O autor gostaria de agradecer a Peter Castro e Mateus Dinday por seus esforços iniciais para estabelecer zebrafish em laboratório. Este trabalho foi financiado pelos Institutos Nacionais de Saúde EUREKA concessão (# R01NS079214-01).

1. Produção de ovos e Cobrança <ol><li> Zebrafish criação segue procedimento padrão descrito anteriormente 8. Resumidamente, zebrafish adultos são criados em tanques de criação com divisórias no lugar. Quando as luzes da sala de vir na manhã seguinte, divisórias são removidas de reprodução tanques e os peixes são permitidos aproximadamente 20 a 60 minutos de tempo de acasalamento imperturbável. </li><li> Ovos de tanques de criação são coletados em uma peneira e lavadas com água de ovo. Os ovos são então transferidos para uma placa de Petri com água de ovo. Ovos não fertilizados e detritos são removidos utilizando uma pipeta de transferência. </li><li> Prato lugar Petri contendo os ovos recolhidos numa incubadora (28-32 ° C). Após eclosão dos ovos, cerca de dois dias após a fertilização (dpf), remova córion e qualquer outros detritos com uma pipeta de transferência. </li><li> No dia desejado pós-fertilização (3-8 dpf) remover uma placa de Petri contendo larvas livremente natação e lugar na bancada do laboratório à temperatura ambiente. </li></ol><ol><li> Usando um extrator micropipeta, puxe de 1,2 mm de diâmetro externo de borosilicato capilar de vidro em duas agulhas e armazenar em uma placa de Petri 150 mm ou caixa de pipeta vazio que coloca sobre rampas bolinha de borracha. As agulhas podem ser puxado com antecedência. Diferente OD capilares de vidro podem ser usados, dependendo do tipo de andar de entrada do amplificador disponível. </li><li> Microeléctrodo protelar o com solução de gravação extracelular (2 M NaCl) utilizando uma seringa de 1 ml com uma seringa de filtro Nalgene (4 mm) e de filamentos Microfil (Warner Instruments Precision) ligado. Agite ou o bolo em direção à ponta da agulha até que haja algumas bolhas ou nenhuma restantes. </li></ol> 3. Imobilização em Agar <ol><li> Prepara-se uma solução fresca de 1,2% de agar baixo ponto de fusão em água ovo. Solução de agar em lugar de um conjunto banho de água a ~ 37 ° C. </li><li> Prepare uma lamela com câmara de gravação e coloque no freezer (5-10 min). A câmara de gravação deve MatCh que é utilizado na plataforma de electrofisiologia. Nós usamos um baixo perfil aberto diamante câmara de banho de imagem da Warner Instruments (modelo RC-26GLP). </li><li> Utilizando uma pipeta de Pasteur ou transferência, um peixe-zebra lugar larval em uma pequena gota de água ovo em uma placa de Petri limpa. Para esta gotícula, adicionar uma gota de uma solução contendo um anestésico (0,02% tricaina) e agente paralisante (1 mg / ml α-bungarotoxina). </li><li> Monitorar zebrafish para perda de movimento (5 a 10 min). </li><li> Retirar as lamelas / câmara de gravação do freezer. Coloque câmara no palco de um estereomicroscópio. Utilizando uma pipeta de transferência, misturar de alguns ml de 1,2% de agarose de ponto de fusão baixo com gota e solução de transferência (com peixe) para a câmara de lamela / gravação. </li><li> Utilizando uma ponta de pipeta ou agulha romba fina, larvas de posição sob o estereomicroscópio, de modo que o dorso do peixe é exposto à superfície do gel de agarose. Permitir agarose para endurecer (5-10 min), em seguida, utilizando uma espátula plana a transferência b agarbloquear a uma câmara de gravação criado em uma plataforma de eletrofisiologia. </li></ol> 3. Gravação de campo extracelular <ol><li> Adicionar 2-5 ml de meio de registo para o peixe-zebra câmara de gravação. Se as mudanças de droga são de câmara, necessário perfundir com solução de gravação a uma velocidade de aproximadamente 1 ml / min. Não oxigenação da solução de gravação é necessária. </li><li> Inserir um microeléctrodo (aproximadamente 1 diâmetro de ponta um, 2-7 MQ) no andar de entrada do amplificador montado num micromanipulador tridimensional. Verificar se o micromanipulador está numa posição apropriada para permitir uma amplitude de movimento e ajustamento. Coloque a extremidade do microeléctrodo para dentro do plano de vista do microscópio, elevado para fora do palco, e utilizando o ajuste grosseiro inferior a um ponto imediatamente acima e ligeiramente à frente da cabeça do peixe-zebra. </li><li> Com o amplificador em &quot;grampo-atual&quot; modo de zero, o eletrodo. </li><li> Usando ajuste grosso inferior ponta do eletrodo para que ele só toca o surface do peixe-zebra, ligeiramente à frente da parte anterior do cérebro. </li><li> Usando etapas de ajuste fino avançar a ponta do eléctrodo até que perfura a pele do peixe-zebra. Deixar depositar e depois avançar o eletrodo vários microns em prosencéfalo. </li><li> Registrar a atividade elétrica no grampo de modo atual usando o software Axoscope. Os dados são adquiridos a uma taxa de amostragem 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>

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O método de gravação extracelular aqui apresentada permite uma análise muito sensível e rápido de atividade cerebral. Estas gravações são análogos a monitorização (EEG) eletroencefalográficos vulgarmente usado para avaliar a presença de descarga eléctrica anormal (isto é, convulsões) em modelos de roedores de epilepsia e 11 pacientes 12. Gravações extracelulares podem ser combinadas com as manipulações farmacológicas, como mostrado aqui. Estes tipos de gravações pod...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Agarose de fusão baixo </td><td> Fisher-Científico </td><td> BP1360-100 </td><td> Dissolver em mídia de embrião em 1,2% </td></tr><tr><td> Mídia de gravação </td><td> Fisher-Científico </td><td> BP3581, P330-3, BP410-1, BP214-500, D16-1, C77-500 </td><td> 1 mM de NaCl, 2,9 mM KCl, 10 mM de HEPES, 1,2 mM de MgCl2, 10 mM de dextrose, 2,1 mM CaCl 2 o pH para aproximadamente 7,3 com NaOH 1 N </td></tr><tr><td> Tricaina </td><td> Argent Labs </td><td> MS-222 </td><td> 0,02% </td></tr><tr><td> α-bungarotoxina </td><td> Tocris Bioscience </td><td> 2133 </td><td> 1 mg / ml </td></tr><tr><td> Um tubo de vidro capilar </td><td> Warner Instruments </td><td> G120TF-3 </td><td> Puxar a uma resistência de 2 -7 MQ </td></tr><tr><td> Amplificador de fixação de membranas </td><td> Warner Instruments </td><td> PC-505B </td><td> Nós usamos um amplificador Warner no modo de fixação de corrente; Ganho fixado em 2 mV / PA e filtro de Bessel fixado em 2K. Modelos comparáveis ​​podem ser usados ​​de acordo com as instruções do fabricante. </td></tr><tr><td> Filtro amplificador / </td><td> Cygnus Tecnologia </td><td> FLA-01 </td><td> Nós usamos um Cygnus pré-amplificador; Ganho fixado em 10-20; Frequência de corte fixada em 1-2K; filtro Notch IN. Modelos comparáveis ​​podem ser usados ​​de acordo com as instruções do fabricante. </td></tr><tr><td> Axon A / D bordo e software Axoscope </td><td> Molecular Devices </td><td> Axon Digidata 1320A; Axoscope 8,2 </td><td> Os dados são coletados em Axoscope usando lacuna livre modo de aquisição; amostragem de 10 kHz. Modelos comparáveis ​​e os programas podem ser usados ​​de acordo com as instruções do fabricante. </td></tr><tr><td> Água ovo </td><td&gt; Instant Oceano </td><td></td><td> 3 g Instantânea Oceano mar de sal, 2 ml de azul de metileno 0,1% em 10 ml de água deionizada </td></tr></tbody></table>

forebrain electrophysiological recording in larval zebrafish

A epilepsia afeta cerca de 3 milhões de pessoas nos Estados Unidos e até 50 milhões de pessoas em todo o mundo. Definido como a ocorrência de convulsões espontâneas não provocados, a epilepsia pode ser adquirido como resultado de um insulto ao cérebro ou a uma mutação genética. Os esforços para convulsões modelo em animais têm utilizado principalmente adquiridos insultos (fármacos convulsivos, estimulação ou lesão cerebral) e manipulações genéticas (knockdown antisense, a recombinação homóloga ou transgénese) em roedores. Zebrafish é um sistema modelo de vertebrados 1-3 que poderia fornecer uma alternativa valiosa para roedor pesquisa baseada em epilepsia. Peixe-zebra são amplamente utilizados no estudo da genética de vertebrados ou de desenvolvimento, exibem um elevado grau de semelhança genética para os mamíferos e expressam homólogos de ~ 85% de humanos conhecidos mutações de um único gene de epilepsia. Devido ao seu tamanho pequeno (4-6 mm de comprimento), larvas de peixe-zebra pode ser mantida em volumes de fluidos tão baixo como 100 ul durante o desenvolvimento inicial e arraYED em multi-poços. Os reagentes podem ser adicionados directamente à solução em que os embriões se desenvolvem, o que simplifica a administração do fármaco e permitindo uma rápida no rastreio in vivo de compostos de teste 4. Os oligonucleótidos sintéticos (morpholinos), mutagénese, dedo de zinco nuclease e abordagens transgénicas podem ser utilizadas para gerar rapidamente knockdown gene ou mutação no peixe-zebra 5-7. Estas propriedades pagar estudos zebrafish uma vantagem sem precedentes análise estatística poder sobre roedores para o estudo de distúrbios neurológicos como a epilepsia. Porque o &quot;padrão ouro&quot; para a pesquisa a epilepsia é monitorar e analisar as descargas elétricas anormais que se originam em uma estrutura cerebral central (isto é, convulsões), um método eficiente para registrar a atividade cerebral em larvas do peixe-zebra é descrito aqui. Este método é uma adaptação de técnicas convencionais de gravação extracelular e permite a monitorização a longo prazo estável de actividade do cérebro em larvas do peixe intacta. Sgravações amplas são mostrados para convulsões agudas induzidas pela aplicação de banho de drogas convulsivas e apreensões espontâneas gravadas em um peixe geneticamente modificado.

Exemplos de descarga do tipo convulsivas electrográfico gravado no cérebro anterior de um agar-embedded larvas do peixe são mostrados na Figura 1. De grande amplitude de descarga explosão multi-pico nas amostras foi evocado por aplicação de banho de um fármaco convulsivo, 40 mM de pilocarpina (em A, 6 dpf) ou 1 mM de picrotoxina (em B, 8 dpf). Nestes registos, imobilizado e de ágar-embedded zebrafish são monitorados continuamente durante até 90 min. Peixe permanecem viáveis ​​nestas condi...

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

Forebrain Electrophysiological Recording in Larval Zebrafish

Um método simples para gravar os potenciais de campo extracelulares no larval do peixe-zebra é descrita prosencéfalo. O método proporciona um forte<em&gt; In vivo</em&gt; Leitura de apreensão como atividade. Esta técnica pode ser utilizada com larvas do peixe geneticamente modificada carregando epilepsia genes relacionados ou convulsões evocadas pela administração de fármacos convulsivos.

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 ...

forebrain-electrophysiological-recording-in-larval-zebrafish

Research

JoVE Journal

Neuroscience

17.0K visualizações.  University of California, San Francisco. Um método simples para gravar os potenciais de campo extracelulares no larval do peixe-zebra é descrita prosencéfalo. O método proporciona um forte In vivo Leitura de apreensão como atividade. Esta técnica pode ser utilizada com larvas do peixe geneticamente modificada carregando epilepsia genes relacionados ou convulsões evocadas pela administração de fármacos convulsivos.

Vídeo: 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.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

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

Neurociência

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.

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 ...

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.

Eletrofisiológica Gravação De<em&gt; Drosophila</em&gt; Labellar Prove 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.

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 ...

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.

Gravação simultânea eletrofisiológica e micro-injeções de agentes inibidores no cérebro do roedor

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.

Gravação eletrofisiológica de<em&gt; Drosophila</em&gt; Sensilla Tricoide em resposta aos odorantes de baixa volatilidade

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.

Na imagem latente de cálcio Vivo de células de cabelo linha Lateral no Zebrafish Larval

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.

Investigando a plasticidade sináptica de longo prazo no hipocampo interlamelar CA1 por gravação de campo eletrofisiológico

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.

Gravação eletrofisiológica sem fio dos neurônios por tetrodes móveis em peixes livremente da natação

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