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.

果蝇（ 果蝇 ）复眼是一个伟大的模型系统来研究神经图像采集和处理感光和中间神经元阵列的功能组织，以及对动物的视力。该系统具有最完整的接线图1,2和是可亲到遗传操作和准确的神经活动监测3-10（高信噪比和时间分辨率）。 果蝇眼睛是模块化的，含有〜750看似普通镜头覆盖的结构，称为小眼，它们共同提供悬挂该占地约它的头几乎每一个方向的全景视野。眼睛的主要信息抽样单位是其rhabdomeric感光7,8,11。每个小眼包含八个感光细胞（R1-R8），它们共享相同的小面透镜，但被对准至七个不同的方向。虽然外光感受器，R1-R6芳E要蓝绿光最为敏感，内细胞R7和R8，它趴在对方并指向同一方向的顶部光谱灵敏度，表现出三个不同的亚型：面色苍白，黄色，背缘区（DRA）12 15。 <img alt="图1" src="/files/ftp_upload/54142/54142fig1.jpg" /> 图 1. 果蝇 眼睛 的功能性组织 。 （A）的两个第一视神经节，视网膜和椎板，以灰色突出显示的蝇眼内。视网膜R1-R6感光细胞和叶片大单极细胞（低收入国家：L1-L3） 在体内常规尖锐的微电极记录容易接近。示意图电极突出，从R1-R6在视网膜记录正常轨道。从低收入国家在椎板记录之一路径是平行的电极转移到左。 （B）叶片是retinotopically器官的矩阵源化墨盒，其中的每一个填充有从在视觉空间中的特定的小区域处理信息的神经元。由于神经叠加，从不同的相邻小眼感光器6送他们的轴突（R1-R6）相同的椎板墨盒，形成组胺输出突触L1-L3和无长突细胞（AM）。 （C）的R1-R6轴突和视觉的interneurons（包括L4，L5，Lawf，C2，C3和T1），内筒层是复杂的神经之间的信息的传播。 （D）R1-R6感光轴突接收来自L2和L4单极细胞突触的反馈。从里维拉阿尔巴等2修改（B）和（C）。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 果蝇眼睛是神经叠加16型。该装置T椎板和髓质：帽子八感光属于7相邻小眼，看起来在空间中的相同点的神经信号，以在接下来的两个髓神经1墨盒汇集在一起​​。而六个外光感受器，R1-R6项目其轴突末梢神经列在薄片（ 图1）中，R7和R8细胞绕过此层，并与它们的相应髓质柱17-19突触联系。这些精确的连线生产用于飞行的早期视觉的视皮层映射神经基板，于是每一个叶片（ 图1A-C）和髓质柱（盒）代表在空间中的一个点。 和无长突细胞（AM）在椎板1,2,20：从R1-R6感光体直接输入由大单极细胞（L1，L2和L3低收入国家）接收。出的这些，L1和L2是最大的细胞，介导主要信息通路（ 图1D），WHI通道响应于接通和关断移动边，并由此形成所述运动检测器21,22的计算基础。行为实验表明，在中间的对比，这两种途径促进相反方向的运动知觉：后端到前在L1和前到后在L2细胞23,24。连接进一步表示：L4神经元可能在相邻的墨盒25,26之间的横向沟通起到至关重要的作用。位于同一L2和L4细胞和两个相邻盒之间发现倒数突触。下游，每个L 2小区和它的三个相关联的L4细胞投射它们的轴突到公共目标，在髓质，其中来自相邻墨盒输入信Tm2的神经元被集成为前到后运动27的处理。虽然L1的神经元通过两个间隙连接和突触接收来自同一盒L2S输入，它们不直接连接到L4S因此相邻薄层墨盒。 <p类=“jove_content”&gt;被属于在L2 / L4电路的神经元只提供突触反馈到R1-R6感光轴突，但不用L1通路1,2（ 图1D）。虽然同一墨盒连接从L2至R1和R2以及从L4到R 5是选择性地，所有的R 1〜R 6的光感受器接收来自任L4或两个相邻的墨盒一样的反馈。此外，还有从上午到R1，R2，R4和R5强突触连接，和神经胶质细胞也突触连接到网络，因此可参与神经图像处理6。最后，轴突间隙路口，连接在相邻的层R1-R6和R6和R7 / R8之间的光感受器，有助于信息不对称表示和处理每个墨盒14,20,28。 从在几乎完整的果蝇个体感光细胞和视觉的interneurons细胞内电压记录提供高的信号-噪音r在亚毫秒分辨率3,5,7-10,29，这是必要用于使连接的神经元之间的快速的神经计算的感ATIO数据。的精度这一级是由目前的光成像技术，这是显著喧闹并且通常在10操作是不可能的 - 100毫秒的分辨率。此外，因为电极具有非常小的和尖锐的提示，该方法不限于细胞体，但可提供从小活性神经结构直接记录;如低收入国家&#39;树突树木或感光体轴突，这是不能由大得多膜片钳电极的前端访问。重要的是，该方法也是在结构上比大多数膜片钳应用侵入性较少，并毁坏等影响少的研究细胞的细胞内环境和信息抽样。因此，传统尖锐的微电极技术作出了贡献，并继续做出贡献，根本发现和原来的洞察神经Infor公司在适当的时间尺度息处理;提高我们的愿景3-10的机械理解。 本文介绍了如何从 果蝇 R1-R6感光细胞和低收入国家体细胞内记录在Juusola实验室中进行。该协议将介绍如何构建一个合适的电装备，准备飞行，并进行录音。一些有代表性的数据被呈现，并且一些共同的问题和潜在的解决方案进行了讨论，可能使用该方法时会遇到。

<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.

锋利微电极记录方法中，作为此处适于果蝇眼睛，可以用于可靠地量化它们4,5,7,8,10,33之间在视网膜和薄层细胞神经信息采样和处理，以及通信。通过使用它来研究不同的野生型股票，突变或基因工程菌蝇编码，该方法已证明了其价值;不仅在量化的突变，温度，饮食或选定表达3,4,6,9,10,14,30,34的影响，而且也揭示了改变的视觉行为14,34机械的原因。该方法也容易适用于其他昆虫的准备工作35,36，赋予神经行为学研究的视野。接下来，我们展示的其成功的应用的几个例子。 <img alt="图7" src="/files/ftp_upload/54142/54142fig7.jpg" /> 图7. 电压一个果蝇R1-R6感光的反应，在20的光脉冲和25℃。由于尖锐的微电极穿透通常非常稳定，所以可以在同一R1-R6感光体电压响应在记录到一个给定的光刺激不同的环境温度下通过加热或冷却该飞。在我们的调校，蝇保持器被置于一个闭环基于珀尔帖元件的温度控制系统。这使我们能够改变飞的头温度为秒。较高的温度加速电压响应和典型降低R1-R6光感受器的静息电位（用红色箭头所示）。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 研究温度对感光输出的影响 25℃（ 图7）记录在同一个R1-R6感光电压响应。由于4,9之前量化，气候变暖降低黑暗感光的静息电位，并加速其电压响应。 <img alt="图8" src="/files/ftp_upload/54142/54142fig8.jpg" /> 图8. 信令性能果蝇R1-R6感光随光照强度提高 （A）感光输出变暗（下图）和光明（以上; 10,000次亮的光）通过重复同样的微电极记录的自然光强度的时间序列在20℃。在同一小区回应明亮的刺激较大，因为它们集成更多的样本，基本反应（碰撞），单光子4,5,7,8。 （B）连续20个一秒长的电压响应叠加。个体反应（浅灰色）后的自适应趋势采取（以A箭头）已消退（在一个虚线框）。相应的响应的装置（的信号）是较暗的痕迹。信号和单独的响应之间的差异是噪声。 （C）将细胞使用标准方法4,5,7,8信号-噪声比（SNR）的&#39;信令性能通过录音量化“。感光体输出具有在明亮的刺激“比在昏暗的（ 明亮 ≥1，高达84赫兹（SNR约64赫兹范围更广可靠的信令的SNR），” 昏暗 ≥1，高达20赫兹），其中signal-信噪比改善大大;从SNR 昏暗 MAX = 87 SNR 亮 MAX = 1868。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 通过反复刺激学习适应和神经编码 的方法中，使在视网膜和薄层结构相对较少损害的无创性，使得它非常适合研究各个细胞在体内其邻近自然生理状态不同的光刺激的信令性能。 图8示出R1-的电压响应R6光感受器昏暗和明亮的反复自然光强度的时间序列刺激在20℃，而图9 </strong&gt;的信息显示，在25 摄氏度的另一个R1-R6光感受器和LMC到不同的自然刺激的反应前和突触后录音被因为同时细胞内记录在同一飞两头尖微电极，一个在视网膜和其它在椎板，太难可行30从两个不同的苍蝇分开执行。 <img alt="图9" src="/files/ftp_upload/54142/54142fig9.jpg" /> 要反复刺激自然主义在25 摄氏度 （A）R1-R6（灰色）和LMC（黑色） 果蝇R1-R6光感受器和LMC 图 9. 电压输出的响应由来自不同苍蝇不同的微电极记录下来。 （B）全光适应连续20前（上图），并与个体反应一样自然的刺激模式突触后（下）的反应，在光显示灰色的D中的相应的响应的装置（的信号）作为暗痕迹。信号和单独的响应之间的差异是噪声。 （C）的细胞信号-噪声比（SNR）的&#39;信令性能通过录音量化“。 LMC输出具有“比R1-R6输出（SNR（LMC≥1，高达104赫兹- [R≥1，高达94赫兹）约10赫兹范围更广的可靠信号的信噪比 ）”。这两个信号与噪声的比值高（SNR LMC MAX = 142， 信噪比读取 最大 = 752），并作为记录噪音低，他们之间的分歧反映了细胞之间的真正的编码区别。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig9large.jpg" target="_blank">请点击此处查看大图这个数字。</a> 刺激发病后，录音典型LY秀快适应了5-6秒内消退主要趋势。从那时起，在细胞产生的每个1秒长刺激呈现（各虚线框包围的这些20）高度一致的反应。当这些叠加（ 图8B和图9B）的响应的可重复性变得明显。个体反应是细灰的痕迹，他们的意思是较厚的深色痕迹。平均响应被作为神经信号 ，而神经噪声的平均值和每个单独的响应4,5,9,37,38之间的差。在频域中的相应的信号对噪声比（ 图8C和图9C）被傅立叶变换的信号和噪声的数据块到功率谱，并除以对应的平均噪声功率谱4的平均信号功率谱得到5,9,37,38。特征，最大信号 - 噪声比öF中的记录，神经输出到自然的刺激是高（100 - 1000），并且在具有非常低记录噪声最稳定的制剂可达到的值&gt;&gt; 1000（ 例如 ， 图8C）。还要注意的是气候变暖扩大了细胞的信号的可靠带宽4（SNR&#39; 光明 ≥1）;例如，在图8 和9两R1-R6S之间的相对差，分别是10赫兹（84在20℃，并在25℃下94赫兹）。 一个可以通过使用香农公式32，或通过三重外推法39计算的响应“熵和噪声熵率之间的差进一步估计距其信噪比信息传输的每个细胞的速率。有关信息理论分析的更多细节，以及它们的使用和限制š专门用这种方法在以前的出版物7,8,39中给出。 <img alt="图10" src="/files/ftp_upload/54142/54142fig10.jpg" /> 杀手粉煤灰，R1-R6光感受器和LMC的 图10 的电压响应反复自然主义刺激在19℃。（A）中，R1-R6（灰色）和LMC（黑色）输出从同一飞同一微电极记录;第一突触后和突触前后，作为电极是在眼推进。 （B）连续20前（上）和突触后（下）的反应（浅灰色的痕迹），以同样的自然刺激模式进行了初步适应（在一个虚线框）后抓获。他们的手段是信号（在顶部的暗的痕迹），而他们各自的个体反应的差异给出的噪音。 （C）的相应的Signa1对噪声比（SNR），计算为在图8C和9C所示 。 LMC输出有关于“比R1-R6输出（LMC MAX≥1，高达234赫兹（SNR 100赫兹范围更广的可靠信号的信噪比 ）&#39;R MAX≥1，最高134赫兹）。两个信号-噪声比是高信噪比（SNR LMC MAX = 137， 信噪比读取 最大 = 627），并作为同一微电极是在录音使用时，它们的差异反映了前和突触后神经输出的实际差别。这些结果表明，该系统记录了低噪音，及其对分析的影响是微不足道的。 <a href="https://www.jove.com/files/ftp_upload/54142/54142fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 神经行为学研究视野 </p&gt; 该方法也可以用来从不同的昆虫物种7,8,35,36的复眼（ 图10）记录前和突触后电压响应，允许可视信息处理的比较神经行为学研究。对于所提出的记录系统中，唯一需要的适应是新制备持，各与用于研究的物种适宜尺寸的开口。这些示范录音是从女杀手飞（ 秽蝇attenuata）的。它们表现出R1〜R6感光体和LMC到相同的重复光刺激的细胞内电压响应，作为用于果蝇对应于图9中 ，但在19℃。在这种情况下，无论是前和突触后的数据是从同一飞行记录;一前一后，用同样的记录电极（填充用3M KCl）中通过侧面薄层第一前进之前进入ING额视网膜。在25℃时， 秽蝇数据相比于果蝇数据-即使在冷却器温度-示出了具有更快的动力学响应;扩大可靠的信令（信噪比&gt;&gt; 1）在较宽的频率范围内的范围。在自然刺激神经 ​​编码这种功能的修改与秽蝇的掠夺性生活36，这需要高精度的时空信息，以实现快速的空中狩猎行为是一致的。

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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.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</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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18.4K 次观看.  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.