Este trabajo fue apoyado por el BRIANBOW Proyecto Europeo (el 7PM Jóvenes Exploradores, Los autores desean agradecer a la Dra Jacopo Tessadori por sus valiosos comentarios sobre el manuscrito, y Silvia Chiappalone por su ayuda en la producción de los gráficos utilizados en el video.

El procedimiento se realizó de acuerdo con las normas del NIH para el cuidado y uso de animales de laboratorio y fue aprobado por el Cuidado y Uso Comité Universidad de Tel Aviv Animal (número de permiso - L-14-019). 1. Elaboración de Instrumentos y PDMS <ol><li> Preparar la oblea (Tabla de materiales, u ordenar la oblea de un laboratorio de microfabricación), un bisturí, y un par de pinzas - no es necesaria la esterilización. </li><li> Hacer poli-D-lisina solución (PDL) de acuerdo con las siguientes condiciones: 4 mg / ml en tampón de borato 0,1 M, pH 8, y se almacena a -20 ° C. </li><li> Preparar plantillas de PDMS de acuerdo con el siguiente procedimiento: <ol><li> Preparar polidimetilsiloxano (PDMS, elastómero de silicona) mezclando entre sí la base y el agente de curado con una proporción de 10: 1, a continuación, se agita la 2 sustancias para aproximadamente 5 min. </li><li> Lugar en la cámara de vacío dos veces durante 15 minutos cada vez y verificar las burbujas se han ido. </li><li> Cuandoel PDMS está listo, preparar recubridora de rotación: perillas de nitrógeno abiertos para permitir el uso de gas, colocar la oblea de silicio (Figura 1) en spinner, y utilizar el vacío para evitar que se mueva. </li><li> Verter PDMS sobre la oblea y activar la spinner durante 1 min a 1000 rpm (crea aproximadamente 100 micras de altura). </li><li> Coloque la oblea en una placa caliente a 100 ° C durante 30 min. </li><li> Cuando el PDMS se ha endurecido, delinear las fronteras de la plantilla con PDMS, usando una pipeta Pasteur. Coloque la oblea en una placa caliente a 100 ° C durante 30 min. </li><li> Cuando las fronteras PDMS se han endurecido, usar un bisturí para cortar plantillas de acuerdo con las fronteras y retire la plantilla de silicio de la oblea (a PDMS cuadrados que contienen un solo patrón). </li></ol></li></ol> 2. Placa de Petri y cubierta Slip Preparación <ol><li> Preparar 23 mm cubreobjetos de vidrio cuadrado y limpiarlas de acuerdo con el siguiente orden: <ol><li> Lavar con agua destilada, etanol 70%, y acetona. </li><html><li> Lavar con isopropanol y secado rápido con nitrógeno. </li></ol></li><li> Coloque estampadas PDMS plantillas en cubreobjetos, y presione suavemente para comprobar que están firmemente unidos a la superficie de cristal. Colocar en la cámara de vacío durante 15 min. </li><li> Aplique 1 ml de PDL en la plantilla. Insertar el cubreobjetos en la cámara de vacío dos veces durante 20 min cada vez, y dejar PDL O / N a secar. </li><li> Preparar placa de Petri para crear una red de células de soporte: <ol><li> Cubrir la superficie de la placa (3,5 cm) con 1 ml de PDL para 2 horas en RT. Retire la caída PDL usando una pipeta. </li><li> Lavar con agua destilada y dejar secar. Coloque una muy pequeña gota de grasa de silicona en cada esquina del cubreobjetos. </li></ol></li><li> Coloque el cubreobjetos en el centro de la placa de Petri (es decir, el PDMS deben estar hacia arriba) y presione suavemente para verificar el apego. </li><li> Retire con cuidado PDMS de cubreobjetos con unas pinzas. Para la esterilización, exponerlo a iluminación UV durante 7 minutos. </li></ol>le &quot;&gt; 3. Multi-Electrodo Array (MEA) Preparación <ol><li> MEA Limpio en este orden (Tabla de Materiales): <ol><li> Lavar con agua debajo del grifo y se somete a ultrasonidos en un detergente enzimático concentrado 3 veces. </li><li> Someter a ultrasonidos en agua destilada 3 veces. </li><li> Lavar con agua destilada (en campana, 1000 l punta) por 3 veces y de lugar en UV durante 30 minutos. </li></ol></li><li> Apoyo a la preparación de la red: <ol><li> Preparar el molde red de apoyo diseñado. <ol><li> Vierta PDMS en una placa de 12 pocillos (22 mm de diámetro). </li><li> Cuando el PDMS se endurece sacar el molde y usando un bisturí, cortar un agujero en el centro para crear una forma de anillo. </li><li> Colocar un molde red de apoyo diseñado en el centro de la MEA (Figura 2A) y cubrir el resto de la superficie con PDL durante 2 horas a RT. </li></ol></li><li> Retire PDL que usa la pipeta y lavar con agua destilada. </li><li> Retire el molde y dejar secar (Figura 2). &lt;/ Li&gt; </ol></li><li> Alinear la plantilla de MEA de la siguiente manera: <ol><li> Coloque la plantilla en manipulador micro designado. Utilice un microscopio invertido para alinear con precisión la estructura modelada a los electrodos, y bajar la plantilla hasta que se coloca en la superficie MEA (Figura 2B). Nota: El contacto con un MEA bien limpia proporciona adhesión de competencia entre los PDMS y la propia AMA. </li><li> Levante el micromanipulador y si es necesario, utilizar pinzas para aplicar una pequeña cantidad de presión por encima de los PDMS para evitar que se separe de la MEA. </li><li> Presione suavemente la plantilla a la superficie MEA, y utilizar el microscopio para verificar que esté bien conectado y bien alineada. (Figura 2C-D). </li></ol></li><li> Coloque MEA en la cámara de vacío durante 15 minutos; poner una gota 1 ml de PDL en la plantilla. </li><li> Inserte en la cámara de vacío 2 veces durante 20 minutos cada uno. Deja PDL para secar O / N en la incubadora. </li><li> Antes de chapado, retire el PDMS plantillas de los AMUMA y lavar con estéril water. Coloque bajo iluminación UV durante 7 minutos. </li></ol> 4. Disección y Cultura <ol><li> Preparar culturas como se describe en Herzog et al. 2011 21. </li></ol> 5. Chapado <ol><li> Calcular el número de células necesarias para el chapado, utilizando un hemocitómetro (densidad óptima de acuerdo con el tamaño del circuito como se discute en el resultado). <ol><li> Asegúrese de que la cámara de recuento (hemocitómetro) es limpio y coloque una hoja de la cubierta en el mismo (el consumo de alcohol para limpiar). </li><li> Diluir 10 l de la suspensión de células en 190 l de medio de baño (dilución 1:20). </li><li> Carga de 10 l de las células diluidas en el borde de la cámara de recuento y lentamente pipetear las células a cabo permitiendo que la cámara se llene. </li><li> Usando un microscopio invertido, visualizar la rejilla hemocitómetro. Determinar el número de células en la cámara por recuento directo (células sanas deben ser redondos). Contar las células dentro de la gran plaza without los que cruzan los bordes. </li><li> Calcular la concentración de las células: Número total de células / 1000 l = células totales contadas × factor de dilución × 10 4 (área del hemocitómetro). Nota: Por ejemplo, si el factor de dilución fue de 20 y las células totales contadas eran 100: 100 × 20 × 10 4 = 0,1 x 10 7 células / 1000 l o 1 x 10 6/100 l. Con el fin de placa 0,75 x 10 6 células, tomar 75 l fuera de las células. </li></ol></li><li> Tome el número de células necesarias para el chapado por MEA simple o placa de Petri, resuspender las células para evitar la agregación y la placa de ellos en el centro, en la parte superior de la zona patternate (si es necesario, es posible diluir las células en el medio) . Para la MEA, coloque una gota de 100 l en el medio. Por hoja de la cubierta, colocar una gota de 1.000 l en el medio. </li><li> Incubar las células sembradas a 37 ° C durante 40 min. Añadir medio en placa para las células cultivadas en placas, hasta 1ml por MEA y 2 ml por hoja de la cubierta. </li><li> Mantenga a 37 ° C y, cada 4 días, se diluye con medio de cultivo fresco enriquecido con 0,5 Pen-Strep, 2% de B-27 y 0,75% glutamax. </li><li> De 6/7 DIV, después de ver las conexiones entre las islas, diluir medio con 10 l / ml FUDR (25 mg desoxiuridina + 62,5 mg de uridina en 12,5 ml de MEM (medio esencial mínimo de Eagle)) o cualquier otro agente anti-mitótico para evitar glial sobrecrecimiento. </li></ol>

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Un protocolo para crecer redes neuronales modulares 2D in vitro compuesto por circuitos funcionalmente interconectados se describe. El procedimiento se basa en modelando una capa de adhesivo celular. Patrones se consigue con PDMS plantillas reproducir la característica negativa de la arquitectura de red deseada. Plantillas de PDMS definen las áreas en las que se deposita la capa adhesiva celular. Una vez que las células se colocan en placas, que espontáneamente se reúnen para las islas recubiertas y auto-o...

Evidencias experimentales y teóricos apoyan la posibilidad de que el cerebro opera a través de la activación coordinada de ensamblajes de pilas de 1-5, que puede considerarse como unidades funcionales dinámicos que interactúan transitoriamente uno con el otro, de conformación y diferentes estados cerebrales subyacentes. Modularidad funcional también es dependiente de y asociado con la organización modular estructural de los circuitos cerebrales 6,7. Cómo función y la estructura de los circuitos cerebrales moldean mutuamente sigue siendo una de las principales cuestiones abiertas en la neurociencia. Para proporcionar una comprensión más profunda de esta cuestión, es importante identificar los marcos experimentales óptimas donde es posible abordar, al menos parcialmente, esas cuestiones. Desde controlado manipulación de la dinámica espacio-temporal de las redes neuronales en experimentos in vivo es difícil, el desarrollo de modelos in vitro redes neuronales es de gran interés debido a su fácil accessibility, el seguimiento, la manipulación y el modelado de 8,9. En los últimos años, en tecnologías in vitro con el apoyo de métodos de modelado sustrato avanzadas han permitido inducir redes neuronales para desarrollar una gama de estructuras modulares predefinidos 3 y estudiar las propiedades funcionales de las redes con topologías impuestas 10. En particular, los métodos han sido recientemente utilizados para organizar redes mediante la imposición de restricciones físicas 4,11. De hecho, para estudiar la relación entre estructura y función en las redes neuronales y para proporcionar una representación simplificada pero plausible de la interacción conjuntos neuronales, sistemas in vitro deben proporcionar subpoblaciones neuronales interconectadas. Estudiados ampliamente cultivos neuronales homogéneos 2D no imponen limitaciones espaciales en el cableado emergente autoorganizada de los circuitos. Por lo tanto un posible enfoque para dar forma a las asambleas de células artificialmente interconectados es posicionar diferentes poblaciones neuronales en disputaially zonas distintas. La distancia entre estos ámbitos no impide las conexiones inter asambleas. Este enfoque, garantizando al mismo tiempo un control considerable sobre complejidad de la red, se ha demostrado proporcionar un repertorio más rico de los modelos de sincronización 6,7,12. Con el fin de facilitar un cultivo reproducible de asambleas neuronales modulares, un protocolo de montar la auto-organización de las redes en conglomerados neuronales conectadas por axones y dendritas se presenta y describe. La estructura polimérica para el confinamiento físico de cultivos neuronales se ha creado a partir de polydimtheylsiloxane (PDMS). PDMS es un elastómero ampliamente utilizado para aplicaciones biomédicas debido a su biocompatibilidad, la transparencia y la permeabilidad a los gases 13. El PDMS se prepara y se excluye del SU8 micromecanizado 2075 14,15 estructuras por spin-coating un PDMS líquido en un &quot;maestro&quot; como se describe anteriormente en Jackman et al. 16 TLogró modelado redes neuronales están compuestos por módulos interconectados de diferentes tamaños y que se han obtenido con éxito en ambos cubreobjetos y Microelectrodo Arrays (AMUMA) 17-20. La densidad de las conexiones entre los módulos puede cambiar las características de la sincronización de la red, desde una red totalmente sincronizado, típico de las culturas uniformes, a los estados transitorios de la sincronización entre los módulos.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>PDMS, Sylgard 184</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Vacuum Chamber</td>
			<td>Thermo</td>
			<td>5305-0609</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine PDL</td>
			<td>Sigma</td>
			<td>P7886</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell - Technologies Corporation</td>
			<td>WS-650-23</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well culture plate</td>
			<td>Sigma</td>
			<td>CLS3336</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8217;-deoxyuridine</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone grease - SILICAID 1010</td>
			<td>&#160;aidchim Ltd</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA1060-Inv-BC</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC02</td>
			<td>Multi Channel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Biological Industries Beit Haemek</td>
			<td>03-033-1c</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>glutaMAX</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Minimum Essential Medium-Eagle</td>
			<td>Biological Industries Beit Haemek</td>
			<td>01-025-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Electrode Arrays 4Q</td>
			<td>Multi Channel Systems</td>
			<td>60-4QMEA1000iR-Ti-pr</td>
			<td>cleaning manual: http://www.multichannelsystems.com</td>
		</tr>
		<tr>
			<td>silicon wafer</td>
			<td>microchem</td>
			<td>SU8-2075</td>
			<td>Preparation protocol: www.microchem.com</td>
		</tr>
	</tbody>
</table>

design, surface treatment, cellular plating, and culturing of modular neuronal networks composed of functionally inter-connected circuits

El cerebro funciona a través de la activación coordinada y la comunicación dinámica de las asambleas neuronales. Una pregunta abierta importante es cómo un vasto repertorio de motivos dinámicos, que subyacen más diversas funciones cerebrales, puede surgir de una organización fija topológica y modular de los circuitos cerebrales. En comparación con los estudios in vivo de los circuitos neuronales que presentan dificultades experimentales intrínsecas, las preparaciones in vitro ofrecen una posibilidad mucho mayor de manipular y probar las propiedades estructurales, dinámicos y químicas de los sistemas neuronales experimentales. Este trabajo describe una metodología experimental in vitro que permite cada vez mayor de las redes modulares compuestas por asambleas neuronales espacialmente distintas, funcionalmente interconectados. El protocolo permite el control de la (2D) de la arquitectura bidimensional de la red neuronal a diferentes niveles de complejidad topológica. Un patrón de red deseada puede serlogrado tanto en cubreobjetos regulares y de sustrato incrustado arrays micro electrodos. Estructuras micromecanizadas están en relieve sobre una oblea de silicio y se utilizaron para crear plantillas poliméricos biocompatibles, que incorporan las características negativas de la arquitectura de red deseada. Las plantillas se colocan sobre los sustratos de cultivo durante el procedimiento de revestimiento de la superficie con una capa molecular para promover la adhesión celular. Después de la eliminación de las plantillas, las neuronas se sembraron y se redirigen de forma espontánea a las áreas recubiertas. Al disminuir la distancia inter-compartimiento, es posible obtener los circuitos neuronales ya sea aislados o interconectados. Para promover la supervivencia celular, las células se co-cultivaron con una red neuronal de soporte que está situado en la periferia de la placa de cultivo. Se presentan los registros electrofisiológicos y ópticas de la actividad de las redes modulares obtenidos respectivamente mediante el uso de sustrato incrustado micro matrices de electrodos y de imágenes de calcio. Si bien cada módulo muestra SPONTsincronizaciones globales aneous, la ocurrencia de sincronización entre módulos está regulado por la densidad de la conexión entre los circuitos.

A SU8-2075 molde sobre una oblea de silicio con un espesor característica de aproximadamente 100 micras se utilizó para dar forma a los PDMS. El patrón se compone de los cuadrados de varias dimensiones, con una longitud de lado y la distancia que varía entre 200 y 700 micras (Figura 1B). El tamaño del cuadrado fue elegido para encajar el campo de visión de un 10X (por islas con una longitud de lado &lt;800 micras) y de un objetivo 20X (por islas con una longitud de lado &lt;400 micras). Tres pará...

Watch this Scientific Journal Video about Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits at JoVE.com

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

Este manuscrito describe un protocolo para crecer en redes modulares vitro consistentes espacialmente confinados, los circuitos neuronales interconectadas funcionalmente. Una máscara polimérico se utiliza para patrón de una capa de proteína para promover la adhesión celular sobre el sustrato de cultivo. Neuronas chapados crecen en zonas recubiertas establecimiento de conexiones espontáneas y que muestran actividad electrofisiológica.

Diseño, tratamiento de superficies, Celular Forrado, y cultivo de Modular Redes Neuronales Compuesto de circuitos conectados-Inter Funcionalmente

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

design-surface-treatment-cellular-plating-culturing-modular-neuronal

Research

JoVE Journal

Neuroscience

8.4K vistas.  Tel-Aviv University.  Este manuscrito describe un protocolo para crecer en redes modulares vitro consistentes espacialmente confinados, los circuitos neuronales interconectadas funcionalmente. Una máscara polimérico se utiliza para patrón de una capa de proteína para promover la adhesión celular sobre el sustrato de cultivo. Neuronas chapados crecen en zonas recubiertas establecimiento de conexiones espontáneas y que muestran actividad electrofisiológica.

Video: Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Mapeo de los circuitos neuronales inhibitorios por Laser Scanning fotoestimulación

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

Investigación

Neurociencias

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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 ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

Experimentos Neuro-robótica de circuito cerrado para comprobar propiedades computacionales de Redes Neuronales

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Rewiring Neuronal Circuits: Un Nuevo Método Para La Extensión Neurítica Rápida Y La Conexión Neuronal Funcional

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Determinación de la expresión de la superficie celular y la tasa endocítica de proteínas en cultivos de astrocitos primarios usando biotinilación

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Estimulación eléctrica transcraneal crónica y grabación Intracortical en ratas

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic de arrastre de las oscilaciones Theta Hippocampal en comportarse ratones

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Imágenes simultáneas de la dinámica microglial y la actividad neuronal en ratones despiertos

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

In vivo Imágenes de calcio de conjuntos neuronales en redes de neuronas sensoriales primarias en ganglios intactos de la raíz dorsal

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...