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.

La mosca della frutta occhio (Drosophila melanogaster) composto è un sistema di grande modello per studiare l&#39;organizzazione funzionale degli array fotorecettori e interneuroni per il campionamento e l&#39;elaborazione di immagini neurali, e per la visione degli animali. Il sistema ha lo schema elettrico più completa 1,2 ed è amabile a manipolazioni genetiche e accurato monitoraggio dell&#39;attività neuronale (di alto rapporto segnale-rumore e risoluzione temporale) 3-10. L&#39;occhio Drosophila è modulare, contenente ~ 750 strutture di lenti-capped apparentemente regolari chiamati ommatidi, che insieme forniscono il campo visivo panoramico che copre quasi tutte le direzioni intorno alla sua testa volare. Informazioni primario della occhio di campionatura unità sono i suoi fotorecettori rabdomerici 7,8,11. Ogni ommatidium contiene otto fotorecettori (R1-R8), che condividono lo stesso obiettivo sfaccettatura, ma sono allineati a sette diverse direzioni. Mentre i fotorecettori esterni R1-R6 are più sensibile alla luce blu-verde, sensibilità spettrale delle celle interne R7 e R8, che si trovano l&#39;uno sopra l&#39;altro e puntare nella stessa direzione, presentano tre sottotipi distintive: pallido, giallo e dorsale zona cerchio (DRA) 12- 15. <img alt="Figura 1" src="/files/ftp_upload/54142/54142fig1.jpg" /> Figura 1. Organizzazione funzionale della Drosophila Eye. (A) I primi due gangli ottico, retina e la lamina, sono evidenziati in grigio all&#39;interno dell&#39;occhio mosca. Fotorecettori Retina R1-R6 e lamina grandi cellule monopolare (MLC: L1-L3) sono facilmente accessibili in vivo alle registrazioni di microelettrodi taglienti convenzionali. L&#39;elettrodo schema evidenzia il percorso normale di registrare da R1-R6 nella retina. Un percorso di registrare da LMCs nella lamina è di spostare parallelamente l&#39;elettrodo a sinistra. (B) Lamina è una matrice di organo retinotopicallycartucce ized, ognuno dei quali è ricco di neuroni che elabora le informazioni da una specifica piccola area nello spazio visivo. A causa della sovrapposizione neurale, sei fotorecettori provenienti da diversi ommatidi vicini inviano i loro assoni (R1-R6) per la stessa cartuccia lamina, formando sinapsi uscita istaminergici a L1-L3 e una cella amacrine (Am). (C) La diffusione delle informazioni neurali tra i terminali degli assoni R1-R6 e le interneuroni visivi (compresi L4, L5, Lawf, C2, C3 e T1), all&#39;interno di una cartuccia lamina è complessa. (D) R1-R6 assoni fotorecettori ricevono una valutazione sinaptiche da cellule monopolare L2 e L4. (B) e (C) modificato da Rivera-alba et al 2. <a href="https://www.jove.com/files/ftp_upload/54142/54142fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> L&#39;occhio Drosophila è del tipo neurale sovrapposizione 16. Questo significa tcappello i segnali neurali di otto fotorecettori appartenenti a sette ommatidi vicina, che guardano allo stesso punto dello spazio, vengono riunite insieme in una cartuccia neurale nei prossimi due neuropils: la lamina e midollo. Mentre i sei fotorecettori esterni progetto R1-R6 loro terminali assoni alle colonne neurali nel lamina (figura 1), cellule R7 e R8 bypassare questo strato e rendere contatti sinaptici con la loro corrispondente colonna medulla 17-19. Questi cablaggi esatte producono il substrato neurale per la mappatura retinotopica di volare visione iniziale, dopo di che ogni lamina (Figure 1A-C) e la colonna midollare (cartuccia) rappresenta un singolo punto nello spazio. Ingressi diretti da fotorecettori R1-R6 sono ricevuti dai grandi cellule monopolare (MLC: L1, L2 e L3) e la cella amacrine (AM) nella lamina 1,2,20. Di questi, L1 e L2 sono i più grandi cellule, che mediano le principali vie di informazione (Figura 1D), WHIch rispondono alla On-Off e bordi off-movimento, e quindi costituiscono la base di calcolo del rilevatore di movimento 21,22. Esperimenti comportamentali suggerisce che in contrasto intermedia, i due percorsi facilitano il movimento percezione di direzioni opposte: back-to-front in L1 e frontale-to-back in cellule L2 23,24. Connettività implica inoltre che i neuroni L4 possono svolgere ruolo fondamentale nella comunicazione laterale tra cartucce vicini 25,26. sinapsi reciproci sono stati trovati tra le cellule L2 e L4 situati nello stesso e due cartucce adiacenti. A valle, ogni cella L2 e le sue tre celle L4 associati proiettano i loro assoni ad un obiettivo comune, il neurone Tm2 nel midollo, dove si ritiene ingressi da cartucce vicini per essere integrato per l&#39;elaborazione di front-to-back motion 27. Anche se i neuroni L1 ricevono input da persone dello stesso cartuccia L2S via sia giunzioni e sinapsi, che non sono direttamente collegati alla cartucce lamina l4s e quindi adiacenti. <pclass = &quot;jove_content&quot;&gt; una valutazione Synaptic per R1-R6 assoni fotorecettori sono forniti solo dai neuroni appartenenti ai circuiti / L4 L2, ma non il percorso di 1,2 L1 (Figura 1D). Mentre i collegamenti dello stesso cartuccia sono selettivamente da L2 a R1 e R2 e da L4 a R5, tutti i fotorecettori R1-R6 ricevono un feedback sinaptica da L4 di una o entrambe le cartucce confinanti. Inoltre, ci sono forti connessioni sinaptiche da Am a R1, R2, R4 e R5, e cellule gliali sono anche sinapticamente collegati alla rete e possono quindi partecipare neurale elaborazione delle immagini 6. Infine, assonale gap giunzioni, che collega la vicina R1-R6 e tra R6 e R7 / R8 fotorecettori nella lamina, contribuiscono alla rappresentazione informazione asimmetrica e la lavorazione in ogni cartuccia 14,20,28. Registrazioni di tensione intracellulari di singoli fotorecettori e interneuroni visivi in quasi intatto Drosophila fornire alta R segnale-rumoredati azio con risoluzione sub-millisecondo 3,5,7-10,29, che è necessaria per dare un senso dei calcoli neurali veloci tra i neuroni connessi. Questo livello di precisione è impossibile dalle attuali tecniche di imaging ottico, che sono significativamente più rumoroso e tipicamente operano a 10-100 risoluzione msec. Inoltre, poiché gli elettrodi sono molto piccoli e taglienti punte, il metodo non è limitato a corpi cellulari, ma può fornire registrazioni diretti da piccole strutture neurali attive; come alberi dendritiche i LMCS &#39;o assoni dei fotorecettori, che non possono essere accessibili da molto più grandi punte di elettrodi di patch-clamp. È importante sottolineare che il metodo è anche strutturalmente meno invasiva e dannosa quanto la maggior parte delle applicazioni di patch-clamp, e quindi incide meno campionamento ambiente e le informazioni intracellulare delle cellule studiate. Così, convenzionali tecniche di microelettrodi taglienti hanno contribuito, e continuare a contribuire, scoperte fondamentali e visione originale in infor neuraleelaborazione mazioni alla scala momento opportuno; migliorare la nostra comprensione meccanicistica della visione 3-10. Questo articolo spiega come nelle registrazioni intracellulari vivo da Drosophila fotorecettori R1-R6 e MLC vengono eseguite in laboratorio Juusola. Questo protocollo descrive come costruire un adeguato impianto di elettrofisiologia, la preparazione al volo, ed eseguire le registrazioni. Alcuni dati rappresentativi è presentato, e alcuni problemi comuni e le possibili soluzioni sono discusse che possono verificarsi quando si utilizza questo metodo.

<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.

Il metodo di registrazione microelettrodo tagliente, adattata qui per l&#39;occhio Drosophila, può essere utilizzato per quantificare attendibilmente neurale campionamento ed elaborazione nelle cellule della retina e lamina, e la comunicazione tra loro 4,5,7,8,10,33. Utilizzando per studiare la codifica in diversi titoli wild-type, mutanti o ceppi mosca geneticamente modificati, il metodo ha dimostrato il suo valore; non solo nel quantificare gli effetti di una mutazione, temperatura, dieta o espressione selezionata 3,4,6,9,10,14,30,34, ma anche nel rivelare motivi meccanicistici per comportamenti visivi alterati 14,34. Il metodo è anche facilmente applicabile ad altri preparati insetti 35,36, che abilita gli studi visione neuroethological. Poi abbiamo vetrina alcuni esempi delle sue applicazioni di successo. <img alt="Figura 7" src="/files/ftp_upload/54142/54142fig7.jpg" /> Figura 7. TensioneRisposte di frutta mosca R1-R6 fotorecettore ad un impulso di luce a 20 e 25 ° C. Poiché le penetrazioni di microelettrodi taglienti sono spesso molto stabile, è possibile registrare le risposte di tensione della stessa fotorecettore R1-R6 ad un dato stimolo luce diverse temperature ambiente tramite riscaldamento o raffreddamento al volo. Nei nostri assetti, fly-custodia viene posta su un sistema di regolazione della temperatura close-loop-based Peltier-elemento. Questo ci permette di cambiare la temperatura testa della mosca in pochi secondi. Temperatura più elevata accelera le risposte di tensione e tipicamente riduce il potenziale di riposo dei fotorecettori R1-R6 (come indicato dalle frecce rosse). <a href="https://www.jove.com/files/ftp_upload/54142/54142fig7large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Studiando l&#39;effetto della temperatura sulla fotorecettori uscita <p class="jove_content" fo:keep-together.within-page = &quot;1&quot;&gt; Con un ben progettato e sistema di registrazione vibrazioni isolato, il metodo può essere utilizzato per misurare l&#39;effetto della temperatura sulla produzione neurali di una singola cella tramite riscaldamento o raffreddamento al volo. La proposta esempio mostra le risposte di tensione ad un luminoso lungo 10 msec impulso, registrata nello stesso fotorecettore R1-R6 a 20 e 25 ° C (Figura 7). Come quantificati prima di 4,9, il riscaldamento riduce il potenziale di riposo di un fotorecettore nel buio, e accelera le sue risposte di tensione. <img alt="Figura 8" src="/files/ftp_upload/54142/54142fig8.jpg" /> Figura 8. segnalazione delle prestazioni di un Fruit Fly R1-R6 fotorecettore migliora con intensità della luce (A) Uscita fotorecettore a dim (sotto) e luminosi. (Di cui sopra; 10.000 volte più luminosa luce) ripetuti naturalistico serie tempo l&#39;intensità della luce registrata dalla stessa microelettrodonella stessa cella a 20 ° C. Le risposte allo stimolo luminoso sono più grandi, perché integrano più campioni, risposte elementari (dossi) a singoli fotoni 4,5,7,8. (B) 20 risposte tensione consecutivi di un secondo-lunghe sono sovrapposti. Risposte individuali (grigio chiaro) sono state prese dopo che le tendenze adattivi (freccia in A) si era ritirato (scatola tratteggiata in A). I mezzi di risposta corrispondente (segnali) sono le tracce più scure. La differenza tra il segnale e le risposte individuali è il rumore. (C) Le cellule &#39;prestazioni di segnalazione è stato quantificato dalle registrazioni&#39; Signal-to-Noise Ratio (SNR) utilizzando i metodi standard di 4,5,7,8. Uscita fotorecettore ha circa 64 Hz più ampia gamma di segnalazione affidabile alla stimolazione luminosa ( &#39;≥1 brillante, fino a 84 Hz rispetto alla dim (SNR SNR)&#39; Dim ≥1, fino a 20 Hz), con signal-to-Noise Ratio migliorando notevolmente; da SNR Dim MAX = 87 a SNR luminoso MAX = 1.868. <a href="https://www.jove.com/files/ftp_upload/54142/54142fig8large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> Studiare Adattamento e Neural codifica per Repetitive stimolazione La non invasività del metodo, causando relativamente pochi danni alle strutture retina e lamina, lo rende ideale per studiare le prestazioni segnalazione di singole celle a differenti stimoli luminosi in loro prossimità stato fisiologico in vivo. La figura 8 mostra le risposte di tensione di un R1- R6 fotorecettore a una ripetuta serie naturalistica stimolo tempo l&#39;intensità della luce fioca e luminoso a 20 ° C, mentre la figura 9 </strong&gt; mostra le risposte di un&#39;altra fotorecettori R1-R6 e LMC a un diverso stimolo naturalistico a 25 ° C. Le registrazioni pre-e post-sinaptici sono state eseguite separatamente da due mosche diverse perché simultanee registrazioni intracellulari di due taglienti microelettrodi nella stessa mosca, uno nella retina e l&#39;altro nella lamina, sono troppo difficili da essere vitali 30. <img alt="Figura 9" src="/files/ftp_upload/54142/54142fig9.jpg" /> Figura 9. Le risposte di tensione di un frutto Fly R1-R6 fotorecettori e LMC a ripetuta stimolazione naturalistico a 25 ° C (A) R1-R6 (grigio) e LMC (nero) uscite registrate da diverse microelettrodi provenienti da diverse mosche. (B) 20 pre completamente luce-adattato consecutivi (sopra) e post-sinaptici (sotto) risposte al stesso modello di stimolo naturalistico con risposte individuali, mostrata in grigio chiaro und i corrispondenti mezzi di risposta (segnali) come le tracce più scure. La differenza tra il segnale e le risposte individuali è il rumore. (C) Le cellule &#39;prestazioni di segnalazione è stato quantificato dalle registrazioni&#39; Signal-to-Noise Ratio (SNR). Uscita LMC ha circa 10 Hz più ampia gamma di segnalazione affidabile (SNR &#39;LMC ≥1, fino a 104 Hz) di uscita R1-R6 (SNR&#39; R ≥1, fino a 94 Hz). Entrambi i rapporti segnale-rumore sono elevati (SNR LMC MAX = 142, SNR R MAX = 752), e come il rumore di registrazione era basso, le loro differenze riflettono le differenze di codifica reali tra le cellule. <a href="https://www.jove.com/files/ftp_upload/54142/54142fig9large.jpg" target="_blank">Cliccate qui per vedere una versione più grande questa figura.</a> Dopo l&#39;esordio di stimolo, le registrazioni tipichely vedi veloce adattando le tendenze che in gran parte regrediscono entro 5-6 secondi. Da allora in poi, le cellule producono risposte altamente coerenti per ogni presentazione stimolo lungo 1 sec (ogni casella tratteggiata racchiude 20 di questi). La ripetibilità delle risposte diventa evidente quando questi sono sovrapposti (Figura 8B e Figura 9B). le risposte individuali sono le sottili tracce di grigio, e la loro media la traccia scura spessa. La risposta media è preso come segnale neurale, mentre il rumore neurale è la differenza tra la media e ogni 4,5,9,37,38 risposta individuale. I rispettivi rapporti segnale-rumore nel dominio della frequenza (Figura 8C e Figura 9C) sono stati ottenuti Fourier-trasformando il segnale e rumore blocchi di dati in spettri di potenza, e dividendo lo spettro di potenza del segnale medio con il corrispondente spettro di potenza del rumore medio 4, 5,9,37,38. Tipicamente, i rapporti massimi segnale-rumore of uscite neurali registrati alla stimolazione naturalistico sono elevate (100 - 1000), e nelle preparazioni più stabili con il rumore di registrazione molto bassa può raggiungere valori &gt;&gt; 1,000 (ad esempio, figura 8C.). Si noti inoltre che il riscaldamento espande le cellule &#39;larghezza di banda affidabile segnalazione 4 (SNR&#39; luminoso ≥ 1); per esempio, la differenza relativa tra i due R1-R6S nelle figure 8 e 9, rispettivamente è 10 Hz (84 a 20 ° C e 94 Hz a 25 ° C). Si può stimare ulteriormente tasso di ogni cellula del trasferimento di informazioni dal rapporto segnale-rumore utilizzando l&#39;equazione Shannon 32, o calcolando la differenza tra entropia e rumorosità tassi entropia le risposte &#39;attraverso il metodo di estrapolazione tripla 39. Maggiori dettagli sulle informazioni analisi teoriche, e il loro uso e limitazionis specificamente con questo metodo sono riportati nelle pubblicazioni precedenti 7,8,39. <img alt="Figura 10" src="/files/ftp_upload/54142/54142fig10.jpg" /> Figura 10. Le risposte di tensione di un Killer Fly R1-R6 fotorecettori e LMC a ripetuta stimolazione naturalistico a 19 o C. (A) R1-R6 (grigio) e LMC (nero) uscite registrate dalla stessa microelettrodo dallo stesso volo; prima postsinaptica e poi presinapticamente, come l&#39;elettrodo è stato avanzato in un occhio. (B) 20 pre consecutive (sopra) e post-sinaptici (sotto) le risposte (tracce grigio chiaro) per lo stesso modello di stimolo naturalistico sono stati catturati dopo l&#39;adattamento iniziale (scatola tratteggiata in A). I loro mezzi sono i segnali (le tracce più scure in alto), mentre le loro rispettive differenze nelle singole risposte danno il rumore. (C) La corrispondente Signal-to-Noise Ratio (SNR) sono stati calcolati come nelle figure 8C e 9C. Uscita LMC ha circa una gamma più ampia di 100 Hz segnalazione affidabile (SNR &#39;LMC MAX ≥1, fino a 234 Hz) di uscita R1-R6 (SNR&#39; R MAX ≥1, fino a 134 Hz). Entrambi i rapporti segnale-rumore sono elevati (SNR LMC MAX = 137, SNR R MAX = 627), e come lo stesso microelettrodo è stato utilizzato nelle registrazioni, le loro differenze riflettono reali differenze nelle uscite neurali pre e post-sinaptici. Questi risultati implicano che il sistema di registrazione ha avuto a basso rumore, e la sua influenza sulle analisi è stato marginale. <a href="https://www.jove.com/files/ftp_upload/54142/54142fig10large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> Neuroethological Studi Vision </p&gt; Il metodo può anche essere usato per registrare le risposte pre e post-sinaptici tensione dagli occhi composti di diverse specie di insetti 7,8,35,36 (Figura 10), permettendo comparativi studi neuroethological di elaborazione delle informazioni visive. Per il sistema di registrazione presentata, l&#39;unico adattamento richiesto è nuovo preparato detentori, ognuna con un&#39;apertura opportunamente dimensionati per le specie studiate. Queste registrazioni sono esemplari da un killer di sesso femminile mosca (Coenosia attenuata). Essi mostrano risposte tensione intracellulari di un fotorecettore R1-R6 e LMC di identica stimolazione luminosa ripetitiva, usati per le controparti Drosophila in figura 9, ma a 19 o C. In questo caso, sia il pre e post-sinaptici dati sono stati registrati dal medesimo fly; uno dopo l&#39;altro, con lo stesso elettrodo di registrazione (riempito con KCl 3 M) prima avanzando attraverso la lamina laterale prima di entrareing retina frontale. Rispetto ai dati Drosophila a 25 ° C, i dati Coenosia - anche alla temperatura cooler - mostra risposte con dinamiche più veloci; ampliando la gamma di segnalazione affidabile (segnale-rumore &gt;&gt; 1) su una gamma di frequenza più ampia. Tali adattamenti funzionali in codifica neurale di stimoli naturalistici sono in linea con lo stile di vita predatori Coenosia &#39;s 36, che richiedono alta precisione le informazioni spazio-temporale per raggiungere i comportamenti di caccia aerei veloci.

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.

Metodo elettrofisiologiche per la registrazione intracellulare di tensione Risposte di<em&gt; Drosophila</em&gt; Fotorecettori e Interneuroni a luce stimoli<em&gt; In Vivo</em

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

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

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18.7K Views.  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.

Metodi elettrofisiologici per la registrazione Potenziali Synaptic dal NMJ Larve di Drosophila

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

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Biologia

Electrophysiological Recording in the Drosophila Embryo

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

Electrophysiological Recording in the Drosophila Embryo

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

Registrazione elettrofisiologiche in Drosophila Embryo

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.

Elettrofisiologici Registrazione da<em&gt; Drosophila</em&gt; Labellar Taste Sensilla

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

Neuroscienze

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.

Misurare risposte fisiologiche di<em&gt; Drosophila</em&gt; Neuroni sensoriali a Lipid feromoni utilizza Live Calcio Imaging

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

Immunologia e infezioni

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.

Determinazione del fotorecettore cellulare Sensibilità spettrale in un modello da insetti<em&gt; In Vivo</em&gt; registrazioni intracellulari

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.

Metodo elettrofisiologico per le registrazioni a morsetto di tensione a cellule intere da<em&gt; Drosophila</em&gt; Fotorecettori

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.

Registrazione elettrofisiologica dell'attività del sistema nervoso centrale di terza-Instar Drosophila Melanogaster

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

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

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

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

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

Metodi elettrofisiologici per misurare i livelli di fotopigmento nei fotorecettori di Drosophila

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

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

Source: Juusola, M., et al. <a href="https://app.jove.com/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.

Registrazione elettrofisiologica delle risposte di tensione dei fotorecettori retinici di Drosophila agli stimoli luminosi

1. Recording from R1-R6 Photoreceptors 

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

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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/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.

Registrazione extracellulare dell'attività elettrica nel sistema nervoso centrale della Drosophila

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

Metodo elettrofisiologiche per la registrazione intracellulare di tensione Risposte di

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Registrazione elettrofisiologiche in Drosophila Embryo

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Determinazione del fotorecettore cellulare Sensibilità spettrale in un modello da insetti

Metodo elettrofisiologico per le registrazioni a morsetto di tensione a cellule intere da

Registrazione elettrofisiologica dell'attività del sistema nervoso centrale di terza-Instar Drosophila Melanogaster

Metodi elettrofisiologici per misurare i livelli di fotopigmento nei fotorecettori di Drosophila

Registrazione elettrofisiologica delle risposte di tensione dei fotorecettori retinici di Drosophila agli stimoli luminosi

Registrazione extracellulare dell'attività elettrica nel sistema nervoso centrale della Drosophila