The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children&#8217;s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.

Este método requiere el uso de sujetos animales, se admiten por el Comité de Ética Animal de la Universidad Laval (protocolo # 2012-170). 1. Instalación y Preparación <ol><li> Soluciones <ol><li> Preparar soluciones madre aCSF de acuerdo con las siguientes recetas 7,17. Otras recetas con variaciones de concentración están disponibles en la literatura. Soluciones tienda de valores a 4 ° C durante hasta un mes. <ol><li> Solución de sal: añadir 75,39 g de NaCl (129 mM final); 2,5 g de KCl (3,35 mM final); 0,81 g de NaH2PO4 (0,58 mM final); 2,33 g de MgCl2 (1,15 mM final); 1,85 g de CaCl2 (1,26 mM). Disolver en 800 ml de agua destilada luego llenar a 1 l con agua destilada. </li><li> Solución de bicarbonato: añadir 17,65 g de NaHCO 3 (21 mM final). Disolver en 900 ml de agua destilada y luego llenar hasta 1 L con agua destilada. Las variaciones de la concentración de bicarbonato resultarán en variaciones de pH. </li><li> Solución de glucosa: añadir 54,06g de glucosa (30 mM final). Disolver en 400 ml de agua destilada y luego llenar hasta 500 ml con agua destilada. </li></ol></li><li> Preparar LCRa por dilución de 100 ml de solución de sal, 100 ml de solución de bicarbonato, y 50 ml de solución de glucosa en 1 L de agua destilada. Almacenar a 4 ° C hasta su uso. </li><li> Preparar un electrodo de vidrio calentando y estirando un tubo de vidrio hasta que se rompe. Lijar la punta antes de su uso. El electrodo se puede reutilizar tantas veces como el tiempo que se mantiene limpio. </li></ol></li><li> Configuración experimental Nota: Los datos de configuración se presentan en la Figura 2. <ol><li> Encienda el amplificador, promediador, sistema de adquisición de datos, control de temperatura y de la bomba en movimiento. Compruebe la amplificación de la señal (ganancia = 10.000), la filtración (de bajo umbral, 10 Hz; umbral alto, 5 kHz), y la tasa de muestreo de la conversión de analógico a digital de la señal en bruto (2,5 kHz). </li><li> Llene una botella con LCRa y la burbuja con carbógeno (95% O 2 </sub&gt;, 5% de CO 2). Inducir un bubbled- y se calentó-LCRa flujo en la cámara de grabación (volumen de 5 ml) a 4 ml / min. Mantener la temperatura en la cámara de registro a 26 (± 1) ° C. pH debe ser 7,4 en estas condiciones estándar. </li><li> Mantenga 50 ml de RT y carbógeno-burbujear LCRa en una jeringa de 50 ml, cerca de la cámara de disección. </li><li> Encienda el ordenador e inicie el software de grabación. </li></ol></li></ol> 2. Disección <ol><li> Pesar y determinar visualmente el sexo del animal (los machos tienen un ano-genital distancia más larga, mientras que las hembras tienen una distancia ano-genital corta; machos genitales son a menudo oscuro, mientras que los genitales femeninos son de color rosa). </li><li> Anestesiar al roedores recién nacidos por una de las siguientes opciones: crioanestesia (sumergir completamente el animal en hielo durante 4 - 5 min 18), inyección (equitensine a 4 ml / kg) 19 o la inhalación de anestésicos volátiles (isoflurano 20 o éter 21). Confirm un plano adecuado de anestesia por la ausencia de un reflejo retirada de la pata. </li><li> Colocar el animal en el banco, la cara ventral hacia abajo. Sección la parte rostral de la cabeza coronalmente con un bisturí en el nivel de bregma (visible a través de la piel y el cráneo). Realice este paso inmediatamente después se anestesia el animal. </li><li> Sección coronal del cuerpo con un bisturí en virtud de los miembros anteriores. </li><li> Quite la piel, los músculos, los tejidos grasos y víscera con tijeras y alicates quirúrgicos y delgadas. Desde los huesos son suaves a esta edad, tenga cuidado de no dañar el sistema nervioso. Realice este paso en el banquillo. </li><li> Colocar la preparación en la cámara de disección. Utilice la LCRa almacenado en la jeringa para oxigenar la preparación. A partir de este punto hasta el final de la grabación, usar un microscopio. </li><li> En la cara dorsal de la preparación, corte el cráneo y las vértebras de la rostral a la parte caudal lo largo del eje medio con tijeras y pinzas quirúrgicas y delgadas. Abra el cráneo cortey vértebras para exponer el tejido nervioso. Utilice la LCRa strored en la jeringa para oxigenar la preparación. </li><li> Con los alicates, quite la membrana aracnoide, un tejido delgado que cubre la superficie del tejido nervioso. Conserve la piamadre y los vasos sanguíneos contra el tejido nervioso. Utilice la LCRa almacenado en la jeringa para oxigenar la preparación. </li><li> Colocar la preparación con el dorsal boca abajo, y cuidadosamente hacer caer el tronco del encéfalo y la médula espinal mediante la reducción de los nervios y el tejido conectivo con las tijeras mientras mantiene la preparación en su lugar. Un enfoque dorsal también es posible. Mantener las raíces y los nervios el mayor tiempo posible. Retire los huesos para aislar el tronco del encéfalo y la médula espinal. Utilice la LCRa almacenado en la jeringa para oxigenar la preparación. </li><li> Retire el cerebelo y el restante estructuras rostrales seccionando con el bisturí. Utilice la LCRa almacenada en la aguja para oxigenar la preparación. </li><li> Opcionalmente dependiendo de experimendiseño tal, retire el puente con el bisturí por una sección anterior de la corona a la arteria cerebelosa inferior 22. Tenga en cuenta que el mantenimiento de todo el puente se ralentizará el ritmo en ratas y totalmente inhibirla en ratones. Las preparaciones que incluyen sólo la parte caudal de la protuberancia muestran una actividad respiratoria como con una frecuencia estable a la raíz C4. </li></ol> 3. Grabación <ol><li> Colocar la preparación en la cámara de grabación, ventral hacia arriba. Fijar la preparación con alfileres en la parte más baja de la médula espinal y el rostral-la mayor parte del tronco cerebral. </li><li> Usando una jeringa vinculado al electrodo por su aguja, inducen una depresión en el electrodo (diámetro de la punta de vidrio: 150-225 micras) tirando de la jeringa de pistón, con el fin de llenar parcialmente el electrodo con LCRa. </li><li> El uso de un micromanipulador, colocar cuidadosamente el electrodo cerca de una de las cuartas raíces ventrales. Otras raíces ventrales también podrían presentar una actividad respiratoria como (e.g., nervio craneal XII, C1 raíz ventral). </li><li> Inducir una depresión en el depósito de electrodo a través de la jeringa tirando del émbolo con el fin de aspirar suavemente el rootlet nervio. A continuación, mueva con cuidado el electrodo para aplicar contra la médula espinal. Nota: Lo ideal sería que el tamaño de la rootlet coincide con el tamaño de la abertura del electrodo y por lo tanto crea un sello entre los compartimentos internos y externos del electrodo. Desde amplificadores diferenciales se utilizan comúnmente para tales grabaciones, cualquier abertura entre el interior del electrodo y la cámara de grabación reduce la calidad de la señal y hace que sea difícil eliminar el ruido de fondo. </li><li> Inicie la grabación. </li><li> Graba el ritmo producido por la preparación en condiciones de normoxia (es decir, LCRa burbujeó con carbógeno: 95% O 2 y 5% de CO 2) durante al menos 20 min para determinar los parámetros basales de la preparación. </li><li> Cambie la perfusión de LCRa carbógeno-burbujear aLCRa estímulo (es decir, burbujeado con 95% N 2 y 5% de CO 2 para el estímulo hipoxia) durante 15 min. Alterar duración de la exposición dependiendo del protocolo experimental. Anote la duración de la exposición en la grabación y animal hoja de datos. Utilice tubos de acero inoxidable entre la botella LCRa y la cámara siempre que sea posible para evitar la difusión del gas al exterior del tubo. </li><li> Cambie la perfusión de nuevo a norma LCRa carbógeno-burbujear durante al menos 15 minutos para una grabación de recuperación. Anota esto en la grabación y animal hoja de datos. </li><li> Finalice la grabación. </li></ol> 4. Análisis estadístico <ol><li> A partir de la señal integrado, calcular la frecuencia como el número de ráfagas por minuto (expresado en ráfagas / min), la amplitud como la diferencia entre la línea de base y el pico de la ráfaga (expresado en mV), la duración de la ráfaga como la duración desde el principio hasta el final de la ráfaga (expresado en s), el área de ráfaga como el área bajo la curve de la ráfaga de la señal integrada (expresado en mV · s) (Figura 3). </li><li> Calcular la frecuencia, amplitud, duración de la ráfaga y la zona de la explosión bajo normóxico (línea de base), hipóxica (estímulo) y post-hipóxico condiciones (post-estímulo) de recuperación como el promedio de los últimos 5 minutos de las grabaciones para cada condición. No analizar la primera porción de cada condición porque hay un retraso entre la conmutación de la perfusión y LCRa homogeneización en la cámara de grabación. </li><li> Expresar entonces estímulo (es decir, la hipoxia) y post-estímulo (es decir, post-hipóxicas) los valores de recuperación en porcentaje de los valores de línea de base para la grabación correspondiente. Exprese los resultados como medias ± SD </li></ol>

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The authors have no competing financial interests or conflicts of interests to disclose.

La cuantificación precisa de la actividad respiratoria puede ser un reto. De hecho, la respiración es una función que puede ser tanto automática y voluntaria, y que se modula en función del entorno, las necesidades del cuerpo, el estado emocional y el comportamiento. La ventaja de esta técnica es el aislamiento de los elementos neurales responsables de producir el comando respiratorio. Por lo tanto, los registros electrofisiológicos de preparaciones de médula espinal y del tronco cerebral-pletismografía son té...

La respiración es una actividad compleja y vital controlada por el cerebro, lo que permite dioxígeno (O 2) la captación y dióxido de carbono (CO 2) la eliminación. El impulso respiratorio central se genera por una compleja red situada en el tronco cerebral en ambos mamíferos 1, 2 anfibios, reptiles, aves 3 4 5 y peces. Incluso si el estudio de la respiración se puede procesar in vivo, las investigaciones mecanicistas precisas requieren el acceso directo a la red de control respiratorio. Con este fin, Adrian y Buytendijk desarrollaron una preparación goldfish reducida, en el que los electrodos colocados en el registro de la superficie del tronco cerebral el ritmo generado asociado con gill ventilación 5. Este enfoque fue posteriormente adaptada por Suzue en 1984 6 para su uso en roedores recién nacidos. La llegada de esta preparación ha dado lugar a importantes avances en neurobiología respiratorio. Dado que es relativamente simple, la técnica presentada hERE es susceptible de una amplia gama de investigaciones básicas de comportamientos motores rítmicos y sus orígenes en roedores recién nacidos. El objetivo general de este método consiste en registrar el correlato neural de la actividad inspiratoria, un ritmo respiratorio similar llamado respiración ficticio, producida por la cadena respiratoria. Este método se puede emplear en una amplia gama de objetivos de la investigación, la orientación respuestas inspiratorios a variaciones respiratorias o farmacología en ambos de tipo salvaje y transgénicos 7 8 animales. Dado que los experimentos se llevan a cabo a baja temperatura, sin aferentes sensoriales, y en condiciones en las concentraciones de glucosa y de O 2 en el LCRa son altos, se han planteado interrogantes sobre la relevancia fisiológica de la señal grabada. Si bien existen diferencias claras entre in vivo e in vitro condiciones (por ejemplo., La frecuencia de ráfagas de inspiración) el hecho es que la presencia delos elementos centrales de la red respiratoria 6 hacen posible el estudio de un ritmo robusto asociado a una función homeostática vitales 9,10. La razón fundamental detrás del desarrollo y el uso de esta técnica es facilitar el acceso directo a los elementos del tronco cerebral de la red respiratoria, que son difícilmente accesibles in vivo, especialmente en los recién nacidos. El tronco encefálico se coloca en condiciones estrictamente controladas: el ritmo grabado no es modulada por impulsos aferentes periféricas de los pulmones o los cuerpos carótidas, lo que permite el estudio se centre en la unidad central de la respiración en sí 11. Por lo tanto, se utiliza este acceso para aplicar estímulos y registrar la señal de salida. En contraste con pletismografía de grabaciones, el ritmo respiratorio es modulada por todos sus componentes en todo el cuerpo (por ejemplo., Distensión de pulmón, sensores químicos periféricos), por lo que es difícil de aplicar estímulos precisos. En unaewborn rata, el protocolo consiste en registrar la cuarta señal ventral raíz en un tronco cerebral aislada y una médula espinal truncado, mantenido en el fluido cerebro-espinal artificial (ACSF). El ritmo generado por los preparativos de la médula espinal, tronco cerebral se compone de ráfagas lentas individuales que están vinculados a la señal inspiratoria 9. Preparaciones de cordón-tronco cerebral médula aislados se pueden grabar fácilmente en ratas desde el primer día postnatal 0-4 (P0 - P4) 7. Este enfoque se utiliza comúnmente para evaluar la respuesta hipóxica de la red respiratoria, y también la respuesta a la hipercapnia, acidosis o drogas. Un protocolo de hipoxia aguda se presenta aquí. Esta estimulación se obtiene por la captación de O 2 en el LCRa; este enfoque se utiliza comúnmente para evaluar la tolerancia y la capacidad de respuesta a insultos hipóxicas. El protocolo induce una depresión ritmo desde el primer minuto hasta el final de la exposición a la hipoxia (Figura 1) 12. Esta depresión se inviertedurante el post-hipóxica 12 de recuperación. Respecto al diseño experimental, es importante darse cuenta de que el puente de Varolio, situados en la parte rostral del tronco cerebral, tiene una acción inhibidora sobre el generador de ritmo 8. Por lo tanto, los preparativos de la plena tronco cerebral y médula espinal rostral muestran un menor ritmo. La inclusión de la protuberancia en la muestra aislada de la grabación se determina de acuerdo con el objetivo del experimento 13; el estudio de la influencia pontina en la red bulbo raquídeo requeriría grabaciones con y sin el puente de Varolio para comparar los resultados 14. Por otra parte, una de las ventajas de esta técnica es la posibilidad de ampliar la parte rostral de la preparación para incluir mesencefálico y / o regiones diencefálicas 15,16, por lo que es posible evaluar el efecto de estas regiones en la red respiratoria-ponto medular.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sylgard</td>
			<td>Sigma Aldrich</td>
			<td>761036-5EA</td>
			<td>Use under hood</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>SOD002</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bioshop</td>
			<td>POC888</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Bioshop</td>
			<td>CCL444</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Bioshop</td>
			<td>MAG510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Bioshop</td>
			<td>SPM306</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Bioshop</td>
			<td>GLU501</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Linde</td>
			<td>343-02-0006&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction electrode</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 573000</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential AC amplifier</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Moving averager</td>
			<td>CWE, Ardmore, PA, USA</td>
			<td>model MA-821</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition system</td>
			<td>Dataq Instruments, Akron, OH, USA</td>
			<td>model DI-720</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart software</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism sofware</td>
			<td>Graphpad, La Jolla, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection chamber</td>
			<td>Plastic box (e.g. petri box) will do</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recording chamber</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instrument Inc, Sarasota, FL, USA</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson, Middleton, WI, USA</td>
			<td>MINIPULS 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

Como se ha mencionado en la introducción, una de las ventajas más importantes de esta técnica es el acceso directo al tronco encefálico para aplicar diversos estímulos. Como ejemplo, la hipoxia se aplicó aquí. Figura 1. AB muestra una grabación protocolo completo, con ambas condiciones de normoxia e hipóxicas. 1.CE Figura muestra el ritmo en condiciones de normoxia registrado (es decir, LCRa burbujeó con 95% O 2 y 5% CO 2 a 26 ° C). Como se ha d...

Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Electrofisiología en aislados Preparativos Cord-tronco cerebral espinal de roedores recién nacidos permite la grabación de salida de red neuronal Respiratorio

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

electrophysiology-on-isolated-brainstem-spinal-cord-preparations-from

Research

JoVE Journal

Neuroscience

8.5K vistas.  Laval University. The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Video: Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Investigación

Neurociencias

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Médula Espinal Electrofisiología II: Fabricación de electrodos de aspiración extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

El examen de procesamiento de la red local usando multi-contacto laminar de grabación de electrodos

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

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

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

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

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

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

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Marcaje fluorescente retrógrada Permite dirigida extracelular sola unidad de grabación de neuronas identificadas<em&gt; En vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

Neural Circuito de grabación de una cucaracha sistema nervioso intacto

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Incremento dependiente del tiempo en la respuesta de la red a la estimulación de cultivos de células neuronales en matrices de microelectrodos

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imágenes de la actividad neuronal en la corteza somatosensorial primaria usando Thy1-ratones transgénicos GCaMP6s

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Preparaciones y protocolos para todo celular parche abrazadera de neuronas Tectal de Xenopus laevis

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...