros Aß 1-42 soluciones más se puede inyectar a cada lado del cerebro que permite realizar comparaciones entre los hemisferios derecho e izquierdo y 1. En este informe vamos a ofrecer una metodología quirúrgica e inyección detallada para adultos de tipo salvaje C57BL / 6J. Esta cepa de ratón se eligió debido a su amplio uso en la investigación. Técnicamente, cualquier región del cerebro puede ser dirigido para perfusión, sin embargo aquí usaremos el giro dentado del hipocampo como el objetivo de ilustrar la técnica.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine HCl (100mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>1049007</td>
			<td>100 mg ketamine per 1 kg animal</td>
		</tr>
		<tr>
			<td>Xylazine (20mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>not available</td>
			<td>10 mg xylazine per 1 kg animal</td>
		</tr>
		<tr>
			<td>Buprenex (0.3mg/ml)</td>
			<td>Henry Schein Medical</td>
			<td>1217793</td>
			<td>0.1 mg buprenex per 1 kg animal</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>David Teplow/UCLA</td>
			<td>not available</td>
			<td>100 &#956;M; This amyloid was used in the paper</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>Bachem</td>
			<td>H-1368</td>
			<td>Can be used in place of amyloid from the Teplow lab</td>
		</tr>
		<tr>
			<td>A&#946;1-42</td>
			<td>American Peptide</td>
			<td>62-0-80B</td>
			<td>Can be used in place of amyloid from the Teplow lab</td>
		</tr>
		<tr>
			<td>Scrambled A&#946;1-42</td>
			<td>American Peptide</td>
			<td>62-0-46B</td>
			<td>Can be used as control peptide for comparing A&#946;1-42</td>
		</tr>
		<tr>
			<td>NU4 Antibody (Oligomeric Amyloid Antibody)</td>
			<td>Gift from William Klein/Northwestern U.</td>
			<td>not available</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Anti-Amyloid Oligomeric Antibody&#160; (Polyclonal Rabbit)</td>
			<td>EMD Millipore</td>
			<td>AB9234</td>
			<td>May be used in place of Nu4; needs to&#160; be tested by the end user</td>
		</tr>
		<tr>
			<td>6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody)</td>
			<td>Biolegend</td>
			<td>sig-39320</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>ChAT Antibody (Polyclonal Goat)</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>DeadEnd Fluorometric TUNEL system</td>
			<td>Promega</td>
			<td>G3250</td>
			<td>Follow manufacturer&#39;s directions for use</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>Use when coverslipping slides</td>
		</tr>
		<tr>
			<td>Fluorogold</td>
			<td>Fluorochrome, LLC</td>
			<td>not available</td>
			<td>2% solution</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>For making 70% ethanol for sanitizing and disinfecting</td>
		</tr>
		<tr>
			<td>Novex 10-20% Tricine gel</td>
			<td>Life Technologies</td>
			<td>EC6625BOX</td>
			<td>For separating A&#946;1-42</td>
		</tr>
		<tr>
			<td>Novex Tricine SDS Running Buffer (10X)</td>
			<td>Life Technologies</td>
			<td>LC1675</td>
			<td>For running 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>Novex Tris-Glycine Transfer Buffer (25X)</td>
			<td>Life Technologies</td>
			<td>LC3675</td>
			<td>For transferring 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>SuperSignal Western Blot Enhancer</td>
			<td>Thermo Scientific</td>
			<td>46640</td>
			<td>For enhancing A&#946;1-42 signal; follow manufacturer&#39;s protocol</td>
		</tr>
		<tr>
			<td>Protran BA79 Nitrocellulose Blotting Membrane, 0.1 &#956;m</td>
			<td>GE Healthcare Life Sciences</td>
			<td>10402088</td>
			<td>For transferring 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>Xcell SureLock Mini-Cell</td>
			<td>Life Technologies</td>
			<td>EI0001</td>
			<td>Electrophoresis aparatus for running 10-20% Tricine gels</td>
		</tr>
		<tr>
			<td>GenTeal Lubricant Eye Gel</td>
			<td>Novartis</td>
			<td>not available</td>
			<td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores</td>
		</tr>
		<tr>
			<td>Refresh Optive Lubricant Eye Drops</td>
			<td>Allergan</td>
			<td>not available</td>
			<td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Stoelting</td>
			<td>50998</td>
			<td>For sanitizing and disinfecting</td>
		</tr>
		<tr>
			<td>Round/Tapered Spatula&#160;</td>
			<td>VWR</td>
			<td>82027-490</td>
			<td>For opening animal mouth</td>
		</tr>
		<tr>
			<td>Bulldog Serrefines Clamps (Jaw Dims. 9X1.6mm; Length 28mm)</td>
			<td>Fine Science Tools</td>
			<td>18050-28</td>
			<td>Optional; For keeping scalp skin apart during injection</td>
		</tr>
		<tr>
			<td>Straight Fine Scissors (Cutting edge 25mm; Length 11.5cm)</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td>For cutting scalp</td>
		</tr>
		<tr>
			<td>#3 Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#11 Surgical Blade</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For making scalp incision</td>
		</tr>
		<tr>
			<td>Student Standard Pattern Forcep (Tip Dims. 2.5x1.5mm; Length 11.5cm)</td>
			<td>Fine Science Tools</td>
			<td>91100-12</td>
			<td>For holding scalp closed during suturing</td>
		</tr>
		<tr>
			<td>Trimmer Combo Kit</td>
			<td>Kent Scientific</td>
			<td>CL9990-1201</td>
			<td>For shaving hair</td>
		</tr>
		<tr>
			<td>T/Pump Warm Water Recirculator&#160;</td>
			<td>Kent Scientific&#160;</td>
			<td>TP-700</td>
			<td>For warming animal during surgery</td>
		</tr>
		<tr>
			<td>Resusable Warmining Pad (5&#34; x 10&#34;)</td>
			<td>Kent Scientific&#160;</td>
			<td>TPZ-0510FEA</td>
			<td>For attaching it to the T/Pump warm water recirculator to warm the animal during surgery</td>
		</tr>
		<tr>
			<td>Cordless Micro Drill</td>
			<td>Stoelting</td>
			<td>58610</td>
			<td>Use 0.8mm steel burrs to drill holes in the skull</td>
		</tr>
		<tr>
			<td>Lab Standard Stereotaxic Instrument with Mouse &#38; Neonatal Rat Adaptor</td>
			<td>Stoelting</td>
			<td>51615</td>
			<td></td>
		</tr>
		<tr>
			<td>Just for Mouse Stereotaxic Instrument</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td>Can use this in place of Stoelting Cat. #51615</td>
		</tr>
		<tr>
			<td>Quintessential Stereotaxic Injector</td>
			<td>Stoelting</td>
			<td>53311</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Glass Bead Sterilizer</td>
			<td>Stoelting</td>
			<td>50287</td>
			<td>For sterilizing stainless steel instruments</td>
		</tr>
		<tr>
			<td>Sterile Surgical Drape (18&#34; x 26&#34;)</td>
			<td>Stoelting</td>
			<td>50981</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Syringe 50 ml, Model 705 RN SYR, NDL</td>
			<td>Hamilton Company</td>
			<td>7637-01</td>
			<td>For brain injection; use different syringes for different solutions</td>
		</tr>
		<tr>
			<td>29 Gauge Needle, Small Hub RN NDL</td>
			<td>Hamilton Company</td>
			<td>7803-06</td>
			<td>For attaching to the Hamilton syringe for brain injection</td>
		</tr>
		<tr>
			<td>1 ml BD Tuberculin Syringes</td>
			<td>VWR</td>
			<td>BD309659</td>
			<td>For administering anesthesia and saline</td>
		</tr>
		<tr>
			<td>30 Gauge Needle (0.5&#34;)</td>
			<td>VWR</td>
			<td>BD305106</td>
			<td>For administering anesthesia and saline</td>
		</tr>
		<tr>
			<td>Portable Electronic CS Series Scale (Ohaus)</td>
			<td>VWR</td>
			<td>65500-202</td>
			<td>For weighing animals to determine anesthesia dose</td>
		</tr>
		<tr>
			<td>Hot plate (Top Plate Dims. 7.25x7.25in)</td>
			<td>VWR</td>
			<td>47751-148</td>
			<td>For warming animals post-surgery</td>
		</tr>
		<tr>
			<td>Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm</td>
			<td>Covidien</td>
			<td>S1173</td>
			<td>For closing wound</td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive (3M)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-361931</td>
			<td>Optional: for aiding in wound closure; Use with suture.</td>
		</tr>
		<tr>
			<td>Cotton-Tipped Wooden-Shaft Sterile Applicators</td>
			<td>Fisher scientific</td>
			<td>22-029-488</td>
			<td>For cleaning and drying surgical wound</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-550-15</td>
			<td>For collecting brain sections</td>
		</tr>
		<tr>
			<td>VWR Micro Cover Glass 24 X 50 mm</td>
			<td>VWR</td>
			<td>48393241</td>
			<td>For mounting microscope slides</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nalgene Syringe Filter 0.2 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>194-2520</td>
			<td>For sterilizing saline solution</td>
		</tr>
		<tr>
			<td>Sterile dual tip skin markers by Viscot Medical</td>
			<td>Medline</td>
			<td>VIS1422SRL91</td>
			<td>For marking coordinates on the skull</td>
		</tr>
	</tbody>
</table>

stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus

La enfermedad de Alzheimer es una enfermedad neurodegenerativa que afecta a la población que envejece. Una característica clave neuropatológico de la enfermedad es la sobre-producción de beta-amiloide y la deposición de placas de amiloide-beta en regiones del cerebro de los individuos afectados. A lo largo de los años los científicos han generado numerosos modelos de ratón de la enfermedad de Alzheimer que intentan replicar la patología amiloide-beta. Por desgracia, los modelos de ratón solamente imitan selectivamente las características de la enfermedad. La muerte neuronal, un efecto prominente en los cerebros de pacientes con enfermedad de Alzheimer, se carece notablemente en estos ratones. Por lo tanto, nosotros y otros han empleado un método de infundir directamente especies oligoméricas solubles de beta-amiloide - formas de beta-amiloide que se han demostrado para ser más tóxico para las neuronas - estereotáxicamente en el cerebro. En este informe utilizamos machos C57BL / 6J para documentar esta técnica quirúrgica de aumento de los niveles de amiloide-beta en una región del cerebro seleccione. Losobjetivo de infusión es el giro dentado del hipocampo, porque esta estructura cerebro, junto con el cerebro anterior basal que está conectada por el circuito colinérgica, representa una de las áreas de degeneración en la enfermedad. Los resultados de la elevación de la beta-amiloide en el giro dentado mediante infusión estereotáxico revelan aumentos en la pérdida de neuronas en el giro dentado dentro de 1 semana, mientras que hay un incremento concomitante en la muerte celular y la pérdida de neuronas colinérgicas en el miembro vertical de la banda diagonal de Broca del cerebro anterior basal. Estos efectos se observan hasta 2 semanas. Nuestros datos sugieren que el actual modelo de infusión de amiloide-beta ofrece un modelo de ratón alternativa para hacer frente a la muerte de neuronas región específica en una base a corto plazo. La ventaja de este modelo es que el amiloide-beta puede ser elevado en una forma espacial y temporal.

El presente método de preparación humana recombinante oligomérica 1-42 produce especies oligoméricas solubles que consiste en monómeros, dímeros, trímeros, y tetrámeros (Figura 1A). Estos especies de bajo peso molecular 1-42, pero no las fibrillas y placas, se ha demostrado en numerosos entornos para ser más tóxico para las neuronas 1,4,9,17-19. Para determinar si es o no oligómeros Aß 1-42 induce la muerte de las neuronas en el cerebro del ratón ...

Watch this Scientific Journal Video about Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus at JoVE.com

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Infusión estereotáxica de Oligomeric beta-amiloide en el hipocampo del ratón

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

stereotaxic-infusion-oligomeric-amyloid-beta-into-mouse

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Neuroscience

17.9K vistas.  Columbia University Medical Center. Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Video: Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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 ...

Investigación

Neurociencias

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 ...

Building An Open-source Robotic Stereotaxic Instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot&#39;s flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8&#176;/Step, geared to 0.346&#176;/Step. A standard stereotax has a resolution of 2.88 &#956;m/step. The maximum recommended cutting speed is 500 &#956;m/sec. The maximum recommended jogging speed is 3,500 &#956;m/sec. The maximum recommended drill bit size is HP 2.

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

La construcción de un código abierto robótica estereotáxica Instrumento

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

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

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

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

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

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

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

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

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Cirugía estereotáctica para manipulación genética en las células estriatales de 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, ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

Grabación espacial restringido a oscilaciones en el hipocampo de ratones de comportarse

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Trasplante de progenitores interneuronas corticales de ingeniería química en cerebros de ratón postnatal temprano

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

Microdisección del cerebro del ratón en regiones funcional y anatómicamente diferentes

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

Cuantificación completa versus subregional de la carga beta amiloide en secciones cerebrales de ratón

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.