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.

Meyve sineği (Drosophila melanogaster) bileşik göz sinir görüntü örnekleme ve işleme için fotoreseptör ve interneuron dizilerin fonksiyonel organizasyonunu araştırmak için büyük bir model sistem ve hayvan vizyonu için. Sistem en eksiksiz bağlantı şemasını 1,2 sahiptir ve genetik manipülasyonlar ve 3-10 (yüksek sinyal-gürültü oranı ve zaman çözünürlük) doğru sinirsel aktivite izleme sevimli. Drosophila gözü hep birlikte başının etrafında hemen hemen her yönünü kapsayan panoramik görüş alanını uçmak sağlayan ommatidia denilen ~ 750 görünüşte normal lens kapaklı yapılar içeren, modüler olduğunu. Birimler kendi rhabdomeric fotoreseptör 7,8,11 olan örnekleme gözün birincil bilgi. Her ommatidium aynı faset lens paylaşırlar, fakat yedi farklı yönlere hizalanır sekiz fotoreseptör hücreleri (R1-R8) içerir. Dış fotoreseptörlerin R1-R6, ar ikene mavi-yeşil ışığa en duyarlı, aynı yönde birbirlerini ve nokta üstünde yalan, iç hücrelerin R7 ve R8, spektral duyarlılık, sergi üç ayrı alt tipi: soluk sarı ve dorsal rim alanı (DRA) 12- 15. <img alt="Şekil 1" src="/files/ftp_upload/54142/54142fig1.jpg" /> Şekil 1. Drosophila Eye Fonksiyonel organizasyonu. (A) ilk iki optik ganglionlar, retina ve lamina, sinek gözün içine gri vurgulanır. Retina R1-R6 fotoreseptör ve lamina Büyük Monopolar Hücreleri (LMCs: L1-L3) geleneksel keskin mikroelektrot kayıtlarına in vivo kolayca erişilebilir. şematik elektrot retinada R1-R6 kayıt normal yolu vurgulamaktadır. lamina içinde LMCs kayıt için bir yol sola paralel elektrot kaydırmaya olduğunu. (B) tabakanın retinotopically organının bir matristirGörsel alan belirli bir küçük bir alanda bilgi işleme nöronlarla paketlendi her biri özelleşmiş kartuşları. Nöral süperpozisyon nedeniyle, farklı komşu ommatidia altı fotoreseptör L1-L3 ve amacrine hücresi (Am) için histaminerjik çıkış sinaps oluşturarak, aynı lamina kartuşun kendi aksonlar (R1-R6) gönderin. (C) R1-R6 akson terminalleri ve lamina kartuş karmaşık iç, (L4, L5, Lawf, C2, C3 ve T1 dahil) görsel internöronlar arasındaki nöral bilgilerin yayılması. (D) R1-R6 fotoreseptör aksonları L2 ve L4 monopolar hücrelerden sinaptik geri dönüşler alıyorsunuz. Rivera-alba ark 2 değiştirilmiş (B) ve (C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Drosophila göz sinir süperpozisyon tip 16 olduğunu. Bu t anlamına gelirlamina ve medulla: şapka uzayda aynı noktada bakmak ommatidia komşu yedi ait sekiz fotoreseptör, nöral sinyaller, önümüzdeki iki neuropils tek sinir kartuşun birlikte bir araya getirilmiştir. Altı dış fotoreseptör lamina sinir sütunlara akson terminalleri R1-R6 projesi (Şekil 1), R7 ve R8 hücreleri bu katman atlamak ve onların medulla sütunu 17-19 karşılık gelen sinaptik temas yaparken. Bu tam kablo bağlantıları her lamina (Şekil 1A-C) bunun üzerine, sinek erken görme retinotopic haritalama için nöral substratı üreten ve medulla kolon (kartuş) uzayda tek bir noktayı temsil etmektedir. Lamina 1,2,20 ve amacrine Celi (AM) R1-R6, fotoreseptör direkt giriş büyük monopolar uydulara (L1, L2 ve L3 LMCs) tarafından alınır. Bunların dışında, L1 ve L2 önemli bilgi yolları (Şekil 1D) aracılık büyük hücreler, WHI vardırch yanıt içi ve kenarları Off-hareket ve böylece hareket dedektörü 21,22 hesaplama temelini oluşturur. L2 hücreleri 23,24 L1 &#39;de-ön arka-ön-arkaya: Davranışsal deneyler orta aksine de, iki yol zıt yönlerde hareket algısını kolaylaştırmak olduğunu göstermektedir. Bağlantı ayrıca L4 nöronlar komşu kartuşları 25,26 arasında yanal iletişimde kritik bir rol oynayabileceğini ima eder. Karşılıklı sinaps L2 ve L4, aynı yer alan hücreler ve iki komşu kartuşları arasında bulunmuştur. Mansap, her L2 hücre ve üç ilişkili L4 hücreleri ortak bir hedefe kendi aksonlarını, komşu kartuşlardan girdiler inanılan medulla içinde Tm2 nöron ön-arka hareket 27 işlenmesi için entegre edilmesi. L1 nöronlar hem gap junction ve sinapsların üzerinden aynı kartuşu L2S girdi alırsınız, ancak doğrudan L4s ve dolayısıyla komşu lamina kartuşları bağlı değildir. <pclass = &quot;jove_content&quot;&gt; R1-R6 fotoreseptör aksonlar için Synaptic geri bildirimler L2 / L4 devreleri ait nöronlar tarafından sadece sağlanmaktadır değil L1 yolu 1,2 (Şekil 1D). Aynı kartuş bağlantıları R1 ve R2 için L2 ve L4 R5 seçici iken, tüm R1-R6 fotoreseptör birinin L4 veya her ikisini komşu kartuşlardan sinaptik geri bildirim almak. Ayrıca, R1, R2, R4 ve R5 Am güçlü sinaptik bağlantıları vardır, ve glia hücreleri de sinaptik ağa bağlı ve bu nedenle sinir görüntü işleme 6 katılabilir. Son olarak, lamina R1-R6 ve R6 ve R7 / R8 arasındaki fotoreseptör komşu bağlayan aksonal boşluk kavşaklar, her bir kartuş 14,20,28 asimetrik bilgi temsili ve işlenmesine katkıda bulunur. Neredeyse sağlam Drosophila bireysel fotoreseptör ve görsel internöronlar hücre içi gerilim kayıtları yüksek sinyal-gürültü r sağlamakBağlı nöronlar arasındaki hızlı sinirsel hesaplamaların içyüzü için gerekli olan alt milisaniye çözünürlük 3,5,7-10,29 en ATIO veri. 100 milisaniye çözünürlük - bu kesinlik düzeyi önemli ölçüde gürültüsü ve genellikle 10 çalışmasına mevcut optik görüntüleme teknikleri ile mümkün değildir. elektrotlar çok küçük ve keskin ipucu var çünkü Dahası, yöntem hücre gövdeleri ile sınırlı değildir, ama küçük aktif nöral yapıların doğrudan kayıtları sağlayabilir; Böyle yama-kelepçe elektrotların çok daha büyük ipuçları ile erişilemez LMCs &#39;dendritik ağaçlar ya da fotoreseptör akson gibi. Önemlisi, yöntem aynı zamanda en yama-kelepçe uygulamalarında daha yapısal az invaziv ve zararlı olduğunu ve bu yüzden daha az çalışılan hücrelerin hücre içi ortamı ve bilgi örnekleme etkiler. Böylece, geleneksel keskin mikroelektrot teknikleri katkıda ve nöral infor içine temel buluşlar ve orijinal fikir katkıda bulunmaya devam varuygun zaman ölçeğinde mation işleme; vizyon 3-10 bizim mekanistik anlayış geliştirmek. Drosophila R1-R6 fotoreseptör ve LMCs gelen vivo hücre içi kayıtlarda Juusola laboratuvarında yapılmaktadır nasıl Bu makalede anlatılmaktadır. Bu protokol, uygun bir elektrofizyoloji teçhizat nasıl oluştuğunu açıklamak sinek hazırlamak ve kayıtları seslendirecek. Bazı temsilci veriler sunulmuştur ve bazı ortak sorunları ve olası çözümleri bu yöntemi kullanırken karşılaşılan olabileceğini tartışılmıştır.

<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.

Drosophila göz için buraya uyarlanmış keskin mikroelektrot kayıt yöntemi, güvenilir onları 4,5,7,8,10,33 arasında sinirsel bilgi örnekleme ve işleme retina ve lamina hücrelerinde ve iletişim ölçmek için kullanılabilir. Farklı vahşi tip hisse senetleri, mutantların veya genetik mühendisliği sinek suşları kodlama incelemek için kullanarak, yöntem değerini kanıtlamıştır; sadece bir mutasyonun, sıcaklık, beslenme ya da seçilen ifade 3,4,6,9,10,14,30,34 etkilerinin miktarının, aynı zamanda değiştirilmiş görsel davranışlar 14,34 için mekanik nedenler ortaya çıkarmakta. Yöntem aynı zamanda neuroethological görme çalışmalarını güçlendirerek diğer böcek hazırlıkları 35,36, kolayca uygulanabilir. Sonraki biz başarılı uygulamalar birkaç örnek vitrin. <img alt="Şekil 7," src="/files/ftp_upload/54142/54142fig7.jpg" /> Şekil 7. GerilimIşık 20 ° C&#39;de Pulse ve bir Meyve Sineği R1-R6 fotoreseptör Cevapları 25 o keskin mikroelektrot girmeler genellikle çok kararlı olduğundan C, en belirli bir ışık uyarısına aynı R1-R6 fotoreseptör gerilim yanıtları kaydetmek mümkündür ısınma veya fly soğutma farklı ortam sıcaklıkları. Bizim set-up, sinek tutucu bir kapalı döngü Peltier elemanı tabanlı sıcaklık kontrol sistemi yerleştirilir. Bu saniye içinde sineğin kafası sıcaklığını değiştirmek için bize sağlar. Daha yüksek sıcaklık gerilim yanıtları hızlandırır ve karakteristik (kırmızı oklarla gösterildiği gibi) R1-R6 fotoreseptör dinlenme potansiyelini düşürür. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig7large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Fotoreseptör Çıktı Sıcaklığın Etkisi incelenmesi <p class="jove_content" fo:keep-together.within-page = &quot;1&quot;&gt; ile iyi tasarlanmış ve titreşim izole kayıt sistemi, yöntem ısınmanın tek bir hücrenin sinir çıkış üzerine sıcaklığın etkisini ölçmek veya fly soğutma için kullanılabilir. Verilen örnek 20 ve 25 ° C (Şekil 7) aynı R1-R6, fotoreseptör kaydedilen parlak 10 msn uzun bir pals, voltaj tepkilerini gösterir. 4,9 önce sayısal olarak, ısınma karanlıkta bir fotoreseptör dinlenme potansiyelini düşürür ve onun gerilim yanıtları hızlandırır. <img alt="Şekil 8," src="/files/ftp_upload/54142/54142fig8.jpg" /> Şekil 8. Sinyal Performansı bir Meyve Sineği R1-R6 fotoreseptör Işık Şiddeti ile Geliştirdi (A) fotoreseptör çıkışı (aşağıda) loş ve parlak. (Yukarıda 10,000 kat daha parlak ışık) aynı mikroelektrot tarafından kaydedilen natüralist ışık yoğunluğu zaman serisini tekrarlanan20 o C&#39;de aynı hücrede Onlar daha fazla numune, tek fotonlar 4,5,7,8 ilköğretim yanıtları (diken) entegre çünkü parlak uyarana Cevapları, büyüktür. (B) 20 ardışık bir saniyelik uzun gerilim yanıtları bindirilmiş. Bireysel tepkiler (açık gri) (A ok) adaptif eğilimleri sonra alınmıştır (A noktalı kutu) gerilemiştir almıştı. İlgili tepki araçları (sinyaller) koyu izleri vardır. sinyal ve bireysel tepkiler arasındaki fark gürültüdür. (C) hücreleri standart yöntemler 4,5,7,8 kullanarak Sinyal-Gürültü Oranları (SNR) &#39;sinyal performans kayıtlarıyla ölçüldü.&#39; Fotoreseptör çıkış s ile, &#39;(20 Hz kadar, Dim ≥1 SNR) dim olduğundan daha (Parlak ≥1, en ​​fazla 84 Hz parlak stimülasyon güvenilir sinyalizasyon yaklaşık 64 Hz geniş yelpazede SNR)&#39; vardırignal-gürültü oranı büyük ölçüde iyileştirilmesi; SNR Dim MAX = SNR Parlak MAX 87 = 1.868 den. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig8large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Tekrarlanan Uyarım tarafından Uyum ve Sinir Kodlama incelenmesi Retina ve lamina yapıları nispeten daha az hasara neden yöntemin girişimsel, in vivo olarak yakın doğal fizyolojik durumu farklı ışık uyaranlara tek tek hücrelerin sinyal performansını incelemek için idealdir. 8 gösteren bir R1- gerilim yanıtları Şekil 20 o C&#39;de loş ve parlak tekrarlanan doğal ışık yoğunluğu zaman serisi uyarana R6 fotoreseptör Şekil 9 ise </strong&gt; 25 o C sıcaklıkta farklı bir doğal uyarana başka R1-R6 fotoreseptör ve LMC tepkilerini gösterir Aynı sinek iki keskin Mikroelektronlar, retinada birinde ve lamina diğer eş zamanlı hücre içi kayıtları, 30 yaşayabilir olması çok zor, çünkü öncesi ve postsinaptik kayıtları iki farklı sinekler ayrı yapıldı. <img alt="Şekil 9," src="/files/ftp_upload/54142/54142fig9.jpg" /> 25 o C (A) (gri) R1-R6 ve LMC (siyah) Tekrarlanan Doğal Stimülasyon bir Meyve Sineği R1-R6 fotoreseptör ve LMC Şekil 9. Gerilim Yanıtları farklı sinekler farklı Mikroelektronlar tarafından kaydedilen çıkışları. (B) ışık gösterilen tamamen ışık adapte ardışık 20 ön- (edilmiş) ve tek tek yanıtları aynı doğal teşvik desenine postsinaptik (aşağıda) tepkileri, bir grikoyu izleri olarak d ilgili yanıt araçları (sinyaller). sinyal ve bireysel tepkiler arasındaki fark gürültüdür. (C) hücreleri Sinyal-Gürültü Oranları (SNR) &#39;sinyal performans kayıtlarıyla ölçüldü.&#39; LMC çıkış &#39;(en fazla 94 Hz R ≥1, SNR) R1-R6 çıkışı daha (104 Hz kadar LMC ≥1, SNR)&#39; güvenilir sinyal yaklaşık 10 Hz geniş bir alanı vardır. Her iki sinyal-gürültü oranı (SNR LMC MAX = 142, SNR R MAX = 752) yüksek ve kayıt gürültü düşük olduğu gibi, aralarındaki farklılıkları hücreler arasındaki gerçek kodlama farklılıkları yansıtacak. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig9large.jpg" target="_blank">Büyük halini görmek için tıklayınız bu figür.</a> uyaranın başlangıcından sonra, kayıtları tipikly hızlı büyük ölçüde 5-6 saniye içinde azalır eğilimleri adapte göstermektedir. O andan itibaren, hücreler her 1 sn uzun uyaran tanıtımı için son derece tutarlı tepkiler (her noktalı kutu bunlardan 20 saran) üretirler. Bu (Şekil 8B ve Şekil 9B) üst üste zaman yanıtların tekrarlanabilirlik belirginleşmektedir. Bireysel tepkiler ince gri izleri vardır ve onların kalın koyu iz anlamına gelir. Sinir Gürültü ortalama ve her bir tepkisi 4,5,9,37,38 arasındaki fark ise, ortalama tepki sinir sinyali olarak alınır. Frekans alanında, ilgili bir sinyal-gürültü oranı (Şekil 8C ve Şekil 9), elektrikli spektrumları halinde sinyal ve ses veri parçaları Fourier dönüştürme ve karşılık gelen ortalama gürültü gücü spektrumunun 4 ortalama sinyal güç spektrumu bölünmesiyle elde edilmiştir 5,9,37,38. Karakteristik olarak, yüksek bir sinyal-gürültü oranı Onatüralist uyarıya kaydedilen sinir çıkışlar (100 - 1000) yüksek f &gt;&gt; değerlere ulaşabilir ve çok düşük kayıt gürültü ile en istikrarlı hazırlıkları 1000 (örneğin, Şekil 8C.). Uyarı Ayrıca ısınma hücrelerin genişletiyor &#39;sinyal güvenilir bant genişliği 4 (SNR&#39; Parlak ≥ 1); Örneğin, Şekil 8 ve 9, iki R1-R6-S arasındaki nispi farkı sırasıyla, 10 Hz (84 de 20 ° C ve 94 Hz, 25 o C&#39;de) &#39;dir. Bir daha Shannon denklemi 32, kullanarak veya üçlü ekstrapolasyon yöntemiyle 39 ile CEVAPLAR &#39;entropi ve gürültü entropi oranları arasındaki farkı hesaplayarak kendi sinyal-gürültü oranı bilgi transferi her hücrenin oranını tahmin edebilirsiniz. Daha fazla bilgi kuramsal analizler hakkında ayrıntılar ve bunların kullanımı ve sınırlamaözellikle, bu yöntemle daha önceki yayınlarda 7,8,39 verilmiştir s. <img alt="Şekil 10," src="/files/ftp_upload/54142/54142fig10.jpg" /> 19 o C (A) R1-R6 (gri) ve LMC (siyah) aynı sinek aynı mikroelektrot tarafından kaydedilen çıkışlar da tekrarlanan Doğal Stimülasyon a Killer Fly R1-R6 fotoreseptör ve LMC Şekil 10. Gerilim Tepkiler; Elektrot göz ileri gibi, daha sonra, ilk presynaptically postsynaptically ve. Aynı doğal teşvik desene (B) (yukarıda), 20 üst üste öncesi ve postsinaptik (aşağıda) cevaplar (açık gri izleri) başlangıçtaki uyarlama (A noktalı kutu) sonra ele geçirildi. Bireysel yanıtları kendi farklılıkları gürültü verirken kendi araçları, sinyalleri (üstte koyu izleri) bulunmaktadır. (C) karşılık gelen SignaL-gürültü oranı (SNR) Şekiller 8C ve 9C&#39;deki gibi hesaplanmıştır. LMC çıkış &#39;(134 Hz kadar R MAX ≥1 SNR) R1-R6 çıkışı daha (en fazla 234 Hz LMC MAX ≥1, SNR)&#39; güvenilir sinyalizasyon 100 Hz daha geniş hakkında sahiptir. Her iki sinyal-gürültü oranı (SNR LMC MAX = 137, SNR R MAX = 627) yüksek ve aynı mikroelektrot kayıtlarında kullanılan gibi, aralarındaki farklılıkları öncesi ve postsinaptik nöral çıkışları gerçek farklılıkları yansıtmaktadır. Bu sonuçlar kayıt sistemi, düşük gürültü vardı ve analizler üzerindeki etkisi marjinal oldu. Ima <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54142/54142fig10large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> Neuroethological Vizyon Çalışmaları </p&gt; Yöntem aynı zamanda görsel bilgi işleme karşılaştırmalı neuroethological çalışmalar izin, farklı böcek türlerinin 7,8,35,36 bileşik göz (Şekil 10) den öncesi ve postsinaptik gerilim yanıtları kaydetmek için kullanılabilir. sunulan kayıt sistemi için, sadece gerekli adaptasyon çalışılan türler için uygun bir büyüklükte açılması ile yeni hazırlık sahipleri, her biri. Bu örnek kayıtlar bir kadın katil sinek (Coenosia attenuata) uzaklık vardır. Onlar Şekil 9&#39;da Drosophila meslektaşları için kullanıldığı gibi, aynı tekrarlayan ışık uyarısına bir R1-R6 fotoreseptör ve LMC hücre içi gerilim tepkileri gösterir, ancak 19 o C sıcaklıkta Bu durumda, ön-ve postsinaptik veriler aynı sinek kaydedildi; bir birinci yanal lamina boyunca ilerleyen (3 M KCl ile dolu) aynı kayıt elektrodu ile, diğer daha önce girdikten sonraFrontal retina ing. Hatta soğutucu sıcaklığı - - 25 o C, Coenosia veri Drosophila verilerine kıyasla daha hızlı dinamikleri ile tepkilerini gösterir; güvenilir sinyalizasyon (sinyal-gürültü oranı &gt;&gt; 1) daha geniş bir frekans aralığında aralığını genişletiyor. Natüralist uyaranlara nöral kodlama Böyle fonksiyonel uyarlamalar hızlı hava av davranışları ulaşmak için yüksek hassasiyetli zamanmekansal bilgi gerektiren Coenosia &#39;ın yırtıcı yaşam tarzı 36, tutarlıdır.

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.

Recording Hücre içi Gerilim Yanıtları için Elektrofizyolojik Yöntem<em&gt; Drosophila</em&gt; Işık Uyaranların için Fotoreseptörler ve internöronlar<em&gt; İn 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 Görüntüleme.  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.

Video: 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.

Drosophila Larva NMJ Synaptic Potansiyeller kayıt için Elektrofizyolojik Yöntemler

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

Biyoloji

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.

Elektrofizyolojik Kayıt Drosophila Embriyo

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.

Elektrofizyolojik Kayıt<em&gt; Drosophila</em&gt; Labellar Sensilla Taste

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Nörobilim

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.

Fizyolojik Yanıtları Ölçüm<em&gt; Drosophila</em&gt; Canlı Kalsiyum Görüntüleme kullanma Lipit Feromonlar Sensory Nöronlar

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

İmmünoloji ve Enfeksiyon

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.

bir Böcek Modeli fotoreseptör hücre spektral Duyarlılık Belirlenmesi<em&gt; İn Vivo</em&gt; Hücre içi Kayıtlar

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.

Tam Hücreli Gerilim Kelepçesi Kayıtları için Elektrofizyolojik Yöntem<em&gt; Drosophila</emPhotoreceptors

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.

Üçüncü-biçim Drosophila Melanogaster merkezi sinir sisteminin etkinliğinin elektrofizyolojik kayıt

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;

Drosophila Fotoreseptörlerinde Fotopigment Düzeylerini Ölçmek için Elektrofizyolojik Yöntemler

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.

Drosophila Retinal Fotoreseptörlerinin Işık Uyaranlarına Voltaj Yanıtlarının Elektrofizyolojik Kaydı

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.

Drosophila Merkezi Sinir Sisteminde Elektriksel Aktivitenin Hücre Dışı Kaydı

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

Recording Hücre içi Gerilim Yanıtları için Elektrofizyolojik Yöntem

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Elektrofizyolojik Kayıt Drosophila Embriyo

Elektrofizyolojik Kayıt

Fizyolojik Yanıtları Ölçüm

bir Böcek Modeli fotoreseptör hücre spektral Duyarlılık Belirlenmesi

Tam Hücreli Gerilim Kelepçesi Kayıtları için Elektrofizyolojik Yöntem

Üçüncü-biçim Drosophila Melanogaster merkezi sinir sisteminin etkinliğinin elektrofizyolojik kayıt

Drosophila Fotoreseptörlerinde Fotopigment Düzeylerini Ölçmek için Elektrofizyolojik Yöntemler

Drosophila Retinal Fotoreseptörlerinin Işık Uyaranlarına Voltaj Yanıtlarının Elektrofizyolojik Kaydı

Drosophila Merkezi Sinir Sisteminde Elektriksel Aktivitenin Hücre Dışı Kaydı