Financiación: Este trabajo fue apoyado por una beca de la Fundación Bertarelli (DJL), un MED-EL subvención (DJL), y los Institutos Nacionales de Salud subvenciones DC01089 (MCB).

NOTA: Todos los procedimientos experimentales se realizan de acuerdo con el Comité de Cuidado de Animales y el empleo de la Massachusetts Eye and Ear Infirmary y la Escuela Médica de Harvard, que siguen las directrices nacionales de cuidado de animales, incluyendo la Política Pública de Salud Servicio de Cuidado Humano y Uso de Animales de laboratorio, la Guía ILAR, y la Ley de Protección de los Animales. Los procedimientos experimentales indican a continuación la exposición detallada de la DCN izquierda. Utilice instrumentos estériles mientras se realiza la cirugía de supervivencia. 1. Craneotomía Primaria y Dorsal coclear Núcleo exposición <ol><li> Anestesia <ol><li> Anestesiar a un ratón, a la edad de 8-12 semanas, con un peso de 18-24 g, con xilazina 20 mg / kg de ketamina y 100 mg / kg a través de una administración intraperitoneal. Confirmar la anestesia adecuada por la tasa de monitorización cardiaca, frecuencia respiratoria, así como reflejo de retirada pizca dedo del pie. Lugar veterinario ungüento en los ojos para evitar la sequedad mientras que bajo anestesia. Una vez adecuadamente bajo anestesia, el pelo afeitado recubre tque el cuero cabelludo para proporcionar un acceso sin obstrucciones a la zona quirúrgica. </li></ol></li><li> Posicionamiento quirúrgico <ol><li> Coloca el ratón de forma segura en un pequeño titular estereotáxica animal, sujeto por una abrazadera hocico. </li><li> Asegúrese de que la abrazadera hocico es lo suficientemente floja para permitir la respiración adecuada, pero lo suficientemente apretado para inmovilizar por completo la cabeza del ratón. Si la cabeza del ratón está suelto, el animal es probable que caiga fuera del soporte de cabeza durante la parte craneotomía de procedimiento. </li><li> Coloque los electrodos del implante auditivo de tronco cerebral en forma estándar, lo que permite la monitorización de la frecuencia cardíaca. La frecuencia respiratoria debe ser supervisada por la visualización. Tenga en cuenta que la frecuencia cardíaca normal y la frecuencia respiratoria variarán dependiendo de la edad del ratón. </li><li> Un termómetro se coloca y euthermia está asegurada a través de la colocación de una manta de calefacción homeotérmico. </li></ol></li><li> Incisión de la piel y la exposición de los huesos interparietales y occipital del cráneo <ol><li> Bajo un Microscopa, hacer una incisión vertical a través de la piel a partir de la línea media, directamente entre el pabellón de la oreja, y se extiende hasta la porción caudal del occipucio. Después de incisión en la piel en la línea media, desplazar lateralmente la piel. Visualizar los músculos que cubren la izquierda de la cara posterior del cráneo. </li><li> Retire el músculo que recubre el parietal izquierdo, interparietal, y los huesos occipitales del cráneo por la desarticulación de las inserciones musculares a sus respectivos huesos con un bisturí o tijera iris. Observar un pequeño grado de sangrado a lo largo de los bordes musculares corte. Minimizar esta por presión suave con un aplicador con punta de algodón para 10-15 seg. </li></ol></li><li> Suprayacente craneotomía coclear Núcleo <ol><li> Identificar las líneas de sutura pertinentes, incluidas las líneas de suturas sagital y lambda (Figura 1, panel izquierdo). </li><li> Usando unas pinzas, hacer una craneotomía sobre el hueso interparietal, izquierda de la línea media, ~ 2 mm caudal a la línea de sutura lambda. Esta región se superpone a la DCN. </li><li> Following craneotomía, observar una fina capa de duramadre que recubre el cerebelo. Usando una hoja de bisturí, retire la duramadre. (Figura 1, el panel medio). NOTA: La eliminación de la duramadre puede causar un pequeño grado de sangrado. Este proceso de usar la hoja de bisturí para eliminar la duramadre está destinado a reducir la cantidad de pérdida de sangre durante la aspiración cerebelo, que será común en la superficie del tronco cerebral y la identificación oscura de la DCN. El volumen de sangre total de ratón es ~ 1,5 ml no deben exceder&gt; al 15%. Si el sangrado es superior a esta cantidad, el ratón debe estar provista con suplementos de fluido. </li><li> Retire la sangre coagulada superponer el cerebelo con el aplicador con punta de algodón. Opcionalmente, escurrir suavemente salina utilizando una punta dental en el cerebelo para limpiar la sangre fuera sangre. </li></ol></li><li> Cerebelosa Aspiración <ol><li> Usando una succión Francés 5, aspirar la porción lateral más del cerebelo izquierdo que recubre el DCN hasta que el DCN es visual (Figura 1, panel derecho). Retire aproximadamente 1/4 a 1/3 del cerebelo izquierdo. El hito principal adyacente a la DCN es la ampolla del canal semicircular superior. NOTA: La aspiración del cerebelo es el paso clave del protocolo. Dirigida aspiración del cerebelo se lleva a cabo con mayor éxito si se produce en un solo intento y no con varias pasadas de la succión, ya que esto causar sangrado. Para ayudar en la aspiración, definir el plano focal del microscopio a la profundidad prevista de la NC, que será ligeramente distal a la superficie del cerebelo. Ajuste del plano focal a la CN mejorará la visualización y asegurar una imagen nítida. </li><li> Se espera que después de la aspiración, más sangrado, el líquido cefalorraquídeo (LCR) la acumulación y el desplazamiento cerebelo en la DCN. Inculcar 0,5 cc de solución salina estéril rápidamente en la craneotomía para prevenir la coagulación de la sangre. Una combinación de dabbing suave con un punto dental y la succión puede ser entoncesutilizado para despejar un camino para la visualización directa de la DCN. No entre en contacto directo con la superficie de la DCN. </li></ol></li></ol> 2. La microinyección de presión para la transferencia génica mediada por virus y la recuperación quirúrgica <ol><li> Después de la DCN es libre de sangre suprayacente y CSF y es claramente visible, hacer microinyecciones de presión en el DCN utilizando una jeringa Hamilton de 10 l durante un período de 2 min. </li><li> Introducir la aguja con un micromanipulador hasta que la punta ya no es visible bajo la superficie de la DCN. </li><li> Para un rendimiento óptimo, utilice una aguja de calibre 33 o 34 (utilizar una jeringa estanca a los gases con la aguja de calibre 34) con un bisel de poca profundidad, tales como 45 °, para reducir al mínimo el traumatismo cerrado y localizar el volumen de inyección dentro de la DCN, que es un delgado y estructura del tronco cerebral profunda (&lt;300 micras de espesor). NOTA: Otros instrumentos de microinyección pueden basarse utilizado. Idealmente, el instrumento de inyección debe ser flexible para permitir una ligera bend, si es necesario apuntar directamente a la DCN. Además, como el ratón se mueve ligeramente durante todo el procedimiento debido a la respiración, el instrumento debe ser lo suficientemente robusta como para soportar pequeñas cantidades de movimiento. </li></ol> 3. Recuperación Quirúrgica <ol><li> Inmediatamente después de la inyección, vuelva a la aproximación de la piel y permitir el ratón para recuperar por procedimiento de recuperación estándar. Restante cerebelo y tejido cicatrizal se llenará en la cavidad causada por la aspiración del cerebelo. Constantemente observar a los animales hasta que la conciencia suficiente se recuperó para mantener decúbito esternal. Monitorear la frecuencia cardiaca, la frecuencia respiratoria, así como la capacidad para alimentarse. NOTA: No animal que ha sido sometido a cirugía es devuelto a una jaula con otros animales hasta que se recupere totalmente. </li><li> Si un ratón comienza a caminar en círculos después de la intervención (&lt;5% de los casos), típicamente 2-4 horas después del cierre de la incisión de la piel, sacrificar inmediatamente el ratón, ya que probablemente tendría una alimentación momento difícil. Tsu efecto secundario es probable debido a la aspiración del cerebelo. El animal debe ser supervisada para el dolor después de la operación, y los narcóticos también se debe prestar para asegurar el confort de los animales sobre la base de las normas institucionales. Los antibióticos pueden ser necesarios, si la evidencia de infección. </li></ol> 4. Secundaria exposición craneotomía y coclear Núcleo <ol><li> Después de 2-4 semanas de curación y el gen de incubación transferencia mediada por virus, volver a anestesiar un ratón y repita el paso 1.1 a 1.5 tiempo de incubación óptimo previamente inyectado varía con el tipo de gen transferido. </li><li> Quitar el tejido de la cicatriz sobre el sitio de la craneotomía por una combinación de pinzas, bisturí, y rongeurs. </li><li> Después de visualizar la craneotomía, identificar una combinación de permanecer cerebelo y tejido cicatrizal que recubre la DCN. Utilice una succión Francés 5 para aspirar cerebelo suprayacente / tejido de la cicatriz (similar a la aspiración cerebelar anterior). </li><li> Después de la aspiración, le espera un sangrado posterior, CSF build-up, y el resto de bits de cerebelo. Introducir rápidamente salina en la craneotomía para evitar la coagulación de la sangre. Use una combinación de suave dabbing punto dental y de succión para despejar el camino para la visualización directa de la DCN. </li><li> Observar la superficie de la DCN. Realizar experimentos de fisiología con sede en la optogenética como DCN es ahora accesible. Introducir un estímulo de luz, por ejemplo, con una fibra óptica (Figura 2), para conducir un experimento basado en la optogenética. </li></ol> 5. Histología <ol><li> Tras la conclusión de los experimentos, sacrificar al ratón con una sobredosis de ketamina. Perfundir el ratón con solución salina normal seguido de paraformaldehído al 4%. </li><li> Extraer el tronco cerebral del cráneo y post-fix durante 2 horas. Cryoprotect el tronco cerebral en el 30% de sacarosa durante 24 a 48 horas. Sección del tronco cerebral usando un criostato estándar con 60 micras secciones. </li></ol>

<ol>
	<li>Lalwani, A., Mhatre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy.">Cochlear gene therapy.</a> Ear Hear. 24 (4), 342-348 (2003).</li><li>Lalwani, A., Jero, J., Mhatre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+issues+in+cochlear+gene+transfer.">Current issues in cochlear gene transfer.</a> Audiol Neurootol. 7 (3), 146-151 (2002).</li><li>Jero, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+delivery+through+an+intact+round+window+membrane+in+mouse.">Cochlear gene delivery through an intact round window membrane in mouse.</a> Hum Gene Ther. 12 (5), 539-548 (2001).</li><li>Koh, S., Pettis, R., Mhatre, A., Lalwani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+microinjection+and+its+effects+upon+auditory+function+in+the+guinea+pig.">Cochlear microinjection and its effects upon auditory function in the guinea pig.</a> Eur Arch Otorhinolaryngol. 257 (9), 469-472 (2000).</li><li>Lalwani, A., Walsh, B., Reilly, P., Muzyczka, N., Mhatre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+in+vivo+gene+therapy+for+hearing+disorders:+introduction+of+adeno-associated+virus+into+the+cochlea+of+the+guinea+pig.">Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.</a> Gene Ther. 3 (7), 588-592 (1996).</li><li>Wareing, M., Lalwani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+gene+therapy:+current+perspectives.">Cochlear gene therapy: current perspectives.</a> Int J Pediatr Otorhinolaryngol. 5 (49), 27-30 (1999).</li><li>Han, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene+expression+in+the+guinea+pig+cochlea+mediated+by+a+lentivirus-derived+gene+transfer+vector.">Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.</a> Hum Gene Ther. 10 (11), 1867-1873 (1999).</li><li>Akil, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+hearing+in+the+VGLUT3+knockout+mouse+using+virally+mediated+gene+therapy.">Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.</a> Neuron. 75 (2), 283-293 (2012).</li><li>Hernandez, V. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+the+auditory+pathway.">Optogenetic stimulation of the auditory pathway.</a> J Clin Invest. 124 (3), 1114-1129 (2014).</li><li>Adamantidis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogation+of+dopaminergic+modulation+of+the+multiple+phases+of+reward-seeking+behavior.">Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior.</a> J Neurosci. 30 (31), 10829-10835 (2011).</li><li>Kim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+mimicry+of+the+transient+activation+of+dopamine+neurons+by+natural+reward+is+sufficient+for+operant+reinforcement.">Optogenetic mimicry of the transient activation of dopamine neurons by natural reward is sufficient for operant reinforcement.</a> PloS One. 7 (4), e33612 (2012).</li><li>Britt, J., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogations+of+the+neural+circuits+underlying+addiction.">Optogenetic interrogations of the neural circuits underlying addiction.</a> Curr Opin Neurobiol. 23 (4), 539-545 (2013).</li><li>Abbott, S., Coates, M., Stornetta, R., Guyenet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+c1+and+retrotrapezoid+nucleus+neurons+causes+sleep+state-dependent+cardiorespiratory+stimulation+and+arousal+in+rats.">Optogenetic stimulation of c1 and retrotrapezoid nucleus neurons causes sleep state-dependent cardiorespiratory stimulation and arousal in rats.</a> Hypertension. 61 (4), 835-841 (2013).</li><li>Carter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+arousal+with+optogenetic+modulation+of+locus+coeruleus+neurons.">Tuning arousal with optogenetic modulation of locus coeruleus neurons.</a> Nat Neurosci. 13 (12), 1526-1533 (2010).</li><li>Darrow, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+central+auditory+neurons.">Optogenetic control of central auditory neurons.</a> Assoc. Res. Otolaryngol. Abstr. (695), (2012).</li><li>Shimano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+AAV-mediated+expression+of+channelrhodopsin-2+and+halorhodopsin+in+brainstem+neurons+mediating+auditory+signaling.">Assessment of the AAV-mediated expression of channelrhodopsin-2 and halorhodopsin in brainstem neurons mediating auditory signaling.</a> Brain Res. 1511, 138-152 (2013).</li><li>Doucet, J., Ryugo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+from+the+ventral+cochlear+nucleus+to+the+dorsal+cochlear+nucleus+in+rats.">Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats.</a> J Comp Neurol. 385, 245-264 (1997).</li><li>Brown, M., Drottar, M., Benson, T., Darrow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissural+axons+of+the+mouse+cochlear+nucleus.">Commissural axons of the mouse cochlear nucleus.</a> J Comp Neurol. 521, 1683-1696 (2013).</li><li>Verma, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+responses+to+electric+and+infrared+neural+stimulation+of+the+rat+cochlear+nucleus.">Auditory responses to electric and infrared neural stimulation of the rat cochlear nucleus.</a> Hear Res. 310, 69-75 (2014).</li><li>Taberner, A. M., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+single+auditory+nerve+fibers+in+the+mouse.">Response properties of single auditory nerve fibers in the mouse.</a> J Neurophysiol. 93 (1), 557-569 (2005).</li><li>Rolls, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+disruption+of+sleep+continuity+impairs+memory+consolidation.">Optogenetic disruption of sleep continuity impairs memory consolidation.</a> Proc Natl Acad Sci. 108 (32), 13305-13310 (2011).</li><li>Huff, M., Miller, R., Deisseroth, K., Moorman, D., LaLumiere, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttraining+optogenetic+manipulations+of+basolateral+amygdala+activity+modulate+consolidation+of+inhibitory+avoidance+memory+in+rats.">Posttraining optogenetic manipulations of basolateral amygdala activity modulate consolidation of inhibitory avoidance memory in rats.</a> Proc Natl Acad Sci. 110 (9), 3597-3602 (2013).</li><li>Stortkuhl, K., Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Smell+of+Blue+Light:+A+New+Approach+toward+Understanding+an+Olfactory+Neuronal+Network.">The Smell of Blue Light: A New Approach toward Understanding an Olfactory Neuronal Network.</a> Front Neurosci. 5 (72), (2011).</li><li>Hira, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+optogenetic+stimulation+for+functional+mapping+of+the+motor+cortex.">Transcranial optogenetic stimulation for functional mapping of the motor cortex.</a> J Neurosci Methods. 179 (2), 258-263 (2009).</li><li>Ayling, O., Harrison, T., Boyd, J., Foroshkov, A., Murphy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+light-based+mapping+of+motor+cortex+by+photoactivation+of+channelrhodopsin-2+transgenic+mice.">Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice.</a> Nat Methods. 6 (3), 219-224 (2009).</li><li>Boyden, E., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nat. Neurosci. 8, 1263-1268 (2005).</li></ol>

Los autores no tienen nada que revelar.

En este trabajo se describe la técnica de la visualización directa de la DCN en el modelo murino para la manipulación del sistema auditivo central. El enfoque esbozado de visualización directa ofrece ventajas significativas sobre la principal alternativa, que son enfoques estereotáxica. Principalmente, la visualización directa de la DCN permite confirmación inmediata del sitio del tronco cerebral, mientras que los enfoques estereotáxicas no proporcionan visualización directa. En experimentos que requieren prolo...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

<table border="0" cellpadding="0" cellspacing="0" width="560">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:197px;">Name of the Material / Equipment</td>
			<td style="width:227px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Stereotaxic holder</td>
			<td style="width:227px;">Stoelting</td>
			<td>51500</td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:197px;">Homeothermic blanket</td>
			<td rowspan="2" style="width:227px;">Harvard</td>
			<td rowspan="2">507214</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Scalpel blade #11</td>
			<td style="width:227px;">Fine Surgical Tools</td>
			<td>10011-00</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Iris scissor</td>
			<td style="width:227px;">Fine Surgical Tools</td>
			<td>14084-08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">5 French suction</td>
			<td style="width:227px;">Symmetry Surgical</td>
			<td>2777914</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:197px;">Dental Points</td>
			<td style="width:227px;">Henry Schein</td>
			<td>100-8170</td>
		</tr>
		<tr height="25">
			<td height="46" rowspan="2" style="height:46px;width:197px;">Bone rongeur</td>
			<td rowspan="2" style="width:227px;">Fine Surgical Tools</td>
			<td rowspan="2">16020-14</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="30">
			<td height="51" rowspan="2" style="height:52px;width:197px;">10 &#181;l Hamilton syringe</td>
			<td rowspan="2" style="width:227px;">Hamilton&#160;</td>
			<td rowspan="2">7633-01</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:197px;">34 gauge, needle</td>
			<td rowspan="2" style="width:227px;">Hamilton&#160;</td>
			<td rowspan="2">207434</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rongeurs</td>
			<td>Fine Surgical Tools</td>
			<td>16021-14</td>
		</tr>
	</tbody>
</table>

direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

Investigación sobre el uso de la transferencia génica mediada por virus para detener o revertir la pérdida de la audición en gran medida ha sido relegado al sistema auditivo periférico. Pocos estudios han examinado la transferencia de genes al sistema auditivo central. El núcleo coclear dorsal (DCN) del tronco cerebral, que contiene las neuronas de segundo orden de la vía auditiva, es un sitio potencial para la transferencia de genes. En este protocolo, una técnica para la exposición directa y máxima de la murino DCN a través de un enfoque de la fosa posterior se demuestra. Este enfoque permite ya sea la cirugía aguda o supervivencia. Después de la visualización directa de la DCN, una serie de experimentos son posibles, incluyendo la inyección de opsinas en el núcleo coclear y la estimulación posterior por una fibra óptica acoplada a un láser de luz azul. Otros experimentos neurofisiología, como la estimulación eléctrica y trazados inyectores neuronales también son factibles. El nivel de Visualización y la duración de la estimulación alcanzable hacen de este enfoque aplicable a una amplia gama de experimentos.

Parcial cerebelosa Aspiración demuestre tener acceso a la coclear Núcleo Después de la piel y el músculo que recubre el cráneo se retiran, superficie cráneo puntos de referencia, tales como las líneas de sutura coronal y Lamda, demostrar la localización aproximada de la craneotomía. Siguiendo craneotomía con gubia, el cerebelo se visualiza. La aspiración cuidadosa de la pequeña porción del cerebelo demuestra la visualización de la CN, que luego se puede inyectar <stron...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

La visualización directa de la murino Dorsal coclear Núcleo de optogenética La estimulación de la vía auditiva

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

direct-visualization-murine-dorsal-cochlear-nucleus-for-optogenetic

Research

JoVE Journal

Neuroscience

9.1K vistas.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

Video: Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Uso del protocolo Theta-Burst para explorar Mechasnism de plasticidad en los individuos con el síndrome de X frágil y el autismo

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Investigación

Neurociencias

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

El dispositivo de TMS NeuroStar: Realización de la FDA aprobó el Protocolo para el tratamiento de la depresión

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

Colocación de los electrodos y montaje en estimulación transcraneal corriente continua

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualización de los propioceptores<em&gt; Drosophila</em&gt; Las larvas y pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

La implantación de fibra óptica para la estimulación optogenético crónica en el tejido cerebral

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenético estimulación de comportamiento de escape en<em&gt; Drosophila melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenético la estimulación del sistema nervioso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Estimulación Optogenética del nervio auditivo

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Análisis de los cambios de expresión génica en la Rata Hippocampus Después de la estimulación cerebral profunda del tálamo anterior Núcleo

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

En Vitro Modelado de canceroso Neural Invasion: Modelo del ganglio de la raíz dorsal

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...