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.
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.
Bananflugan (Drosophila melanogaster) förening ögat är en stor modell för att undersöka den funktionella organisationen av fotoreceptorer och interneuronen arrayer för neurala bild provtagning och behandling, samt för djur vision. Systemet har den mest kompletta kopplingsschemat 1,2 och är älskvärd till genetiska manipulationer och korrekt neural aktivitet övervakning (av hög signal-till-brusförhållande och tidsupplösning) 3-10.
Drosophila ögat är modulärt, innehållande ~ 750 till synes vanliga lins tak strukturer som kallas ommatidia, som tillsammans ger flyga ett panorama synfält som täcker nästan varje riktning runt huvudet. Ögat primära uppgifter provtagning enheter är dess rhabdomeric fotoreceptorer 7,8,11. Varje ommatidium innehåller åtta ljusmätare celler (R1-R8), som delar samma aspekt objektiv men är i linje med sju olika riktningar. Medan de yttre fotoreceptorer R1-R6 are mest känsliga för blått-grönt ljus, spektrala känsligheter inre cellerna R7 och R8, som ligger ovanpå varandra och pekar på samma riktning, uppvisar tre distinkta subtyper: blek, gul och dorsala kantområdet (DRA) 12- 15.
Figur 1. funktionella organisation Drosophila Eye. (A) De två första optisk ganglier, näthinnan och lamina, är markerade i grått i farten ögat. Retina R1-R6 fotoreceptorer och lamina Stora Monopolär Cells (LMC: L1-L3) är lätt tillgängliga in vivo med konventionella vassa mikroelektrod inspelningar. Den schematiska elektroden belyser den normala väg att spela in från R1-R6 i näthinnan. En väg att spela in från LMC i lamina är att skifta parallellt elektroden till vänster. (B) Lamina är en matris av retinotopically organized kassetter, vilka vardera är packad med neuroner som bearbetar information från ett visst litet område i den visuella utrymme. På grund av neural super sex fotoreceptorer från olika grann ommatidia skicka sina axoner (R1-R6) till samma lamina patronen bildar histaminerga utgångs synapser till L1-L3 och en amakrina cell (Am). (C) Spridningen av neural information mellan R1-R6 axon terminaler och visuella intern (inklusive L4, L5, Lawf, C2, C3 och T1), i en lamina patronen är komplex. (D) R1-R6 ljusmätare axoner får synaptiska återkopplingar från L2 och L4 monopolära celler. (B) och (C) modifierad från Rivera-alba et al 2. Klicka här för att se en större version av denna siffra.
Drosophila ögat är av neural super typ 16. Detta betyder thatt neurala signaler från åtta fotoreceptorer som hör till sju grann ommatidia, som tittar på samma punkt i rymden, slås samman till en neural patron under de kommande två neuropils: lamina och märgen. Medan de sex yttre fotoreceptorer R1-R6 projekt sina axon terminaler till neurala kolumner i lamina (Figur 1), R7 och R8 celler kringgå detta skikt och göra synaptiska kontakter med deras motsvarande förlängda kolumn 17-19. Dessa exakta wirings producerar den neurala substrat för retinotopic kartläggning av flyg tidiga vision, varefter varje lamell (figurerna 1A-C) och medulla-kolonn (patron) representerar en enda punkt i rymden.
Direkta insignaler från R1-R6 fotoreceptorer tas emot av Stora Monopolär Cells (LMC: L1, L2 och L3) och amakrina Cell (Am) i lamina 1,2,20. Av dessa, L1 och L2 är de största cellerna, förmedla viktiga informationsvägar (figur 1D), WHIch svara på på och utanför rörliga kanter, och på så sätt bilda beräknings grund av rörelsedetektorn 21,22. Beteendeförsök tyder på att vid mellan Däremot två vägar underlättar rörelse uppfattning om motsatta riktningar: back-to-front i L1 och främre-to-back i L2-celler 23,24. Anslutning innebär vidare att L4 nervceller kan spela avgörande roll i sidled kommunikation mellan angränsande patroner 25,26. Ömsesidiga synapser påträffades mellan L2 och L4 celler som ligger i samma och två angränsande patroner. Nedströms, varje L2 cell och dess tre tillhörande L4 celler projicera sina axoner till ett gemensamt mål, att Tm2 neuron i märgen, där ingångar från angränsande patroner tros integreras för bearbetning av front-to-back rörelse 27. Även L1 nervceller får input från samma kassett L2S via både kanalförbindelser och synapser, de är inte direkt kopplad till L4s och därmed angränsande lamina patroner.
<pclass = "jove_content"> Synaptic kopplingar till R1-R6 ljusmätare axoner endast tillhandahålls av neuroner som tillhör de L2 / L4 kretsar men inte L1 vägen 1,2 (figur 1D). Medan samma patron anslutningar är selektivt från L2 till R1 och R2 och från L4 till R5, alla R1-R6 fotoreceptorer får synaptiska feedback från L4 av endera eller båda grann patroner. Dessutom finns det starka synapsförbindelser från Am till R1, R2, R4 och R5, och gliaceller är också synaptically ansluten till nätverket och kan därmed delta i neural bildbehandling 6. Slutligen, axonal gap-junctions, som förbinder angränsande R1-R6 och mellan R6 och R7 / R8 fotoreceptorer i lamina, bidra till asymmetrisk information representation och behandling i varje patron 14,20,28.Intracellulära spännings inspelningar från enskilda fotoreceptorer och visuella intern i nästan intakt Drosophila ger hög signal-brus-rIG data vid sub-millisekund upplösning 3,5,7-10,29, vilket är nödvändigt för att göra känsla av de snabba neurala beräkningar mellan de anslutna nervceller. Denna precisionsnivå är omöjligt med dagens optiska avbildningstekniker, som är betydligt bullrigare och vanligtvis arbetar vid 10-100 ms upplösning. På grund elektroderna har mycket små och vassa spetsar, metoden är inte begränsad till cellkroppar, men kan ge direkta inspelningar från små aktiva neurala strukturer; såsom LMC "dendritiska träd eller fotoreceptor axoner, som inte kan nås av mycket större tips av patch-clamp elektroder. Viktigt är metoden också strukturellt mindre invasiv och skadliga än de flesta patch-clamp applikationer, och så förekommer hos färre de studerade cellernas intracellulär miljö och informations provtagning. Således har konventionella skarpa mikroelektrodteknik bidragit, och hålla på att bidra, grundläggande upptäckter och original inblick i neural information bearbetning vid den lämpliga tidsskala; förbättra vår mekanistisk förståelse av synen 3-10.
Den här artikeln förklarar hur in vivo intracellulära inspelningar från Drosophila R1-R6 fotoreceptorer och LMC utförs i Juusola laboratorium. Detta protokoll kommer att beskriva hur man konstruerar en lämplig elektro rigg, förbereda flugan, och utföra inspelningarna. Några representativa data presenteras, och några vanliga problem och möjliga lösningar diskuteras som kan uppstå vid användning av denna metod.
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.
The authors have nothing to disclose.
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).
Stereo Zoom Microscope for making the fly preparation | Olympus | SZX12 DFPLFL1.6x PF eyepieces: WHN30x-H/22 | Capable of ~150X magnification with long working distance; bespoke heavy steel table mount stand |
Stereomicroscope in the intracellular set-up | · Olympus | Olympus SZX7; eyepieces: WHN30x-H/22 | 30x eyepieces are needed for seeing the electrode tip reflections well when driving it through the small corneal hole into the eye |
· Nikon | Nikon SMZ645; eyepieces: C-W30x/7 | ||
Anti-vibration Table | · Melles Griot | With metric M6 holes on the breadboard | 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 |
· Newport | |||
Micromanipulators | · Narishige | · Narishige NMN-21 | 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. |
· Huxley Bertram | · Huxley xyz-axis with fine manual control | ||
· Sensapex | · Sensapex triple axis | ||
· Märzhäuser | · Märzhäuser DC-3K with additional x-axis piezo stepper and MS 314 controller | ||
Magnetic Stands | Any magnetic base with on/off switch will do | For example, to manage cables inside the Faraday cage | |
Electrode Holders | Harvard Apparatus | ESP/W-F10N | |
Silver Wire | World Precision Instruments | AGW1510 | 0.3-0.5 mm diameter; needs to be chloridized for the electrode holders |
Fiber Optic Light Source | Many different, including Olympus | ||
Fiber Optic Bundles | · UltraFine Technology | To deliver the LED light stimulus to the Cardan arm system. We use both liquid and quartz light guides (range from UV to IR) | |
· Thorn Labs | |||
Fly Cathing Tube | P80-50P 50ml Cent. Tube PP., Pack of 100 Pcs | Cut the conical bottom off from 50 ml Plastic Centrifuge Tube and glue a 1 ml pipette tip on it. | |
Digital Acquisition System | National Instruments | ||
Single-electrode current/voltage-clamp microelectrode amplifier | npi SEC-10LX | http://www.npielectronic.de/products/amplifiers/sec-single-electrode-clamp/sec-10lx.html | Outstanding performer! |
Head-stage | Standard (+/- 150 nA) | For npi SEC-10LX | |
LED light sources and drivers | · 2-channel OptoLED (Cairn Research Ltd., UK) | Many of our stimulus systems are in-house built | |
· Self-designed and constructed | |||
Acquisition and Analyses Software | Many companies to choose from | Biosyst; custom written Matlab-based system for experimental and theoretical work in the Juusola laboratory | |
Personal Computer or Mac | Ensure that PC or Mac is compatible with data acquisition system and software | ||
Cardan arm system | Self-designed and constructed | Providing accurate x,y,z-positioning of the light stimuli | |
Peltier temperature control system | Self-designed and constructed | ||
Faraday Cage | Self-constructed | Electromagnetic noise shielding | |
Filamented Borosilicate Glass Capillaries | Outer diameter: 1 mm | ||
Inner diameter: 0.5-0.7 mm | |||
Filamented Quartz Glass Capillaries | Outer diameter: 1 mm | ||
Inner diameter: 0.5-0.7 mm | |||
Pipette Puller | Sutter Instrument Company | Model P-2000 laser Flaming/Brown Micropipette Puller | 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 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’ preset parameters serve as useful starting points for systematic modifications to generate electrodes with good penetration success and low recording noise. |
Extracellular Ringer Solution for the reference electrode | Chemicals from Fisher Scientific | 10326390, NaCl 10010310, KCl 10147753, TES 10161800, CaCl2 10159872, MgCl2 10000430, sucrose | See the recipe in the protocol section |
3 M KCl solution for filling the filamented recording microelectrode | Salts from Fisher Scientific | 10010310, KCl | |
Petroleum jelly | Vaselin | ||
Non-stainless steel razor blades | |||
Blade holder/breaker | Fine Science Tools By Dumont | 10053-09 | 9 cm |
Blu-tack | Bostik | Alternatively, use molding clay | |
Forceps | Fine Science Tools By Dumont | 11252-00 | #5SF (super-fine tips) |