This research was supported by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (2010-0022362) and (2012R1A2A2A02046812).

Declaración de Ética: Este experimento fue aprobado por el Comité Institucional Cuidado de Animales y el Empleo de la Universidad Nacional de Kyungpook, y se realizó de acuerdo a la Guía para el Cuidado y Uso de Animales de Laboratorio (Instituto Nacional de la Salud, 1996). Después de experimento, los animales fueron sacrificados bajo la eutanasia dióxido de carbono. 1. Diseño de la barra de la Cabeza-fijación para ratas NOTA: Este paso es una guía general para un diseño de barra de fijación. Cualquier modificación de la dimensión y el material es posible en función de los animales o implantes. El diseño de una barra de cabeza de fijación utilizando software de CAD se recomienda pero no es necesario. <ol><li> Utilice software de CAD para diseñar una barra de fijación. <ol><li> Diseñar una barra de fijación de una forma rectangular (123 × 35 × 6 mm) (Figura 1). Hacer una muesca rectangular (16 × 20 mm; puerto) en el medio de la barra de fijación. Añadir pistas o hendiduras which servir como un carril de guía en las fronteras del puerto (Figura 1-A, Figura 1B -arrows). NOTA: Asegúrese de que el área del puerto cubre el rectángulo prevista de cráneo de rata sobre el cerebro para permitir accesos verticales usando un manipulador estereotáxica para la mayoría de las áreas del cerebro a través de una craneotomía. </li><li> Adjuntar placas (50 × 20 × 6 mm) con dos agujeros alargados de anclaje a ambos lados de la barra de fijación. Asegúrese de que estos agujeros pasan tornillos de 6 mm de diámetro (Figura 1-d). Hacer un agujero de 3 mm de diámetro en la parte delantera del puerto para un gancho de bloqueo (Figura 1-b). </li><li> Hacer una caja que tiene un piso (135 × 305 mm) y paredes laterales (305 × 76 mm). Perforar dos agujeros de diámetro 6 mm como se muestra en la figura 1A (recuadro). Añadir muros de soporte de la barra de fijación (Figura 1-e). </li></ol></li><li> Fabricación del bar diseñado y caja y ensamblar cada una de ellas utilizando cianoacrilato comose muestra en la Figura 1A (recuadro). Montar la barra en la caja con los tornillos de 6 mm de diámetro. Aplicar pendiente ajustando la posición del tornillo de la barra para la fijación de cabeza ergonómico (Figura 1A-inserción). NOTA: Para los ratones, la dimensión de la barra de fijación es 60 × 20 × 4 mm (que consta de dos capas de 2 mm de espesor) paneles de acrílico. El puerto es cuadrada de 8 mm de ancho (superior) y 9 x 10 mm (inferior), como se muestra en la Figura 1C. Placas de anclaje son del mismo tamaño de las ratas &#39;. La placa de caja y las paredes laterales son 100 × 72 mm × 50 y 100 mm, respectivamente. </li></ol> 2. Cabeza Mounter construcción durante la cirugía de implantación PRECAUCIÓN: Tenga cuidado al tratar con resina acrílica dental. El líquido puede causar irritación de la piel. Póngase una máscara y usar guantes. Trabajar en un lugar bien ventilado. <ol><li> Preparación antes de la cirugía <ol><li> Envuelva bien la frontera del puerto de la barra de fijación utilizando aparaffin película para evitar la adhesión de resina acrílica aplicada a la barra (véase la Figura 1B y la Figura 2A). Escudo de la frontera del puerto cubierta por la película de parafina con una fina capa de resina acrílica. </li><li> Utilice instrumentos quirúrgicos esterilizados. Completamente desinfecte marco estereotáxico y sus alrededores usando 70% de alcohol etílico. </li></ol></li><li> Realizar cirugía de implantación estereotáxica <ol><li> Anestesiar a un animal por inyección intraperitoneal de una mezcla de cóctel (2 ml / kg) de ketamina 10 ml (50 mg / ml), clorhidrato de xilazina 1,5 ml (23,32 mg / ml), y la solución salina de 2,5 ml. Confirme que el animal totalmente anestesiado y no responde a la cola y de los pies pellizcos. Inyectar mezcla de cóctel de 1 ml / kg durante la cirugía cuando sea necesario. </li><li> Fijar el animal al aparato estereotáxico. Aplicar pomada oftálmica en los ojos para evitar la sequedad durante la cirugía. Afeitado y limpiar el cuero cabelludo con etanol al 70%. Déle la inyección subcutánea de lidocaína al 2% y la incisión del scalp lo largo de la línea media del cráneo. Completamente raspar periostio. Enjuague con solución salina varias veces. </li><li> Hacer perforación rebabas hoyos cerca del borde del cráneo. Tornillos de implantes 4-5 vueltas (hasta 1 mm de profundidad) de la superficie del cráneo para evitar un incumplimiento de la duramadre. Asegúrese de que no hay fugas de LCR y sangre en la superficie del cráneo después de la implantación del tornillo. Enjuague con solución salina y se seca la superficie del cráneo usando aire soplo, especialmente alrededor de los tornillos. </li><li> Superficie del cráneo Escudo con una capa delgada de &quot;adhesivo dental (Super-Bond C &amp; B)&quot;. No cubra por completo la superficie del cráneo, incluidos el bregma si la implantación de electrodos adicionales está programada. Utilice el bregma para el marcado de coordenadas craneotomía. Electrodos y / o Microdrive implantes y fijarlo en el cráneo mediante la aplicación de resina acrílica a su alrededor. </li><li> Aplicar resina acrílica adicional en la parte superior del cemento dental. Utilice la cantidad apropiada de resina acrílica de dejar suficiente espacio relleno de espera entre la barra recubiertos de una resinaresina de montaje inicial d cuando fijando los tornillos y / o electrodos (Figura 2A). </li></ol></li><li> Coloque la barra de frente fijación 5 mm o más por encima de la superficie del cráneo y alinee su centro (Figura 2A -Izquierda). </li><li> Aplique resina acrílica en el espacio entre la base del cráneo y resinas de montaje iniciales. (Figura 2A -Centro). Espere hasta que la resina se endurecen. </li><li> Retire el oído-bar y tire de la montadora moldeado por el puerto de la barra de fijación. (Figura 2A -Derecho). Aplique cuidadosamente resina acrílica adicional alrededor de los raíles de borde de la montadora moldeado si es necesario. </li><li> Enjuague la superficie de la herida del el cuero cabelludo con solución salina estéril. Inyectar un antibiótico (enrofloxacina, 5 mg / kg sc) y un (tartrato de butorfanol, de 2 mg / kg sc) analgésico inmediatamente después de la cirugía. Casa del animal de forma individual en una sola jaula. </li></ol> 3. habituar Animales de Head-fijación Nota: Este paso deescribas horarios habituación basadas en evaluaciones empíricamente exitosos autores. Para una visión general de procedimiento de habituación para la cabeza de fijación, ver 11 la obra de 10 Schwartz o de Guo. <ol><li> Dar suficiente tiempo de recuperación (una semana o más) a los animales. El día 1, la manija animales con frecuencia en sesiones cortas divididas. </li><li> Al día siguiente (Día 2), exponer el animal para el medio ambiente de fijación (recuadro). No inserte su montador cabeza en el puerto de la barra de fijación. Deja animales para acostumbrarse al medio ambiente. Segura agarrar el montador cabeza con la mano para 1 a 2 seg y liberarlo inmediatamente. Repita la fijación a corto plazo con la mano-agarrar varias veces. </li><li> El día 3, habituar a los animales a la cabeza de fijación. <ol><li> Traiga los animales de las jaulas y asentarse en la caja de la dirección se dirige hacia el puerto de la barra de fijación. Coge el montador jefe y la inserta en el puerto de la barra de fijación. </li><li> Enlazar el montador (animal decabeza) al gancho de bloqueo de la barra de fijación utilizando una goma elástica. Soporte para 5 min. NOTA: No toque el tronco o la cola del animal después de la fijación, lo que lleva al animal a chirriar y luchar aumentar el estrés del animal. Cuando el animal continúa luchando más de 1 min, liberarlo y tratar más tarde. A partir de este paso, los animales muestran excreción de orina y heces siempre expuesto a la cabeza de fijación, pero esto poco a poco se reduce a medida que avanza de habituación. </li><li> Recuperar la banda elástica de bloqueo y expulsar el montador del puerto. Ponga los animales de vuelta a la jaula y proporcionar un pedazo de cereal o una semilla de girasol en la jaula como recompensa. La siguiente prueba empieza en 30 min. Repita 3.4.1 a 3.4.4 cinco o más dentro de un día. </li></ol></li><li> En el Día 4 a 7, exponer el animal a la cabeza de fijación como se describe en el Protocolo 3.4. Poco a poco aumentar la duración de la fijación de la cabeza (10 min, 20 min, 30 min, 50 min y como producto de habituación). NOTA: Si los animales no hacent alimentar el premio de comida, puede ser un signo de un alto nivel de estrés. No aumente el tiempo de fijación. Si los animales muestran repiten luchando comportamiento, habituar a ellos por un período más largo. </li><li> Compruebe el peso corporal de animales diariamente. Considere la habituación sin éxito cuando el peso corporal disminuye. Continúe con las tareas de comportamiento con la fijación de cabeza después de la habituación éxito. NOTA: El éxito de la habituación facilita la formación del comportamiento posterior (Figura 3E y F). </li></ol>

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	<li>Mayrhofer, J. 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=Novel+two-alternative+forced+choice+paradigm+for+bilateral+vibrotactile+whisker+frequency+discrimination+in+head-fixed+mice+and+rats.">Novel two-alternative forced choice paradigm for bilateral vibrotactile whisker frequency discrimination in head-fixed mice and rats.</a> J Neurophysiol. 109, 273-284 (2013).</li><li>Bryant, J. L., Roy, S., Heck, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+stereotaxic+recordings+of+neuronal+activity+in+awake,+head-restrained+mice.">A technique for stereotaxic recordings of neuronal activity in awake, head-restrained mice.</a> J Neurosci Methods. 178, 75-79 (2009).</li><li>Ollerenshaw, D. 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=Detection+of+tactile+inputs+in+the+rat+vibrissa+pathway.">Detection of tactile inputs in the rat vibrissa pathway.</a> J Neurophysiol. 108, 479-490 (2012).</li><li>Lee, M. G., Manns, I. D., Alonso, A., Jones, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep-wake+related+discharge+properties+of+basal+forebrain+neurons+recorded+with+micropipettes+in+head-fixed+rats.">Sleep-wake related discharge properties of basal forebrain neurons recorded with micropipettes in head-fixed rats.</a> J Neurophysiol. 92, 1182-1198 (2004).</li><li>Lutcke, 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=Optical+recording+of+neuronal+activity+with+a+genetically-encoded+calcium+indicator+in+anesthetized+and+freely+moving+mice.">Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice.</a> Front Neural Circuits. 4, 9 (2010).</li><li>Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+large-scale+neural+activity+with+cellular+resolution+in+awake,+mobile+mice.">Imaging large-scale neural activity with cellular resolution in awake, mobile mice.</a> Neuron. 56, 43-57 (2007).</li><li>Kimura, 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=Reinforcing+operandum:+rapid+and+reliable+learning+of+skilled+forelimb+movements+by+head-fixed+rodents.">Reinforcing operandum: rapid and reliable learning of skilled forelimb movements by head-fixed rodents.</a> Journal of neurophysiology. 108, 1781-1792 (2012).</li><li>Hadlock, T., Kowaleski, J., Mackinnon, S., Heaton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+head+fixation+for+the+study+of+rodent+facial+function.">A novel method of head fixation for the study of rodent facial function.</a> Experimental neurology. 205, 279-282 (2007).</li><li>Roh, M., Jang, I. S., Lee, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Push-in+Head+Restraining+Apparatus+for+Intracranial+Self+Stimulation+Tasks+in+Rats.">Push-in Head Restraining Apparatus for Intracranial Self Stimulation Tasks in Rats.</a> Korean J Physiol Pharmacol. 18, 103-108 (2014).</li><li>Schwarz, C., 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=The+head-fixed+behaving+rat--procedures+and+pitfalls.">The head-fixed behaving rat--procedures and pitfalls.</a> Somatosensory &amp; motor research. 27, 131-148 (2010).</li><li>Guo, Z. V., 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=Procedures+for+Behavioral+Experiments+in+Head-Fixed+Mice.">Procedures for Behavioral Experiments in Head-Fixed Mice.</a> PLoS ONE. 9, (2014).</li><li>Osborne, J. E., Dudman, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIVETS:+a+mechanical+system+for+in+vivo+and+in+vitro+electrophysiology+and+imaging.">RIVETS: a mechanical system for in vivo and in vitro electrophysiology and imaging.</a> PLoS One. 9, e89007 (2014).</li></ol>

The authors have nothing to disclose.

This protocol demonstrates a simple and convenient head fixation method using materials at hand. For physically successful head fixation, the first critical steps during surgery is to rinse and dry the skull surface clearly before applying the dental adhesive resin cement. Second, this method does not uses screws implanting under temporal muscles and only depends on the screws on the skull, their perpendicular implantation against the surface of skull rim is crucial10. This reduces the possibility of accidenta...

Cabeza de fijación o la cabeza de restricción es una técnica de inmovilizar la cabeza del animal uniendo un marco o placa en el cráneo para la sujeción rígida. La técnica se ha adoptado para la estimulación sensorial precisa, detección de comportamiento con o sin grabación neural en animales despiertos 1-3 y para el etiquetado juxtacellular durante el ciclo de sueño-vigilia 4. También se utiliza para in vivo de imágenes cerebrales 5,6. Ahora técnica de fijación de cabeza es una herramienta de uso común en la neurociencia y la investigación del comportamiento. Básicamente, la fijación de la cabeza tradicional requiere unión quirúrgica de metales cabeza postes en la parte superior del cráneo 6-8. Los archivos adjuntos se sujetan a continuación a una plataforma estacionaria, lo que resulta en la inmovilización de la cabeza. La investigación neurofisiológica animales despiertos en neural tales como y / o grabación óptica, cerebro intervención circuitos, imágenes del cerebro se acompaña de cirugía de implantación con acrílicos dentales. Por lo tanto,parece una tarea sencilla para aplicar la fijación de la cabeza en esos laboratorios cuando es experimentalmente necesario. Los marcos deben ser diseñados para ser ligero y lo suficientemente pequeño como para no perturbar el comportamiento natural de los animales en sus jaulas. También proporciona rigidez mecánica cuando sujeta a la plataforma estacionaria. Tiene más biocompatibilidad dependiendo de los fines experimentales. Por lo tanto, es técnicamente difícil diseñar y fabricar este tipo de cabeza cargos en el equipo general de laboratorio. En este estudio, un método de fijación de la cabeza novela se describe, proporcionando rigidez mecánica a través de sujeción cómoda y sin emplear dispositivos metálicos cabeceras de mensajes. Al utilizar el método de fijación de cabeza podría hacerse en condiciones de laboratorio en general, donde la cirugía de implantación se realiza generalmente en roedores para el estudio del cerebro. Las cabeceras de postes de metal son reemplazadas con cemento de resina acrílica moldeada parcialmente por una barra de fijación de la cabeza a medida y el proceso de moldeo se realiza durante IMPLANcirugía tación. La barra de fijación proporciona una alta compatibilidad espacial con otros implantes, tales como sondas de grabación neuronales, electrodos de estimulación. También simplifica los pasos de sujeción la cabeza del animal en la plataforma de fijación quitando el tornillo de sujeción. Se utilizó este método en el estudio del comportamiento anterior y llegamos a la conclusión de que este método es fácil de implementar y conveniente en la experimentación práctica 9.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dental acrylic resin</td>
			<td style="width:199px;">Lang Dental Manufacturing</td>
			<td style="width:129px;">Ortho-Jet</td>
			<td style="width:343px;">to make molded mounter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Paraffin film</td>
			<td style="width:199px;">Bemis</td>
			<td style="width:129px;">Parafilm</td>
			<td>to prevent adhesion of acrylic resin to the bar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dental adhesive</td>
			<td style="width:199px;">Sun Medical</td>
			<td style="width:129px;">Super-Bond C&#38;B</td>
			<td>to bond between acrylic resin and skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Plexglass or other acrylic plate</td>
			<td style="width:199px;"></td>
			<td style="width:129px;"></td>
			<td>to make fixation bar and box platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Cyanoacrylate</td>
			<td style="width:199px;"></td>
			<td style="width:129px;"></td>
			<td>to bond acrylic plates</td>
		</tr>
	</tbody>
</table>

acrylic resin molding based head fixation technique in rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Este método de fijación de cabeza es aplicable tanto a ratas y ratones. Totales ocho ratas recibieron implantación bilateral de electrodos de estimulación en el haz medial del cerebro anterior. Para cuatro de ellos, electrodos de alambre de tungsteno personalizados (Microdrive) fueron implantados, además, en el área de la extremidad anterior M1 (Figura 2C). Para los ratones (n = 2), cuatro electrodos de alambre de acero inoxidable se implantaron en áreas corticales frontales / occipitales. Durant...

Watch this Scientific Journal Video about Acrylic Resin Molding Based Head Fixation Technique in Rodents at JoVE.com

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

Técnica de fijación en el acrílico resina de moldeo basado en Jefe en roedores

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

acrylic-resin-molding-based-head-fixation-technique-in-rodents

Research

JoVE Journal

Neuroscience

11.7K vistas.  Kyungpook National University. Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

Video: Acrylic Resin Molding Based Head Fixation Technique in Rodents

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

La preparación de modelos Recorte de Epileptogénesis postraumático en roedores

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

Investigación

Neurociencias

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

Animal entero fijación por perfusión de los roedores

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A gran escala de grabación de las neuronas por medio de sondas de silicio móviles en comportarse roedores

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registro y análisis de los ritmos circadianos en funcionamiento las ruedas Actividad en roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

El trasplante de células ensheathing olfativo para evaluar la recuperación funcional después de la lesión del nervio periférico

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

La estimulación cerebral profunda con simultánea fMRI en roedores

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Estimulación eléctrica cerebral transcraneal en roedores alerta

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Análisis de dolor en roedores y farmacoterapia intracraneal

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

El poder del Interstimulus intervalo para la evaluación de proceso Temporal en roedores

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Un enfoque quirúrgico revisado para inducir hidrops endoifáticos en el cerdo de Guinea

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...