Name: zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons
Uploaded: 2017-04-18T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

Este trabajo fue apoyado por subvenciones del NIH (F32 NS059120 a RLM y R01NS25217 y P30NS048154 a ABR).

Los autores declaran no tener intereses financieros en competencia.

Los métodos descritos aquí permiten la caracterización eléctrica y morfológica de las neuronas sensoriales y motoras de embriones de pez cebra después de la disección mínima de la médula espinal. Las neuronas permanecen sanos durante al menos 1 h, el límite de tiempo impuesto en estas grabaciones. Las neuronas han sido registrados utilizando la configuración de célula completa estándar, así como de parches nucleadas; el último método minimiza los problemas de espacio-clamp que se opone a que un estudio b...

<html> George Streisinger fue pionera en el uso de Danio rerio, conocido comúnmente como el pez cebra, como un sistema modelo para el análisis genético de desarrollo de los vertebrados 10. El modelo ofrece varias ventajas, incluyendo: (1) relativamente simple y barato cría de animales; (2) fertilización externa, lo que permite un fácil acceso a los embriones de las etapas de desarrollo más tempranas; y (3) un embrión transparente, permitiendo la observación directa y repetidas de células, tejidos y órganos, ya que forman. En las décadas siguientes, varios avances aumentaron aún más el poder del modelo de pez cebra. En particular, las pantallas adelante genéticos y los esfuerzos de secuenciación de todo el genoma jugaron un papel clave en la identificación de mutaciones y genes críticos para muchos procesos de desarrollo 11, 12, 13, 14,&quot;&gt; 15, métodos de clonación 16. Puerta de enlace han permitido la aplicación rutinaria de transgénicos se aproxima a 17, 18. Los recientes avances en la edición genoma, ejemplificada por la transcripción del tipo activador de (Talens) y agrupados repeticiones intercaladas regularmente cortos palindrómicas (CRISPR) -Cas9 nucleasas, permitir la introducción selectiva de mutaciones, así como knock-out y knock-in se aproxima a 19, 20, 21, 22. combinados, estos métodos hacen que el pez cebra un poderoso modelo para el estudio de los mecanismos genéticos que subyacen a los comportamientos específicos y varias enfermedades humanas 23, 24, 25, 26, 27. Este trabajo se centra en el desarrollola regulación mental y el papel de la actividad eléctrica en el desarrollo neuronal. La atención se centra en la médula espinal, para el que el modelo de pez cebra ofrece varias ventajas. En primer lugar, es relativamente fácil de acceder pez cebra en estadios embrionario y larval; Por lo tanto, se puede estudiar la función de la médula espinal durante las etapas de desarrollo que tienen menos neuronas y circuitos simples 28, 29. Por otra parte, la médula espinal pez cebra tiene un conjunto diverso de neuronas, similar a otros vertebrados, como se demuestra por los patrones característicos y distintivos de factores de transcripción 30, 31, 32, 33, 34, 35. La mayoría de los estudios en el pez cebra que tienen como objetivo descubrir los mecanismos que subyacen a la función de los circuitos de la médula espinal, especialmentelos que apoyan la locomoción, se centran comprensiblemente en estadios larvarios 36, 37, 38, 39, 40, 41, 42, 43. Sin embargo, muchas de las neuronas que forman las redes de locomotoras espinales iniciado su diferenciación en las etapas embrionarias tempranas, ~ 9-10 h después de la fertilización (HPF) 44, 45, 46, 47, 48, 49, 50, 51. En vista de esto, la comprensión de cómo surgen las propiedades morfológicas y eléctricas de las neuronas espinales y el cambio entre las etapas embrionario y larval es importante para un overacomprensión ll de la formación y la función de circuito locomotor. Los métodos de disección descritas aquí permiten grabaciones de patch clamp de neuronas de la médula y se han aplicado con éxito en las etapas embrionarias (~ 17-48 HPF) y estadios larvarios (~ 3-7 días después de la fertilización [dpf]). Este enfoque limita la cantidad de disección requerida para proporcionar acceso a las neuronas de interés. El protocolo se diferencia de la mayoría de los otros métodos publicados para la grabación de las neuronas espinales de pez cebra en que pegamento veterinario de sutura se utiliza, en lugar de un pasador de tungsteno bien, para unir el embrión o larva a la cámara de registro. La disponibilidad de dos enfoques diferentes (es decir, cola de sutura contra el pasador de tungsteno) para el montaje de los embriones de pez cebra o larvas para el análisis electrofisiológico proporciona a los investigadores con opciones alternativas para alcanzar sus objetivos experimentales específicas. En primer lugar, los procedimientos de acceso y grabación en una emergente<html> ulación de las neuronas sensoriales primarias, células Rohon-Barba, se describen. Los cuerpos celulares de estas neuronas se encuentran dentro de la médula espinal dorsal. Existen células Rohon-Barba en numerosas especies de vertebrados, se diferencian en el desarrollo temprano, y subyacen a la respuesta táctil embrionaria 6, 44, 47, 48. En segundo lugar, los procedimientos de acceso y registro de las neuronas motoras espinales se detallan. surgen de pez cebra neuronas motoras espinales durante dos oleadas de la neurogénesis. Surgen las neuronas motoras primarias nacidos antes-al final de la gastrulación (~ 9-16 hpf), con sólo 3-4 neuronas motoras primaria presentes por hemisegment 45, 46, 49. En contraste, la población nacida después de las neuronas motoras secundarias es más numeroso y se produce durante un período prolongado, a partir de ~ 14 HPFef &quot;&gt; 45, 50. génesis secundaria de neuronas motoras en los segmentos de mediados de tronco se ha completado todo por 51 HPF 50. neuronas motoras secundarias se considera que son la contrapartida de las neuronas motoras en amniotas 46. Curiosamente, las neuronas supraespinales, a través de la dopamina, regulan la locomoción en la larva y el motor secundario génesis neurona en el embrión y larva joven 50, 51. neuronas motoras primarias y secundarias comprenden cada uno varios subtipos diferentes. cada motor primario proyectos subtipo neurona un axón periférico que inerva un grupo muscular característica, lo que resulta en un estereotipo, la identificación de trayectoria axonal. Generalmente, las neuronas motoras secundarias siguen las vías axonales previamente establecidos por las neuronas motoras primarias. por lo tanto, con respecto a las trayectorias axonales, neuronas motoras primarias y secundarias son similares, con la excepción de que el espesor axonal y somata tamaño unare mayor para las neuronas motoras primarias 45. En tercer lugar, se discuten los métodos para la grabación de algunos tipos de interneuronas. Sin embargo, en estos casos, se requiere una cantidad limitada de la eliminación de otras células de la médula espinal, y por lo tanto la médula espinal es menos intacta que para las grabaciones de células Rohon-Barba o neuronas motoras.

<table><tbody><tr><td>Vacuum filter/Storage bottle, 0.22 mm pore</td><td>Corning</td><td>431096</td><td></td></tr><tr><td>Syringe filter 0.2 mm</td><td>Whatman</td><td>6780-2502</td><td></td></tr><tr><td>Tricaine</td><td>Sigma</td><td>A-5040</td><td>Ethyl 3-aminobenzoate methanesulfonate salt&amp;nbsp;</td></tr><tr><td>&amp;alpha;-bugarotoxin</td><td>Tocris</td><td>11032-79-4</td><td></td></tr><tr><td>Tetrodotoxin</td><td>Tocris</td><td>4368-28-9</td><td></td></tr><tr><td>Alexa-549 hydrazine salt</td><td>Molecular Probes</td><td>A-10438</td><td>fluorescent dye</td></tr><tr><td>Spin-X centrifuge tube filter</td><td>Corning</td><td>8161</td><td></td></tr><tr><td>Glass microscope slide</td><td>Fisher</td><td>12-550C</td><td></td></tr><tr><td>Sylgard silicone elastomer kit</td><td>Dow Corning</td><td>184</td><td>silicone elastomer</td></tr><tr><td>Petri dishes</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Harvard Apparatus</td><td>30-0038</td><td>inner and outer diameters of 0.78 and 1.0 mm (thin walled glass capillaries)</td></tr><tr><td>Borosilicate glass capillaries</td><td>Drummond Scientific</td><td>1-000-1000-100</td><td>inner and outer diameters of 1.13 and 1.55 mm (thick walled glass capillaries)</td></tr><tr><td>Miniature barbed polypropylene fitting&amp;nbsp;</td><td>Cole-Palmer</td><td>6365-90</td><td></td></tr><tr><td>Vetbond tissue adhesive&amp;nbsp;</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Collagenase XI</td><td>Sigma</td><td>C7657</td><td></td></tr><tr><td>Microelectrode puller</td><td>Sutter Instruments</td><td>&amp;nbsp;Model P-97&amp;nbsp;</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axopatch 200B</td><td></td></tr><tr><td>Head stage</td><td>Molecular Devices</td><td>CV203BU</td><td></td></tr><tr><td>Motorized micromanipulator&amp;nbsp;&amp;nbsp;</td><td>Sutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Tygon tubing</td><td>Fisher</td><td>14-169-1B</td><td>ID 1/16 IN, OD 1/8 IN and WALL 1/32 IN (flexible laboratory tubing)</td></tr><tr><td>Electrode holder</td><td>Molecular Devices</td><td>1-HC-U</td><td></td></tr><tr><td>Pharmaseal Three-Way Stopcocks&amp;nbsp;</td><td>Baxter</td><td>K75</td><td></td></tr><tr><td>Digitizer</td><td>Axon Instruments</td><td>Digidata 1440A</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Zeiss</td><td>Axioskop2 FS plus</td><td></td></tr><tr><td>40X/0.80W Achroplan objective</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Data acquisition and analysis software&amp;nbsp;</td><td>Axon Instruments</td><td></td><td>PClamp 10 - Clampex and Clampfit&amp;nbsp;</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>Model P-97</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Dissection and Recording Solutions (in mM)&lt;/strong&gt;</td></tr><tr><td>All solutions, except the intracellular, are stable for ~2-3 months when filtered (0.22 mm filter cups) and stored at room temperature (RT).</td><td></td><td></td><td></td></tr><tr><td>The intracellular solution is filtered (0.2 mm syringe filters) and stored frozen (-20 &amp;deg;C) in small aliquots that are individually thawed on the day of use.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Dissection/Ringer&amp;rsquo;s solution</td><td></td><td></td><td>145 NaCl, 3 KCl, 1.8 CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 10 HEPES; pH 7.4 (with NaOH)</td></tr><tr><td>Pipette (intracellular) recording solution</td><td></td><td></td><td>135 KCl, 10 EGTA-acid, 10 HEPES; pH 7.4 (with KOH).</td></tr><tr><td>Bath (extracellular) recording solution/voltage and current-clamp</td><td></td><td></td><td>125 NaCl, 2 KCl, 10 CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O, 5 HEPES; pH 7.4 (with NaOH).</td></tr><tr><td>Alexa-594 hydrazine salt stock solution.&amp;nbsp;</td><td></td><td></td><td>Prepare a 13.2 mM stock in ddH&lt;sub&gt;2&lt;/sub&gt;O, aliquot (~100 &amp;micro;l) and store at -20 &amp;deg;C. For use, dilute the stock solutiond 132 fold with pipette solution to a final concentration of 100 mM. After dilution, filter the Alexa-594 containing pipette solution&amp;nbsp; with a centrifuge tube filter.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immobilizing agents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.4% ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td></td><td></td><td>Prepare a 0.4% stock solution in 0.2 M Tris, pH 9 (0.4 g Tricaine/100 mL 0.2 M Tris</td></tr><tr><td colspan="4">Adjust pH to 7 with NaOH and store at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute the stock solution ~25 fold in embryo media</td></tr><tr><td>250 mM &amp;alpha;-bungarotoxin</td><td></td><td></td><td>Prepare a 250 mM stock in ddH&lt;sub&gt;2&lt;/sub&gt;O (1 mg/500 mL), prepare 100 &amp;micro;L aliquots, and sotre at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute 2,500-fold with extracellular solution to a final concentration of 100 nM.</td></tr><tr><td>1 mM Tetrodotoxin</td><td></td><td></td><td>Prepare a 1 mM stock in ddH2O (1 mg/3 mL), prepare 100 &amp;micro;L aliquots, and sotre at -20 &amp;deg;C.</td></tr><tr><td colspan="4">For use, dilute 2,000-fold with extracellular solution to a final concentration of 500 nM.</td></tr></tbody></table>

zebrafish in situ spinal cord preparation for electrophysiological recordings from spinal sensory and motor neurons

El pez cebra, introducido por primera vez como un modelo de desarrollo, han ganado popularidad en muchos otros campos. La facilidad de la cría de un gran número de organismos que se desarrollan rápidamente, combinada con la claridad óptica embrionario, sirvió como atributos de peso iniciales de este modelo. Durante las últimas dos décadas, el éxito de este modelo ha sido impulsado además por su susceptibilidad a las pantallas de mutagénesis a gran escala y por la facilidad de transgénesis. Más recientemente, los enfoques de edición gen haber aumentado la potencia del modelo. Para los estudios del desarrollo neurológico, el embrión de pez cebra y larva proporcionan un modelo al que se pueden aplicar varios métodos. Aquí, nos centramos en los métodos que permiten el estudio de una propiedad esencial de las neuronas, excitabilidad eléctrica. Nuestra preparación para el estudio electrofisiológico de neuronas de la médula de pez cebra implica el uso de pegamento veterinario de sutura para asegurar la preparación de una cámara de registro. Los métodos alternativos para la grabacióna partir de embriones de pez cebra y larvas implicar la unión de la preparación a la cámara utilizando un alfiler fino de tungsteno 1, 2, 3, 4, 5. Un pasador de tungsteno es la más utilizada para montar la preparación en una orientación lateral, aunque se ha usado para montar larvas dorsal del lado hasta 4. El pegamento de sutura se ha usado para montar embriones y larvas en ambas orientaciones. Usando el pegamento, una disección mínima se puede realizar, permitiendo el acceso a las neuronas espinales sin el uso de un tratamiento enzimático, evitando así cualquier daño resultante. Sin embargo, para las larvas, es necesario aplicar un tratamiento enzimático breve para eliminar el tejido muscular que rodea la médula espinal. Los métodos descritos aquí se han utilizado para estudiar las propiedades eléctricas intrínsecas de las neuronas motoras, interneuronas, y las neuronas sensoriales en varios developmental etapas 6, 7, 8, 9.

Hemos registrado con éxito de las neuronas Rohon-barba en 17 hpf embriones a través de 7 dpf larvas (Figura 5A y 5B). Cuando se registraron células Rohon-barba, la preparación se monta en el lado dorsal hacia arriba. Dicho montaje permite la identificación inequívoca de células Rohon-Barba sobre la base de sus posiciones dorsal superficial y grandes tamaños soma. La identificación se confirmó adicionalmente por el potencial de membrana en repos...

Watch this Scientific Journal Video about Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons at JoVE.com

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Este manuscrito describe métodos para registros electrofisiológicos de neuronas espinales de embriones de pez cebra y larvas. La preparación mantiene las neuronas in situ y a menudo implica la disección mínimo. Estos métodos permiten para el estudio electrofisiológico de una variedad de neuronas de la médula, de la adquisición inicial de la excitabilidad eléctrica a través de las primeras etapas larvales.

El pez cebra<em&gt; In Situ</em&gt; Médula Espinal Preparación para Registros electrofisiológicos de Spinal Sensory y neuronas motoras

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

zebrafish-situ-spinal-cord-preparation-for-electrophysiological

Research

JoVE Journal

Neuroscience

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

11.4K vistas.  University of Colorado Anschutz Medical Campus (UCAMC).  Este manuscrito describe métodos para registros electrofisiológicos de neuronas espinales de embriones de pez cebra y larvas. La preparación mantiene las neuronas in situ y a menudo implica la disección mínimo. Estos métodos permiten para el estudio electrofisiológico de una variedad de neuronas de la médula, de la adquisición inicial de la excitabilidad eléctrica a través de las primeras etapas larvales. 

Video: Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Emparejados grabaciones patch clamp de neurona motora y el músculo esquelético de destino en el pez cebra

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Investigación

Neurociencias

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Potencial de membrana de la neurona motora de Biosensing en los embriones vivos de pez cebra

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Biología del Desarrollo

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

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 ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch Clamp Las grabaciones de las células embrionarias de pez cebra Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

La disección y de montaje lateral de embriones de pez cebra: Análisis para el Desarrollo de la médula espinal

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

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 ...

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 ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

Actividad De Las Neuronas Aferentes De La Línea Lateral Posterior Durante La Natación En El Pez Cebra

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Lesiones controladas semiautomatizadas inducidas por láser para estudiar la regeneración de la médula espinal en larvas de pez cebra

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

Medicina

A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos

Source: Benedetti, L., et al. <a href="https://app.jove.com/v/55297">Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos.</a> J. Vis. Exp. (2017).
This video demonstrates a non-invasive assay to study electrophysiology of motor neurons in zebrafish embryos. Upon measuring tail coiling in the embryos as a result of spontaneous depolarization of the spinal cord motor neurons, a sodium channel blocker is applied to reduce the coiling frequency. Next, the embryos are immobilized in agarose and placed under a fluorescence microscope. The genetically engineered motor neurons express a voltage-sensing biosensor with a donor and an acceptor fluorophore. A decrease in acceptor fluorophore emission indicates a reduction in the spontaneous depolarization of motor neurons due to the sodium channel blocker.

Un enfoque no invasivo para estudiar la electrofisiología de las neuronas motoras en embriones de pez cebra

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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