Agradecemos do fundo de Vida, Universidade de Oklahoma Graduação de Pesquisa do Programa Oportunidades, e do Departamento de Zoologia para o financiamento deste trabalho. Agradecemos também Hoefnagels Dr. Marielle para ajudar a editorial.

1. Pré-gravação, Preparação Animais Vivos <ol><li> Remova o escorpião do seu jar casa e colocá-lo em um recipiente de vidro pré-refrigerados; seguinte, coloque o jar contendo o escorpião em um ambiente de congelamento (-5 ° C) por um período mínimo de dois minutos. Este tempo pode variar de acordo com a idade, tamanho, espécies, etc do escorpião específica em estudo. Mas, geralmente, a fase de ambiente de congelamento é completo quando o animal está imóvel. </li><li> Depois de dois minutos, retire o escorpião imobilizado do recipiente de vidro frio, e colocá-lo lado ventral para cima sobre uma lâmina de microscópio. Segura o ferrão, cauda, ​​pernas e pedipalpos com barro moldável (à base de óleo, Van Aken Plastalina). </li><li> Tornar uma plataforma na qual a colocar pectinas do escorpião. Ajustar o comprimento da plataforma e largura de acordo com o tamanho do pecten. Tipicamente, as nossas plataformas são construídos a partir de tampa de vidro do microscópio e são aproximadamente 10 (L) X 18 mm (W). </li><li> Aderem fita dupla face adesiva para a plataforma pecten, aparando a fita (se necessário) para se adequar. </li><li> Para fazer com que uma câmara pectínios, coloque três dos quatro lados da plataforma, em pré-fundido de cera (que usam chips de cera de abelha 100%) a uma profundidade de 3-4 mm. Nós derreter a cera, colocando as fichas em uma placa de Petri de vidro, localizada em uma chapa quente. Uma vez que a cera nas bordas da câmara de pectínios é resfriado a temperatura ambiente, este deve criar paredes de cera cerca de 1 mm de altura. </li><li> Sobre o animal, localizar o ponto de inserção pectínios, e coloque a borda un-encerado da plataforma (fita up-side) imediatamente posterior a ele. Depois, estabilizar a plataforma (para que ele não se moverá mais tarde) pressionando suavemente a sua direita e esquerda bordas periféricas contra o barro utilizado para limitar as pernas do animal. </li><li> Use uma pinça de ponta fina, coloque cuidadosamente a pecten pectinas ou no interior da câmara. Especificamente, usando uma pinça, segure o pecten ao longo da espinha pectínios, e gentilmente trazer o pecten de baixo da tampa de vidro e abaixá-lo para o chão plataforma adesivo. Estabelecer um vínculo pecten-para-fita com cuidado de aplicar pressão sobre a coluna pectínios. </li><li> Agora, aplique cera derretida (nós usamos uma espátula de metal pré-aquecido) até a borda un-encerado da plataforma. O objetivo é duplo, para garantir a pecten e para envolver completamente a câmara para futura introdução de óleo mineral. Tenha cuidado ao aplicar a cera sobre pecten do animal, como é possível danificar o pecten com cera que é muito quente. </li><li> A construção da câmara pecten está completa. Em seguida, estabeleça uma conexão eletrodo indiferente com hemolinfa do animal através da inserção de um fio de prata entre dois segmentos da cauda. </li></ol> 2. Gravação simultânea Extracelular e Estimulação Química <ol><li> Nós preparamos o animal 24 horas antes da realização extracelular, tip-gravações, que, entre outras razões (ou seja, ajuste de posição fixa para animais), dá tempo para fazer custom-pipetas de um estimulante específico (sensillum-dependente) da ponta de diâmetro. Usamos um extrator micropipeta para se alcançar tais especificações. Idealmente, o diâmetro das pipetas é ≈ 2 M maior do que o diâmetro dos poros sensillar. Nós ajustamos nossas especificações micropipeta de acordo para cada animal. </li><li> 1 h antes da gravação, adicionar 5 mL de óleo mineral para a câmara de pectínios, submergindo o pecten sob óleo. Então, a posição do animal debaixo de um microscópio de alta potência, que há muito trabalho a distância e objetivos epi-iluminação. </li><li> Para estimular quimicamente peg sensilla, introduzimos tastants aquoso (ou seja, ácido cítrico e sal) por meio de óleo. Primeiro, usamos um enchimento micropipeta para injetar o estimulante para a pipeta estimulante. Nota: todas as soluções estimulante deve conter um eletrólito de condutância elétrica. </li><li> Em seguida, após o posicionamento da pipeta estimulante em um micromanipulador (no equipamento de gravação), insira o eletrodo de registro para o fim, sem corte aberto da pipeta estimulante. É importante para garantir o contato entre o eletrodo de metal e uma solução eletrolítica estimulante. </li><li> Uma vez que a preparação é eletricamente aterrado (através do eletrodo indiferente), simplesmente diminuir a pipeta através do meio de petróleo e em um poro sensillar. A duração da estimulação química por sensillum pectínios pode variar de tanto quanto 30 minutos para apenas um segundo. Geralmente, podemos amostra pinos múltiplos (&gt; 30) por pipeta. No caso em que a pipeta estimulantes torna-se obstruídas por escombros minuto, simplesmente trocar a pipeta entupidos de uma pipeta, nova sem uso. </li></ol> 3. Preparação de um animal por vários dias de Experimentação <ol><li> Se o animal deve ser usado durante um período de tempo prolongado, recomendamos a reutilização da câmara pectínios e substituindo a gota de óleo mineral para cada sessão de gravação. Após a sessão de um determinado dia de gravação estiver concluída, remova o animal a partir da gravação de set-up e fazer uma lavagem de óleo mineral. </li><li> Specifically, adicionar uma gota (5 L) de óleo mineral para a câmara, e remover todo o óleo. Isso ajuda a minimizar a presença de estimulantes residual, o que pode ter vazado para fora da pipeta durante a sessão de gravação. </li><li> Antes da próxima sessão, adicione uma gota de óleo como descrito anteriormente. </li></ol> 4. Resultados representativos: Dependendo do equipamento de gravação utilizado (por exemplo, o amplificador particular, digitalização hardware, etc), um resultado representativo é uma gravação extracelular com um S: N proporção de pelo menos 3:1. Sucesso na construção da câmara de óleo mineral é necessário para isolar a estimulação química sensilla individual, uma vez que fornece um meio para a entrega tastants química polar (à base de água) 8. Se houver óleo muito pouco ou mesmo nenhum, a solução vai se espalhar estimulante do sensillum gravadas ao seu sensilla vizinhos, o que é susceptível de causar um ataque prolongado e descontrolado de químicos estimulação 8.

<ol>
	<li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+Primary+Chemoreceptor+unit.">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li><li>Newland, P. L., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+processing+in+the+insect+nervous+system.">Taste processing in the insect nervous system.</a> Methods in Insect Sensory Neuroscience. , 289-318 (2005).</li><li>Foelix, R. F., M&#252;ller-Vorholt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla.">The fine structure of scorpion sensory organs II. Pecten sensilla.</a> Bull. Br. Arachnol. Soc. 6, 68-74 (1983).</li><li>Carthy, 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=Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten.">Fine structure and function of the sensory pegs on the scorpion pecten.</a> Experientia. 22, 89-91 (1966).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions.">Response properties of chemosensory peg sensilla on the pectines of scorpions.</a> J. Comp. Physiol. A. 181, 291-300 (1997).</li><li>Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pectine+organs+of+the+scorpion,+Vaejovis+spinigerus:+Structure+and+(glomerular)+central+projections.">The pectine organs of the scorpion, Vaejovis spinigerus: Structure and (glomerular) central projections.</a> Arthropod Struct. Dev. 37, 67-80 (2008).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines.">Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines.</a> J. Comp. Physiol. A. 181, 301-307 (1997).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+examining+scorpion+peg+sensilla:+the+mineral+oil+flood+technique.">A new approach to examining scorpion peg sensilla: the mineral oil flood technique.</a> J. Arachnol. 37, 379-382 (2009).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants.">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , .</li><li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+sucrose+perception+in+the+honey+bee+(Apis+mellifera+L.):+behaviour+and+electrophysiology.">Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology.</a> J. Comp. Physiol. A. 190, 735-745 (2004).</li><li>Bland, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensilla+on+the+antennae,+mouthparts,+and+body+of+the+larva+of+the+alfalfa+weevil,+hypera+postica+(gyllenhal)+(Coleoptera+:+Curculionidae).">Sensilla on the antennae, mouthparts, and body of the larva of the alfalfa weevil, hypera postica (gyllenhal) (Coleoptera : Curculionidae).</a> Int. J. Insect Morphol. Embryol. 12, 261-272 (1983).</li><li>Cate, H. S., Derby, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooded+Sensilla+Homologues:+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum.">Hooded Sensilla Homologues: Structural Variations of a Widely Distributed Bimodal Chemomechanosensillum.</a> J. Comp. Neurol. 444, 345-357 (2002).</li></ol>

Temos publicados anteriormente estes métodos em dois jornais, Knowlton e Gaffin (2009) e Knowlton e Gaffin (no prelo).

O protocolo acima descreve como preparar um deserto pastagens escorpião (Paruroctonus utahensis) para estudo eletrofisiológico. Especificamente, mostramos como construir uma câmara de controle extracelular ponta gravações de pectínios neurônios chemosensory sob óleo. Porque água e óleo não se misturam, é possível isolar a estimulação química (com base de água estimulantes) para sensilla único. Deve-se ressaltar que P. utahensis, é um animal relativamente pequeno (≈ 2,5-5 cm) e aplicando este protocolo para animais maiores podem exigir ajustes de tamanho de muitos, tais como o tamanho da plataforma do animal, de câmara, e quantidade de óleo aplicado. Recomendamos a realização de estudos-piloto para testar a quantidade mínima de óleo necessária para liberação controlada de estimulantes à base de água química. Além disso, não houve efeito do óleo sobre a atividade neural de base dentro pectínios sensilla 8. Este deve ser confirmado para sistemas de outro modelo, bem como, antes de avaliar chemoresponsiveness. Este método pode ser usado em conjunto com outro método para testar efeitos de interação entre chemoresponse sensilla no campo sensorial. Por exemplo, é possível registrar-ponta (em óleo) a partir de uma base de sensillum como registro (através de um eletrodo de tungstênio) de uma sensillum vizinhos 9. Essa configuração de gravação pode ser usada para avaliar se deve ou não quimicamente estimulando uma sensillum afeta a atividade neural do sensillum-base registrados. Até o momento, ninguém testou isso por escorpião pectínios sensilla, e permanece uma questão em aberto para outros sistemas chemosensory também. Para resumir, nós achamos que o método de gravação de ponta em avanços de petróleo do âmbito de investigações eletrofisiológicas para a base neural da gustação artrópodes. Neste manuscrito, nós fornecemos um protocolo para a preparação de animais para este método, e esperamos que fornece uma base sólida para um estudo mais aprofundado de periféricos sensoriais sistema nervoso.

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electrophysiology of scorpion peg sensilla

Nós descrevemos uma modificação de uma técnica existente 1,2 ponta de gravação para eletrofisiologicamente investigando curto, peg-like sensilla sensoriais 3,4. Na superfície ventral de todos os escorpiões são dois apêndices chamados pectinas, que têm campos densos de mecano-peg e chemosensory sensilla 5,6. Um método para avaliar chemoresponsiveness destes sensilla usa um eletrodo de tungstênio para extracelularmente gravação atividade neural dentro de um sensillum como um odorant volátil é introduzido no campo sensorial 5,7. As limitações deste método incluem a coleta de dados lento e estimulante introdução não controlada, e remoção de, no campo peg. Para superar essas limitações, desenvolvemos uma técnica de ponta de gravação de novo que usa óleo mineral apolares como um meio através do qual a entregar à base de água tastants para indivíduo peg sensilla 8,9. Nós temos aplicado com sucesso este método para obter chemoresponses sensillar ao ácido cítrico, etanol e sal. Aqui nós descrevemos o protocolo experimental para tal estudo 9. Nós pensamos que este novo método pode ser útil para estudar as propriedades de resposta de outros artrópodes chemosensory sistemas, incluindo os dos insetos 10, 11 e 12 de crustáceos.

Watch this Scientific Journal Video about Electrophysiology of Scorpion Peg Sensilla at JoVE.com

Electrophysiology of Scorpion Peg Sensilla

Este artigo descreve um método eletrofisiológico para isolar a estimulação química sensilla individuais via extracelular, tip-gravações sob óleo mineral.

Eletrofisiologia da Scorpion Peg sensilla

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

electrophysiology-of-scorpion-peg-sensilla

Universidad San Sebastian

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Neuroscience

8.9K visualizações.  University of Oklahoma. Este artigo descreve um método eletrofisiológico para isolar a estimulação química sensilla individuais via extracelular, tip-gravações sob óleo mineral.

Vídeo: Electrophysiology of Scorpion Peg Sensilla

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Eletrofisiologia II: Fabricação Eletrodo de Sucção Extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Cultura e Eletrofisiologia de células em NRCC patch-clamp-Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

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 ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Eletrofisiologia em tempo real: Usando protocolos de circuito fechado para sondar Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Do gânglio espiral Neuron Explant Cultura e Eletrofisiologia em multi-eléctrodos Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

A incubação prolongada de aguda Neuronal Tissue de Eletrofisiologia e cálcio-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

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, ...

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

Único Sensillum gravações para Locust palpo sensilas Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

Avaliação da multiplicidade sináptica usando células inteiras Patch-clamp eletrofisiologia

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...