El autor desea dar las gracias a Pedro Castro y Dinday Mateo para sus primeros esfuerzos por establecer pez cebra en el laboratorio. Este trabajo fue financiado por los Institutos Nacionales de Salud de subvención EUREKA (# R01NS079214-01).

1. Huevo de Producción y Recolección <ol><li> El pez cebra cría sigue los procedimientos estándar descritos previamente 8. En pocas palabras, el pez cebra adultos se instalan en acuarios de cría con separadores en su lugar. Cuando las luces de la habitación se enciende a la mañana siguiente, los divisores son retirados de los tanques de cría y los peces se permiten aproximadamente 20 a 60 minutos de tiempo de apareamiento sin ser molestados. </li><li> Los huevos procedentes de tanques de cría se recogen en un filtro y se enjuagó con agua huevo. Los huevos se transfieren entonces a una placa de Petri con agua huevo. Huevos no fertilizados y desechos se eliminan utilizando una pipeta de transferencia. </li><li> Coloque una placa de Petri que contiene los huevos recolectados en una incubadora (28-32 ° C). Después de incubar huevos, unos dos días después de la fertilización (dpf), retire cualquier residuo corion y otro con una pipeta de transferencia. </li><li> En el día deseado después de la fertilización (3 a 8 dpf) eliminar placa de Petri que contiene las larvas libremente natación y el lugar en el banco de laboratorio a temperatura ambiente. </li></ol><ol><li> El uso de un extractor de micropipeta, tire de 1,2 mm de diámetro externo de borosilicato de vidrio capilar en dos agujas y guárdelos en un plato de Petri de 150 mm o caja pipeta vacía por encima de las rampas de plastilina. Las agujas pueden ser tirado por adelantado. Diferente OD capilares de vidrio se pueden utilizar dependiendo del tipo de headstage amplificador disponible. </li><li> Retrocarga el microelectrodo con solución de registro extracelular (2 M NaCl) utilizando una jeringa de 1 ml con un filtro de jeringa Nalgene (4-mm) y Microfil filamento (Warner Precision Instruments) conectado. Agitar o golpear ligeramente el bolo hacia la punta de la aguja hasta que no haya burbujas de pocos o ningún restantes. </li></ol> 3. Inmovilización en Agar <ol><li> Preparar una solución fresca de agar 1,2% bajo punto de fusión en agua huevo. Place solución de agar en un conjunto de baño de agua a ~ 37 ° C. </li><li> Preparar un cubreobjetos con cámara de registro y el lugar en el congelador (min 5-10). La cámara de grabación debe MATCh que se utiliza en la plataforma de electrofisiología. Usamos un bajo perfil baño abierto diamante cámara de imágenes de Warner Instruments (modelo RC-26GLP). </li><li> El uso de un Pasteur o pipeta de transferencia, coloque una larvas de pez cebra en una pequeña gota de agua de huevo en una placa de Petri plato limpio. Para esta gota, añadir una gota de solución que contiene un anestésico (0,02% tricaína) y agente paralizante (1 mg / ml de α-bungarotoxina). </li><li> Monitorear el pez cebra para la pérdida de movimiento (5 a 10 min). </li><li> Quite el cubreobjetos / cámara de registro del congelador. Coloque la cámara en la etapa de un microscopio estereoscópico. Usando una pipeta de transferencia, se mezcla con algunos mililitros de bajo punto de fusión 1,2% de agarosa con la gotita y solución de transferencia (de pescado) a la cámara de cubreobjetos / grabación. </li><li> Utilizando una punta de pipeta o aguja fina romo, larvas posición bajo el estereomicroscopio de modo que la cara dorsal del pescado se expone a la superficie del gel de agarosa. Permita que se endurezca agarosa (min 5-10), y luego usando una espátula plana la transferencia b agarbloquear a una cámara de grabación puede configurarse en una plataforma de electrofisiología. </li></ol> 3. Grabación de campo extracelular <ol><li> Añadir 2-5 ml de medio de grabación de pez cebra para la cámara de grabación. Si los cambios son drogas cámara necesaria, perfundir con solución de grabación a una velocidad de aproximadamente 1 ml / min. No oxigenación de la solución de grabación es necesario. </li><li> Insertar un microelectrodo (aproximadamente 1 m diámetro de la punta, 2-7 mW) en el headstage amplificador montado en un micromanipulador de tres dimensiones. Comprobar que el micromanipulador se encuentra en una posición adecuada para permitir un rango de movimiento y ajuste. Llevar la punta microelectrodo en el plano de visión del microscopio, alta de la etapa, y el uso de ajuste grueso inferior a un punto justo por encima y ligeramente por delante de la cabeza del pez cebra. </li><li> Con el amplificador en &quot;corriente-clamp&quot; modo cero del electrodo. </li><li> Uso del ajuste del grueso inferior de la punta del electrodo para que apenas toque el surface del pez cebra, ligeramente por delante del prosencéfalo. </li><li> El uso de pasos de ajuste fino avanzar la punta del electrodo hasta que perfora la piel del pez cebra. Deje que se asiente y luego avanzar el electrodo varias micras en cerebro anterior. </li><li> Registrar la actividad eléctrica en modo de corriente-clamp utilizando software AxoScope. Los datos se adquirieron a una velocidad de muestreo de 10 kHz. </li></ol>

<ol>
	<li>Clark, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressing+zebrafish+for+behavioral+genetics.">Stressing zebrafish for behavioral genetics.</a> Reviews in Neuroscience. 22 (1), 49 (2011).</li><li>Rinkwitz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+an+integrative+system+for+neurogenomics+and+neurosciences.">Zebrafish: an integrative system for neurogenomics and neurosciences.</a> Progress in Neurobiology. 93 (2), 231 (2011).</li><li>Penberthy, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Frontiers in Bioscience. 7, d1439 (2002).</li><li>Letamendia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+automated+high-throughput+system+for+zebrafish+in+vivo+screenings.">Development and validation of an automated high-throughput system for zebrafish in vivo screenings.</a> PLoS One. 7, e36690 (2012).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nature Genetics. 26 (2), 216 (2000).</li><li>Haffter, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+development+of+the+zebrafish+inner+ear+and+lateral+line.">Mutations affecting development of the zebrafish inner ear and lateral line.</a> Development. 123, 1 (1996).</li><li>Suster, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Molecular Biology. 561, 41 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, 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=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pentylenetetrazole+induced+changes+in+zebrafish+behavior,+neural+activity+and+c-fos+expression.">Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.</a> Neuroscience. 131 (3), 759 (2005).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+mutagenesis+screen+to+identify+seizure-resistant+zebrafish.">A large-scale mutagenesis screen to identify seizure-resistant zebrafish.</a> Epilepsia. 48 (6), 1151 (2007).</li><li>Williams, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+radiotelemetry+to+evaluate+electrographic+seizures+in+rats+with+kainate-induced+epilepsy.">The use of radiotelemetry to evaluate electrographic seizures in rats with kainate-induced epilepsy.</a> Journal of Neuroscience Methods. 155 (1), 39 (2006).</li><li>Marsh, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+EEG+spikes+identify+the+region+of+electrographic+seizure+onset+in+some,+but+not+all,+pediatric+epilepsy+patients.">Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients.</a> Epilepsia. 51 (4), 592 (2010).</li><li>Zhu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+application+of+modularly+assembled+zinc-finger+nucleases+in+zebrafish.">Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.</a> Development. 138 (20), 4555 (2011).</li></ol>

No hay conflictos de interés declarado.

El método de registro extracelular presentado aquí permite un análisis muy sensible y rápida de la actividad cerebral. Estas grabaciones son análogos a electroencefalográfica (EEG) de seguimiento comúnmente utilizado para evaluar la presencia de descarga eléctrica anormal (es decir, convulsiones) en modelos de roedores de epilepsia y 11 pacientes 12. Grabaciones extracelular se puede combinar con las manipulaciones farmacológicas, como se muestra aquí. Estos tipos de grabaciones ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Agarosa de bajo punto de fusión </td><td> Fisher Scientific- </td><td> BP1360-100 </td><td> Disolver en los medios de embriones en el 1,2% </td></tr><tr><td> Soporte de grabación </td><td> Fisher Scientific- </td><td> BP3581, P330-3, BP410-1, BP214-500, D16-1, C77-500 </td><td> 1 mM NaCl, 2,9 mM KCl, 10 mM HEPES, 1,2 mM de MgCl 2, 10 mM de dextrosa, 2,1 mM CaCl 2 pH a aproximadamente 7,3 con 1 N NaOH </td></tr><tr><td> Tricaína </td><td> Argent Labs </td><td> MS-222 </td><td> 0,02% </td></tr><tr><td> α-bungarotoxina </td><td> Bioscience Tocris </td><td> 2133 </td><td> 1 mg / ml </td></tr><tr><td> Tubo capilar de vidrio </td><td> Warner Instruments </td><td> G120TF-3 </td><td> Tire a una resistencia de 2 -7 mW </td></tr><tr><td> Patch amplificador de pinzamiento </td><td> Warner Instruments </td><td> PC-505B </td><td> Nosotros usamos un amplificador de Warner en modo de corriente-clamp, ganancia fijó en 2 mV / Pa y filtro de Bessel fijado en 2K. Modelos comparables se pueden utilizar de acuerdo con las instrucciones del fabricante. </td></tr><tr><td> Filtro / amplificador </td><td> Cygnus Tecnología </td><td> FLA-01 </td><td> Usamos un pre-amplificador de Cygnus, ganancia ajustada a 10-20; Frecuencia límite fijado en el 1-2K, filtro Notch IN. Modelos comparables se pueden utilizar de acuerdo con las instrucciones del fabricante. </td></tr><tr><td> Axon A / D bordo y software AxoScope </td><td> Molecular Devices </td><td> Axon Digidata 1320A; AxoScope 8,2 </td><td> Los datos se recogen en AxoScope utilizando brecha sin modo de adquisición, el muestreo a 10 kHz. Modelos comparables y programas pueden ser utilizados de acuerdo con las instrucciones del fabricante. </td></tr><tr><td> Huevo agua </td><td&gt; Instant Ocean </td><td></td><td> 3 g de sal Instant Ocean mar, 2 ml 0,1% de azul de metileno en 10 ml de agua desionizada </td></tr></tbody></table>

forebrain electrophysiological recording in larval zebrafish

La epilepsia afecta a casi 3 millones de personas en los Estados Unidos y hasta 50 millones de personas en todo el mundo. Se define como la aparición de convulsiones no provocadas espontáneos, la epilepsia puede ser adquirido como resultado de un daño cerebral o una mutación genética. Los esfuerzos para convulsiones modelo en animales han utilizado principalmente adquirido insultos (fármacos anticonvulsivos, estimulación o lesión cerebral) y las manipulaciones genéticas (knockdown antisentido, la recombinación homóloga o transgénesis) en roedores. El pez cebra son un sistema modelo vertebrado 1-3 que podría proporcionar una valiosa alternativa a los roedores investigación basada en la epilepsia. El pez cebra se utilizan extensivamente en el estudio de la genética de vertebrados o de desarrollo, presentan un alto grado de similitud genética para los mamíferos y expresar homólogos de ~ 85% de las mutaciones conocidas humanos epilepsia de un solo gen. Debido a su pequeño tamaño (4-6 mm de largo), larvas de pez cebra puede ser mantenido en volúmenes de fluido tan bajo como 100 l durante el desarrollo temprano y arraYED en múltiples pocillos. Los reactivos se pueden añadir directamente a la solución en la que se desarrollan los embriones, la simplificación de la administración del fármaco y que permite una rápida selección in vivo de compuestos de ensayo 4. Los oligonucleótidos sintéticos (morfolinos), mutagénesis, dedo de zinc nucleasa y enfoques transgénicos se pueden utilizar para generar rápidamente desmontables gen o mutación en el pez cebra 5-7. Estas propiedades costear los estudios de pez cebra sin precedentes ventaja estadística sobre el análisis del poder roedores en el estudio de los trastornos neurológicos como la epilepsia. Debido a que el &quot;estándar de oro&quot; para la investigación epilepsia es monitorear y analizar las descargas eléctricas anormales que se originan en una estructura del cerebro central (por ejemplo, convulsiones), un método para grabar de manera eficiente la actividad cerebral en larvas de pez cebra se describe aquí. Este método es una adaptación de las técnicas convencionales de registro extracelular y permite una estabilidad a largo plazo de vigilancia de la actividad cerebral en larvas de pez cebra intacto. Sgrabaciones amplias se muestran para las convulsiones agudas inducidas por la aplicación de fármacos anticonvulsivos baño y convulsiones espontáneas grabadas en un pez modificado genéticamente.

Ejemplos de electrográfica convulsiva de descarga registrada en el cerebro anterior de un agar-incrustado larvas de pez cebra se muestra en la figura 1. De gran amplitud multi-pico de descarga ráfaga en estas muestras fue evocado por baño de aplicación de un fármaco anticonvulsivo, pilocarpina 40 mM (en A; 6 dpf) o 1 mM picrotoxina (en B; 8 dpf). En estas grabaciones, y inmovilizada agar-encajados pez cebra se controlan continuamente durante un máximo de 90 min. Fish siendo viable bajo estas condi...

Watch this Scientific Journal Video about Forebrain Electrophysiological Recording in Larval Zebrafish at JoVE.com

Forebrain Electrophysiological Recording in Larval Zebrafish

Un método simple para registrar los potenciales de campo extracelular en el cerebro anterior de las larvas de pez cebra se describe. El método proporciona una robusta<em&gt; In vivo</em&gt; Lectura de actividades similares a convulsiones. Esta técnica se puede utilizar con larvas de pez cebra transgénico que lleva genes relacionados con la epilepsia o las convulsiones provocadas por la administración de fármacos anticonvulsivos.

Prosencéfalo electrofisiológico de grabación en larvas de pez cebra

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

forebrain-electrophysiological-recording-in-larval-zebrafish

Research

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Neuroscience

17.0K vistas.  University of California, San Francisco. Un método simple para registrar los potenciales de campo extracelular en el cerebro anterior de las larvas de pez cebra se describe. El método proporciona una robusta In vivo Lectura de actividades similares a convulsiones. Esta técnica se puede utilizar con larvas de pez cebra transgénico que lleva genes relacionados con la epilepsia o las convulsiones provocadas por la administración de fármacos anticonvulsivos.

Video: Forebrain Electrophysiological Recording in Larval Zebrafish

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse de imágenes en vivo de la relación clonal con células progenitoras neurales en el cerebro anterior El desarrollo de pez cebra

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Investigación

Neurociencias

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 ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Registro electrofisiológico en el cerebro del pez cebra adultos Intacto

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

La transección de la médula espinal en el pez cebra larval

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Grabación simultánea electrofisiológico y micro-inyecciones de agentes inhibitorios en el cerebro de roedores

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Registro electrofisiológico de<em&gt; Drosophila</em&gt; Tricoide Sensilla en Respuesta a Odorantes de Baja Volatilidad

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

En proyección de imagen del calcio Vivo de las células de pelo de la línea Lateral en el pez cebra larvas

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.

Investigación de la plasticidad sináptica a largo plazo en Interlamellar Hippocampus CA1 por grabación de campo electrofisiológica

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

Grabación electrofisiológica inalámbrica de las neuronas por tetrodes móviles en la natación libre de peces

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...