Damos las gracias al fondo de la vida, de la Universidad de Oklahoma Programa de Licenciatura de Investigación de Oportunidades, y el Departamento de Zoología de la financiación de este trabajo. También agradecemos al Dr. Hoefnagels Mariëlle ayuda editorial.

1. Pre-registro, preparación de animales vivos <ol><li> Quitar el escorpión de su frasco en casa y colocarlo en un recipiente de vidrio pre-enfriado, al lado, coloque el frasco que contiene el escorpión en un entorno de congelación (-5 ° C) durante un mínimo de dos minutos. Este tiempo puede variar de acuerdo a la edad, tamaño, especie, etc de los específicos de escorpión en estudio. Pero, en general, la fase de congelación medio ambiente se completa una vez que el animal no se mueve. </li><li> Después de dos minutos, retire el inmovilizado escorpión del envase de vidrio en frío y luego colocar la parte ventral hacia arriba en un portaobjetos de microscopio. Asegurar el aguijón, cola, piernas y pedipalpos con la arcilla moldeable (a base de aceite, Van Aken Plastalina). </li><li> Hacer una plataforma en la que poner pectinas del escorpión. Ajustar la longitud de la plataforma y el ancho de acuerdo al tamaño de los géneros Pecten. Por lo general, nuestras plataformas están construidas con microscopio de vidrio cubierta y son aproximadamente 10 (L) x 18 (W) mm. </li><li> Se adhieren a doble cara cinta adhesiva a la plataforma de Pecten, el recorte de la cinta (si es necesario) para que quepa. </li><li> Para hacer que una cámara de pectinal, poner tres de los cuatro lados de la plataforma en la pre-fundido de cera (nosotros usamos la cera de abejas 100% de los chips) a una profundidad de 3-4 mm. Nos derretir la cera mediante la colocación de los chips en una placa de Petri de vidrio, que se encuentra en un plato caliente. Una vez que la cera en los bordes de la cámara de pectinal se enfría a temperatura ambiente, debe crear las paredes de cera alrededor de 1 mm de alto. </li><li> En el animal, localizar el punto de inserción pectinal, y coloque el borde sin encerar de la plataforma (la cinta de lado hacia arriba) justo por detrás de él. Después, estabilizar la plataforma (para que no se mueva más adelante) presionando suavemente su derecha e izquierda bordes periféricos en contra de la arcilla utilizada para restringir las patas del animal. </li><li> Use unas pinzas de punta fina para colocar con cuidado el Pecten o pectinas dentro de la cámara. En concreto, el uso de fórceps, mantenga el Pecten a lo largo de la columna vertebral pectinal, y pase suavemente el Pecten de debajo de la cubierta de vidrio y lo baja en el suelo de la plataforma adhesiva. Establecer un vínculo pecten-a-cinta con cuidado la aplicación de presión a la columna vertebral pectinal. </li><li> Ahora aplique la cera derretida (se utiliza una espátula de metal previamente calentado) hasta el borde sin encerar de la plataforma. El objetivo es doble, para asegurar el Pecten y para encerrar totalmente la cámara para la futura introducción de aceite mineral. Tenga cuidado al aplicar la cera sobre Pecten del animal, ya que es posible dañar el Pecten con la cera que está demasiado caliente. </li><li> La construcción de la cámara de Pecten se ha completado. A continuación, establecer una conexión con el electrodo indiferente hemolinfa de los animales mediante la inserción de un alambre de plata entre dos segmentos de la cola. </li></ol> 2. Grabación simultánea extracelular y estimulación química <ol><li> Nosotros preparamos los animales 24 horas antes de realizar extracelular, punta de las grabaciones, que, entre otras razones (es decir, el ajuste de los animales de posición fija), da tiempo a medida que las pipetas de un estimulante específico (sensillum-dependiente) la punta de diámetro. Nosotros usamos un extractor de micropipeta para el logro de dichas especificaciones. Idealmente, el diámetro de las pipetas es ≈ 2 m mayor que el diámetro de los poros sensillas. Nos ajustamos nuestras especificaciones micropipeta en consecuencia, para cada animal. </li><li> 1 h antes de la grabación, se añade 5 l de aceite mineral a la cámara de pectinal, sumergiendo el Pecten en aceite. Entonces, la posición del animal debajo de un microscopio de alta potencia, que durante mucho tiempo de trabajo los objetivos de la distancia y el epi-iluminación. </li><li> Para estimular la vinculación química sensilla, se introducen sustancias sápidas acuosa (es decir, ácido cítrico y sal) a través del medio de aceite. En primer lugar, se utiliza una micropipeta de relleno para inyectar el estimulante dentro de la pipeta estimulante. Nota: todas las soluciones estimulantes deben contener un electrolito de la conductancia eléctrica. </li><li> A continuación, después de colocar la pipeta estimulante en un micromanipulador (en el equipo de grabación), insertar el electrodo de registro en el extremo romo, abierto de la pipeta estimulante. Es importante asegurar el contacto entre el electrodo de metal y una solución electrolítica estimulante. </li><li> Una vez que la preparación es una toma de tierra (a través del electrodo indiferente), sólo inferior de la pipeta a través del medio del petróleo y en un poro sensillas. La duración de la estimulación química por sensillum pectinal puede variar de tanto como treinta minutos a tan sólo un segundo. Por lo general, se pueden degustar las clavijas múltiples (&gt; 30) por la pipeta. En el caso de que la pipeta estimulante se tapa con escombros minutos, simplemente el intercambio de la pipeta obstruida por una pipeta nueva, sin uso. </li></ol> 3. La preparación de un animal durante varios días de experimentación <ol><li> Si el animal se va a utilizar durante un período de tiempo prolongado, se recomienda volver a utilizar la cámara pectinal y sustitución de la gota de aceite mineral para cada sesión de grabación. Después de la sesión de un día determinado de la grabación, retire el animal de la grabación de puesta a punto y realizar un lavado de aceite mineral. </li><li> Especificacionesficamente, añadir una gota (5 l) de aceite mineral a la cámara, y retirar todo el aceite. Esto ayuda a minimizar la presencia del estimulante residual, que puede haberse escapado hacia fuera de la pipeta durante la sesión de grabación. </li><li> Antes de la sesión siguiente, agregue una gota de aceite como se describió anteriormente. </li></ol> 4. Los resultados representativos: Dependiendo del aparato de control utilizado (por ejemplo, el amplificador en particular, la digitalización de hardware, etc), un resultado representativo es un registro extracelular con una S: N de al menos 3:1. Construcción exitosa de la cámara de aceite mineral es necesario para aislar a la estimulación química sensilla individual, ya que proporciona un medio para la entrega de químicos estimulantes del gusto polar (a base de agua) 8. Si hay muy poco aceite o ninguno en absoluto, la solución estimulante se extenderá desde el sensillum registrado a su sensilla vecinos, lo que es probable que cause una enfermedad prolongada y sin control de la estimulación química 8.

<ol>
	<li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+Primary+Chemoreceptor+unit.">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li><li>Newland, P. L., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+processing+in+the+insect+nervous+system.">Taste processing in the insect nervous system.</a> Methods in Insect Sensory Neuroscience. , 289-318 (2005).</li><li>Foelix, R. F., M&#252;ller-Vorholt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla.">The fine structure of scorpion sensory organs II. Pecten sensilla.</a> Bull. Br. Arachnol. Soc. 6, 68-74 (1983).</li><li>Carthy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten.">Fine structure and function of the sensory pegs on the scorpion pecten.</a> Experientia. 22, 89-91 (1966).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions.">Response properties of chemosensory peg sensilla on the pectines of scorpions.</a> J. Comp. Physiol. A. 181, 291-300 (1997).</li><li>Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pectine+organs+of+the+scorpion,+Vaejovis+spinigerus:+Structure+and+(glomerular)+central+projections.">The pectine organs of the scorpion, Vaejovis spinigerus: Structure and (glomerular) central projections.</a> Arthropod Struct. Dev. 37, 67-80 (2008).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines.">Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines.</a> J. Comp. Physiol. A. 181, 301-307 (1997).</li><li>Knowlton, E. D., Gaffin, D. D. <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+approach+to+examining+scorpion+peg+sensilla:+the+mineral+oil+flood+technique.">A new approach to examining scorpion peg sensilla: the mineral oil flood technique.</a> J. Arachnol. 37, 379-382 (2009).</li><li>Knowlton, E. D., Gaffin, D. D. <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+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants.">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , .</li><li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+sucrose+perception+in+the+honey+bee+(Apis+mellifera+L.):+behaviour+and+electrophysiology.">Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology.</a> J. Comp. Physiol. A. 190, 735-745 (2004).</li><li>Bland, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensilla+on+the+antennae,+mouthparts,+and+body+of+the+larva+of+the+alfalfa+weevil,+hypera+postica+(gyllenhal)+(Coleoptera+:+Curculionidae).">Sensilla on the antennae, mouthparts, and body of the larva of the alfalfa weevil, hypera postica (gyllenhal) (Coleoptera : Curculionidae).</a> Int. J. Insect Morphol. Embryol. 12, 261-272 (1983).</li><li>Cate, H. S., Derby, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooded+Sensilla+Homologues:+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum.">Hooded Sensilla Homologues: Structural Variations of a Widely Distributed Bimodal Chemomechanosensillum.</a> J. Comp. Neurol. 444, 345-357 (2002).</li></ol>

Anteriormente hemos publicado estos métodos en dos revistas, Knowlton y Gaffin (2009) y Knowlton y Gaffin (en prensa).

El protocolo anterior describe cómo preparar un desierto de pastizales escorpión (Paruroctonus utahensis) para el estudio electrofisiológico. En concreto, se muestra cómo construir una cámara para controlar extracelular punta de las grabaciones de las neuronas pectinal quimiosensoriales en aceite. Debido a que el petróleo y el agua no se mezclan, es posible aislar la estimulación química (con base de agua, estimulantes) para sensilla solo. Cabe destacar que P. utahensis, es un animal relativamente pequeño (≈ 2,5 a 5 cm) y la aplicación de este protocolo para los animales más grandes pueden requerir muchos ajustes de tamaño, tales como el tamaño de la plataforma del animal, de cámara, y la cantidad de aceite aplicada. Recomendamos la realización de estudios piloto para probar la mínima cantidad de aceite necesaria para la entrega controlada de los estimulantes químicos a base de agua. Además, no se encontraron efectos del petróleo sobre la actividad neural de referencia dentro de pectinal sensilla 8. Esto debe ser confirmado por los sistemas de otro modelo, así, antes de evaluar chemoresponsiveness. Este método puede ser utilizado en combinación con otro método para probar los efectos de interacción entre chemoresponse sensilla en el campo sensorial. Por ejemplo, es posible punta de registro (en aceite) de una sensillum como base-registro (a través de un electrodo de tungsteno) a partir de una sensillum vecinos 9. Tal configuración de la grabación se puede utilizar para evaluar si o no químicamente estimular una sensillum afecta a la actividad neuronal de la base de sensillum grabado. Hasta la fecha, nadie ha probado esto de escorpión pectinal sensilla, y sigue siendo una pregunta abierta para otros sistemas quimiosensoriales así. En resumen, creemos que el método de la punta de grabación en los avances de petróleo del alcance de las investigaciones electrofisiológicas en la base neural de gustation artrópodos. En este manuscrito, que proporcionan un protocolo para la preparación de animales para este método, y esperamos que proporciona una base sólida para un mayor estudio de los sistemas nervioso periférico sensorial.

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electrophysiology of scorpion peg sensilla

Se describe una modificación de un 1,2 técnica existente punta de grabación para la investigación electrofisiológica corto, como clavijas sensorial sensilla 3,4. En la superficie ventral de la mitad de todos los escorpiones son dos apéndices llamados pectinas, que han densos campos de mecano-y quimiosensoriales clavija 5,6 sensilla. Un método para evaluar chemoresponsiveness de estos sensilla utiliza un electrodo de tungsteno para la grabación de la actividad neuronal extracelular dentro de un sensillum como un olor volátiles se introduce en el campo sensorial 5,7. Las limitaciones de este método incluyen la entrega de datos es lenta y la introducción descontrolada de estimulantes, y la eliminación de, el campo de tipo de cambio. Para superar estas limitaciones, hemos desarrollado una nueva punta de grabación de la técnica que utiliza el aceite mineral no polar como un medio a través del cual puedan entregar a base de agua estimulantes del gusto de cada clavija de 8,9 sensilla. Hemos aplicado con éxito este método para obtener chemoresponses sensillas al ácido cítrico, etanol, y la sal. A continuación se describe el protocolo experimental para un estudio de 9. Creemos que este nuevo método puede ser útil para el estudio de las propiedades de respuesta de otros sistemas de artrópodos quimiosensoriales, incluidos los de los insectos 10, 11 y 12 de crustáceos.

Watch this Scientific Journal Video about Electrophysiology of Scorpion Peg Sensilla at JoVE.com

Electrophysiology of Scorpion Peg Sensilla

Este artículo describe un método electrofisiológico para aislar a la estimulación química sensilla individuales a través extracelular, punta-grabaciones en aceite mineral.

Electrofisiología del Escorpión Peg sensilla

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

electrophysiology-of-scorpion-peg-sensilla

Research

JoVE Journal

Neuroscience

8.9K vistas.  University of Oklahoma. Este artículo describe un método electrofisiológico para aislar a la estimulación química sensilla individuales a través extracelular, punta-grabaciones en aceite mineral.

Video: Electrophysiology of Scorpion Peg Sensilla

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

FMRI simultánea y electrofisiología en el cerebro de roedores

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

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 ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

El cultivo y la electrofisiología de las células en NRCC de patch-clamp chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection parejas y Ensayo de electrofisiología para la detección de bioactividad de compuestos utilizando el Drosophila melanogaster Sistema de fibra gigante

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Una preparación semi-intacta aislada del ratón epitelio sensorial vestibular de Electrofisiología y de alta resolución de la microscopía de dos fotones

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Electrofisiológico Grabación desde<em&gt; Drosophila</em&gt; Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

En tiempo real Electrofisiología: Usando protocolos de bucle cerrado para sondear neuronales Dinámica y allá

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

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.

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 ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Ganglio espiral neurona explante cultura y Electrofisiología de multi electrodos matrices

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

La incubación prolongado de aguda neuronal de tejidos para la Sección de Electrofisiología y calcio de imágenes

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...