Agradecemos el apoyo de los NIH (NS047073 a HJC). También estamos agradecidos a Catherine Kehl por sus primeros experimentos con Kwik-Sil, que nos animó a usarlo para nuestra aplicación.

1. Fabricación de electrodos <ol><li> Para crear el electrodo, use tijeras para cortar un trozo de esmaltado 0.001 alambre de acero inoxidable de diámetro alrededor de 2 pies de largo. Coloque una pequeña bola de masilla a cada extremo del cable, doble el alambre a la mitad, y el giro de los extremos para crear un par trenzado, llamado electrodo diferencial. </li><li> Tape ambos extremos del alambre para evitar que desentrañar. Hacer cuatro electrodos diferenciales, usando un color diferente de la cinta para cada electrodo diferencial para fines de identificación (Figura 1). </li><li> Giro de dos de los electrodos diferenciales juntos, dejando dos pulgadas de torsión en un extremo y 1 pulgada en el otro extremo. Repita con el resto de par de electrodos diferenciales (fig. 1B). </li><li> Gire los dos conjuntos de dos electrodos diferenciales juntos, asegurándose de que los extremos quedan sin torsión. Levante el conjunto de electrodos y aplicar la goma de pegar de silicona en la unión de los cuatro electrodos diferenciales, dejando los extremos libres. Cuelgue y deje que el pegamento se seque por lo menos durante 4 horas (fig. 1C). </li><li> Para aislar las ramas, estaba el conjunto de electrodos hacia abajo sobre una superficie plana, muebles, un trozo de madera. Fan de los extremos y fijarlos con cinta adhesiva. Use una pequeña pieza de plástico para elevar el conjunto de electrodos justo antes de que las ramas, y seguro que con la cinta. </li><li> En un microscopio de disección, el uso de fórceps para cubrir las ramas con pegamento de silicona. Los extremos de 1 pulgada de largo será en el interior del animal, por lo que debe tener una capa muy fina de pegamento. Deje que el pegamento se seque por lo menos 3 horas. </li><li> Acortar el extremo largo de 1 pulgada de electrodo diferencial de uno a dos centímetros. En cada alambre, quite 1 cm de silicona y el esmalte. Curl un cable pelado en un gancho para la grabación. Doble el otro cable hacia arriba y lejos del gancho, lo que servirá como cable a tierra. Repita el procedimiento para las otras tres electrodos. </li><li> Seleccione uno de los cuatro electrodos en la parte 2 pulgadas. El uso de fórceps para raspar el esmalte de silicona y apagado, la exposición de 1 cm de cable desnudo de acero inoxidable en cada uno de los dos cables. Soldar un conector de oro a cada uno de los dos cables. Use un pedazo de cinta de laboratorio para asegurar el final y mantener una separación entre el suelo y los pasadores de grabación. Repita el procedimiento para las otras tres electrodos. </li><li> Coloque una bola de 1-2cm de la cola de silicona para el cable, alrededor de 1,5 pulgadas del extremo del gancho para fijar los electrodos dentro del animal. Deje secar por lo menos durante una hora (Figura 1D). <img src="/files/ftp_upload/1791/1791fig1.jpg" alt="Figura 1" /> Figura 1. Fabricación de electrodos de la Asamblea. (A) Electrodo diferencial único, compuesto de un alambre doblado y retorcido, con sus extremos asegurados con cinta adhesiva. (B) dos electrodos diferenciales entrelazadas, dejando un espacio sin torsión en los extremos. Cintas de diferentes colores o marcado otro debe ser utilizado para identificar los extremos. (C) Todos los electrodos diferenciales cuatro combinados en un único conjunto de electrodos que forman un solo cable, con ramas separadas de los electrodos individuales en cada extremo, marcados con distintos colores de la cinta. (D) Forma definitiva del conjunto de electrodos. A la izquierda, no se enroscan los ganchos para las grabaciones y los cables de tierra para cada electrodo diferencial. A la derecha, alfileres de oro conector han sido soldadas a los cables para la conexión al amplificador. Una bola de anclaje de silicona pegamento también se ha colocado hacia el lado izquierdo de la asamblea. </li></ol> 2. Preparación de animales para la Implantación <ol><li> Seleccione un animal sano y pesa alrededor de 350-450g. En respuesta a pequeños trozos de algas marinas, el animal debe generar las picaduras a intervalos de entre 3 a 5 segundos. La cirugía completa con la implantación de electrodos debe tomar alrededor de una hora. </li><li> Para anestesiar al animal, que se inyectan con un 30% w / v isotónica MgCl 2. Para evitar la pérdida de hemolinfa y MgCl 2, el sitio de inyección se pueden revestir de pegamento antes de retirar la aguja. <img src="/files/ftp_upload/1791/1791fig2.jpg" alt="Figura 2" /> Figura 2. Diagrama esquemático que muestra cómo el animal se fija para la implantación de electrodos, y la ubicación de la incisión (indicado por una barra). </li><li> Coloque el animal anestesiado en una bandeja de disección con su pie en el suelo. Frente a la cabeza de animal en, desplace el área de enmalle a la izquierda tal que el animal es parte de su lado, apoyándose de distancia. Precisar los tentáculos anterior (Figura 2). </li><li> Coloque la bandeja en el escenario de un microscopio de disección. Sostener la bandeja en un ángulo de 20 grados para elevar la cabeza y recoger la hemolinfa en la cola, lejos de la incisión. </li><li> Usando tijeras de disección, iniciar una incisión ligeramente a la derecha de la mancha ocular y su ampliación del 1 al 1,5 cm hacia delante. Utilice separadores unido a los bordes de la bandeja para mantener la incisión abierta (Figura 2, un bar cerca de la incisión nota indicando mancha ocular). </li><li> Para exponer la masa bucal, el uso de fórcepss para levantar la membrana transparente debajo de la piel y las tijeras para cortar el uso de una abertura correspondiente de la incisión principal. Meta los bordes de la membrana en los separadores. </li></ol> 3. La implantación de electrodos <ol><li> Coloque el conjunto de electrodos en un micromanipulador y moverlo sobre la incisión del animal. </li><li> El nervio rádula es de más difícil acceso, y se debe abordar en primer lugar. Hay dos opciones para acceder a los nervios radular. </li><li> La primera opción, que es preferible, es buscar en el ganglio vestibular entre BN1 y BN2. Si una rama del nervio radular es visible, use unas pinzas de punta fina doblada en forma de gancho para levantar la rama (Figura 3, el bucle de RN en ganglio marcado 1). </li><li> Si una rama del nervio radular no es accesible por encima del músculo, una segunda opción es acceder a ella a través de una pequeña incisión en I2 al lado del tejido conectivo en la base del ganglio vestibular (Figura 3, RN por debajo de los músculos marcados 2). La incisión en I2 debe pasar por dos capas del músculo I2. Sostenga el borde de la incisión del músculo con pinzas rectas y el uso de las pinzas de gancho para tirar de una rama de RN. </li><li> Mientras mantiene el nervio con la pinza de gancho, utilizar un conjunto diferente de pinzas para manipular el gancho del electrodo en el nervio. </li><li> Utilice el micromanipulador para elevar el gancho para que el nervio se levanta lejos de la masa bucal y libre de cualquier otros nervios o el tejido. No estire demasiado el nervio. </li><li> Seque el electrodo con un Kim-Wipe o succión de el exceso de líquido con una jeringa. Tensión del agua puede causar que el nervio se derrumbe sobre sí misma, o un sello de agua para formar en el nervio. En cualquier caso, el área debe estar completamente seca antes de pegamento se aplica. </li><li> Aplicar una cantidad muy pequeña de pegamento en la base del gancho está en contacto con la parte inferior del nervio. Aplique una gota de solución salina o hemolinfa Aplysia para establecer el pegamento. Cuando el pegamento no es húmedo se debe mover como conseguir unión entre el cable y el nervio se bloquea la conexión eléctrica. </li><li> Seque el gancho. Mezcla de Kwik-Sil pegamento en una bolsa de hielo. </li><li> Use la punta de un alfiler para aplicar una pequeña cantidad de pegamento Kwik-Sil, cubriendo completamente los cables expuestos del gancho, pero no el cable a tierra. </li><li> Deje que el pegamento para fijar durante unos 5 minutos, el reajuste de la cola si se aleja de las secciones del gancho. La cola se establece cuando se pega cuando ya no se tocó con el pasador (Figura 4 muestra la configuración final). </li><li> Una vez que el electrodo nervio rádula está establecido, el músculo I2 se puede acceder. En la zona entre BN1 y BN2, utilice las pinzas gancho para conectar y separar dos bandas del músculo I2 del resto de los músculos tirando suavemente de las bandas hacia arriba. Conecte el electrodo utilizando el mismo procedimiento. </li><li> Nervios bucales 2 y 3 son de fácil acceso y los electrodos están conectados con el mismo procedimiento. </li><li> Una vez que todos se coloquen los electrodos, deslice el conjunto de electrodos en el animal hasta el anclaje de silicona. </li><li> Sutura la incisión se cierra alrededor del cable del electrodo que sobresale, manteniendo el anclaje en el interior del animal. Superglue los puntos para asegurar un buen sello. </li><li> Inserte las puntas del conector de oro en el otro extremo del electrodo en un pequeño bloque de espuma de poliestireno. Devolver el animal al tanque principal en un contenedor de aislamiento celular durante la noche para la recuperación. </li></ol><img src="/files/ftp_upload/1791/1791fig3.jpg" alt="Figura 3" /> Figura 3. Un diagrama del aparato de alimentación Aplysia se conoce como la masa bucal. Se muestran los nervios vestibulares (BN1, BN2, BN3), el nervio rádula (RN) que sale de la parte inferior del ganglio vestibular, luego se sumerge por debajo del músculo, y la ubicación en el músculo I2 donde está conectado el electrodo. <img src="/files/ftp_upload/1791/1791fig4.jpg" alt="Figura 4" /> Figura 4. Esquema de electrodo conectado a un nervio (o músculo) y pegados en su lugar. Tenga en cuenta que la región de aislamiento del gancho curvado está en contacto directo con el nervio (o músculo), anclado en su lugar con pegamento, y aislados eléctricamente del medio líquido utilizando Kwik-Sil. Tenga en cuenta también que la punta deinsulated del electrodo de masa está expuesto al medio líquido. 4. Grabación <ol><li> Aclimatar al animal en un recipiente aireado mientras se está conectado al aparato de control durante al menos 1,5 horas. Monitorizar la señal mientras el animal está aclimatando a verificar que todos los electrodos están funcionando y que los niveles de ruido son bajos. </li><li> Utilice una cámara de video para registrar el comportamiento de alimentación mientras se utiliza el ordenador para grabar las señales de los electrodos neurales y musculares. Sincronizar las grabaciones de vídeo y el electrodo con una señal de onda cuadrada que controla un contador digital en la vista de la cámara mientras se graban simultáneamente como un canal adicional en el equipo en Axoscope. </li><li> Comportamientos de alimentación son provocadas por las técnicas descritas en Morton y Chiel 1993. </li></ol> Aunque los animales invertebrados no requieren la aprobación formal por parte de un uso de los animales y el comité institucional de atención, nos hemos asegurado de que todos los tratamientos de Aplysia minimizar el daño y sufrimiento a los animales, y que todas las técnicas quirúrgicas se realizan mientras el animal esté totalmente anestesiado. Resultados representante <img src="/files/ftp_upload/1791/1791fig5.jpg" alt="Figura 5" /> Figura 5. (A) En forma simultánea las grabaciones de cuatro canales a partir de un músculo (el músculo del transportador I2), y de tres nervios vestibulares (el nervio radular, RN, nervio bucal 2, BN2, y el nervio vestibular 3, BN3) durante la respuesta a la deglución. (B) Una imagen fija de la grabación simultánea de vídeo del animal libre se comportan como se traga una tira de algas precortadas (0,5 cm de ancho), que ha estado marcada a intervalos de 0,5 cm con bandas blancas para que sus movimientos pueden ser fácilmente visualizados como el animal se alimenta.

<ol>
	<li>Lu, H., Chestek, C. A., Shaw, K. M., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extracellular+stimulation+of+individual+neurons+in+ganglia.">Selective extracellular stimulation of individual neurons in ganglia.</a> J. Neural Eng. 5, 287-309 (2008).</li><li>Morton, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+buccal+nerve+activity+that+distinguishes+ingestion+from+rejection+can+be+used+to+predict+behavioral+transitions+in+Aplysia.">In vivo buccal nerve activity that distinguishes ingestion from rejection can be used to predict behavioral transitions in Aplysia.</a> J Comp. Physiol. A. 172, 17-32 (1993).</li><li>Warman, E. N., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+chronic+single+extracellular+recording+in+freely+behaving+animals+using+pipette+electrodes.">A new technique for chronic single extracellular recording in freely behaving animals using pipette electrodes.</a> J. Neurosci. Methods. 57, 161-169 (1995).</li><li>Ye, H., Morton, D. W., Chiel, H. J., J, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nbsp;Neuromechanics+of+multifunctionality+during+rejection+in+Aplysia+californica.">nbsp;Neuromechanics of multifunctionality during rejection in Aplysia californica.</a> J. Neurosci. 26, 10743-10755 (2006).</li></ol>

Una innovación clave que ha hecho de estas grabaciones de éxito es la combinación de los electrodos en un cable. Dos grabaciones de electrodos se han usado antes, pero ampliando a cuatro electrodos individuales dio lugar a la formación de adherencias quirúrgicas que a menudo impide al animal hacer los movimientos normales de alimentación. La combinación de los electrodos en un cable reduce la mayor parte de los electrodos en el interior del animal, que a su vez reduce la respuesta inmune y permite que el animal s...

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electrode fabrication and implantation in aplysia californica for multi-channel neural and muscular recordings in intact, freely behaving animals

Grabación de los nervios y los músculos clave de<em&gt; Aplysia</em&gt; En el comportamiento de alimentación nos permite estudiar los patrones de control de los nervios en un animal intacto. Al mismo tiempo de grabación de múltiples nervios y los músculos nos da información precisa sobre el calendario de la actividad neuronal. Los métodos anteriores han trabajado para la grabación de dos electrodos, pero el estudio de los nervios o los músculos adicionales necesarios combinando y un promedio de las grabaciones de varios animales, lo que hacía difícil determinar los detalles finos de la oportunidad y la eliminación gradual, debido a la variabilidad de la respuesta a la respuesta, y de un animal a otro. La implantación de cuatro electrodos individuo tiene una tasa de éxito muy baja debido a la formación de adherencias que impiden que los animales de la realización de movimientos normales de alimentación. Hemos desarrollado un nuevo método de fabricación de electrodos, que reduce la mayor parte de los electrodos en el interior del animal teniendo en cuenta los movimientos de la alimentación normal. Usando una combinación de gomas para sujetar los resultados de los electrodos en un aislamiento más confiable del electrodo, que dura más tiempo, por lo que es posible grabar por periodos de hasta una semana. La técnica de fabricación que describimos podría ser ampliado para incorporar varios electrodos adicionales, y que se aplique a los animales vertebrados.

Watch this Scientific Journal Video about Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals at JoVE.com

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Una técnica se describe para la implantación de cuatro<em&gt; In vivo</em&gt; Electrodos para monitorear el control neuromuscular de la conducta alimenticia en<em&gt; Aplysia californica</em&gt;.

Electrodo de fabricación e implantación de Aplysia californica Para grabaciones multi-canal neural y muscular en los animales intactos, libremente Comportarse

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

electrode-fabrication-implantation-aplysia-californica-for-multi

Research

JoVE Journal

Neuroscience

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

13.0K vistas.  Case Western Reserve University. Una técnica se describe para la implantación de cuatro In vivo Electrodos para monitorear el control neuromuscular de la conducta alimenticia en Aplysia californica.

Video: Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

La combinación del comportamiento de Endocrinología Experimental y Economía: Decisión de la testosterona y sociales que

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Investigación

Neurociencias

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Alta densidad de registros de EEG de los ratones se mueven libremente con poliamida basado microelectrodos

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

Diseño y montaje de un Microdrive ultraligero motorizado para crónicos Grabaciones neuronales en Pequeños Animales

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

Un<em&gt; In Vitro</em&gt; Preparación para Obtener y grabar programas de alimentación del motor con los movimientos fisiológicos en<em&gt; Aplysia californica</em

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dos registros electrofisiológicos de sinápticamente respuestas evocadas neuronal y astroglial en agudas rodajas de hipocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracelularmente Identificar las neuronas motoras de un músculo en la piscina Motor<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

El aislamiento de las neuronas sensoriales de<em&gt; Aplysia californica</em&gt; Se Patch clamp grabaciones de Corrientes glutamatérgicos

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

Un procedimiento para la implantación de matrices Organizados de Microhilos para grabaciones de una sola unidad en Awake, Comportarse Animales

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

Diseño y fabricación de Ultralight Peso, ajustables Sondas Multi-electrodos para electrofisiológicos grabaciones en ratones

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

Grabaciones de potenciales evocadas visuales en ratones usando un multicanal no invasivo seco del cuero cabelludo EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...