Este trabalho foi apoiado por bolsas do NIH (F32 NS059120 para RLM e R01NS25217 e P30NS048154 a ABR).

Os autores declaram não haver interesses financeiros concorrentes.

Os métodos aqui descritos permitem a caracterização eléctrica e morfológica dos neurónios sensoriais e motores de embriões de peixes-zebra após dissecção mínima da medula espinhal. Neurônios permanecem saudáveis ​​por pelo menos 1 h, o limite de tempo imposto sobre essas gravações. Os neurónios foram registados utilizando a configuração de célula inteira padrão, bem como a partir de manchas nucleadas; este último método minimiza os problemas de espaço-de braçadeira que pode impedir um estudo ...

<html> George Streisinger pioneira no uso de Danio rerio, vulgarmente conhecido como peixe-zebra, como um sistema modelo para a análise genética do desenvolvimento dos vertebrados 10. O modelo oferece várias vantagens, incluindo: (1) criação de animais relativamente simples e de baixo custo; (2) a fertilização externa, permitindo um acesso fácil aos embriões desde os primeiros estádios de desenvolvimento; e (3) um embrião transparente, permitindo observações directas e repetidas de células, tecidos e órgãos, como eles formam. Ao longo das décadas que se seguiram, vários avanços aumentou ainda mais o poder do modelo peixe-zebra. Em particular, telas genéticos para a frente e os esforços de sequenciação de todo o genoma desempenharam um papel-chave na identificação de mutações e genes críticos para muitos processos de desenvolvimento 11, 12, 13, 14,&quot;&gt; 15, os métodos de clonagem 16. Gateway permitiram a aplicação de rotina transgénico se aproxima de 17, 18. Recentes avanços na edição genoma, exemplificado pelo activador da transcrição do tipo (TALENS) e agrupados repete regularmente interspaced curtas palindrómicos (CRISPR) -Cas9 nucleases, permitir a introdução alvo de mutações, bem como knock-out e knock-in se aproxima de 19, 20, 21, 22. Combinada, estes métodos fazem peixe-zebra um poderoso modelo para o estudo dos mecanismos genéticos subjacentes comportamentos específicos e várias doenças humanas 23, 24, 25, 26, 27. Este trabalho se concentra em desenvolverregulação mental e o papel da atividade elétrica no desenvolvimento neuronal. O foco é sobre a medula espinal, para os quais o modelo de peixe-zebra proporciona várias vantagens. Em primeiro lugar, é relativamente fácil de acessar peixe-zebra em estágios embrionários e larval; portanto, pode-se estudar a função da medula espinal durante os estágios de desenvolvimento que têm menos neurónios e circuitos mais simples 28, 29. Além disso, a espinal medula de peixe-zebra tem um conjunto diversificado de neurónios, semelhante a outros vertebrados, como demonstrou padrões característicos e distintivas de factores de transcrição 30, 31, 32, 33, 34, 35. A maioria dos estudos em peixes-zebra que têm como objectivo de desvendar os mecanismos subjacentes à função de circuitos da medula espinhal, especialmenteaqueles que suportam a locomoção, são compreensivelmente focada em estágios larvais 36, 37, 38, 39, 40, 41, 42, 43. No entanto, muitos dos neurónios que formam as redes locomotiva espinhais iniciar a sua diferenciação em estágios embrionários iniciais, ~ 9-10 h pós-fertilização (HPF) 44, 45, 46, 47, 48, 49, 50, 51. Em vista disso, a compreensão de como as propriedades morfológicas e eléctricas dos neurónios espinais e surgem alterações entre os estágios embrionários e larval é importante para uma overacompreensão ll de formação e função circuito locomotor. Os métodos aqui descritos permitem dissecção gravações de patch clamp de neurónios espinais e têm sido aplicados com sucesso em estágios embrionários (~ 17-48 HPF) e as fases larvares (~ 3-7 dias após a fertilização [dpf]). Esta abordagem limita a quantidade de dissecção requerida para fornecer acesso aos neurônios de interesse. O protocolo difere da maioria dos outros métodos publicados para a gravação de neurónios espinais zebrafish em que a cola veterinário sutura é utilizado, em vez de um pino de tungsténio finos, para fixar o embrião ou larva para a câmara de registo. A disponibilidade de duas abordagens diferentes (isto é, cola de sutura contra o pino de tungsténio) para a montagem dos embriões de peixe-zebra ou larvas para análise electrofisiológica fornece investigadores com opções alternativas para alcançar os seus objectivos específicos experimentais. Em primeiro lugar, os procedimentos para acessar e gravar a partir de um pop<html> ulação de neurónios sensoriais primários, células Rohon-Beard, são descritos. Os corpos celulares destes neurónios se encontram dentro da espinal medula dorsal. Existem células Rohon-Beard em numerosas espécies de vertebrados, diferenciar no início do desenvolvimento, e estão subjacentes à resposta ao toque embrionário 6, 44, 47, 48. Em segundo lugar, os procedimentos de acesso e gravação de neurónios motores espinais são detalhados. Zebrafish neurónios motores espinais surgir durante duas ondas de neurogese. Os neurónios motores primários anterior-nascidos surgem no final da gastrulação (~ 9-16 HPF), com apenas 3-4 neurónios motores primária presentes por hemisegmento 45, 46, 49. Em contraste, a população depois-nascido de neurónios motores secundários é mais numerosos e surge durante um período prolongado, a partir das ~ 14 hpfef &quot;&gt; 45, 50. Secundário neurônio motor gênese nos segmentos mid-tronco é principalmente concluída até 51 hpf 50. neurônios motores secundários são considerados a contrapartida dos neurônios motores em amniotes 46. Curiosamente, os neurônios supraespinhais, via dopamina, regular locomoção na larva e motor secundário neurónio génese no embrião e jovem larva 50, 51. neurónios motores primários e secundários compreendem cada um vários subtipos diferentes. cada motor primário projectos neurónio subtipo a de axónios periférica que inerva um grupo muscular característico, resultando em um estereotipada, identificar trajectória axonal. Geralmente, os neurónios motores secundários seguir as vias axonais previamente estabelecidos por neurónios motores primários. Assim, no que diz respeito a trajectórias axonal, os neurónios motores primária e secundária são semelhantes, com a excepção de que a espessura axonal e uma somata tamanhore maior para os neurónios motores primários 45. Em terceiro lugar, são discutidos métodos para gravação de alguns tipos de interneurônios. No entanto, nestes casos, é necessária uma limitada quantidade de remoção de outras células do cordão espinal, e, assim, a medula espinal é menos intacta do que para as gravações de células Rohon-barba ou neurónios motores.

<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

Peixe-zebra, introduzido pela primeira vez como um modelo de desenvolvimento, ganharam popularidade em muitos outros campos. A facilidade de criação de um grande número de organismos em rápido desenvolvimento, combinada com a clareza óptica embrionário, serviu como atributos atraentes iniciais deste modelo. Ao longo das últimas duas décadas, o sucesso deste modelo foi ainda mais impulsionado por sua receptividade às telas de mutagênese em larga escala e pela facilidade de transgenia. Mais recentemente, de edição de gene abordagens ter aumentado a potência do modelo. Para os estudos do neurodesenvolvimento, o embrião do peixe-zebra e larva de fornecer um modelo para que vários métodos podem ser aplicados. Aqui, vamos nos concentrar em métodos que permitem o estudo de uma propriedade essencial de neurônios, excitabilidade elétrica. A nossa preparação para o estudo electrofisiológico de neurónios espinais zebrafish envolve o uso de cola veterinário de sutura para fixar a preparação para uma câmara de registo. Métodos alternativos para gravaçãoa partir de embriões de peixe-zebra e larvas envolver a fixação da preparação para a câmara utilizando um pino fina de tungsténio de 1, 2, 3, 4, 5. Um pino de tungsténio é mais frequentemente usado para montar a preparação de uma orientação lateral, embora tenha sido usado para montar dorso-lateral larvas até 4. A cola de sutura tem sido utilizado para montar os embriões e larvas em ambas as orientações. Usando a cola, uma dissecção mínima pode ser realizada, permitindo o acesso a neurónios espinais sem o uso de um tratamento enzimático, evitando assim qualquer dano resultante. No entanto, para as larvas, é necessário aplicar um breve tratamento com enzima para remover o tecido muscular em torno da medula espinhal. Os métodos aqui descritos têm sido utilizados para estudar as propriedades eléctricas intrínsecas de neurónios motores, interneurónios, e neurónios sensoriais em vários DevelopmentAl módulos 6, 7, 8, 9.

Temos registos com sucesso a partir de neurónios Rohon-Beard em 17 hpf embriões através 7 dpf larvas (Figura 5A e 5B). Quando as células Rohon-Beard foram registados, a preparação foi montada do lado dorsal para cima. Essa montagem permite a identificação inequívoca de células Rohon-Beard com base nas suas posições dorsais superficiais e tamanhos grandes soma. A identificação é adicionalmente confirmada pelo potencial de membrana em repous...

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 descreve métodos para registos electrofisiolicos de neurónios espinais de embriões de peixe-zebra e larvas. A preparação mantém neurónios in situ e muitas vezes envolve a dissecção mínima. Estes métodos permitem o estudo electrofisiológico de uma variedade de neurónios espinais, a partir da aquisição inicial excitabilidade eléctrica através dos primeiros estádios larvares.

zebrafish<em&gt; In Situ</em&gt; Spinal Cord Preparação para gravações eletrofisiológicas da Spinal Sensorial e Motor Neurons

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 visualizações.  University of Colorado Anschutz Medical Campus (UCAMC).  Este manuscrito descreve métodos para registos electrofisiolicos de neurónios espinais de embriões de peixe-zebra e larvas. A preparação mantém neurónios in situ e muitas vezes envolve a dissecção mínima. Estes métodos permitem o estudo electrofisiológico de uma variedade de neurónios espinais, a partir da aquisição inicial excitabilidade eléctrica através dos primeiros estádios larvares. 

Vídeo: 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.

Gravações patch emparelhado Grampo da Motor-neurônio e músculo alvo ósseas em Zebrafish

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

Neurociência

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 neurônio motor biossensível em embriões vivos de peixe-zebra

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

Biologia do Desenvolvimento

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 gravação eletrofisiológico em 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 ...

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.

Grampo patch Gravações de Embryonic Cells Zebrafish 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.

Dissecção e montagem lateral de embriões Zebrafish: Análise de Desenvolvimento da Medula Espinhal

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.

Gravação eletrofisiológica no cérebro de Intacta Zebrafish Adulto

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.

Transecção medular nos 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 ...

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.

Atividade da Linha Lateral Posterior Neurônios Diferentes durante a Natação em Zebrafish

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.

Lesões semi-automatizadas semi-automatizadas controladas por laser para estudar a regeneração da medula espinhal em larvas de zebrafish

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.

Uma abordagem não invasiva para estudar a eletrofisiologia dos neurônios motores em embriões de peixe-zebra

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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