Este trabajo fue apoyado por el NIH / NINDS NS subvención 4R00 057.940, y la concesión SCBI 200-12 de la médula espinal y lesión cerebral Fondo de Investigación del Departamento de Salud del Estado de Indiana.

1. Haciendo un simple dispositivo para la cirugía de socavar <ol><li> El dispositivo de socavar hemos creado consta de tres partes (Fig. 1): (1) una placa de soporte de acero inoxidable o de plástico que permite la unión de un tubo guía y una aguja, y se sienta a través de la ventana craneal durante la cirugía, (2) un tubo guía que sostiene una aguja en su posición y permite la rotación de agujas, y (3) una aguja que es un ángulo de 90 grados en ~ 3 mm de la punta, y se puede mover por la rotación y la inserción. </li><li> Corte un pedazo de 1 a 1,5 mm de espesor, de 7 a 10 x 30 mm rectangular de acero inoxidable o de plástico transparente (1) para hacer la placa de soporte. Preparar un 1,5 pulgadas de calibre 22 (BD empresa, # 305156) y un 1,5 pulgadas de calibre 25 (BD empresa, # 305127) aguja de la jeringa. Para hacer un tubo de guía, cortar el extremo de plástico y la punta de la aguja inferior de la aguja de la jeringa de calibre 22 para que su longitud total es de unos 31-32 mm (2). Arena al final de metal para que sea plana y lisa. La longitud final de este tubo guía debe ser de unos 5-6 mm más corta que la aguja de la jeringa de calibre 25. </li><li> Utilice pegamento de cianoacrilato para fijar el tubo guía en la placa de soporte, asegurándose de que la aguja es perpendicular a la orilla del metal. </li><li> Inserte la aguja de la jeringa de calibre 25 en el tubo guía, y doblar la aguja de 90 grados a 2,5-3 mm de la punta (Fig. 1). </li><li> Ajustar el rango vertical que se desplaza de la aguja, pegando un pequeño tubo de plástico en el extremo de la aguja (Fig. 1, parada de la aguja). El rango final de movimiento de la aguja que determina la profundidad de la aguja se puede insertar en la corteza, y debe ser 1.6-1.8 mm de P21 ratas y 1.3-1.5 mm de P21 ratones. Esta longitud debe ser ajustado para diferentes especies y edades de los animales. </li><li> Conecte un cable de cobre pequeña o un pedazo pequeño de cinta adhesiva en el extremo superior de la aguja en la misma dirección que la punta doblada (Fig. 1 indicador de aguja). El cable o cinta indica el ángulo de giro de la aguja durante la cirugía de socavar (Fig. 1). </li></ol> 2. Animal preparación <ol><li> Todos los instrumentos quirúrgicos deben ser esterilizados en autoclave, esterilizado con un esterilizador de bolas de cristal, o desinfectada con etanol al 70%. </li><li> Utilizamos ratas Sprague / Dawley a la edad postnatal 20 a 22 días (P20-22) o ratones CD1 a la misma edad. Las diferentes cepas de ratones o ratas pueden ser utilizados para proyectos específicos. </li><li> Anestesiar a los ratones con una inyección ip de ketamina 80 mg / kg y xilacina 8 mg / kg. El procedimiento quirúrgico se inicia después de que el animal no responde al pellizcar la cola. Durante la cirugía, si es necesario, una dosis de refuerzo de un tercio de la dosis original del cóctel anestésico se puede dar para restablecer el estado original de la anestesia. </li><li> Cortar el cabello en el cuero cabelludo del animal con una cortadora de cabello eléctrica. Aplique una pequeña cantidad de ungüento oftálmico en los ojos de los animales para protegerse de la sequedad durante la anestesia. Desinfectar el cuero cabelludo con un 10% de solución de povidona yodada, seguido de 70% de etanol. </li><li> Monte el animal en un aparato estereotáxico para mantener la cabeza en una posición fija estable. A lo largo de la cirugía, que mantener al animal en un cojín de la calefacción para prevenir la hipotermia. </li><li> Haga una línea media anterior-posterior incisión en el cuero cabelludo con un bisturí, que se extiende desde lambda de entre los ojos. Use pinzas hemostáticas para tirar de la piel a un lado y exponer el cráneo de la izquierda lo suficiente. </li><li> Haga un corte rectangular en el cráneo a la izquierda, y el uso del bisturí para raspar el periostio. Este paso será reducir el sangrado y facilitar la perforación en el cráneo. </li></ol> 3. Lo que socava <ol><li> Agregue una pequeña cantidad de solución salina estéril en la zona del cráneo expuesto, a continuación, utilizar varios Q-tips para limpiar la sangre y seca el área. </li><li> Bajo un microscopio quirúrgico, iniciar la perforación de una ranura rectangular (~ 5 x 7 mm en las ratas, 4 x 5 mm en los ratones) en el centro del cráneo, a la izquierda (aproximadamente por encima de la corteza sensoriomotora izquierda). La aplicación de una gota de solución salina en el cráneo facilita la perforación y el calor se disipa. Después de aproximadamente 2 / 3 de profundidad del hueso se perfora, eliminar el exceso de solución salina y limpiar el área de perforación con un Q-tip. Despacio y con cuidado perforar más profundo hasta que la pieza central de los huesos se puede mover a tocarlo con un fórceps. </li><li> Retire con cuidado la pieza central de hueso mediante la inserción de una punta afilada de una pinza en el borde del hueso y levantando lentamente las pinzas para exponer el hemisferio izquierdo. </li><li> Bajo el microscopio quirúrgico, mantenga el dispositivo de socavar, y orientar la aguja en una dirección parasagital y la placa de soporte perpendicular a la línea media, la inclinación del dispositivo ligeramente caudal a fin de mantener la visualización de la punta de la aguja y la región de destino cortical. Dirija la punta de la aguja a una zona 1-2 mm lateral a la sutura sagital superior, en el centro de la ventana del cráneo, y evitar la penetración directa de los grandes vasos. </li><li> Insertar la aguja en dirección horizontal a través de la duramadre y en el PIA para evitar a los vasos sanguíneos. Sentarse la parte inferior del dispositivo socavaren ambos bordes de la ventana del cráneo para que la aguja se coloca perpendicular a la superficie cortical (Fig. 1). Descansando el dispositivo en los bordes de la ventana craneal tanto a mano de manera significativa a reducir o eliminar la mano temblorosa en el siguiente procedimiento. Levante la aguja por debajo de la pia, y luego baja lentamente para crear una corte transcortical hasta que la aguja no puede ir más profundo. Gire ~ 135 ° de la línea media para crear un tema de medio círculo blanco / capa profunda VI vender más barato. A continuación, levantar la aguja de nuevo a por debajo de la pia. Incline el dispositivo hacia atrás y retirar la aguja. </li><li> Opcionalmente, se puede repetir el procedimiento anterior (paso 2.5) pero sin volver la aguja a fin de crear un recorte adicional transcortical en los bordes laterales de la ventana. Esto creará un aislamiento cortical más completa. </li><li> Coloque un trozo de película de plástico (6 x 6 mm) en la ventana del cráneo de protección, y la sutura en el cuero cabelludo. Colocar el animal sobre un cojín calentado hasta que esté completamente recuperado de la anestesia. </li></ol> 4. Los resultados representativos: Coronal rodajas cortical se puede preparar para confirmar el éxito de la cirugía de vender más barato. En rodajas preparado&gt; 2 semanas después de la cirugía, los recortes transcortical y socavar son discernibles en el objetivo de baja potencia de un microscopio (Fig. 2). La corteza parcialmente aislados por lo general se hace un poco más delgada, y la actividad epileptiforme se puede detectar en la mayoría de las rebanadas corticales con la grabación de campo de potencial (Fig. 3). Por el contrario, la formación de grandes agujeros en la sustancia blanca o en profundas capas corticales, dramático adelgazamiento de la corteza lesionada, o un corte errante por encima o por debajo de la sustancia blanca del cerebro se hacen inutilizables para otros experimentos. <img src="/files/ftp_upload/2840/2840fig1.jpg" alt="Figura 1"/> Figura 1. Estructura y la aplicación de un dispositivo de retención. Un tubo de guía (2) está pegada sobre una placa de apoyo (1) que es de acero inoxidable o de plástico transparente. Una aguja de la jeringa (3) se inserta a través del tubo guía y se inclinó a ~ 3 mm de la punta. Una aguja deje de tubo de plástico pegado a la parte superior de la aguja de modo que el rango vertical en movimiento de la aguja se limita a ~ ~ 1,2 mm y 1,5 mm para su uso en P21 ratones y ratas, respectivamente. Un segmento de alambre de cobre pequeños se fija por debajo de la manija como aguja del indicador de la orientación de la aguja doblada. Tenga en cuenta que el dispositivo está inclinado y en contacto con los bordes de la ventana del cráneo para que el corte puede hacerse en paralelo a la superficie pial. <img src="/files/ftp_upload/2840/2840fig2.jpg" alt="Figura 2"/> Figura 2. Una imagen representativa de la rebanada de vender más barato. A. fluorescentes imagen de una sección que se preparó dos semanas después de la lesión socavado en una rata P48. La herida de corte fue marcado por fluorescentes DII tinte. Las flechas blancas a la derecha indican corte transcortical, y las flechas en la parte inferior indica que socavar pasó a través de la frontera entre la capa IV y la materia blanca. <img src="/files/ftp_upload/2840/2840fig3.jpg" alt="Figura 3"/> Figura 3. Grabación de campo potencial de una rebanada de vender más barato. Campo de registro de potenciales de una rebanada del cerebro mostraron socavar la actividad epileptiforme, lo que sugiere la hiperexcitabilidad cortical del tejido lesionado.

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	<li>Hoffman, S. N., Salin, P. A., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neocortical+epileptogenesis+in+vitro.">Chronic neocortical epileptogenesis in vitro.</a> J Neurophysiol. 71, 1762-1773 (1994).</li><li>Topolnik, L., Steriade, M., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperexcitability+of+intact+neurons+underlies+acute+development+of+trauma-related+electrographic+seizures+in+cats+in+vivo.">Hyperexcitability of intact neurons underlies acute development of trauma-related electrographic seizures in cats in vivo.</a> Eur J Neurosci. 18, 486-496 (2003).</li><li>Graber, K., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Models of Seizures and Epilepsy. , 477-493 (2005).</li><li>Nita, D. A., Cisse, Y., Timofeev, I., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+propensity+to+seizures+after+chronic+cortical+deafferentation+in+vivo.">Increased propensity to seizures after chronic cortical deafferentation in vivo.</a> J Neurophysiol. 95, 902-913 (2006).</li><li>Sharpless, S. K., Halpern, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrical+excitability+of+chronically+isolated+cortex+studied+by+means+of+permanently+implanted+electrodes.">The electrical excitability of chronically isolated cortex studied by means of permanently implanted electrodes.</a> Electroencephalogr Clin Neurophysiol. 14, 244-255 (1962).</li><li>Echlin, F. A., Battista, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptiform+Seizures+from+Chronic+Isolated+Cortex.">Epileptiform Seizures from Chronic Isolated Cortex.</a> Arch Neurol. 9, 154-170 (1963).</li><li>Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptogenic+neurons+and+circuits.">Epileptogenic neurons and circuits.</a> Adv Neurol. 79, 665-684 (1999).</li><li>Marin-Padilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+neuropathology+and+impact+of+perinatal+brain+damage.+II:+white+matter+lesions+of+the+neocortex.">Developmental neuropathology and impact of perinatal brain damage. II: white matter lesions of the neocortex.</a> J Neuropathol Exp Neurol. 56, 219-235 (1997).</li><li>Jin, X., Prince, D. A., Huguenard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitatory+synaptic+connectivity+in+layer+v+pyramidal+neurons+of+chronically+injured+epileptogenic+neocortex+in+rats.">Enhanced excitatory synaptic connectivity in layer v pyramidal neurons of chronically injured epileptogenic neocortex in rats.</a> J Neurosci. 26, 4891-4900 (2006).</li><li>Li, H., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+in+chronically+injured,+epileptogenic+sensory-motor+neocortex.">Synaptic activity in chronically injured, epileptogenic sensory-motor neocortex.</a> J Neurophysiol. 88, 2-12 (2002).</li><li>Salin, P., Tseng, G. F., Hoffman, S., Parada, I., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+sprouting+in+layer+V+pyramidal+neurons+of+chronically+injured+cerebral+cortex.">Axonal sprouting in layer V pyramidal neurons of chronically injured cerebral cortex.</a> J Neurosci. 15, 8234-8245 (1995).</li><li>Avramescu, S., Nita, D. A., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortical+post-traumatic+epileptogenesis+is+associated+with+loss+of+GABAergic+neurons.">Neocortical post-traumatic epileptogenesis is associated with loss of GABAergic neurons.</a> J Neurotrauma. 26, 799-812 (2009).</li><li>Avramescu, S., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+strength+modulation+after+cortical+trauma:+a+role+in+epileptogenesis.">Synaptic strength modulation after cortical trauma: a role in epileptogenesis.</a> J Neurosci. 28, 6760-6772 (2008).</li></ol>

No hay conflictos de interés declarado.

El modelo de socavar un sistema altamente eficiente para el estudio de epileptogénesis postraumático. Una cirugía típica sólo toma unos 20-30 minutos para el final, y evocó la actividad epileptiforme espontánea o se pueden grabar en rodajas de la mayoría de los animales dos semanas después de la cirugía 1-2. Más importante aún, este modelo reproduce los aspectos de los cambios después de una lesión traumática del cerebro, tales como sangrado, inflamación, edema, axotomía y la muerte neuronal ...

<table border="1"><tbody><tr><td> Nombre del Material </td><td> Tipo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Foredom micromotor kit </td><td> equipo </td><td> Foredom </td><td> K.1070 </td></tr><tr><td> 1,5 pulgadas de calibre 22 aguja de la jeringa </td><td> material </td><td> BD empresa </td><td> 305156 </td></tr><tr><td> 1,5 pulgadas de calibre 25 aguja de la jeringa </td><td> material </td><td> BD empresa </td><td> 305127 </td></tr><tr><td> Pegamento de cianoacrilato </td><td> material </td><td> Ted Pella </td><td> 14450 </td></tr></tbody></table>

preparing undercut model of posttraumatic epileptogenesis in rodents

Parcialmente aisladas corteza (&quot;socavar&quot;) es un modelo animal de la epileptogénesis postraumático. El procedimiento quirúrgico consiste en cortar a través de la corteza sensorio-motor y la parte de abajo la sustancia blanca (debilitar) a fin de que una región específica de la corteza cerebral es en gran parte aislado de la corteza vecinos y regiones subcorticales 1-3. Después de una latencia de dos o más semanas después de la cirugía, las descargas epileptiformes se pueden grabar en secciones de cerebro de los roedores una, y ataques eléctricos o de comportamiento se puede observar en vivo a partir de otras especies como el gato y el mono 4-6. Este modelo animal bien establecido es eficiente para generar e imita varias características importantes de la lesión cerebral traumática. Sin embargo, es técnicamente difícil tratar de precisar las lesiones corticales en el cerebro de roedores pequeños con las manos libres. Con base en el procedimiento establecido inicialmente en el laboratorio del Dr. David príncipe en la Universidad de Stanford 1, aquí le presentamos una técnica mejorada para realizar una cirugía para la preparación de este modelo en ratones y ratas. Nos muestra cómo crear un dispositivo quirúrgico simple y lo utilizan para obtener un mejor control de la profundidad de corte y el ángulo de generar resultados más precisos y consistentes. El dispositivo es fácil de hacer, y el procedimiento es rápido de aprender. La generación de este modelo animal proporciona un sistema eficiente para el estudio de los mecanismos de epileptogénesis postraumático.

Watch this Scientific Journal Video about Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents at JoVE.com

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Parcialmente aisladas corteza (&quot;socavar&quot;) es un modelo animal eficiente de epileptogénesis postraumático. Aquí se demuestra cómo hacer que un dispositivo quirúrgico novedoso y lo utilizan para hacer que las lesiones más preciso y consistente para generar este modelo.

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 ...

preparing-undercut-model-of-posttraumatic-epileptogenesis-in-rodents

Research

JoVE Journal

Neuroscience

9.3K vistas.  Indiana University School of Medicine. Parcialmente aisladas corteza ("socavar") es un modelo animal eficiente de epileptogénesis postraumático. Aquí se demuestra cómo hacer que un dispositivo quirúrgico novedoso y lo utilizan para hacer que las lesiones más preciso y consistente para generar este modelo.

Video: Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

Habituación y la inhibición prepulso de sobresalto acústico en roedores

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

Investigación

Neurociencias

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 ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

Una instalación de Bajo Costo para audiometría conductual en roedores

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

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, ...

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 ...

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.

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 ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Extrusión hidráulica de la médula espinal y aislamiento de los ganglios de la raíz dorsal en roedores

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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 ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Implantación de la cánula en la Cisterna Magna de roedores

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

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 ...