Name: electrophysiological method for recording intracellular voltage responses of drosophila photoreceptors and interneurons to light stimuli in vivo
Uploaded: 2016-06-19T15:00:00.000+00:00
Duration: 11 min 42 s
Description: Watch this Scientific Journal Video about Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo at JoVE.com

The authors thank Mick Swann, Chris Askham and Martin Gautrey for their important contributions in designing and building many electrical and mechanical components of the rigs. MJ's current research is supported by the Biotechnology and Biological Sciences Research Council (BBSRC Grant: BB/M009564/1), the State Key Laboratory of Cognitive Neuroscience and Learning open research fund (China), High-End Foreign Expert Grant (China), Jane and Aatos Erkko Foundation Fellowship (Finland), and the Leverhulme Trust grant (RPG-2012-567).

The authors have nothing to disclose

We have presented the basic key steps of how to use sharp conventional microelectrodes to record intracellular responses of R1-R6 photoreceptors and LMCs in intact fly eyes. This method has been optimized, together with bespoke hardware and software tools, over the last 18 years to provide high-quality long-lasting recordings to answer a wide range of experimental questions. By investing time and resources to construct robust and precise experimental set-ups, and to produce microelectrodes with favorable electrical properties, high-quality recordings can become the norm in any laboratory working on Drosophila visual neurophysiology. Whilst well-designed recording and light stimulation systems are important for swift execution of different experimental paradigms, there are three procedural steps that are even more critical to achieving successful recordings: (i) to make the fly preparation with minimal eye damage, (ii) to pull microelectrodes with the right electrical properties, and (iii) to drive the recording electrode into the eye without breaking its tip. Ultimately, to record meaningful data, the investigator has to understand the physical basis of electrophysiology and how to fabricate suitable microelectrodes for the targeted cell-types.
Therefore, the limitations of this technique are primarily set by the patience, experience and technical ability of the investigator. Because this technique can take a long time to master for small Drosophila cells, it is advisable for trainee electrophysiologists to first practice with larger insect eyes, such as the blowfly36 or locust35, using the same rig. Once performing high-quality intracellular recordings from the larger photoreceptors and interneurons becomes routine, it is time to move on to the Drosophila eye. Another limitation of the technique concerns cellular identification. Penetrated Drosophila cells can be loaded electrophoretically with dyes, including Lucifer yellow or neurobiotin. However, because of the small tip size of the microelectrodes, electrophoresis works less efficiently than with lower resistance electrodes, such as patch-electrodes. Furthermore, the dye-filled microelectrodes characteristically have less favorable electrical properties, making it much harder to record high-quality responses with them from Drosophila photoreceptors and LMCs.
A technical problem that occurs sometimes is unstable input signal, or a complete lack of it. This is often associated with the voltage signal being either constantly drifting or higher/lower than the amplifier's recording range. On most occasions, this behavior is caused by the recording electrode being blocked (or its tip being too fine - having too high a resistance or intramural capacitance - to properly conduct fast signal changes). Although one can try to unblock the tip by buzzing the electrode capacitance, which sometimes works, often the situation is best resolved by simply changing the recording electrode. This may further require parameter adjustments in the microelectrode puller instrument to lower the tip resistance of the new electrodes. The electrode tip can also become blocked in preparations, for which it took too much time to cover the corneal hole by petroleum jelly. Prolonged air-contact can dry up the freshly exposed retinal tissue, turning its surface layer into a glue-like substance. If this is the case, the investigator typically sees a red blob of tissue stuck on the recording electrode when pulling it out of the eye. The only solution here is to make a new preparation. Petroleum jelly may provide many benefits for electrophysiological recordings: (i) it prevents the coagulation of the hemolymph that could break the electrode tip; (ii) it coats the electrode tip reducing its intramural capacitance, which lowers the electrode's time constant, and thus has the potential to improve the temporal resolution of the recorded neural signals40,41; (iii) it keeps the electrode tip clean, facilitating penetrations; and after penetration, (iv) it may even help to seal the electrode tip to the cell membrane42. 
The signal can further be unstable or lost when the silver-chloride wire of the electrode-holder is broken or dechloridized; in which case just replace or rechloridize the old wire. The missing signal can also result from one (or both) of the electrode-holders not being securely connected to their jacks. However, it is extremely unusual that a piece of equipment would be malfunctioning. If signal is undetectable and all other possibilities have been exhausted, test that each part of the recording apparatus, including the headstage, amplifier, low-pass filters and AD/DA-converters, are connected properly and functioning normally. One way to achieve this is to replace each instrument with another from a rig that is known to operate normally. Alternatively, use a signal generator to check the performance of the electronic components one by one.
But perhaps the most common technical problem facing the electrophysiologist is that of recording noise. Broadly, recording noise is the observed electrical activity other than the direct neuronal response to a given stimulus. Because the fly preparation, when properly done, is very stable, the observed noise (beyond the natural variably of the responses) most often results from ground-loops in the recording equipment, or is picked up from nearby electrical devices. Such noise is typically 50/60 Hz mains hum and its harmonics; but sometimes composed of more complex waveforms. To work out the origin of the noise, remove the fly preparation holder from the set-up, connect the recording and reference electrodes through a drop of fly Ringer (or place them in a small Ringer's solution bath; see step 1.2.6) and record the signal in CC- or bridge-mode. If noise is observable on the recorded signal, this likely means that the noise is external to the fly preparation.
Another good test for identifying the origin of noise is to replace the electrode-holders with an electric cell model connected to the amplifier. In an ideally configured and grounded set-up, the recorded signal should now be practically noise-free, showing only stochastic bit-noise from the AD-converter (in the best case not even that!). If noise is still present, then recheck that all rig equipment is properly grounded. A convenient approach to detect ground-loops is to: (i) disconnect all the grounding wires from all the parts within the rig; (ii) ensure that, after doing this, every single part is actually isolated from ground, by means of an ohm-meter; (iii) connect the parts, one by one, to the central ground directly, not through any other part of the rig. Try also changing the equipment configurations. For example, sometimes moving the computer and monitor further away from the rig can reduce noise; yet at other times, moving the computer inside the equipment rack reduces noise. It is also worth unplugging nearby equipment to see if noise is reduced, or shield additional components. Furthermore, try unplugging or replacing different components of the recording equipment, especially BNC cables (which can have faulty ground connections). If only bit-noise is observed when using the cell model, the initial noise source is either the electrodes or the fly preparation itself. For example, it could be that the reference electrode is inadvertently touching a motor nerve or active muscle fibers inside the head capsule (or disturbing flight muscles in the thorax - if placed there). It is usually simplest to prepare a new fly for recording, taking care to minimize damage to the fly. But if the noise persists and is broadband, it is likely that the electrodes are suboptimal for the experiments; too sharp/fine (hence too noisy) or just wrong for the purpose; we have even seen quartz-electrodes acting as antennas - picking up faint broadcasting signals! Although iteration of the puller-instrument parameter settings to generate the just right microelectrodes for consistent high-quality recordings from specific cell-types can take a lot of effort, it is worth it. Once the recording electrodes are well-tailored for the experiments, they can provide long-lasting recordings of outstanding quality.
Sharp microelectrode recording techniques can be similarly applied to study neural information processing in multitude of preparations, including different processing layers in the insect eyes and brain43,44. Because the microelectrode tips can be made very fine, these typically damage the studied cells less than most patch-clamp applications. Importantly, the modern sample-and-hold microelectrode amplifiers enable good control of the tips' electrical properties40,45-47. Thus, when correctly applied, this technique can provide reliable data from both in vivo3,5,7-10,44 or in vitro48 preparations with high signal-to-noise ratio at sub-millisecond resolution. Such precision would be impossible with today's optical imaging techniques, which are noisier and slower. Moreover, the method can be used to characterize small cells' electrical membrane properties both in current- and voltage-clamp configurations5,29,33,36,40-42,49, providing valuable data for biophysical and empirical modeling approaches7,8,11,33,49-54 that link experiments to theory.

A mosca da fruta (Drosophila melanogaster) olho composto é um grande sistema modelo para investigar a organização funcional dos fotorreceptores e interneurônio matrizes para a amostragem de imagem neural e processamento, e para a visão animal. O sistema tem o esquema de ligações mais completa 1,2 e é amável a manipulações genéticas e monitoramento da atividade neural preciso (de alta relação sinal-ruído e hora resolução) 3-10. O olho Drosophila é modular, contendo ~ 750 estruturas cobertas de lente aparentemente regulares chamados ommatidia, que juntos fornecem a um campo visual panorâmico que cobre quase todas as direções ao redor de sua cabeça voar. Informação primária do olho amostragem unidades são seus fotorreceptores rabdoméricos 7,8,11. Cada omatídeo contém oito células fotorreceptoras (R1-R8), que compartilham a mesma lente faceta, mas estão alinhados com sete direções diferentes. Embora os fotorreceptores exteriores R1-R6 ARe mais sensível à luz azul-verde, sensibilidades espectrais das células internas R7 e R8, que se encontram em cima uns dos outros e apontam para a mesma direcção, apresentam três subtipos distintos: pálido, amarelo e a área de rebordo dorsal (DRA) 12- 15. <img alt="figura 1" src="/files/ftp_upload/54142/54142fig1.jpg" /> Figura 1. Organização Funcional do Olho Drosophila. (A) Os dois primeiros gânglios óptico, retina e lâmina, são destacadas em cinza no interior do olho da mosca. Fotorreceptores da retina R1-R6 e lâmina grandes células monopolar (LMCs: L1-L3) são facilmente acessíveis in vivo para gravações microeletrodos afiada convencionais. O eletrodo esquemática destaca o caminho normal para gravar a partir de R1-R6 na retina. Um caminho para gravar a partir de LMCs na lâmina é deslocar paralelamente o eletrodo para a esquerda. (B) lâmina é uma matriz de órgão retinotopicallyized cartuchos, cada um dos quais é embalado com neurónios que processa informação a partir de uma pequena área específica no espaço visual. Devido à superposição neural, seis fotorreceptores de diferentes ommatidia vizinhos enviam seus axónios (R1-R6) para o mesmo cartucho de lâmina, formando sinapses de saída histaminï¿½gicos para L1-L3 e uma célula amacrine (Am). (C) A difusão de informação entre os terminais de axónios neuronais R1-R6 e os interneurónios visuais (incluindo L4, L5, Lawf, C2, C3 e T1), no interior de um cartucho de lâmina é complexa. (D) R1-R6 axônios fotorreceptoras receber feedbacks sinápticos de células monopolares L2 e L4. (B) e (C) modificado a partir de Rivera-alba et al 2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> O olho Drosophila é do tipo neural superposição 16. Isto significa tchapéu de sinais neurais de oito fotorreceptores pertencentes a sete ommatidia vizinha, que olham para o mesmo ponto no espaço, são agrupados em um cartucho de neural nos próximos dois neuropils: a lâmina e medula. Enquanto os seis fotorreceptores exteriores projecto R1-R6 seus terminais de axónios para colunas neurais na lâmina (Figura 1), as células R7 e R8 ignorar essa camada e fazer contactos sinápticos com a sua coluna correspondente medula 17-19. Estas fiações exactas produzir o substrato neural para o mapeamento de mosca retinotópica visão inicial, após o que cada lâmina (Figuras 1A-C) e medula coluna (cartucho) representa um único ponto no espaço. Entradas directas de fotorreceptores R1-R6 são recebidos pelos grandes células monopolar (LMCs: L1, L2 e L3) ea célula amácrina (AM) na lâmina 1,2,20. Destes, L1 e L2 são as maiores células, mediadores principais vias de informação (Figura 1D), WHIch respondem a on e bordas Off-movimento, e, assim, formar a base computacional do detector de movimento 21,22. Experimentos comportamentais sugere que pelo contraste intermediária, as duas vias facilitar a percepção do movimento de sentidos opostos: back-to-frente em L1 e para frente e para trás em células L2 23,24. Conectividade implica ainda que os neurônios L4 pode desempenhar papel fundamental na comunicação lateral entre cartuchos vizinhos 25,26. sinapses recíprocas foram encontradas entre as células L2 e L4 localizados na mesma e dois cartuchos adjacentes. Jusante, cada célula L2 e seus três células L4 associados projetam seus axônios para um objectivo comum, o neurônio Tm2 na medula, onde se acredita entradas de cartuchos vizinhos para ser integrado para o processamento do movimento 27 de front-to-back. Embora os neurônios L1 receber entrada a partir do mesmo cartucho L2s via ambas as junções comunicantes e sinapses, eles não estão diretamente ligados à cartuchos de lâmina L4s e, portanto adjacentes. <pclasse = &quot;jove_content&quot;&gt; Comentários Synaptic para axónios fotorreceptoras R1-R6 são fornecidas apenas pelos neurónios pertencentes aos circuitos / L4 L2 mas não a via de 1,2 L1 (Figura 1D). Enquanto as conexões do mesmo cartucho são seletivamente de L2 a R1 e R2 e de L4 a R5, todos os fotorreceptores R1-R6 receber feedback sináptica de L4 de um ou de ambos os cartuchos vizinhos. Além disso, existem fortes ligações sinápticas de AM para R1, R2, R4 e R5, e células gliais, também estão sinapticamente ligado à rede e pode, assim, participar na neural de processamento de imagem 6. Finalmente, axonais-gap junctions, ligando vizinha R1-R6 e entre R6 e R7 / R8 fotorreceptores na lâmina, contribuir para a representação da informação assimétrica e o processamento em cada cartucho 14,20,28. Gravações de tensão intracelulares de fotorreceptores individuais e interneurônios visuais em Drosophila quase intacto fornecer alta r sinal-ruídodados ATIO com resolução sub-milissegundo 3,5,7-10,29, que é necessário para dar sentido às computações neurais rápidas entre os neurônios conectados. Este nível de precisão é impossível por técnicas de imagem ópticos atuais, que são significativamente mais ruidoso e tipicamente operam em 10-100 resolução ms. Além disso, porque os eletrodos têm muito pequenas e afiadas pontas, o método não é restrito aos corpos celulares, mas pode fornecer gravações diretas de estruturas neurais ativas pequenas; tais como árvores dendríticas os LMCs &#39;ou axônios fotorreceptoras, que não podem ser acessados ​​por muito maiores pontas dos eletrodos de patch-clamp. É importante ressaltar que o método também é estruturalmente menos invasivo e prejudicial do que a maioria das aplicações de patch-clamp, e assim afectam menos intracelular meio e informações de amostragem das células estudadas. Assim, as técnicas de microeletrodos afiada convencionais têm contribuído, e continuar a contribuir, descobertas fundamentais e uma visão original em infor neuralprocessamento de infor- na escala de tempo apropriado; melhorar o nosso entendimento mecanicista de visão 3-10. Este artigo explica como em gravações intracelulares vivo a partir de Drosophila fotorreceptores e LMCs R1-R6 são realizados no laboratório Juusola. Este protocolo irá descrever como construir uma plataforma de eletrofisiologia adequada, preparar a mosca, e realizar as gravações. Alguns dados representativo é apresentado, e alguns problemas comuns e as possíveis soluções são discutidas que podem ser encontrados quando utilizando este método.

<table><tbody><tr><td>Stereo Zoom Microscope for making the fly preparation</td><td>Olympus</td><td>SZX12 DFPLFL1.6x PF eyepieces: WHN30x-H/22</td><td>Capable of ~150X magnification with long working distance; bespoke heavy steel table mount stand</td></tr><tr><td>Stereomicroscope in the intracellular set-up</td><td>&amp;middot;&amp;nbsp; Olympus</td><td>Olympus SZX7; eyepieces: WHN30x-H/22</td><td>30X eyepieces are needed for seeing the electrode tip reflections well when driving it through the small corneal hole into the eye</td></tr><tr><td>Nikon microscope</td><td>Nikon SMZ645; eyepieces: C-W30x/7</td><td></td><td></td></tr><tr><td>Anti-vibration Table</td><td>Melles Griot</td><td>With metric M6 holes on the breadboard</td><td>Our bespoke rigs have a large hole drilled through the thick breadboard that lets in the fly preparation platform pole (houses a copper heatsink with electronics) from below</td></tr><tr><td>Newport</td><td></td><td></td><td></td></tr><tr><td>Micromanipulators</td><td>Narishige</td><td>Narishige NMN-21</td><td>In our intracellular set-ups, different micromanipulator systems are used for driving the shap recording electrodes into the fly eye. All the listed manipulators are succesfully providing long-lasting stable recordings from Drosophila photoreceptors and LMCs.&amp;nbsp;</td></tr><tr><td>Huxley Bertram</td><td>Huxley xyz-axis with fine manual control</td><td></td><td></td></tr><tr><td>Sensapex</td><td>Sensapex triple axis</td><td></td><td></td></tr><tr><td>M&amp;auml;rzh&amp;auml;user</td><td>M&amp;auml;rzh&amp;auml;user DC-3K with additional x-axis piezo stepper and MS 314 controller</td><td></td><td></td></tr><tr><td>Magnetic Stands</td><td>Any magnetic base with on/off switch will do</td><td></td><td>For example, to manage cables inside the Faraday cage</td></tr><tr><td>Electrode Holders</td><td>Harvard Apparatus</td><td>ESP/W-F10N</td><td></td></tr><tr><td>Silver Wire</td><td>World Precision Instruments&amp;nbsp;</td><td>AGW1510</td><td>0.3 - 0.5 mm diameter; needs to be chloridized for the electrode holders</td></tr><tr><td>Fiber Optic Light Source</td><td>Many different, including Olympus</td><td></td><td></td></tr><tr><td>Fiber Optic Bundles</td><td>UltraFine Technology</td><td></td><td>To deliver the LED light stimulus to the Cardan arm system. We use both liquid and quartz light guides (range from UV to IR)</td></tr><tr><td>Thorn Labs</td><td></td><td></td><td></td></tr><tr><td>Fly Cathing Tube</td><td></td><td>P80-50P 50ml Cent. Tube PP., Pack of 100 Pcs</td><td>Cut the conical bottom off from 50 ml Plastic Centrifuge Tube and glue a 1 ml pipette tip on it.</td></tr><tr><td>Digital Acquisition System</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Single-electrode current/voltage-clamp microelectrode amplifier</td><td>npi SEC-10LX</td><td>http://www.npielectronic.de/products/amplifiers/sec-single-electrode-clamp/sec-10lx.html</td><td>Outstanding performer!</td></tr><tr><td>Head-stage</td><td>Standard (+/- 150 nA)</td><td>For npi SEC-10LX</td><td></td></tr><tr><td>LED light sources and drivers</td><td>2-channel OptoLED (Cairn Research Ltd., UK)</td><td></td><td>Many of our stimulus systems are in-house built&amp;nbsp;</td></tr><tr><td>Self-designed and constructed</td><td></td><td></td><td></td></tr><tr><td>Acquisition and Analyses Software</td><td>Many companies to choose from</td><td></td><td>Biosyst; custom written Matlab-based system for experimental and theoretical work in the Juusola laboratory</td></tr><tr><td>Personal Computer or Mac</td><td></td><td></td><td>Ensure that PC or Mac is compatible with data acquisition system and software</td></tr><tr><td>Cardan arm system&amp;nbsp;</td><td>Self-designed and constructed</td><td></td><td>Providing accurate x,y,z-positioning of the light stimuli</td></tr><tr><td>Peltier temperature control system&amp;nbsp;</td><td>Self-designed and constructed</td><td></td><td></td></tr><tr><td>Faraday Cage</td><td>Self-constructed</td><td></td><td>Electromagnetic noise shielding</td></tr><tr><td>Filamented Borosilicate Glass Capillaries</td><td></td><td>Outer diameter: 1 mm</td><td></td></tr><tr><td>Inner diameter: 0.5 - 0.7 mm</td><td></td><td></td><td></td></tr><tr><td>Filamented Quartz Glass Capillaries</td><td></td><td>Outer diameter: 1 mm</td><td></td></tr><tr><td>Inner diameter: 0.5 - 0.7 mm</td><td></td><td></td><td></td></tr><tr><td>Pipette Puller</td><td>Sutter Instrument Company</td><td>Model P-2000 laser Flaming/Brown Micropipette Puller</td><td>For borosilicate reference electrodes, use the preset program #11 (patch electrodes): Heat = 350; Filament = 4; Velocity 36; Delay = 200).1.2.1). For borosilicate recording electrodes, use the preset program #12 (this typically pulls good conventional sharps&amp;nbsp; for photoreceptor recordings): Heat = 355; Filament = 4; Velocity 50; Delay = 225; Pull = 150. For LMC recordings, which require electrodes with finer tips, these values need to be adjusted. For pulling quartz capillaries, P-2000 manual suggests programs for fine tipped microelectrodes. These programs&amp;rsquo; preset parameters serve as useful starting points for systematic modifications to generate electrodes with good penetration success and low recording noise.</td></tr><tr><td>Extracellular Ringer Solution for the reference electrode</td><td>Chemicals from Fisher Scientific</td><td>10326390, NaCl 10010310, KCl 10147753, TES 10161800, CaCl2 10159872, MgCl2 10000430, sucrose</td><td>See the recipe in the protocol section</td></tr><tr><td>3 M KCl solution for filling the filamented recording microelectrode</td><td>Salts from Fisher Scientific</td><td>10010310, KCl</td><td></td></tr><tr><td>Petroleum jelly</td><td>Vaselin</td><td></td><td></td></tr><tr><td>Non-stainless steel razor blades</td><td></td><td></td><td></td></tr><tr><td>Blade holder/breaker</td><td>Fine Science Tools By Dumont</td><td>10053-09</td><td>9 cm</td></tr><tr><td>Blu-tack</td><td>Bostik</td><td></td><td>Alternatively, use molding clay</td></tr><tr><td>Forceps</td><td>Fine Science Tools By Dumont</td><td>11252-00</td><td>#5SF (super-fine tips)</td></tr></tbody></table>

electrophysiological method for recording intracellular voltage responses of drosophila photoreceptors and interneurons to light stimuli in vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

O método de gravação de microeletrodos afiada, tal como adaptado aqui para o olho Drosophila, pode ser usado para quantificar de forma fiável de amostragem neural de informação e processamento nas células da retina e lâmina, e comunicação entre eles 4,5,7,8,10,33. Ao usá-lo para estudar a codificação em diferentes estoques de tipo selvagem, mutantes ou linhagens de moscas geneticamente modificadas, o método tem provado o seu valor; não só para quantificar os efeitos de uma mutação, temperatura, dieta ou de expressão seleccionado 3,4,6,9,10,14,30,34, mas também em revelar razões mecanística para comportamentos visuais alteradas 14,34. O método também é facilmente aplicável a outras preparações de insetos 35,36, capacitando estudos visão neuroetológica. Em seguida nós mostrar alguns exemplos de suas aplicações bem sucedidas. <img alt="Figura 7" src="/files/ftp_upload/54142/54142fig7.jpg" /> Figura 7. TensãoRespostas de uma mosca da fruta R1-R6 fotorreceptores a um pulso de luz a 20 e 25 ° C. Uma vez que as penetrações microeléctrodo afiadas são, frequentemente, muito estável, é possível gravar as respostas tensão da mesma fotorreceptores R1-R6 a um dado estímulo a luz diferentes temperaturas ambiente pelo aquecimento ou arrefecimento da mosca. Em nossos set-ups, fly-titular da é colocada em um sistema de controle de temperatura de circuito fechado baseada Peltier-element-. Isso nos permite alterar a temperatura da cabeça da mosca em segundos. Temperatura mais elevada acelera as respostas de tensão e caracteristicamente reduz o potencial de repouso de fotorreceptores R1-R6 (como indicado pelas setas vermelhas). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig7large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Estudando o efeito da temperatura na saída de fotorreceptores <p class="jove_content" fo:kEEP-together.within-page = &quot;1&quot;&gt; Com um bem concebido e sistema de gravação isolada de vibrações, o método pode ser utilizado para medir o efeito da temperatura na saída de uma célula neural indivíduo por aquecimento ou arrefecimento da mosca. O exemplo dado mostra as respostas de tensão a um pulso de 10 ms de comprimento brilhante, registrados no mesmo fotorreceptor R1-R6 em 20 e 25 o C (Figura 7). Quantificada antes de 4,9, o aquecimento reduz potencial de repouso de um fotorreceptor na escuridão, e acelera as respostas de tensão. <img alt="Figura 8" src="/files/ftp_upload/54142/54142fig8.jpg" /> Figura 8. Desempenho sinalizando uma Fruit Fly R1-R6 fotorreceptores melhora com a intensidade de luz (A) a saída de fotorreceptores para escurecer (abaixo) e brilhantes. (Acima; 10.000 vezes uma luz mais brilhante) repetiu naturalista séries temporais de intensidade de luz registado pela mesma microeletrodosna mesma célula a 20 ° C. As respostas ao estímulo luminoso são maiores, porque integram mais amostras, respostas elementares (solavancos) para fótons individuais 4,5,7,8. (B) 20 respostas tensão de um segundo de duração consecutivos são sobrepostos. As respostas individuais (cinza claro) foram tomadas após as tendências adaptativas (seta em A) tinha diminuído (caixa pontilhada em A). Os meios de resposta correspondente (os sinais) são os vestígios mais escuras. A diferença entre o sinal e as respostas individuais é o ruído. (C) As células &#39;desempenho sinalização foi quantificada pelas gravações de&#39; relações sinal-ruído (SNR) usando os métodos padrão 4,5,7,8. Saída de fotorreceptores tem cerca de 64 Hz mais ampla gama de sinalização de confiança no estímulo luminoso ( &#39;≥1 brilhante, até 84 Hz do que no dim (SNR SNR)&#39; Dim ≥1, até 20 Hz), com signal-to-noise ratio melhorando consideravelmente; de SNR Dim MAX = 87 a SNR brilhante MAX = 1.868. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig8large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Estudar Adaptação e codificação neural por repetitivo Estimulação O noninvasiveness do método, causando relativamente pouco dano nas estruturas da retina e da lâmina, faz com que seja ideal para estudar o desempenho de sinalização de células individuais a diferentes estímulos luminosos no seu próximo estado fisiológico natural in vivo. A Figura 8 mostra respostas de tensão de um R1- R6 fotorreceptor a um repetido naturalista série estímulo luminoso tempo de intensidade fraca e brilhante a 20 ° C, enquanto que a Figura 9 </strong&gt; mostra as respostas de outro fotorreceptor R1-R6 e uma LMC a um estímulo naturalista diferente a 25 o C. As gravações pré e pós-sinápticos foram realizadas separadamente a partir de duas moscas diferentes, porque as gravações intracelulares simultâneos entre dois microeletrodos afiada na mesma mosca, um na retina e o outro na lâmina, são muito difíceis de ser viável 30. <img alt="Figura 9" src="/files/ftp_upload/54142/54142fig9.jpg" /> Figura 9. Respostas tensão de uma mosca de fruto R1-R6 fotorreceptores e LMC à Estimulação naturalista repetida a 25 o C. (A) R1-R6 (cinza) e LMC (preto) Saídas gravada por diferentes microeletrodos de diferentes moscas. (B) 20 totalmente pré-luz adaptado consecutivo (acima) e pós-sinápticos (abaixo) as respostas ao mesmo padrão de estímulo naturalista com respostas individuais, mostrado em cinzento claro de umd os correspondentes meios de resposta (os sinais) como os traços mais escuros. A diferença entre o sinal e as respostas individuais é o ruído. (C) As células &#39;desempenho sinalização foi quantificada pelas gravações de&#39; relações sinal-ruído (SNR). Saída LMC tem cerca de 10 Hz mais ampla gama de sinalização confiável (SNR &#39;LMC ≥1, até 104 Hz) do que a produção R1-R6 (SNR&#39; R ≥1, até 94 Hz). Ambos os rácios de sinal-para-ruído são elevados (SNR LMC MAX = 142, SNR R MAX = 752), e como o ruído de gravação era baixa, as suas diferenças refletem diferenças de codificação reais entre as células. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig9large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior esta figura.</a> Após o início do estímulo, as gravações típicoly mostrar rápida adaptação tendências que em grande parte desaparecem dentro de 5-6 seg. A partir de então, as células produzem respostas altamente consistentes para cada apresentação longa estímulo 1 segundo (cada caixa pontilhada inclui 20 destes). A reprodutibilidade das medidas torna-se óbvio quando estes são sobrepostos (Figura 8B e Figura 9B). As respostas individuais são os traços cinzas finas, e sua significa o traço mais escura mais grosso. A resposta média é tomada como o sinal neuronal, ao passo que o ruído neural é a diferença entre a média e cada resposta individual 4,5,9,37,38. As respectivas relações sinal-para-ruído no domínio da frequência (Figura 8C e Figura 9C) foram obtidos por Fourier-transformar os fragmentos de dados de sinal e de ruído na espectros de potência, e dividindo-se o espectro de potência do sinal significativo com o correspondente espectro de potência média de ruído 4, 5,9,37,38. Caracteristicamente, as proporções máximas de sinal-para-ruído óf as saídas neurais registados para estimulação naturalista são elevadas (100 - 1000), e nas preparações mais estáveis ​​com muito baixo ruído de gravação pode atingir valores &gt;&gt; 1000 (por exemplo, Figura 8C.). Observe também que o aquecimento expande as células &quot;largura de banda de confiança sinalização 4 (SNR &#39;brilhante ≥ 1); por exemplo, a diferença relativa entre os dois R1-R6s nas Figuras 8 e 9, respectivamente, é de 10 Hz (84 a 20 o C e 94 Hz a 25 o C). Pode-se estimar a taxa de mais de cada célula de transferência de informação a partir da sua relação sinal-ruído através da utilização da equação de Shannon 32, ou através do cálculo da diferença entre as taxas de entropia entropia e de ruído das respostas através do método de extrapolação triplo 39. Mais detalhes sobre as informações de análises teóricas, e seu uso e limitaçõess especificamente com este método são apresentados nas publicações anteriores 7,8,39. <img alt="Figura 10" src="/files/ftp_upload/54142/54142fig10.jpg" /> Figura 10. Respostas tensão de uma mosca assassina R1-R6 fotorreceptores e LMC à Estimulação naturalista repetida a 19 o C. (A) R1-R6 (cinza) e LMC (preto) envia registado pela mesma microeletrodos da mesma mosca; primeira pós-sinapticamente e, posteriormente, pré-sináptico, como eletrodo foi avançado no olho. (B) 20 pré consecutiva (acima) e pós-sinápticos (abaixo) as respostas (traços cinza claro) para o mesmo padrão de estímulo naturalista foram capturados após a adaptação inicial (caixa pontilhada em A). Seus meios são os sinais (os traços mais escuros no topo), enquanto as suas respectivas diferenças para as respostas individuais dão o barulho. (C) O correspondente SignaRácios l-a-ruído (SNR) foram calculados como nas Figuras 8C e 9C. Saída LMC tem cerca de uma faixa de 100 Hz mais amplo de sinalização confiável (SNR &#39;LMC MAX ≥1, até 234 Hz) do que a produção R1-R6 (SNR&#39; R MAX ≥1, até 134 Hz). Ambos os rácios de sinal-para-ruído são elevados (SNR LMC MAX = 137, SNR R MAX = 627), e como o mesmo microeletrodos foi utilizado nas gravações, suas diferenças refletem diferenças reais nas saídas pré e pós-sinápticos neurais. Estes resultados implicam que o sistema de gravação tinha baixo nível de ruído, e sua influência sobre as análises foi marginal. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig10large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Estudos Visão neuroetológica </p&gt; O método também pode ser utilizado para registar as respostas pré- e pós-sinápticos de tensão dos olhos compostos de diferentes espécies de insectos 7,8,35,36 (Figura 10), que permite estudos comparativos neuroetológica de processamento de informações visuais. Para o sistema de gravação apresentada, a única adaptação necessária é de preparação titulares novos, cada um com uma abertura de dimensões adequadas para as espécies estudadas. Estas gravações exemplares são provenientes de um assassino fêmea de mosca (Coenosia attenuata). Eles mostram respostas intracelulares de tensão de um fotorreceptor R1-R6 e LMC à estimulação repetitiva de luz idêntica, tal como se utiliza para os homólogos de Drosophila na Figura 9, mas a 19 o C. Neste caso, tanto o pré- e pós-sinápticos, os dados foram registados a partir do mesmo mosca; uma após a outra, com o mesmo eléctrodo de registo (cheia com 3 M KCl) primeiro avançando através da lâmina lateral, antes de entrarção da retina frontal. Em comparação com os dados de Drosophila a 25 ° C, os dados Coenosia - até à temperatura de refrigerador - mostra as respostas com dinâmica mais rápidos; expandir a gama de sinalização fiável (relação sinal-ruído &gt;&gt; 1) ao longo de uma gama de frequência mais ampla. Essas adaptações funcionais na codificação neural de estímulos naturalistas são consistentes com estilo de vida predatório &#39;s Coenosia 36, que exigem alta precisão informações espaço-temporal para alcançar rápidos comportamentos caça aérea.

Watch this Scientific Journal Video about Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo at JoVE.com

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Método eletrofisiológico para respostas gravação intracelular de tensão<em&gt; Drosophila</em&gt; Fotorreceptores e Interneurônios à luz Estímulos<em&gt; In Vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

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18.7K visualizações.  Beijing Normal University. Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Vídeo: Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Métodos eletrofisiológicos de gravação Potenciais Synaptic do MNJ de larvas de Drosophila

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Biologia

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Gravação eletrofisiológicas na Drosophila Embrião

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

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 ...

Neurociência

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Medindo respostas fisiológicas de<em&gt; Drosophila</em&gt; Neurônios sensoriais para Lipid feromonas Usando Imagem de cálcio ao vivo

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Imunologia e Infecção

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

Determinação dos fotorreceptores celular Sensibilidade espectral em um inseto modelo de<em&gt; In Vivo</em&gt; Gravações intracelulares

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Método eletrofisiológico para gravações de tensão de célula inteira de<em&gt; Drosophila</em&gt; Fotorreceptores

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

Eletrofisiológica gravação da atividade do sistema nervoso Central de terceiro ínstar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Métodos Eletrofisiológicos para Medir Níveis de Fotopigment em Fotorreceptores de Drosophila

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli

Source: Juusola, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54142">Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo.</a> J. Vis. Exp. (2016).
This video outlines the protocol for electrophysiological recording of photoreceptor activity in the eye of an immobilized Drosophila, enabling precise measurement of voltage changes in response to light stimuli.

Registro eletrofisiológico das respostas de voltagem de fotorreceptores retinianos de Drosophila a estímulos luminosos

1. Recording from R1-R6 Photoreceptors 

	Always be grounded when operating the microelectrode amplifier (for example by touching the metal surface of the ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Extracellular Recording of Electrical Activity in the Drosophila Central Nervous System

Source: Swale, D. R., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58375">Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster.</a> J. Vis. Exp. (2018)
The video demonstrates the extracellular recording of neuronal activity from the central nervous system (CNS) of Drosophila larvae. The transected CNS is positioned in a recording chamber, and the electrical activity from a descending nerve is recorded to establish the baseline firing frequency. A test agent is then introduced, and the change in firing frequency is recorded to determine the agent&#39;s excitatory or inhibitory effect.

Registro extracelular da atividade elétrica no sistema nervoso central da drosófila

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Dissect and Prepare the Larval ...

Método eletrofisiológico para respostas gravação intracelular de tensão

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