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.

ミバエ（ キイロショウジョウバエ ）複眼は、神経画像サンプリングおよび処理のための感光体と介在ニューロンの配列の機能的な組織を調査するのに最適なモデルシステムであり、動物のビジョンのために。システムは、最も完全な配線図1,2を有しており、遺伝子操作および3-10（高い信号対雑音比と時間分解能の）正確な神経活動の監視に愛想です。 ショウジョウバエの目は一緒にその頭の周りのほぼすべての方向をカバーするパノラマ視野を飛ぶ提供個眼と呼ばれる〜750一見、通常のレンズキャップされた構造を含む、モジュール化されています。目の一次情報サンプリングユニットは、そのrhabdomeric光受容体7,8,11です。それぞれの個眼は、同じファセットレンズを共有する8光受容体細胞（R1-R8）を含むが、7つの異なる方向に整列されます。外光受容体R1-R6 ARながら青緑色の光、同じ方向に互いの上部と点に横たわる内側の細胞R7およびR8の分光感度、展示3独特のサブタイプに電子最も敏感：、淡い黄色と背側リムエリア（DRA）12- 15。 <img alt="図1" src="/files/ftp_upload/54142/54142fig1.jpg" /> 図1. ショウジョウバエ の目 の機能組織 。 （A）は、2つの第1の光学要素神経節、網膜およびラミナは、フライアイ内側の灰色で強調されています。網膜R1-R6の光受容体とラミナ大モノポーラ細胞（LMCS：L1-L3）は、従来の鋭い微小電極記録に生体内で容易にアクセスできます。模式電極は、網膜にR1-R6から記録するために通常のパスを強調しています。ラミナにLMCSから録音するための一つのパスを左に平行電極にシフトすることです。 （B）ラミナはretinotopically臓器のマトリックスであります視覚空間内の特定の小さな領域からの情報を処理するニューロンが詰め込まれて、それぞれが化さカートリッジ。神経重ね合わせのために、異なる隣接個眼から6の感光体は、L1〜L3とアマクリン細胞（アム）のヒスタミン出力シナプスを形成し、同じラミナカートリッジにその軸索（R1-R6）を送信します。ラミナカートリッジ内の（C）R1-R6の軸索末端と（L4、L5、Lawf、C2、C3とT1を含む）視覚介在ニューロン間の神経情報の普及は、複雑です。 （D）R1-R6の感光体の軸索は、L2とL4単極細胞からシナプスフィードバックを受けます。リベラ・アルバら2から変更された（B）及び（C）。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> ショウジョウバエの目は、神経重畳型16です。これは、トンを意味します帽子空間内の同じ点を見て7、隣接個眼に属する8の光受容体の神経信号は、次の2 neuropils内の1つの神経カートリッジで一緒にプールされていますラミナおよび髄質。 6外光受容体R1-R6プロジェクトラミナにおける神経の列への軸索の端末（ 図1）が、R7およびR8細胞は、この層をバイパスし、それらに対応する髄質列17-19とのシナプス接触を行います。これらの正確な配線はすべてのラミナ（ 図1A-C）その後、フライ初期視覚の網膜マッピングのための神経基盤を生成し、髄質カラム（カートリッジ）は、空間内の単一の点を表します。 ラミナ1,2,20およびアマクリン細胞（アム）：R1-R6の光受容体からの直接入力が大型単極細胞（L1、L2とL3 LMCS）によって受信されます。これらのうち、L1、L2は、（ 図1D）の主要な情報伝達経路を媒介する、WHIの最大の細胞でありますchのエッジをオンとオフの移動に応答し、したがって、動き検出器21,22の計算基礎を形成します。 L2細胞23,24におけるL1中にフロント、バックとフロントツーバック：行動実験は、中間対照的に、二つの経路が反対方向の運動知覚を促進することを示唆しています。接続は、さらにL4ニューロンは、隣接カートリッジ25,26間の横方向の通信において重要な役割を果たし得ることを意味します。逆数シナプスは、L2とL4同じに位置するセルと隣接する二つのカートリッジの間で発見されました。下流では、各L2細胞およびその関連する3つのL4細胞は、共通の目標に隣接カートリッジからの入力は、前面から背面への運動27の処理のために統合されると考えられている髄質、中Tm2がニューロンをその軸索を投射します。 L1のニューロンは、両方のギャップ結合とシナプスを介して、同じカートリッジL2Sからの入力を受け取るが、それらは直接L4sので、隣接するラミナカートリッジに接続されていません。 <pクラス= &quot;jove_content&quot;&gt; R1-R6の感光体の軸索へのシナプスのフィードバックは、L2 / L4回路に属するニューロンによってのみ提供されていなく、L1経路1,2（ 図1D）。同じカートリッジの接続は、L2からR1とR2およびL4からR5に選択されている一方で、すべてのR1-R6の光受容体は、いずれかのL4または両方の隣接カートリッジからシナプスフィードバックを受けます。また、R1、R2、R4及びR5の時から強いシナプス結合が存在し、およびグリア細胞は、シナプスネットワークに接続され、従って、神経画像処理6に参加することができます。最後に、薄層に隣接したR1-R6をリンクし、R6およびR7 / R8光受容体との間に、ギャップジャンクション軸索は、各カートリッジ14,20,28における非対称情報の表現と処理に貢献しています。 ほぼ無傷のショウジョウバエの個々の光受容体と視覚介在ニューロンからの細胞内の電圧の記録は高い信号対雑音Rを提供します接続されたニューロン間の高速神経計算の意味を理解するために必要なサブミリ秒の分解能3,5,7-10,29、のデータatio。 100ミリ秒の解像度 - 高精度のこのレベルは、有意に騒々しいであり、典型的には10で動作する現在の光イメージング技術によっては不可能です。電極は非常に小さく、鋭い先端を有するのでさらに、方法は、細胞体に限定されるものではないが、小さなアクティブ神経構造からの直接録音を提供することができます。このようなパッチクランプ電極のはるかに大きなヒントがアクセスすることはできませんLMCS「樹状突起や感光体の軸索、など。重要なことは、この方法は、ほとんどのパッチクランプのアプリケーションよりも構造的に低侵襲性および損傷であるので、あまり研究され、細胞の細胞内環境と情報のサンプリングに影響を与えます。このように、従来の鋭い微小電極技術が貢献し、かつ神経フォアに寄与し、基本的な発見やオリジナルの洞察に維持しています適切な時間スケールでの報処理;ビジョン3-10の我々のメカニズムの理解を向上させることができます。 この記事では、ショウジョウバエ R1-R6の光受容体とLMCSからin vivoでの細胞内記録はJuusolaの実験室で行われている方法について説明します。このプロトコルは、適切な電気生理学リグを構築する方法について説明フライを準備し、録音を行います。いくつかの代表的なデータが提示され、いくつかの一般的な問題や潜在的な解決策は、この方法を使用するときに遭遇する可能性があるという議論されています。

<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>
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</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.

鋭い微小電極記録方法は、 ショウジョウバエの眼のためにここに適合したように、確実に神経情報サンプリングおよび処理網膜および薄層細胞において、それらの間の通信4,5,7,8,10,33を定量化するために使用することができます。別の野生型株、変異体または遺伝子操作フライ株における符号化を研究するためにそれを使用することにより、この方法は、その価値を証明しています。だけでなく、突然変異、温度、食事または選択した式3,4,6,9,10,14,30,34の効果を定量化ではなく、変更された視覚的な行動14,34のための機構的な理由を明らかにしました。この方法はまた、neuroethologicalビジョンの研究に力を与える他の昆虫の製剤35,36、に容易に適用可能です。次に、その成功したアプリケーションのいくつかの例を紹介しました。 <img alt="図7" src="/files/ftp_upload/54142/54142fig7.jpg" /> 図7. 電圧20および25 O℃で光パルスにミバエR1-R6感光体の応答鋭い微小電極の貫通部には、多くの場合、非常に安定しているので、で与えられた光刺激に対して同じR1-R6の感光体の電圧応答を記録することができますハエを温めるまたは冷却することにより、異なる周囲温度。我々のセットアップでは、フライホルダーは閉ループペルチェ素子に基づく温度制御システムに配置されています。これは、秒単位でハエの頭の温度を変更することを可能にします。より高い温度は、電圧応答を加速し、特徴（赤い矢印で示されているように）R1-R6の光受容体の静止電位を低下させる。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig7large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 感光体の出力に及ぼす温度の影響を研究 25°Cの （ 図7）で同じR1-R6の感光体に記録された明るい10ミリ秒の長パルス、に電圧応答を示します。 4.9前に定量化されるように、温暖化は暗闇の中で光受容体の静止電位を低下させ、その電圧応答を加速します。 <img alt="図8" src="/files/ftp_upload/54142/54142fig8.jpg" /> フルーツフライR1-R6感光体は、光強度に向上しました 図8. シグナリングパフォーマンス （A）感光体の出力（下）暗くし、明るい（上記; 10,000倍明るい光）。同じ微小電極によって記録された自然光強度の時系列を繰り返します20 O℃で同一セル内彼らはより多くのサンプル、単一光子4,5,7,8に基本応答（バンプ）を統合するので、明るい刺激に対する応答は、大きくなっています。 （B）20の連続した1秒の長電圧応答が重畳されています。適応傾向は（Aの矢印）（Aの破線ボックス）後退した後、個々の応答（ライトグレー）を採取しました。対応する応答手段（信号）が暗くトレースです。信号と個々の応答との間の差は、ノイズです。 （C）細胞を、標準的な方法4,5,7,8を用いて信号対雑音比（SNR）」シグナリング性能を記録することによって定量化しました」。感光体の出力がsで、 &#39;（20ヘルツまでの薄暗い ≥1、薄暗いSNR）よりも（84ヘルツまでの明るい ≥1、明るい刺激SNR）&#39;での信頼性のシグナル伝達の約64 Hzの広い範囲を持っていますignal対雑音比が大幅に向上します。 SNR 薄暗い MAX = 87からのSNR ブライト MAX = 1868へ。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig8large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 反復刺激によって適応と神経エンコーディングを学びます 網膜およびラミナ構造で比較的少ない損傷を引き起こす方法の非侵襲性は、in vivoでの彼らの近くに自然の生理的状態の異なる光刺激に対する個々の細胞の信号伝達性 ​​能を研究するために理想的です。8に示すR1-の電圧応答を図 20°Cので暗く、明るい繰り返さ自然光強度の時系列刺激に対するR6の感光体、 図9のに対し、 </stronグラム&gt; 25 O℃で異なる自然刺激に別のR1-R6の感光体とLMCの応答を示します同じフライで2鋭い微小電極、網膜内の1とラミナ内の他の同時細胞内記録は、30生存可能であるには余りにも困難であるため、前とシナプス後録音は、二つの異なるハエとは別に行われました。 <img alt="図9" src="/files/ftp_upload/54142/54142fig9.jpg" /> 別のハエとは異なる微小電極によって記録された25℃。（A）R1-R6（灰色）およびLMC（黒）出力で繰り返さ自然主義的刺激へのミバエR1-R6感光体とLMCの 図9 の電圧応答 。 （B）光に示す完全に明順応連続20前（上）と個々の応答と同じ自然な刺激パターンにシナプス後（下）の応答を、灰色暗いトレースとしてDに対応する応答手段（信号）。信号と個々の応答との間の差は、ノイズです。 （C）細胞信号対雑音比（SNR）」シグナリング性能を記録することによって定量化しました」。 LMC出力は&#39;（R≥1、94ヘルツまでのR1-R6の出力SNR）よりも（104ヘルツまでLMC≥1、信頼性の高いシグナリングSNR）」の約10 Hzの広い範囲を持っています。両方の信号対雑音比が高い（SNR LMC MAX = 142、SNR R MAX = 752）、記録ノイズが低かったとして、その違いは、細胞間の実際のエンコーディングの違いを反映している。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig9large.jpg" target="_blank">の拡大版をご覧になるにはこちらをクリックしてください。この図。</a> 刺激開始後、録音典型的なLY大部分は5-6秒以内に治まる高速適応傾向を示します。その時から、細胞が（各点線のボックスは、これらの20を囲む）各1秒長い刺激提示に非常に一貫性のある応答を生成します。これらは（ 図8Bおよび図9B）重畳されたときの応答の再現性が明白になります。個々の応答は、薄い灰色の痕跡であり、その厚い暗いトレースを意味します。 神経雑音は、平均、各個々の応答4,5,9,37,38との差であるのに対し、平均応答は、 神経信号として取り出されます。周波数領域でそれぞれの信号対雑音比（ 図8C及び図9C）は 、パワースペクトルに信号とノイズデータチャンクをフーリエ変換し、対応する平均雑音パワースペクトル4との平均信号パワースペクトルを分割することにより得られました5,9,37,38。特徴的に、最大の信号対雑音比O自然な刺激に記録された神経の出力は（100 -千）高いF（。 例えば 、 図8C）、および非常に低い記録ノイズで最も安定した製剤中の値&gt;&gt;千に達することができます。通知はまた、温暖化は&#39;（ ブライト ≥1セル信頼性のシグナル4 SNR）の帯域幅」を展開します。例えば、 図8 及び図9中の2つのR1-R6sとの間の相対的な差は、それぞれ、10ヘルツ（84、20 O Cと94ヘルツ25 O Cで）です。 一つは、さらに、シャノンの式32を使用することによって、または三重外挿法39を介して応答&#39;エントロピー及び雑音エントロピーレートの差を計算することにより、その信号対雑音比の情報転送の各セルの速度を推定することができます。情報理論解析、およびそれらの使用および制限に関する詳細具体的には、この方法では、Sは、以前の刊行物7,8,39に示されています。 <img alt="図10" src="/files/ftp_upload/54142/54142fig10.jpg" /> 19℃。（A）R1-R6（灰色）およびLMC同じフライから同じ微小電極によって記録（黒）出力で繰り返さ自然主義的刺激へのキラーフライR1-R6感光体とLMCの 図10 の電圧応答 。電極が目に進めたとして、後シナプス前最初のシナプス後部と、。同じ自然な刺激パターンに（B）20の連続した前（上）とシナプス後（下）の応答（ライトグレーのトレース）は、最初の適応（Aの点線のボックス）の後に捕獲されました。個々の応答に、それぞれの違いは、ノイズを与えながら、その手段は、信号（上部の暗いトレース）です。 （C）に対応するシグナリットル対ノイズ比（SNR）は 、図8C及び図9Cのように計算しました。 LMC出力が&#39;（134ヘルツまでのR MAX≥1、SNR）R1-R6の出力よりも（234ヘルツまでのLMC MAX≥1、信頼性の高いシグナリングSNR）」の100 Hzの広い範囲について持っています。両方の信号対雑音比は、（SNR LMC MAX = 137、SNR R MAX = 627）高く、同じ微小電極がレコーディングに使用されたとして、その違いは、前と後シナプス神経出力の本当の違いを反映しています。これらの結果は、記録システムは、低ノイズを持っていた、と分析への影響が限界であったことを示唆している。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig10large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> Neuroethologicalビジョン研究 </p&gt; この方法はまた、視覚情報の処理の比較neuroethological研究を可能にする、異なる昆虫種の化合物の眼7,8,35,36（ 図10）から前およびシナプス後電位の応答を記録するために使用することができます。提示記録システムの場合、唯一の必要な適応を検討した種のための適切なサイズの開口部を有する新規作成ホルダー、それぞれです。これらの例示的な録音は女性キラーフライ（Coenosiaのattenuata）のからのものです。彼らは、 図9のショウジョウバエの対応のために使用されるように、同一の繰り返し光刺激に対するR1-R6の感光体とLMCの細胞内の電圧応答を示すが、19℃。でこの場合、前およびシナプス後データの両方を同じ場から記録しました。 1第1の横の薄層を進行（3 M KClで満たされた）同じ記録電極と、他の前に入力した後、正面網膜る。でも、クーラーの温度で- - 25°Cの 、Coenosiaデータでのショウジョウバエのデータと比較してより高速なダイナミクスと応答を示しています。広い周波数範囲にわたって信頼できるシグナリング（信号対雑音比&gt;&gt; 1）の範囲を拡大します。自然刺激の神経符号化におけるこのような機能的適応は高速空中狩猟行動を達成するために高精度な時空間情報を必要とCoenosiaの捕食ライフスタイル36、と一致しています。

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.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</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 ビュー.  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.

ビデオ: 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.

急性海馬スライスにおけるシナプス誘発アストログリアとニューロン活動の二重の電気生理学的記録

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

神経科学

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.

神経電位電極の体系的電気化学的および電気生理学的評価方法

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.

から電気生理学的記録<em&gt;ショウジョウバエ</em&gt; Labellar味感覚器

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.

へのガイド<em&gt;インビボ</emOptogenetically同定された皮質抑制性介在から&gt;シングル単位記録

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.

のシナプス変調を分析<em&gt;キイロショウジョウバエ</em&gt;長時間の光照射後の感光体

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.

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

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.

からの電気生理学的記録<em&gt;ショウジョウバエ</em&gt;低揮発性の臭気物質に対するトリコイドセンシラ

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ショウジョウバエにおける内分泌シグナル伝達する脳の反応を可視化するためのカルシウム イメージング

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.

行動と刺激反応の間に全脳イメージングのための成人ショ ウジョウバエメラノガスター を準備する

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.

マウスの神経節神経節ニューロン反応のインビボカルシウムイメージングが味覚刺激に反応する

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