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/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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 rowspan="2">Stereomicroscope in the intracellular set-up</td>
			<td>&#183;&#160; Olympus</td>
			<td>Olympus SZX7; eyepieces: WHN30x-H/22</td>
			<td rowspan="2">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>&#183;&#160; Nikon</td>
			<td>Nikon SMZ645; eyepieces: C-W30x/7</td>
		</tr>
		<tr>
			<td rowspan="2">Anti-vibration Table</td>
			<td>&#183;&#160; Melles Griot</td>
			<td rowspan="2">With metric M6 holes on the breadboard</td>
			<td rowspan="2">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>&#183;&#160; Newport</td>
		</tr>
		<tr>
			<td rowspan="4">Micromanipulators</td>
			<td>&#183;&#160; Narishige</td>
			<td>&#183;&#160; Narishige NMN-21</td>
			<td rowspan="4">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.&#160;</td>
		</tr>
		<tr>
			<td>&#183;&#160; Huxley Bertram</td>
			<td>&#183;&#160; Huxley xyz-axis with fine manual control</td>
		</tr>
		<tr>
			<td>&#183;&#160; Sensapex</td>
			<td>&#183;&#160; Sensapex triple axis</td>
		</tr>
		<tr>
			<td>&#183;&#160; M&#228;rzh&#228;user</td>
			<td>&#183;&#160; M&#228;rzh&#228;user DC-3K with additional x-axis piezo stepper and MS 314 controller</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&#160;</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 rowspan="2">Fiber Optic Bundles</td>
			<td>&#183;&#160; UltraFine Technology</td>
			<td rowspan="2"></td>
			<td rowspan="2">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>&#183;&#160; Thorn Labs</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 rowspan="2">LED light sources and drivers</td>
			<td>&#183;&#160; 2-channel OptoLED (Cairn Research Ltd., UK)</td>
			<td rowspan="2"></td>
			<td rowspan="2">Many of our stimulus systems are in-house built&#160;</td>
		</tr>
		<tr>
			<td>&#183;&#160; Self-designed and constructed</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&#160;</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&#160;</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 rowspan="2">Filamented Borosilicate Glass Capillaries</td>
			<td rowspan="2"></td>
			<td>Outer diameter: 1 mm</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Inner diameter: 0.5-0.7 mm</td>
		</tr>
		<tr>
			<td rowspan="2">Filamented Quartz Glass Capillaries</td>
			<td rowspan="2"></td>
			<td>Outer diameter: 1 mm</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Inner diameter: 0.5-0.7 mm</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&#160; 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&#8217; 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/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/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/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/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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18.5K 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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Neurociência

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

Um Método para Sistemática de Eletroquímica e eletrofisiológica Avaliação do Neurais gravação Eletrodos

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

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 ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Um Guia para<em&gt; In vivo</em&gt; Gravação única unidade de Optogenetically Identificado Cortical Inibitória Interneurônios

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analisando Synaptic Modulação da<em&gt; Drosophila melanogaster</em&gt; Fotorreceptores após exposição à luz prolongada

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

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 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, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Imagem de cálcio para visualizar as respostas do cérebro a sinalização endócrina em Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

Preparando melanogaster de Drosophila adulto para imagem cerebral inteira durante respostas comportamentais e estímulos

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

In vivo Imagem de cálcio do rato Geniculado Gânglio Respostas ao Sabor Estímulo

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...