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.

Die Fruchtfliege (Drosophila melanogaster) -Verbindung Auge ist ein großes Modellsystem die funktionelle Organisation des Photorezeptors und Euron - Arrays für die neuronale Bild Probenahme und Verarbeitung sowie für die Tier Vision zu untersuchen. Das System verfügt über das umfassendste Schaltplan 1,2 und ist liebenswürdig zu genetischen Manipulationen und präzise neuronale Aktivitätsüberwachung (von hohen Signal-zu-Rausch - Verhältnis und Zeitauflösung) 10.03. Das Drosophila Auge ist modular aufgebaut, mit ~ 750 scheinbar normalen Linse bedeckten Strukturen Ommatidien genannt, die zusammen das eine Panorama - Sichtfeld fliegen sorgen , dass fast jede Richtung um seinen Kopf bedeckt. Das Auge des primären Informationsstichprobeneinheiten sind seine rhabdomer Photorezeptoren 7,8,11. Jede Ommatidium enthält acht Sehzellen (R1-R8), welche die gleiche Facettenlinse teilen, sind jedoch sieben verschiedene Richtungen ausgerichtet sind. Während die äußeren Photorezeptoren R1-R6 are am empfindlichsten auf blau-grünes Licht, spektralen Empfindlichkeiten der inneren Zellen R7 und R8, die in die gleiche Richtung liegen übereinander und zeigen, zeigen drei verschiedene Subtypen: blass, gelb und dorsalen Randbereich (DRA) 12- 15. <img alt="Abbildung 1" src="/files/ftp_upload/54142/54142fig1.jpg" /> Abbildung 1. Funktionelle Organisation des Drosophila Auge. (A) Die beiden ersten Opticusganglien, Retina und Lamina, sind grau im Inneren des Fliegenaugen hervorgehoben. Retina R1-R6 und Photorezeptoren Lamina Große monopolare Zellen (LMCs: L1-L3) sind leicht zugänglich in vivo zu herkömmlichen scharfen Mikroelektroden - Aufnahmen. Die schematische Elektrode hebt den normalen Weg von R1-R6 in der Netzhaut aufzuzeichnen. Ein Weg aus LMCs in der Lamina zu erfassen ist parallel der Elektrode zu verschieben nach links. (B) Lamina ist eine Matrix von retinotop organized Patronen, von denen jede mit Neuronen gepackt, die Informationen von einem bestimmten kleinen Bereich in dem Bildraum verarbeitet. Aufgrund neuralen Überlagerung von sechs Photorezeptoren aus verschiedenen benachbarten Ommatidien senden ihre Axone (R1-R6) auf den gleichen Plättchen Patrone histaminerge Ausgangs Synapsen L1-L3 und einer amacrine Zelle (Am) bilden. (C) Die Ausbreitung der neuronalen Informationen zwischen R1-R6 Axonterminalen und die visuellen Inter (einschließlich L4, L5, Lawf, C2, C3 und T1), innerhalb einer Lamina Patrone komplex ist. (D) R1-R6 Photorezeptor Axone erhalten synaptischen Feedbacks von L2 und L4 monopolare Zellen. (B) und (C) modifiziert von Rivera-alba et al 2. <a href="https://www.jove.com/files/ftp_upload/54142/54142fig1large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/54142/54142fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Das Drosophila Auge des neuronalen Überlagerung Typ 16. Dies bedeutet, tHut die neuronalen Signale von acht Photorezeptoren zu sieben gehör Ommatidien Nachbar, die im Raum an der gleichen Stelle suchen, werden zusammen in den nächsten zwei Neuropile an einer neuralen Patrone gepoolt: die Lamina und Medulla. Während die sechs äußeren Photorezeptoren R1-R6 Projekt ihre Axonterminalen zu neuronalen Spalten in der Lamina (Abbildung 1), R7 und R8 Zellen umgehen diese Schicht und synaptische Kontakte mit ihren Medulla Spalte 17-19 entspricht. Diese exakten Verdrahtungen erzeugen , das neuronale Substrat für die retinotopic Abbildung von Fly frühe Vision, wonach jede Lamelle (1A-C) und der Medulla Säule (Patrone) stellt einen einzigen Punkt im Raum. Direkte Eingänge von R1-R6 Photorezeptoren sind durch die großen monopolare Zellen erhalten (LMCs: L1, L2 und L3) und die Amakrinzell (Am) in der Lamina 1,2,20. Von diesen L1 und L2 sind die größten Zellen, zu vermitteln wichtige Informationswege (1D), which reagieren auf On- und Off-bewegten Kanten und bilden somit die Berechnungsgrundlage des Bewegungsmelders 21,22. Verhaltensexperimente legen nahe , dass bei mittleren dagegen die beiden Bahnen Bewegungswahrnehmung von entgegengesetzten Richtungen zu erleichtern: Back-to-Front in L1 und Front-to-Back in L2 - Zellen 23,24. Konnektivität bedeutet ferner , dass L4 - Neuronen entscheidende Rolle zwischen benachbarten Patronen 25,26 in der seitlichen Kommunikation spielen kann. Gegenseitige Synapsen wurden zwischen L2 und L4-Zellen befindet sich in der gleichen und zwei benachbarte Patronen gefunden. Downstream jede L2 - Zelle und die drei zugeordneten L4 Zellen ihre Axone mit einem gemeinsamen Ziel - Projekt, das Tm2 Neuron in der Medulla, wobei Eingänge von benachbarten Patronen geglaubt werden , integriert werden zur Verarbeitung von vorne nach hinten Bewegung 27. Obwohl L1 Neuronen über beide gap junctions und Synapsen Eingang von gleich Patrone L2s erhalten, sind sie nicht direkt mit L4s und damit benachbarte Lamina Cartridges. <pclass = &quot;jove_content&quot;&gt; Synaptische Feedbacks zu R1-R6 Photorezeptor Axone werden nur von Neuronen, die zu den L2 / L4 - Schaltungen aber nicht die L1 - Weg 1,2 (1D) zur Verfügung gestellt. Während gleichen Patronen Verbindungen selektiv von L2 auf R1 und R2 und von L4 bis R5 alle R1-R6 Photorezeptoren erhalten synaptische Rückkopplung von L4 von einem oder beiden benachbarten Patronen. Darüber hinaus gibt es starke synaptische Verbindungen von Am zu R1, R2, R4 und R5 und Glia - Zellen werden auch synaptisch mit dem Netzwerk verbunden und kann somit in neuronalen Bildverarbeitung 6 teilnehmen. Schließlich tragen axonalen spalt Kreuzungen verbindet benachbarte R1-R6 und zwischen R6 und R7 / R8 Photorezeptoren in der Lamina, die asymmetrische Informationsdarstellung und -verarbeitung in jeder Patrone 14,20,28. Intrazellulärer Spannung Aufnahmen von einzelnen Photorezeptoren und visuelle Inter in nahezu intakten Drosophila bieten hohen Signal-zu-Rausch - ratio Daten an Sub-Millisekunden - Auflösung 3,5,7-10,29, die zur Herstellung von Sinn der schnellen neuronalen Berechnungen zwischen den verbundenen Neuronen notwendig ist. Dieser Grad an Genauigkeit ist durch aktuelle optische Abbildungstechniken unmöglich, die wesentlich geräuschvoller sind und typischerweise bei 10 arbeiten - 100 msec Auflösung. Darüber hinaus, da die Elektroden sehr klein und scharfe Spitzen haben, ist das Verfahren nicht auf Zellkörper beschränkt, sondern direkte Aufnahmen von kleinen aktiven neuronalen Strukturen zur Verfügung stellen kann; wie die dendritischen Bäume oder Photorezeptor Axone &#39;LMCs, die von viel größeren Spitzen Patch-Clamp-Elektroden nicht zugegriffen werden kann. Wichtig ist, dass das Verfahren auch strukturell weniger invasive und schädlich als die meisten Patch-Clamp-Anwendungen, und wirkt sich so weniger die intrazelluläre Milieu und Informationen Sampling &#39;studierte Zellen. So haben herkömmliche scharfe Mikroelektroden-Techniken beigetragen, und halten Sie auf einen Beitrag, grundlegenden Entdeckungen und originelle Einblicke in neuronale information Verarbeitung bei der entsprechenden Zeitskala; unser mechanistisches Verständnis der Vision Verbesserung 10.03. Dieser Artikel beschreibt , wie in vivo intrazelluläre Aufnahmen von Drosophila R1-R6 und Photorezeptoren LMCs im Juusola Labor durchgeführt werden. Dieses Protokoll wird beschrieben, wie ein geeignetes Elektro rig zu konstruieren, bereiten die Fliege, und die Aufnahmen durchführen. Einige repräsentative Daten vorgestellt, und einige häufig auftretende Probleme und mögliche Lösungen diskutiert, die auftreten können, wenn mit dieser Methode.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stereo Zoom Microscope for making the fly preparation</td>
			<td>Olympus</td>
			<td>SZX12 DFPLFL1.6x PF eyepieces: WHN30x-H/22</td>
			<td>Capable of ~150X magnification with long working distance; bespoke heavy steel table mount stand</td>
		</tr>
		<tr>
			<td rowspan="2">Stereomicroscope in the intracellular set-up</td>
			<td>&#183;&#160; Olympus</td>
			<td>Olympus SZX7; eyepieces: WHN30x-H/22</td>
			<td rowspan="2">30x eyepieces are needed for seeing the electrode tip reflections well when driving it through the small corneal hole into the eye</td>
		</tr>
		<tr>
			<td>&#183;&#160; Nikon</td>
			<td>Nikon SMZ645; eyepieces: C-W30x/7</td>
		</tr>
		<tr>
			<td rowspan="2">Anti-vibration Table</td>
			<td>&#183;&#160; Melles Griot</td>
			<td rowspan="2">With metric M6 holes on the breadboard</td>
			<td rowspan="2">Our bespoke rigs have a large hole drilled through the thick breadboard that lets in the fly preparation platform pole (houses a copper heatsink with electronics) from below</td>
		</tr>
		<tr>
			<td>&#183;&#160; Newport</td>
		</tr>
		<tr>
			<td rowspan="4">Micromanipulators</td>
			<td>&#183;&#160; Narishige</td>
			<td>&#183;&#160; Narishige NMN-21</td>
			<td rowspan="4">In our intracellular set-ups, different micromanipulator systems are used for driving the shap recording electrodes into the fly eye. All the listed manipulators are succesfully providing long-lasting stable recordings from Drosophila photoreceptors and LMCs.&#160;</td>
		</tr>
		<tr>
			<td>&#183;&#160; Huxley Bertram</td>
			<td>&#183;&#160; Huxley xyz-axis with fine manual control</td>
		</tr>
		<tr>
			<td>&#183;&#160; Sensapex</td>
			<td>&#183;&#160; Sensapex triple axis</td>
		</tr>
		<tr>
			<td>&#183;&#160; M&#228;rzh&#228;user</td>
			<td>&#183;&#160; M&#228;rzh&#228;user DC-3K with additional x-axis piezo stepper and MS 314 controller</td>
		</tr>
		<tr>
			<td>Magnetic Stands</td>
			<td>Any magnetic base with on/off switch will do</td>
			<td></td>
			<td>For example, to manage cables inside the Faraday cage</td>
		</tr>
		<tr>
			<td>Electrode Holders</td>
			<td>Harvard Apparatus</td>
			<td>ESP/W-F10N</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Wire</td>
			<td>World Precision Instruments&#160;</td>
			<td>AGW1510</td>
			<td>0.3-0.5 mm diameter; needs to be chloridized for the electrode holders</td>
		</tr>
		<tr>
			<td>Fiber Optic Light Source</td>
			<td>Many different, including Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Fiber Optic Bundles</td>
			<td>&#183;&#160; UltraFine Technology</td>
			<td rowspan="2"></td>
			<td rowspan="2">To deliver the LED light stimulus to the Cardan arm system. We use both liquid and quartz light guides (range from UV to IR)</td>
		</tr>
		<tr>
			<td>&#183;&#160; Thorn Labs</td>
		</tr>
		<tr>
			<td>Fly Cathing Tube</td>
			<td></td>
			<td>P80-50P 50ml Cent. Tube PP., Pack of 100 Pcs</td>
			<td>Cut the conical bottom off from 50 ml Plastic Centrifuge Tube and glue a 1 ml pipette tip on it.</td>
		</tr>
		<tr>
			<td>Digital Acquisition System</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single-electrode current/voltage-clamp microelectrode amplifier</td>
			<td>npi SEC-10LX</td>
			<td>http://www.npielectronic.de/products/amplifiers/sec-single-electrode-clamp/sec-10lx.html</td>
			<td>Outstanding performer!</td>
		</tr>
		<tr>
			<td>Head-stage</td>
			<td>Standard (+/- 150 nA)</td>
			<td>For npi SEC-10LX</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">LED light sources and drivers</td>
			<td>&#183;&#160; 2-channel OptoLED (Cairn Research Ltd., UK)</td>
			<td rowspan="2"></td>
			<td rowspan="2">Many of our stimulus systems are in-house built&#160;</td>
		</tr>
		<tr>
			<td>&#183;&#160; Self-designed and constructed</td>
		</tr>
		<tr>
			<td>Acquisition and Analyses Software</td>
			<td>Many companies to choose from</td>
			<td></td>
			<td>Biosyst; custom written Matlab-based system for experimental and theoretical work in the Juusola laboratory</td>
		</tr>
		<tr>
			<td>Personal Computer or Mac</td>
			<td></td>
			<td></td>
			<td>Ensure that PC or Mac is compatible with data acquisition system and software</td>
		</tr>
		<tr>
			<td>Cardan arm system&#160;</td>
			<td>Self-designed and constructed</td>
			<td></td>
			<td>Providing accurate x,y,z-positioning of the light stimuli</td>
		</tr>
		<tr>
			<td>Peltier temperature control system&#160;</td>
			<td>Self-designed and constructed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Self-constructed</td>
			<td></td>
			<td>Electromagnetic noise shielding</td>
		</tr>
		<tr>
			<td rowspan="2">Filamented Borosilicate Glass Capillaries</td>
			<td rowspan="2"></td>
			<td>Outer diameter: 1 mm</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Inner diameter: 0.5-0.7 mm</td>
		</tr>
		<tr>
			<td rowspan="2">Filamented Quartz Glass Capillaries</td>
			<td rowspan="2"></td>
			<td>Outer diameter: 1 mm</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Inner diameter: 0.5-0.7 mm</td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>Model P-2000 laser Flaming/Brown Micropipette Puller</td>
			<td>For borosilicate reference electrodes, use the preset program #11 (patch electrodes): Heat = 350; Filament = 4; Velocity 36; Delay = 200).1.2.1). For borosilicate recording electrodes, use the preset program #12 (this typically pulls good conventional sharps&#160; for photoreceptor recordings): Heat = 355; Filament = 4; Velocity 50; Delay = 225; Pull = 150. For LMC recordings, which require electrodes with finer tips, these values need to be adjusted. For pulling quartz capillaries, P-2000 manual suggests programs for fine tipped microelectrodes. These programs&#8217; preset parameters serve as useful starting points for systematic modifications to generate electrodes with good penetration success and low recording noise.</td>
		</tr>
		<tr>
			<td>Extracellular Ringer Solution for the reference electrode</td>
			<td>Chemicals from Fisher Scientific</td>
			<td>10326390, NaCl 10010310, KCl 10147753, TES 10161800, CaCl2 10159872, MgCl2 10000430, sucrose</td>
			<td>See the recipe in the protocol section</td>
		</tr>
		<tr>
			<td>3 M KCl solution for filling the filamented recording microelectrode</td>
			<td>Salts from Fisher Scientific</td>
			<td>10010310, KCl</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum jelly</td>
			<td>Vaselin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-stainless steel razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blade holder/breaker</td>
			<td>Fine Science Tools By Dumont</td>
			<td>10053-09</td>
			<td>9 cm</td>
		</tr>
		<tr>
			<td>Blu-tack</td>
			<td>Bostik</td>
			<td></td>
			<td>Alternatively, use molding clay</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools By Dumont</td>
			<td>11252-00</td>
			<td>#5SF (super-fine tips)</td>
		</tr>
	</tbody>
</table>

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

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

Die scharfe Mikroelektroden - Aufzeichnungsverfahren, wie hier für das Auge Drosophila angepasst, kann zuverlässig verwendet werden , um neuronale Informations Probenahme und Verarbeitung in der Netzhaut und Lamina Zellen, und die Kommunikation zwischen ihnen 4,5,7,8,10,33 quantifizieren. Durch die Verwendung Codierung in verschiedenen Wildtyp-Bestände zu untersuchen, Mutanten oder genetisch Fliegenstämme entwickelt, hat das Verfahren sich bewährt; nicht nur in die Auswirkungen einer Mutation, die Temperatur, Ernährung oder ausgewählten Ausdruck Quantifizierung 3,4,6,9,10,14,30,34, sondern auch mechanistischen Gründe für veränderte visuelle Verhalten 14,34 in enthüllt. Das Verfahren ist auch ohne weiteres auf andere Insekten Zubereitungen 35,36, Befähigung neuroethologischen Vision Studien. wir präsentieren Next einige Beispiele für seine erfolgreiche Anwendungen. <img alt="7" src="/files/ftp_upload/54142/54142fig7.jpg" /> Abbildung 7. SpannungsAntworten eines Fruchtfliegen - R1-R6 Photorezeptor auf einen Lichtimpuls bei 20 und 25 ° C. Da die scharfen Mikroelektroden - Penetrationen oft sehr stabil sind, ist es möglich , Spannungsantworten des gleichen R1-R6 Photorezeptor zu einem gegebenen Lichtreiz aufnehmen zu unterschiedlichen Umgebungstemperaturen durch Erwärmen oder Abkühlen der Fliege. In unserem Set-ups ist die Fly-Halter auf einem geschlossenen Regelkreis-Peltier-Element-basierte Temperatur-Kontrollsystem gegeben. Dies ermöglicht uns, die Kopftemperatur in Sekunden Fliege ändern. Eine höhere Temperatur beschleunigt die Spannungsantworten und in charakteristischer Weise senkt das Ruhepotential der R1-R6 Photorezeptoren (wie durch rote Pfeile gekennzeichnet). <a href="https://www.jove.com/files/ftp_upload/54142/54142fig7large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www.jove.com/files/ftp_upload/54142/54142fig7large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Die Untersuchung der Einfluss der Temperatur auf dem Photorezeptor Output <p class="jove_content" fo:kEEP-together.within-page = &quot;1&quot;&gt; Mit einem gut gestalteten und schwingungsisolierten Aufnahmesystem kann das Verfahren verwendet werden, um den Einfluss der Temperatur auf einer einzelnen Zelle des neuronalen Ausgang durch Erwärmung messen oder Fliege Kühlung. Das gegebene Beispiel zeigt Spannungsantworten zu einer hellen 10 msec langen Impuls, in der gleichen R1-R6 Photorezeptor aufgezeichnet bei 20 und 25 o C (Abbildung 7). Wie quantifiziert vor 4,9 senkt Erwärmung ein Ruhepotential des Photorezeptors in der Finsternis, und beschleunigt seine Spannung Antworten. <img alt="Abbildung 8" src="/files/ftp_upload/54142/54142fig8.jpg" /> Abbildung 8. Signalleistung eine Fruchtfliegen - R1-R6 Photorezeptor Verbessert mit der Lichtintensität (A) Photorezeptor Ausgang zu dimmen (unten) und hell. (Oben; 10.000-mal heller Licht) wiederholt naturalistischen Lichtintensität Zeitreihe mit dem gleichen Mikroelektroden aufgezeichnetin der gleichen Zelle bei 20 o C. Die Reaktionen auf den hellen Reiz größer sind, weil sie mehr Proben, elementare Antworten (Bumps) auf einzelne Photonen 4,5,7,8 integrieren. (B) 20 aufeinanderfolgende Ein-Sekunden langen Spannungsantworten überlagert werden. Individuelle Antworten (hellgrau) wurden nach den adaptiven Trends genommen (in Pfeil A) hatte zurückgegangen (gepunktete Box in A). Die entsprechenden Antwortmittel (die Signale) sind die dunkleren Spuren. Die Differenz zwischen dem Signal und den individuellen Reaktionen ist das Rauschen. (C) Die Zellen Signal-zu-Rausch - Verhältnisse &quot;Signalisierungsleistung wurde durch die Aufnahmen quantifiziert&quot; (SNR) unter Verwendung der Standardmethoden 4,5,7,8. Der Photorezeptor - Ausgang hat etwa 64 Hz breiteres Spektrum an zuverlässigen Signalisierung an der hellen Stimulation (SNR &#39;Bright ≥1, bis zu 84 Hz) als an der dim (SNR&#39; Dim ≥1, bis zu 20 Hz), mit signal-zu-Rausch-Verhältnis erheblich zu verbessern; von SNR Dim MAX = 87 bis SNR Hell MAX = 1.868. <a href="https://www.jove.com/files/ftp_upload/54142/54142fig8large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/54142/54142fig8large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Studieren Anpassung und Neuronale Kodierung durch repetitive Stimulation Die Nichtinvasivität des Verfahrens, was relativ wenig Schaden in der Retina und Lamina Strukturen, macht es ideal für die Signalisierung Leistung der einzelnen Zellen auf unterschiedliche Lichtreize in ihrer nahen natürlichen physiologischen Zustand in vivo zu studieren. 8 zeigt Spannungsantworten eines R1- R6 Photorezeptor zu einem dunklen und hellen wiederholt naturalistischen Serie Stimulus Lichtintensität bei 20 ° C, während 9 </strong&gt; zeigt Reaktionen eines anderen R1-R6 Photorezeptor und einer LMC zu einem anderen naturalistischen Reiz bei 25 o C. Die prä - und postsynaptischen Aufnahmen wurden getrennt von zwei verschiedenen Fliegen durchgeführt , weil die gleichzeitige intrazelluläre Aufnahme von zwei scharfen Mikroelektroden in der gleichen Fliege, eine in der Netzhaut und die andere in der Lamina, zu schwierig sind 30 lebensfähig zu sein. <img alt="9" src="/files/ftp_upload/54142/54142fig9.jpg" /> Abbildung 9. Spannungsantworten einer Fruchtfliege R1-R6 Photorezeptor und LMC Wiederholte Naturalistische Stimulation bei 25 o C (A) R1-R6 (grau) und LMC (schwarz) Ausgänge von verschiedenen Mikroelektroden aus verschiedenen Fliegen aufgezeichnet. (B) Völlig Licht angepasst 20 aufeinander folgenden Vor- (oben) und postsynaptischen (unten) Antworten auf die gleiche naturalistischen Reizmuster mit individuellen Antworten, in Licht gezeigt , dass ein graud die entsprechenden Antwortmittel (die Signale) als die dunkleren Spuren. Die Differenz zwischen dem Signal und den individuellen Reaktionen ist das Rauschen. (C) Die Zellen Signal-Rausch - Verhältnis (SNR) &quot;Signalisierungsleistung wurde durch die Aufnahmen quantifiziert&quot;. LMC - Ausgang hat etwa 10 Hz breiteres Spektrum an zuverlässigen Signalisierung (SNR &#39;LMC ≥1, bis zu 104 Hz) als R1-R6 - Ausgang (SNR&#39; R ≥1, bis zu 94 Hz). Beide Signal-zu-Rausch - Verhältnisse sind hoch (SNR LMC MAX = 142, SNR R MAX = 752), und als das Aufzeichnungsrauschen gering war, ihre Unterschiede spiegeln reale Codierung Unterschiede zwischen den Zellen. <a href="https://www.jove.com/files/ftp_upload/54142/54142fig9large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/54142/54142fig9large.jpg" target="_blank">eine größere Version zu sehen diese Figur.</a> Nach dem Beginn des Stimulus, die Aufnahmen typischly zeigen schnelle Trends anzupassen, die innerhalb von 5-6 sec weitgehend abklingen. Von da an produzieren die Zellen sehr konsistente Antworten auf jede 1 Sekunde lang Stimuluspräsentation (jede gepunktete Box 20 davon umschließt). Die Wiederholbarkeit der Antworten wird offensichtlich , wenn diese überlagert sind (8B und 9B). Die individuellen Antworten sind die dünnen grauen Spuren, und ihre die dicker dunkler Spur bedeuten. Die mittlere Antwort wird als neuronales Signal genommen, während das neuronale Rauschen die Differenz zwischen dem Mittelwert und jeder einzelnen Antwort 4,5,9,37,38 ist. Die jeweiligen Signal-zu-Rausch - Verhältnis im Frequenzbereich (8C und 9C) wurden durch erhaltenen Fourier-Transformieren der Signal- und Rauschdatenabschnitte in Leistungsspektren, und Dividieren des mittleren Signalleistungsspektrum mit dem entsprechenden mittleren Rauschleistungsspektrum 4, 5,9,37,38. Charakteristischer die maximalen Signal-zu-Rausch-Verhältnisse of die aufgezeichneten neuronalen Ausgänge auf naturalistische Stimulation sind hoch (100 - 1000), und in den stabilsten Zubereitungen mit sehr niedrigen Aufzeichnungsrauschen kann Werte erreichen &gt;&gt; 1000 ( zum Beispiel 8C.). Beachten Sie auch , dass die Erwärmung dehnt sich die Zellen der Bandbreite von zuverlässigen Signalisierung 4 (SNR &#39;Bright ≥ 1); die relative Differenz zwischen den beiden R1-R6s beispielsweise in den Figuren 8 bzw. 9, 10 Hz (84 bei 20 o C und 94 Hz bei 25 o C). Man kann weiter jede Rate der Zelle Schätzung der Informationsübertragung von seinem Signal-zu-Rausch - Verhältnis durch die Gleichung Shannon mit 32, oder durch den Unterschied zwischen den Antworten der Entropie und Lärm Entropierate durch die dreifache Extrapolationsmethode 39 zu berechnen. Weitere Einzelheiten zu den Informationen theoretischen Analysen und deren Verwendung und Beschränkungs speziell mit dieser Methode sind in den früheren Veröffentlichungen 7,8,39 gegeben. <img alt="10" src="/files/ftp_upload/54142/54142fig10.jpg" /> Abbildung 10. Spannungsantworten eines Killer - Fly R1-R6 Photorezeptor und LMC Wiederholte Naturalistische Stimulation bei 19 o C (A) R1-R6 (grau) und LMC (schwarz) gibt mit dem gleichen Mikroelektrode aus dem gleichen Fliege aufgezeichnet; erste postsynaptisch und später präsynaptisch, da die Elektrode im Auge fortgeschritten war. (B) 20 aufeinanderfolgende prä- (oben) und postsynaptischen (unten) Antworten (hellgrau Spuren) auf denselben naturalistischen Reizmuster wurden nach der ersten Anpassung (gepunktete Box in A) erfasst. Ihre Mittel sind die Signale (die dunkleren Spuren oben), während ihre jeweiligen Differenzen zu den einzelnen Antworten der Lärm geben. (C) Das entsprechende Signal-zu-Rausch - Verhältnis (SNR) wurden , wie in den 8C und 9C berechnet. LMC - Ausgang über ein 100 Hz breiteres Spektrum an zuverlässigen Signalisierung (SNR &#39;LMC MAX ≥1 bis zu 234 Hz) als R1-R6 - Ausgang (SNR&#39; R MAX ≥1, bis 134 Hz). Beide Signal-zu-Rausch - Verhältnisse sind hoch (SNR LMC MAX = 137, SNR R MAX = 627) und als die gleiche Mikroelektrode in die Aufnahmen eingesetzt, deren Unterschiede spiegeln wirkliche Unterschiede in den prä- und postsynaptischen neuronalen Ausgänge. Diese Ergebnisse implizieren , dass das Aufzeichnungssystem geringes Rauschen hatte, und dessen Einfluss auf die Analysen war marginal. <a href="https://www.jove.com/files/ftp_upload/54142/54142fig10large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www.jove.com/files/ftp_upload/54142/54142fig10large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Neuroethologischen Vision - Studien </p&gt; Das Verfahren kann auch prä - und postsynaptischen Spannung Antworten von den Facettenaugen verschiedener Insektenarten 7,8,35,36 (Abbildung 10), ermöglicht vergleichende neuroethologischen Studien der visuellen Informationsverarbeitung zur Aufzeichnung verwendet werden. Für die dargestellten Aufzeichnungssystem ist die einzige erforderliche Anpassung neuer Vorbereitung Halter, die jeweils mit einem angemessen dimensionierten Öffnung für die untersuchten Arten. Diese beispielhaften Aufnahmen sind aus einer weiblichen Killer fliegen (Coenosia attenuata). Sie zeigen intrazellulärem Spannungsantworten eines R1-R6 Photorezeptor und LMC identische sich wiederholende Lichtstimulation, wie sie für die Drosophila Pendants in Figur 9 verwendet, aber bei 19 o C. In diesem Fall sind sowohl die prä- und postsynaptischen Daten wurden aus der gleichen fly aufgezeichnet; einer nach dem anderen, zuerst mit dem gleichen Aufzeichnungselektrode (gefüllt mit 3 M KCl) durch die seitliche lamina Vorschieben vor eingebening die frontale Retina. Im Vergleich zu den Drosophila - Daten bei 25 o C, die Coenosia Daten - selbst bei der Kühlertemperatur - zeigt Antworten mit schnelleren Dynamik; die Erweiterung der Palette an zuverlässigen Signalisierung (Signal-zu-Rausch-Verhältnis &gt;&gt; 1) über einen breiteren Frequenzbereich. Derartige funktionelle Anpassungen in neuronale Codierung der naturalistischen Reize sind im Einklang mit Coenosia &#39;s räuberische Lebensweise 36, die hochpräzise Raumzeit - Informations erfordern schnelle Luftjagdverhalten zu erreichen.

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.

Elektrophysiologische Verfahren zur Aufzeichnung von intrazellulärer Spannung Antworten von<em&gt; Drosophila</em&gt; Fotorezeptoren und Inter auf Lichtreize<em&gt; In Vivo</em

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

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

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18.5K Aufrufe.  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.

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

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

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

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

Dual-Elektrophysiologische Recordings synaptisch-evozierte astrogliale und neuronale Antworten in Acute Hippocampusschnitte

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

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

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

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

Eine Methode zur systematischen Elektrochemische und elektrophysiologische Bewertung der Neural Aufzeichnungselektroden

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

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Elektrophysiologische Aufnehmen von<em&gt; Drosophila</em&gt; Labellar Geschmack Sensillen

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

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

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

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

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

Ein Leitfaden zur<em&gt; In vivo</em&gt; Single-Unit-Aufnahme von Optogenetically Identifizierte kortikalen inhibitorischen Inter

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

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

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

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

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

Analyse Synaptische Modulation von<em&gt; Drosophila melanogaster</em&gt; Photorezeptoren nach der Einwirkung von Längerer Licht

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

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

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

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

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

Elektrophysiologische Methode für Ganzzell-Spannungsklemmen Aufnahmen von<em&gt; Drosophila</em&gt; Fotorezeptoren

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

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Elektrophysiologische Aufzeichnung von<em&gt; Drosophila</em&gt; Trichoid Sensilla in Reaktion auf Odoranten der niedrigen Volatilität

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

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

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

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

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

Ex-Vivo Calcium Imaging zur Visualisierung von Gehirn Reaktionen auf endokrine Signalisierung bei Drosophila

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

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

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

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

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

Vorbereitung von Adult Drosophila melanogaster für die Bildgebung des gesamten Gehirns während des Verhaltens und der Stimuli-Reaktionen

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

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

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

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

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Reaktionen auf Geschmacksreize

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