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 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.

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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18.5K 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

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.

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

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.

Un metodo per la sistematica elettrochimici ed elettrofisiologiche di valutazione dei neurali registrazione elettrodi

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.

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 ...

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.

Una guida per<em&gt; In vivo</em&gt; Singola unità di registrazione da Optogenetically Identificato corticale inibitorio interneuroni

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.

Analizzando Synaptic Modulazione di<em&gt; Drosophila melanogaster</em&gt; Fotorecettori dopo l&#39;esposizione alla luce prolungata

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.

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 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.

Registrazione elettrofisiologica da<em&gt; Drosophila</em&gt; Senso Trichoide in risposta a Odoranti di bassa volatilità

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 Calcio Imaging per visualizzare le risposte del cervello alla segnalazione endocrina 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 ...

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.

Preparazione della Drosophila melanogaster adulta per l'imaging cerebrale intero durante il comportamento e le risposte agli stimoli

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.

Imaging in vivo del calcio delle risposte del neurone ganglio genicolato di topo agli stimoli del gusto

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