Supported by grants from CIHR (MOP231122) and NSERC (RGPIN-2014-06503). We would like to thank Genevi&#232;ve Cyr for her help preparing experiments and supervising laboratory work. MAL received a scholarship from The Natural Sciences and Engineering Research Council of Canada (NSERC).

Nota: Todos os procedimentos foram realizados em conformidade com as diretrizes do Conselho Canadense para a Proteção dos Animais e do conselho de revisão Ética da Université de Montréal. 1. Assembleia da pipeta de gravação de injecção <ol><li> Puxar um capilar de vidro a cerca de 7 cm de comprimento (diâmetro exterior 1 mm), utilizando um puxador de pipeta. </li><li> Quebrar a ponta do capilar e verificar a abertura sob um microscópio de luz. Confirmar que o diâmetro interno é entre 30 um e 40 um. </li><li> Inserir um longo fio de prata 7 centímetros para o capilar de vidro com cerca de 1 cm protuberantes a partir da extremidade não-cónica da pipeta de vidro. </li><li> Dobre o excesso de filamentos perpendicularmente ao capilar de vidro. </li><li> Aplicar uma gota de adesivo plástico flexível sobre o eixo de uma agulha hipodérmica 30 G. </li><li> Inserir uma agulha hipodérmica 30 G na pipeta de vidro de acordo com os esquemas apresentados na Figura 1 </strong&gt;. </li><li> Adicionar uma segunda camada de cola para assegurar uma vedação adequada a partir da junção entre a pipeta de vidro e a agulha hipodérmica. </li><li> Deixar secar a pipeta com a ponta virada para cima, durante cerca de 12 h para assegurar uma cura adequada da cola. O resultado final é mostrado na Figura 2. </li></ol> NOTA: Este procedimento é feito em experiências agudas e esterilização da ponta da pipeta não é necessária. 2. Preparação de animais <ol><li> Coloque o rato em uma caixa de anestesia. </li><li> Induzir anestesia, utilizando 4% de isoflurano durante 5 a 10 min. </li><li> Colocar o animal sobre uma mesa estereotáxico com almofada de aquecimento e uma sonda rectal para manter uma temperatura corporal de 37 ° C. Use um cone do nariz para manter a anestesia com isoflurano a 2%. Fixe a cabeça do rato, utilizando barras de ouvido e suporte de dentes. </li><li> Aplicar pomada ou colírio oftalmológicas, que incluem 1% Atropina cai para ajudar na dilatação da pupila. Para evitar o ressecamento, aplicar lubrificante quedas aproximadamente a cada 30 min. </li><li> Raspar a cabeça e limpe-o com 10% de iodopovidona. </li><li> Para a anestesia local, injectar 0,5 ml de lidocaína a 2% no âmbito do couro cabeludo em 2-3 locais, levantando-se a pele e inserir a ponta da agulha. </li><li> Confirmar nível adequado da anestesia através da realização de um aperto do dedo do pé e observando-se a falta de movimento. Além disso, controlar o ritmo cardíaco para garantir que ele é dentro dos valores normais (de 300 a 400 batimentos / min). </li><li> Inciso o couro cabeludo em uma linha reta ao longo da mediana, com uma lâmina de bisturi nº 10 para expor ambas as suturas coronal e sagital. </li><li> Revelar o lambda e Bregma pontos, empurrando de lado o tecido que está cobrindo o crânio com uma espátula cirúrgica. </li><li> Nível do crânio para que posições bregma e lambda são no mesmo plano. </li><li> Para definir o ponto de referência, use um dispositivo estereotáxico com um tubo de vidro montada para defini-la bem acima Bregma. Este será o &quot;zero&quot; para o antero-posterior e medcoordena medições ial-laterais. </li><li> Defina o ponto de interesse, movendo o estereotáxico para montar as coordenadas necessárias, observe as coordenadas estereotáxica e desenhar um quadrado ao redor da área-alvo que marca onde a craniotomia será realizada. </li><li> Use uma furadeira com uma broca cirúrgica esterilizada ao longo da praça marcada lentamente, sem pressão para remover lentamente o material ósseo. Tenha cuidado para não perfurar muito tempo na mesma área, uma vez que irá produzir calor e causar lesões no córtex. </li><li> Quando o osso que delimita a craniotomia tornou-se suficientemente fina, retire cuidadosamente a seção cranial com pinça para expor o córtex. </li><li> Irrigar freqüentemente o córtex exposto com fluido cerebrospinal artificial para evitar a dessecação dos tecidos. NOTA: a remoção dura-máter é desnecessário no rato como a ponta do injectrode é robusto o suficiente para penetrar. </li></ol> 3. Preenchimento e Montagem do Sistema de Injeção <ol><li> Encha o5-10 ul micro por aspiração com óleo mineral. </li><li> Encher a agulha hipodérmica com a pipeta de vidro fixado com uma solução de 0,5% de Chicago Sky Blue (CSB) e 300 uM de ácido γ-aminobutírico (GABA) ou uma solução de lidocaína a 2% com 0,5% de CSB 9. Dilui-se todas as soluções com solução salina. <ol><li> No caso de uma substância abundante, preencher usando uma seringa regular e utilizar as técnicas usuais de precaução para evitar a formação de bolhas de ar. </li><li> No caso de substâncias mais caros, usar óleo mineral para preencher o injectrode e o agente químico pode então ser introduzido por aspiração. À medida que a diferença de densidade entre o óleo mineral e água é relativamente elevada, esta substância é um bom candidato para a injecção de soluções aquosas. NOTA: Um corante pode ser adicionado para confirmar a separação entre os dois líquidos. </li></ol></li><li> Para encher a pipeta de injecção, encher uma seringa de 1 ml com a solução por aspiração e depois injectar a solução lentamentepara a pipeta de injecção. </li><li> Preste muita atenção para vazamentos em regiões indicadas na figura 1 limpando essas áreas limpas e observando as fugas pela injeção ainda mais a solução lentamente com a seringa. </li><li> Remover a seringa de 1 ml. Ao fazê-lo, certifique-se de manter uma leve pressão sobre o êmbolo de modo que o vácuo não remover a solução da pipeta de injeção. </li><li> Preencha a micro com óleo mineral por aspiração e anexá-lo firmemente à pipeta de injeção preenchido, em seguida, limpe cuidadosamente qualquer excesso de solução na injectrode montado com gaze. </li><li> Verificar que a ponta não é bloqueado pela injecção de um volume muito reduzido, o suficiente para ver uma pequena gota formando na ponta da pipeta de vidro. </li><li> Monte o injectrode no sistema micropump e garantir que ele está bem fixo. </li><li> Posicionar cuidadosamente a ponta injectrode nas coordenadas alvo e diminuir a ponta para a superfície do córtex. </li><li> Lentamente, abaixe o injectrode utilizar o aparelho estereotáxico para a estrutura de alvo (colículo superior, neste caso), utilizando as coordenadas anatómicas apropriadas. </li><li> Cubra o córtex exposto com agar quente para evitar a dessecação dos tecidos. </li></ol> 4. Injeção e inativação reversível <ol><li> Defina a bomba de microinjeção para injetar 400 a 800 nl em 40 nl / min e pressione Executar para iniciar a injeção. Note-se que taxa de pico vai mostrar redução durante a injecção. NOTA: Na configuração experimental, a atividade neural recuperado dentro de uma hora após o fim da entrega GABA. Qualquer dispositivo mecânico calibrado pode ser utilizado para aplicar pressão sobre a seringa de micro-injecção, a fim de realizar a injecção. </li><li> Após a aquisição dos dados eletrofisiológicos, eutanásia usando um método aprovado pela comunidade local animal Ética. </li></ol>

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	<li>Martin, J. H., Ghez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+inactivation+in+the+analysis+of+the+central+control+of+movement.">Pharmacological inactivation in the analysis of the central control of movement.</a> Journal Of Neuroscience Methods. 86 (2), 145-159 (1999).</li><li>Ponce, C. R., Hunter, J. N., Pack, C. C., Lomber, S. G., Born, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+indirect+pathways+to+visual+response+properties+in+macaque+middle+temporal+area+MT.">Contributions of indirect pathways to visual response properties in macaque middle temporal area MT.</a> The Journal Of Neuroscience The Official Journal Of The Society For Neuroscience. 31 (10), 3894-3903 (2011).</li><li>Lomber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantages+and+limitations+of+permanent+or+reversible+deactivation+techniques+in+the+assessment+of+neural+function.">The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function.</a> Journal Of Neuroscience Methods. 86 (2), 109-117 (1999).</li><li>Malpeli, J., Schiller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+reversible+inactivation+of+small+regions+of+brain+tissue.">A method of reversible inactivation of small regions of brain tissue.</a> Journal Of Neuroscience Methods. 1 (2), 143-151 (1979).</li><li>Lomber, S. 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(46), (2010).</li><li>Hup&#233;, J., Chouvet, G., Bullier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+parameters+of+cortical+inactivation+by+GABA.">Spatial and temporal parameters of cortical inactivation by GABA.</a> Journal Of Neuroscience Methods. 86 (2), 129-143 (1999).</li><li>Casanova, C., McKinley, P., Molotchnikofff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsiveness+of+Reorganized+Primary+Somatosensory+(SI)+Cortex+after+Local+Inactivation+of+Normal+SI+Cortex+in+Chronic+Spinal+Cats.">Responsiveness of Reorganized Primary Somatosensory (SI) Cortex after Local Inactivation of Normal SI Cortex in Chronic Spinal Cats.</a> Somatosensory &amp; Motor Research. 8 (1), 65-76 (1991).</li><li>Malpeli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+inactivation+of+subcortical+sites+by+drug+injection.">Reversible inactivation of subcortical sites by drug injection.</a> Journal Of Neuroscience Methods. 86 (2), 119-128 (1999).</li><li>Minville, K., Casanova, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+frequency+processing+in+posteromedial+lateral+suprasylvian+cortex+does+not+depend+on+the+projections+from+the+striate-recipient+zone+of+the+cat&#8217;s+lateral+posterior-pulvinar+complex.">Spatial frequency processing in posteromedial lateral suprasylvian cortex does not depend on the projections from the striate-recipient zone of the cat&#8217;s lateral posterior-pulvinar complex.</a> Neuroscience. 84 (3), 699-711 (1998).</li><li>Diao, Y., Wang, Y., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representation+of+the+binocular+visual+field+in+the+superior+colliculus+of+the+albino+rat.">Representation of the binocular visual field in the superior colliculus of the albino rat.</a> Experimental Brain Research. 52 (1), 67-72 (1983).</li></ol>

We have nothing to disclose.

O protocolo proposto foi projetado para resolver os desafios decorrentes de métodos atuais de inativação reversíveis. Especificamente, este projeto visa aperfeiçoar os métodos utilizados para microinjeções químicas das substâncias que modulam a atividade neural, particularmente em estruturas cerebrais profundas. Um desafio técnico que emerge a partir deste tipo de instalação é a necessidade de ambas as sondas para ser co-localizada no mesmo espaço restrito in vivo a fim de obter gravações preci...

A inactivação de áreas corticais e núcleos sub-corticais é importante no estudo das relações funcionais entre diversas estruturas cerebrais 2-4. A literatura recente empregue química perda de função ou técnicas criogénicas para estudar o papel das estruturas cerebrais 2,5. No que diz respeito a micro-injecções farmacológicas, pequenos volumes de medicamentos podem ser administrados em uma região do cérebro, a uma taxa controlada ao mesmo tempo minimizando os danos colaterais ao tecido circundante 6,7. Esta técnica pode ser usada para fornecer agonistas específicos, agonistas inversos ou antagonistas para estudar o efeito de diferentes alvos farmacológicos na actividade neuronal. Tais efeitos também pode ser estudado através da medição alterações nas respostas neuronais a partir de locais distantes, permitindo que os investigadores a estudar as relações entre as diferentes estruturas corticais e sub-corticais. Aqui, nós demonstramos a montagem de um dispositivo, o injectrode, capaz de both gravar sinais eletrofisiológicos e entrega de pequenas quantidades de drogas no local de destino. Nós demonstramos as capacidades deste sistema através da injecção de GABA, um inibidor comum de actividade neuronal, no colículo superior do rato. Esta região é sensível à estimulação visual, o que nos permitiu usar a atividade multiunit visualmente evocados para confirmar injectrode localização. A reversibilidade da inativação foi avaliada pela recuperação da atividade neuronal normal após o final da injeção GABA. A capacidade para monitorizar a actividade de multi-unidade a partir do local de injecção permite o ajuste fino das taxas de injecção e dos volumes necessários para se conseguir a resposta farmacodinâmica desejada. Portanto, uma vantagem desta técnica é a de limitar o potencial dano tecidual causado pela microperfusão, uma vez que os menores volumes eficazes são injectados. O protocolo proposto fornece um método eficiente de custos para gerar o necessar hardware descartávely para a realização de experiências onde a entrega de drogas e gravação atividade neuronal local está desejados.

<table border="0" cellpadding="0" cellspacing="0" width="981">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Name of the reagent</td>
			<td style="width:209px;">Company</td>
			<td style="width:184px;">Catalogue number</td>
			<td style="width:308px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection pump (UltraMicroPump III)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#UMP3</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Injection console (Micro4 Controller)</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#SYS-MICRO4</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Hamilton syringe</td>
			<td style="width:209px;">Hamliton</td>
			<td style="width:184px;">(80301) 701LT 10 &#181;L SYR</td>
			<td style="width:308px;">Syringes between 5 and 10 &#956;L used</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Gel cyanoacrylate adhesive</td>
			<td style="width:209px;">Krazy Glue</td>
			<td style="width:184px;">KG86648R</td>
			<td style="width:308px;">The gel form is easier to apply on the shaft of the 30G hypodermic needle</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:280px;">Glass pipettes</td>
			<td style="width:209px;">WPI</td>
			<td style="width:184px;">#TW100F-4</td>
			<td style="width:308px;">Thin wall, 1mm OD, 0.75mm ID with filament pipettes used</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">720 Needle Pipette Puller</td>
			<td style="width:209px;">Kopf</td>
			<td style="width:184px;">720</td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:280px;">Silver wire</td>
			<td style="width:209px;">A-M Systems, Inc.</td>
			<td style="width:184px;">782500</td>
			<td style="width:308px;">Bare 0.010&#8221;</td>
		</tr>
	</tbody>
</table>

simultaneous electrophysiological recording and micro-injections of inhibitory agents in the rodent brain

Aqui nós descrevemos um método para a construção de um uso único &quot;injectrode&quot; usando peças comercialmente acessíveis e económicas. Um sistema de sondagem foi desenvolvido que permite a injecção de um fármaco durante a gravação de sinais electrofisiológicos da população de neurónios afectados. Este método fornece uma alternativa simples e económica para soluções comerciais. Uma pipeta de vidro foi modificado combinando-o com uma agulha hipodérmica e um filamento de prata. O injectrode está ligado a bomba de micro comercial para entrega da droga. Isso resulta em uma técnica que fornece feedback em tempo real através de sinais extracelulares farmacodinâmica de múltiplas unidades provenientes do local de entrega de drogas. Como prova de conceito, registramos a atividade neuronal do colículo superior provocada por flashes de luz em ratos, concomitantemente com a entrega de drogas através da injectrode. A capacidade de gravação injectrode permite a caracterização funcional do injsite de exão favorecendo o controle preciso sobre a localização de entrega da droga. A aplicação deste método também se estende muito além do que é demonstrado aqui, como a escolha de substância química carregada no injectrode é vasto, incluindo o rastreamento de marcadores para experimentos anatômicos.

A construção do injectrode é ilustrada na Figura 1. Um fio de prata (C) é alimentado para dentro de uma pipeta de vidro de (D) com uma porção do arame dobrado e projectando-se para fora a partir da abertura. Uma agulha 30 G (B) está ligado e selado para a abertura da pipeta de vidro com cola. Após a pipeta foi cheia com a substância de injecção, uma micro seringa de vidro (A) está ligado à agulha. É importante que exista uma boa vedação onde o micro liga seringa com a agulha (E) e em que...

Watch this Scientific Journal Video about Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain at JoVE.com

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

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 ...

simultaneous-electrophysiological-recording-micro-injections

Research

JoVE Journal

Neuroscience

8.0K visualizações.  Université de Montréal. 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.

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

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

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

FMRI simultânea e Eletrofisiologia no cérebro de roedores

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

Neurociência

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Concepção e Construção de um Headstage Custo Efetivo de Estimulação Neural Simultânea e Gravação no labirinto aquático

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

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 ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Simultânea de pré e pós-sináptica gravação eletrofisiológico de<em&gt; Xenopus</em&gt; Nervo músculo-Co-culturas

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

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 ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Gravação de plasticidade sináptica em fatias Hippocampal agudas, mantidas em um pequeno volume reciclagem - perfusão- e sistema de câmara de submersão-tipo

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Gravação a atividade eletromagnética cerebral durante a administração de agentes anestésicos gasosos Xenon e óxido nitroso em voluntários saudáveis

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Gravação e modulação da atividade epileptiforme em fatias de cérebro de roedor acoplado para matrizes de microeletrodos

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Cryosectioning simultânea de vários cérebros de roedores

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...