We wish to thank Mr Wabbel for preparing the wires and Mr Tietsch for constructing the plugs and cages according to our plans. This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 688). Felix Fluri holds a fellowship of the Interdisziplin&#228;re Zentrum f&#252;r Klinische Forschung (IZKF), University Clinics W&#252;rzburg, Germany.

Los experimentos con animales fueron aprobados por la Universidad de Würzburg y las autoridades estatales legales (Baja Franconia, número de homologación: 54-2531.01-102 / 13) y realizado de acuerdo con las recomendaciones para la investigación en el accidente cerebrovascular experimental estudia 17 y la corriente de Investigación de Animales: Notificación de En Vivo Directrices Experimentos (http://www.nc3rs.org.uk/arrive-guidelines). 1. Anestesia <ol><li> Compruebe el sistema de anestesia para garantizar cantidades adecuadas de suministro de gas (oxígeno) y el isoflurano durante la duración del procedimiento. Conecte el cono de nariz con la barra incisivo del instrumento estereotáxico y poner la barra de incisión en -3.3 mm. </li><li> Encienda el suministro de gas (2 l / min). Coloque la rata en una caja y sellar la parte superior. Encienda el vaporizador isoflurano al 3,5%. </li><li> Cuando la rata es reclinada, conmutar el sistema de modo que el gas anestésico fluye hacia el cono de nariz que se fija a la barra de incisión. </li><li> Retire la rat de la cámara de caja y afeitar el área entre las orejas y los ojos; utilizando un bastoncillo de algodón empapado con Jodosept PVP, limpiar la zona afeitada para quitar cualquier pelo suelto. </li><li> Coloque la rata en el cono de nariz (Figura 1) y continuar la anestesia con isoflurano 2,5% en O 2 (1 L / min). Compruebe el nivel de anestesia pellizcando la zona interdigital. Si la rata se anestesia adecuada, los reflejos defensivos son abolidos (es decir, la retirada de los pies). </li><li> Monitorear la respiración y la respuesta a la estimulación durante el procedimiento y ajustar el vaporizador según sea necesario. </li><li> Aplique un ungüento veterinario en los ojos para evitar la sequedad mientras que bajo anestesia. Controlar y mantener la temperatura corporal a 37 ± 0,5 ° C por un sistema de calefacción de retroalimentación controlada. </li></ol> 2. Cirugía <ol><li> Mantenga el campo quirúrgico estéril durante toda la cirugía. Una vez que las manos del cirujano son estériles y el campo operatorio es estéril, mover sólo carefully y recordar para no romper la esterilidad. Esto incluye tener también un campo estéril (es decir, cortinas impermeables estériles) en el que uno puede establecer instrumentos. </li><li> Inyectar 0,2 ml por vía subcutánea en la mepivacaína el centro de la zona afeitada. La mepivacaína es un anestésico local que tiene una duración de acción de hasta 3 hr. Será más anestesiar el área quirúrgica. </li><li> Usando un bisturí, hacer una incisión en la línea media de inicio entre las orejas y se extiende hacia 2 cm. Asegúrese de que el periostio (membrana brillante bajo la piel) también se realiza una incisión. Exponer el cráneo con cuatro abrazaderas (Figura 2). </li><li> El uso de un bastoncillo de algodón, retire suavemente el periostio hasta que se exponen las suturas coronal y sagital; a partir de entonces, restañar la sangre con un algodón. </li><li> Determinar las coordenadas del bregma utilizando una aguja fijada en un soporte de la sonda, y luego marcar la punta de la aguja con un rotulador negro. Utilizando el anterior / posterior (AP), la línea media / lateral(ML) y los tornillos (DV) de accionamiento dorsoventral, coloque la punta de la aguja directamente sobre el bregma. </li><li> Tome la AP y ML lecturas de la escala vernier: restar 3,6 mm de la lectura AP y 2,5 mm de la lectura ML para la implantación de electrodos en el STN derecha, o añadir 2,5 mm para la implantación de electrodos en el STN izquierda. Esta posición estará marcado por el tinte del rotulador después de bajar la punta de la aguja a la superficie del cráneo. </li><li> Sujete el taladro dental en el soporte de la sonda grande del instrumento estereotáxico. Mueva el taladro dental a la superficie calculada - es decir, el punto marcado en el cráneo. Mirando a través del microscopio, perforar un agujero (diámetro de aproximadamente 1 mm) a través del cráneo hasta la duramadre es visible (el cráneo es de aproximadamente 1 mm de espesor). Repliegue la duramadre utilizando pinzas de micro-disección o una aguja estéril. La duramadre es lo suficientemente fuerte como para destruir la punta del electrodo. </li><li> Perforar un agujero con el taladro dental en cada escama frontal, y en el interparietal squama opuesta al agujero electrodo. Desconecte el titular de la sonda del instrumento estereotáxico. No taladre en una sutura del cráneo como vasos venosos siguen las suturas bajo el cráneo. </li><li> Atornillar un tornillo de hueso en cada uno de los cinco agujeros. Evite enhebrar los tornillos en las zonas profundas. Para tornillos de acero inoxidable (M1.6), 2-3 vueltas de tuerca llevará a cabo de manera adecuada el tornillo sin poner presión sobre el cerebro. El número de vueltas dependerá del paso del tornillo. Fije el soporte de la sonda con el electrodo en el micromanipulador (Figura 3). </li><li> Utilizando los tornillos de la unidad de AP, ML y DV, mueva el soporte de la sonda con el electrodo hasta que la punta está casi tocando el bregma. Tenga en cuenta las lecturas de la escala vernier AP, ML y DV en el bregma. Cuando se realizan las lecturas, elevar el electrodo de unos pocos milímetros para evitar que el electrodo de raspar el cráneo durante el movimiento. Para determinar las coordenadas de la posición en la que el electrodo tiene que ser insertado enal agujero, añada 3,6 mm a la lectura AP y añadir (o restar) 2,5 mm a la lectura ML. </li><li> Con los tornillos de accionamiento AP y ML, mover el electrodo a la posición calculada. En este punto, la punta del electrodo debe estar situado directamente sobre el agujero perforado electrodo. Entonces, mirando a través del microscopio, bajar el electrodo al nivel de la duramadre (Figura 4). Este nivel sirve como nivel cero en la dirección DV. A partir de entonces, inserte suavemente la punta del electrodo en el cerebro mirando a través del microscopio. </li><li> Conectar el pasador de electrodo al conector del sistema de grabación. Ponga una jaula de Faraday (o sustituirlo con papel de aluminio) sobre la rata en el instrumento estereotáxico (Figura 5). Conecte a tierra el instrumento estereotáxica con el contrapeso de la habitación que se está trabajando en. </li><li> Inicie el sistema de grabación. Si está disponible, también utilizar un altavoz para obtener una señal acústica de las descargas / bálsamos de unidades individuales durante advancing electrodo. </li><li> Lentamente insertar el electrodo en el cerebro mediante el registro de la actividad eléctrica durante el avance del electrodo. A una profundidad de entre 7,5 y 8,1 mm desde la duramadre, la actividad eléctrica específica de la STN es generalmente detectable (Figura 6). 18: La actividad típica de neuronas en el STN se caracteriza por un patrón de disparo irregular y una alta tasa de disparo (40,9 ± 12,9 Hz de frecuencia media). </li><li> Durante la grabación, la anestesia reducir tanto como sea posible (por ejemplo, a 0,8 a 1,0%); animales anestesiados con bajas muestran una actividad cerebral eléctrica más clara. </li><li> Hisopo basura cualquier sangre o líquido cefalorraquídeo que se desplaza en la superficie del cráneo al bajar el electrodo. </li><li> Mezcle una pequeña cantidad de cemento dental y aplicarlo alrededor del electrodo y alrededor de cuatro de los cinco tornillos con una pequeña espátula (Figura 7). El quinto tornillo se utiliza para fijar el cable a tierra de la clavija. </li><li> Desconectar el pasador de electrodo de la pinza portaelectrodos y el conector del sistema de grabación cuando el cemento dental es fijo. </li><li> Aflojar el tornillo que no se fija con cemento dental. Ponga el tapón en el pasador de electrodo. Fije el cable de tierra de la clavija con el quinto tornillo (Figura 8). </li><li> Mezcle cemento dental y aplicarlo alrededor del tapón. Como el cemento se espesa, moldearla alrededor del tapón para formar una tapa. Evite los bordes afilados del cemento dental que puede dañar al animal y eliminarlos durante el endurecimiento (Figura 9A y B). </li><li> Desbride bordes de la herida y cerca de ellos con una sutura en la parte delantera y detrás de la tapa. Desinfectar los bordes de la herida. </li><li> Conecte el enchufe de la cabeza al cable que se fija sobre un pivote. Retire la rata del instrumento estereotáxico. </li><li> Aplicar tramadol (12,5 mg / kg, por vía intraperitoneal) en el final de la intervención y luego una vez al día durante 2-3 días. Coloque la rata en una jaula limpia con Thermal apoyo, fijar la pieza giratoria en esta jaula (Figura 10) e inspeccione cuidadosamente durante 1 hora. </li><li> No deje a un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. No devuelva un animal que se ha sometido a una cirugía para la compañía de otros animales hasta que se recupere totalmente. </li></ol> 3. Estimulación <ol><li> Determinar la resistencia en el animal antes de la estimulación utilizando un medidor de impedancia. </li><li> Conectar el enchufe del eslabón giratorio con un alambre y los tapones en el otro extremo del alambre con la salida de corriente y la salida para el cable de tierra del estimulador. Conecte el estimulador con un ordenador con el fin de programar el estimulador. </li><li> Elija los parámetros de estimulación en el programa; por ejemplo, los parámetros utilizados en la enfermedad de Parkinson son la longitud de pulso: 60 microsegundos; Frecuencia: 130 Hz. Estimular la rata con una amplitud de la corriente cada vez mayor hasta que se reconocen discinesia. Reducir ªe intensidad eléctrica en un 10-20% por debajo de la intensidad que provocó discinesia o hasta que desaparezcan los signos neurológicos y el animal es cómodo. Pulsos rectangulares monofásicos se utilizaron en este estudio. </li><li> Después de completar el experimento, la eutanasia a los animales con isoflurano: Ajuste el caudal isofurane o concentración de 5% o más. Continúa la exposición isoflurano hasta 1 minuto después de la respiración se detiene. </li></ol>

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The authors declare that they have no competing financial interests.

<html> Este estudio presenta un conjunto paso a paso las instrucciones para la implantación de un electrodo monopolar crónica en el STN de ratas. Aunque electrodos de tungsteno con baja impedancia se utilizan a menudo para DBS 18,19, un electrodo monopolar de platino / iridio (Pt / Ir) fue empleado que tenía una impedancia de aproximadamente 1 MW. Electrodos de Pt / Ir también se utilizan en pacientes con la enfermedad de Parkinson debido a sus propiedades favorables: demuestran la erosión mínima 20...

La estimulación cerebral profunda (DBS) es un tratamiento basado en la entrega de los impulsos eléctricos a través de electrodos implantados en las estructuras cerebrales específicas, tales como el globo pálido interno globus 1, el núcleo subtalámico (STN) 2-4 o el tálamo ventral intermedio 5. En las dos últimas décadas, este tratamiento se ha establecido como una herramienta terapéutica potente para la enfermedad de Parkinson 1 - 4, distonía 6 y el temblor 7, y también se utiliza para modular el dolor crónico 7, trastornos psiquiátricos (por ejemplo, trastorno obsesivo-compulsivo 8, depresión mayor 9) o epilepsia intratable 10,11. Además, DBS podría, en un futuro, convertirse en una opción de tratamiento para la hipertensión arterial refractaria 12 o hipotensión ortostática 13. Los mecanismos fisiológicos que subyacen a los efectosde DBS siguen siendo poco conocidos. Los estudios realizados en roedores anestesiados han proporcionado información sobre las respuestas neuronales a la estimulación de alta frecuencia que imitan clínicamente aplica DBS 14. Sin embargo, estos estudios no sólo carecen de corroboración del comportamiento del efecto DBS, pero también dan lugar a una considerable variabilidad en función de los parámetros de estimulación aplicada 14. Para investigar más concisa los efectos conductuales y mecanismos subyacentes de DBS en roedores conscientes, se necesita una estimulación configuración que cumple con los requisitos específicos. DBS se utiliza principalmente como un tratamiento a largo plazo (por ejemplo, enfermedad de Parkinson, el dolor crónico). Por lo tanto, la estimulación configuración en roedores debe ser diseñado de modo que la unidad consta de un electrodo con un enchufe, así como un cable desde el enchufe a un estimulador externo; y esta unidad debe ser ligero pero irrompible cuando se fija en el cráneo. Por otra parte, la libertad de movimiento es indispensable para ratas durante STIMULAción durante un período prolongado. Las estructuras diana de DBS son pequeñas; Por ejemplo, el STN en ratas tiene una longitud de 1,2 mm y un volumen de 0,8 mm 3,15. Por lo tanto, los electrodos deben estar diseñados de tal manera que el núcleo no está lesionada durante la inserción y la orientación necesidades para ser precisos. Como la mayoría de los estudios llevados a cabo en roedores DBS han utilizado señal basada en la inserción estereotáxica del electrodo a la estructura de destino, la tasa de error puede ser relativamente alta, incluso cuando se utilizan las coordenadas de acuerdo con Paxinos y Watson 16. Esto se traduce en un mayor número de animales necesarios para llegar a un resultado estadísticamente significativo. En el presente estudio se introduce una técnica de implantación de electrodos, que se dirige a la STN con gran precisión mediante el uso de un sistema microrregistro mientras se avanza en el electrodo. Además, un sistema de estimulación se presenta que no sólo permite un alto grado de movilidad para el animal estimulado pero también garantiza stimulati continuaen medio de una fijación segura del cable de estimulación (que está protegida por un resorte de acero inoxidable) en la cabeza de la rata.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pt/Ir electrode</td>
			<td>FHC Inc.</td>
			<td>UE</td>
			<td>Custom-made: Specification: UEPSEGSECN1M</td>
		</tr>
		<tr>
			<td>Plugs</td>
			<td>GT Labortechnik (Arnstein/Germany)</td>
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			<td>Custom-made</td>
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			<td>Pin header</td>
			<td>DISTRELEC</td>
			<td>143-95-324</td>
			<td>single-row, 90&#176; 1x3 datamate, Type M80-8420342</td>
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			<td>Socket</td>
			<td>DISTRELEC</td>
			<td>143-95-621</td>
			<td>single-row,straight 2 mm pole no.1x3 datamate, Type M80-8400342</td>
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			<td>Stainless steel spring</td>
			<td>Plastics ONE</td>
			<td>SS0102</td>
			<td>Part-#: .120 X .156 Spring ID (mm): 3.0&#160; Spring OD (mm): 4.0</td>
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			<td>Dental cement/Paladur</td>
			<td>Heraeus Kulzer</td>
			<td>64707938</td>
			<td>Liquid, 500 ml</td>
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			<td>Dental cement/Paladur</td>
			<td>Heraeus Kulzer</td>
			<td>64707954</td>
			<td>Powder, rose, 500g</td>
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			<td>Head screw</td>
			<td>Hummer &#38; Reiss</td>
			<td>V2ADIN84 M1.6x3</td>
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			<td>Jodosept PVP</td>
			<td>Vetoquinol</td>
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			<td>435678/E04</td>
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			<td>Mepivacain 1%</td>
			<td>AstraZeneca</td>
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			<td>Epinephrine</td>
			<td>Sanofi-Aventis</td>
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			<td>PZN00176118</td>
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			<td>Tramadolhydrochloride</td>
			<td>Rotexmedica</td>
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microelectrode guided implantation of electrodes into the subthalamic nucleus of rats for long-term deep brain stimulation

La estimulación cerebral profunda (DBS) es una terapia ampliamente utilizado y efectivo para varios trastornos neurológicos, como la enfermedad, la distonía o temblor de Parkinson idiopática. DBS se basa en el suministro de estímulos eléctricos a lo profundo estructuras anatómicas específicas del sistema nervioso central. Sin embargo, los mecanismos subyacentes el efecto de DBS siguen siendo enigmática. Esto ha dado lugar a un interés en investigar el impacto de DBS en modelos animales, especialmente en las ratas. Como DBS es un tratamiento a largo plazo, la investigación debe centrarse en los cambios moleculares genéticos de los circuitos neuronales que se producen varias semanas después de DBS. A largo plazo DBS en ratas es un reto porque las ratas se mueven alrededor de su jaula, lo que causa problemas en mantener en su lugar el cable que va desde la cabeza del animal al estimulador. Además, las estructuras diana para la estimulación en el cerebro de rata son pequeñas y por lo tanto los electrodos no pueden ser fácilmente colocados en la posición requerida. Así, una puesta a punto para larga duración STIMULAción de ratas utilizando electrodos de platino / iridio con una impedancia de aproximadamente 1 MW fue desarrollado para este estudio. Un electrodo con estas especificaciones permite no sólo la estimulación adecuada, sino también la grabación de las estructuras cerebrales profundos para identificar el área objetivo para DBS. En nuestra puesta a punto, un electrodo con un enchufe para el cable se ha incrustado en el cemento dental con cuatro tornillos de anclaje garantizados en el cráneo. El cable desde el enchufe para el estimulador estaba protegido por un resorte de acero inoxidable. A giratoria se conecta al circuito para evitar que el alambre se enrede. En general, esta estimulación configuración ofrece un alto grado de movilidad libre para la rata y permite que el tapón de cabeza, así como la conexión del cable entre el enchufe y el estimulador, para retener la fuerza de larga duración.

La implantación de un electrodo dentro de la STN de una rata utilizando un sistema de grabación - tal como se presenta aquí - es un procedimiento eficaz y preciso para DBS que tarda aproximadamente 1 hr por animal. Este modelo es un procedimiento bastante menor: de cada 10 ratas sometidas a cirugía, todos sobrevivieron a la intervención. Veinticuatro horas después de la intervención, el estado de cada rata se monitorizó y ningún animal logra más de 1 de 3 puntos según el código de gravedad. Durante el perío...

Watch this Scientific Journal Video about Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation at JoVE.com

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Microelectrodos guiada implantación de electrodos en el Subtalámico Núcleo de ratas de largo plazo estimulación cerebral profunda

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

microelectrode-guided-implantation-electrodes-into-subthalamic

Research

JoVE Journal

Neuroscience

19.4K vistas.  University Hospital Wuerzburg. A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Video: Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

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 ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

Un pulido y reforzado Diluido cráneo-Ventana para a largo plazo de imágenes del cerebro de ratón

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

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 ...

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 ...

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 ...

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 ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Implantación crónica de todo cortical matriz electrocorticográfica en el tití común

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Evaluación de la inducción a largo plazo de la depresión en rodajas de cerebelosores adultos

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Preparación de electrodos de estimulación nerviosa periférica para la implantación crónica en ratas

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...