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Electroretinogram (ERG) Recordings from Drosophila
果蝇视网膜电图(ERG)记录   

Elisheva Rhodes-MordovElisheva Rhodes-Mordov*Hadar SamraHadar Samra*Baruch MinkeBaruch Minke  (*contributed equally to this work)
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Abstract

Phototransduction is a process in which light is converted into electrical signals used by the central nervous system. Invertebrate phototransduction is a process mediated by the phosphoinositide signaling cascade, characterized by Phospholipase C (PLC) as the effector enzyme and the Transient Receptor Potential (TRP) channels as its target. The great advantage of using invertebrate photoreceptors is the simplicity of the preparation, the ease of light stimulation, the robust expression of key molecular components, and most importantly, the ability to apply the power of molecular genetics. This last feature is mainly attributed to Drosophila melanogaster as a preferred animal model.

The Electroretinogram (ERG) is an extracellular voltage recording from the entire eye, which reflects the total electrical activity arising from the retina in response to a light stimulation. The Drosophila ERG light response is robust and easily obtained, thus making it a convenient method to identify defects in the light response as a result of mutations. The Prolonged Depolarizing Afterpotential (PDA) is a useful ERG phenomenon that can be recorded from white-eyed flies following intense blue light. It is induced by a massive photo-conversion of the photopigment rhodopsin to its dark stable state called metarhodopsin, due to failure of light response termination. Unlike the light coincident ERG recording, which declines quickly to the dark baseline after the cessation of the light stimulus, the PDA response continues long (hours) after light offset. However, this response can be suppressed to the dark baseline at any time by photo-conversion of metarhodopsin back to rhodopsin, by application of an intense orange light stimulus (see Figure 7; Minke, 2012). The PDA has been used as an important tool to screen for visual defective mutant (Minke, 2012).

Keywords: Drosophila photoreceptors(果蝇的感光), Electroretinogram (ERG)(视网膜电图(ERG)), Photoresponse(响应), Drosophila mutants(果蝇突变体), in vivo electrical recordings(体内电记录)

Materials and Reagents

  1. Sterile disposable filter (0.2 μm pore size, aPES membrane, 75 mm diameter) [such as NalgeneTM Rapid-FlowTM Sterile Disposable Filter Units (Thermo Fisher Scientific)]
  2. 1 borosilicate glass capillary (OD 1.0 mm, ID 0.58 mm) (Harvard Apparatus)
  3. 3 silver filaments (1 mm diameter, 10 cm length) [such as Electrode AG/AGCL 1 mm DIA (WPI)]
  4. 1 ml/2 ml Syringe + 1 ml/2 ml Syringe with elongated tip [such as MEDI-PLUS 1ml without needle (KDL Medical Product Company)]
  5. Syringe filter (0.22 μm pore size, suitable for a 4 mm syringe tip) with PVDF membrane [such as Millex-GV Syringe Driven Filter Unit (Merck Millipore Corporation)]
  6. White-eyed Drosophila flies (available for purchase at Bloomington Drosophila Stock Center)
    Note: Red-eyed Drosophila flies are also suitable for ERG recordings, but a PDA cannot be induced in these flies.
  7. Homemade low temperature melting wax composed of mixture of paraffin and bee wax
  8. Redux cream for electrocardiography [such as Redux Electrolyte Crème (Parker Laboratories)]
  9. Ringer’s solution (see Recipes)

Equipment

  1. Horizontal Micropipette puller [such as programmable Flaming/Brown type micropipette puller (Sutter Instrument Company, model: P-97 )] with platinum filament. Less expensive pullers [such as Narishige vertical puller (NARISHIGE Group, model: PP-830 )] can also be used
  2. Pulsed gas flow anesthesia system with injector [such as the Sleeper system (Inject+Matic Sleeper)] connected to CO2 tank
  3. Fly stand [such as Alnico Shallow Pot Magnet (Eclipse Magnetics)] (Figure 1, #2, Figure 2, #2 and Video 1)
  4. Fine tweezers [such as tweezers (Biologie, model: number 5 )] and fine paint brush [such as paint brush (Rekab, model: series 40, number 3 )]
  5. Homemade Wax filament heater + soldering iron composed of a platinum-iridium (0.25 mm diameter) filament and a holder (Figure 3)
  6. Dissecting Stereoscopic zoom Microscope [such as Nikon Stereozoom Microscope (Nikon Corporation, model: SMZ-2 )]
  7.  “Cold” illuminator [such as KL 1500 LCD (SCHOTT North America, model: KL 1500 LCD )] with heat and red filters [such as KG3 and RG620, respectively (SCHOTT North America, models: KG3 and RG620 )]
  8. Computer with electric signal processor (such as Clampex) and data analysis program (such as Clampfit)
  9. Stereo-microscope with at least 4 different magnification settings [such as the Wild M5 (Leica Microsystems) with 6, 12, 25 and 50 magnification settings] (see Figure 1, #3 and Video 1)
  10. Vibration Isolated Table
  11. Dark Faraday cage covered in black cloth (Figure 1, #6 and Video 1).
  12. On/Off magnet block to provide convenient placement of the fly stand [such as complete Magnetic Base (Eclipse Magnetics, model: E905 )] (Figure 2, #5 and Video 1)
  13. 4 triple axis coarse mechanical micromanipulators [such as the Three-Axis Compact Coarse Micromanipulator (Tritech Research, Narishige, model: M-2 )] (Figure 1, #4, Figure 2, #3 and Video 1)
  14. 1 triple axis, fine, very stable mechanical micromanipulator for holding the recording electrode [such as the Leitz Mechanical Micromanipulator (Leica Microsystems)] (Figure 1, #8)
  15. Light detector phototransistor for light monitoring (to be fixed on one of the micromanipulators) (Figure 1, #5, Figure 2, #4 and Video 1).
  16. Shutter system [such as LS2 2 mm Uni-stable Shutters (Uniblitz, Vincent Associates®)] + shutter driver [such as VCM-D1 Single Channel Uni-stable (Uniblitz, Vincent Associates®)]
  17. High-pressure ozone-free 75 W Xenon lamp (operating on 50 W) + Schott KG3 heat filter + 2 condenser lenses + fiber optic (3 mm diameter, 1.3 m long) conducting light into the Faraday cage for light stimulation
  18. Lamp power supply (such as PTI LPS-220)
  19. Data acquisition system [such as Digidata 1550 digitizer (Molecular Devices, model: Digidata 1550 digitizer )]
  20. 2 glass electrode holders suitable for capillary O.D. 1 mm (Figure 1, #1, Figure 2, #1 and Video 1)
  21. Microelectrode preamplifier system with head-stage impedance tester, at least x10 amplification [such as Extracellular Preamplifier Current Pump System (Dagan, model: 2400A )] (Figure 1, #7 and Video 1)
  22. Pulse generator [such as Master 8 and Master 9 (A.M.P.I, models: Master 8 and Master 9 )]
  23. Neutral Density (ND) filters [such as ND filters (Balzer) and color filters including Red (SCHOTT North America, model: RG610 ), Orange (SCHOTT North America, model: OG590 ) and Blue (SCHOTT North America, model: BG63 )]


    Figure 1. An overview of the ERG setup. #1-electrode holder, #2-fly stand, #3- stereo-microscope, #4-micromanipulator, #5-light detector phototransistor, #6- Faraday cage, #7-amplifier head-stage, #8-Leica-Leitz Mechanical Micromanipulator.


    Figure 2. A magnified view of the ERG setup. #1-electrode holder, #2-fly stand, #3- micromanipulator, #4-light detector probe, #5-magnet block.


    Figure 3. A homemade instrument for immobilizing the living fly. A platinum-iridium (0.25 mm diameter) filament is electrically connected to a power supply with a controlled current output. The filament is attached via insolating plastic to a copper rod holder (heater and soldering iron).

    Video 1. A general overview of the ERG set

Procedure

The main components of the light response recorded by the ERG are (1) the extracellularly recorded photoreceptor potential, (2) the “on” and “off” transients, at the beginning and the end of the light pulse, arising from the second order lamina neurons, and (3) the slow response of the pigment (glia) cells. The photoreceptor potential is the physiological response to light arising from the light-induced openings of the Transient Receptor Potential (TRP) and TRP-Like (TRPL) channels. In response to intense step of light, the photoreceptor’s component of the ERG is composed of an initial corneal negative fast transient, which declines to a lower steady state phase due to light adaptation. The depolarization of the photoreceptor cell triggers the induction of the corneal positive “on” transient, via sign inverting synapse between the photoreceptor axon and the large monopolar neurons of the lamina. The response of the pigment cells appears as a corneal negative slow rise and slow decay of the ERG after light on and off, respectively. These slow components arise from an increase in extracellular K+, resulting from K+ efflux from the photoreceptor cells via the TRP and TRPL channels, which depolarize the pigment cells surrounding the photoreceptor cells (see Figure 6; Minke, 1982).

  1. Preparing electrodes.
    1. Insert each of the two silver-chloride filaments into the electrode holders, ensuring that the end of the filament makes contact with the metal bottom of the holder.
    2. Carefully insert glass capillary into micropipette puller, and set puller program to create pipettes with adequate tip size (approximately 1 µm), taper (approximately 4 mm) shape and resistance of 5-10 MΩ. These parameters may be achieved by using three heating cycles on the programmable P-97 Flaming/Brown puller (note that these settings are not required in cheaper pullers).
    3. Load the elongated tip syringe with Ringer's solution, using an additional syringe, filtering the solution through a syringe filter as you do so. Fill each glass capillary with Ringer's solution, using the elongated syringe (ensure that the capillary is at least half full).
    4. Slide the silver-chloride filament (attached to the electrode holder) into the glass capillary.
    5. Insert one electrode holder with the recording pipette into the electrode micromanipulator that will be used for grounding (ground electrode, as shown in Figure 2, #1), and insert the other electrode holder with the recording pipette into the preamplifier head-stage (active electrode, as shown in Figure 1, #7 and Video 1).
  2. Ensure that the work-set is properly connected and turned on (as shown in Figure 1, Figure 3 and Video 1).
  3. Preparing a live fly for the experiment: All the below procedure should be done under deep red illumination to keep the fly under dark adapted conditions. To obtain deep red illumination insert red filters into the “Cold” illuminator and into the Xenon lamp.
    1. Switch on the "Cold" illuminator to provide red light during the fixation of the fly.
    2. Anesthetize flies in the vial using the fly sleeper injector.
    3. Pour flies out of the vial, on to the sleeper container, while pressing down on the CO2 emitting pedal.
    4. Choose one fly for the experiment, and cover the rest with a Petri dish.
    5. Ensure the fly is in the proper orientation-lying on its side, with its back towards the hand that will be used to fix the fly on the fly holder (as shown in Figure 4).
    6. Place a drop of wax on the soldering iron.
    7. Lift the fly from its wings with the tweezers, and use the soldering iron to fix its wings to the fly stand (see Figure 4).
    8. Using the soldering iron and wax drops, fix the following parts of the fly (see Figure 4):
      1. The back: Connect most of the fly’s back to the stand surface with wax.
      2.  The legs: Lower the tip of the soldering iron onto the joining point of the legs, wait as the fly brings its legs to that point, and melt the wax to cover all the legs together.
        *Take care not to cover the thorax and abdominal sides of the fly, where the fly trachea openings are located, for that will prevent it from breathing.
      3.  The head: carefully place a drop of low temperature melting wax between the head and the back, in the neck area.
        *Take care not to overheat the head but to cover the upward facing eye with wax as you fix the head to the back. Overheating the head would not kill the fly but would eliminate the light response.
      4. The trunk-optional: Using a small drop of wax, connect the trunk to the upper end of the torso.
        *Ensure that the fly head, thorax and legs are properly fixed, and are unable to move during the experiment.
        *Even small movements of these organs would cause large electrical artifacts.


      Figure 4. A living fly properly fixed to the fly stand with wax


    9. Place a drop of Redux cream on the lower end of the fly's body.
    10. Place the stand in the Faraday cage, on the magnet block (when in the ON mode), and ensure that the fly is approximately 0.5 cm from the end of the light guide.
    11. Look through the smallest amplification of the stereo-microscope to roughly position the recording electrode above the fly's eye, and the ground electrode over the fly's upper torso/upper back, using the micromanipulators.
    12. Gradually enlarge the magnification of the stereo-microscope (usually x 25 is sufficient magnification), ensuring that the fly is still in the center of the field of view (as shown in Figure 5).
    13. Insert the ground electrode into the upper torso/back of the fly, using the micromanipulator (as shown in Figure 5).
    14. Preferably, insert the recording active electrode into the outer periphery of the fly's eye (the area where the tissue is a bit sturdier), using the micromanipulator.
      *The ERG amplitude is roughly similar at all corneal regions. Therefore, the exact location of the recording electrode on the cornea is not critical.


      Figure 5. Fly with electrodes placed in the eye and torso at high (left) and low (right) magnifications

  4. Close the Faraday cage, ensuring it is completely dark.
    *It is preferable to adjust the amplifier DC offset to 0 before performing the recordings, but it is not critical since the experimental objective is to measure the ERG amplitude relative to the dark baseline.
    *Ensure good electrical connections between all metal parts of the setup and ground. This is important to eliminate pickup of the 50/60 Hz of the lab electricity.
  5. ERG Intensity Response protocol:
    1. Start documenting the experiment, using the electric signal processor computer program.
    2. Place an orange filter in front of the Xenon lamp, together with an ND -log6 filter.
    3. Wait at least 30 sec in the dark, and then apply a 5 sec light pulse, using the pulse generator.
    4. Replace the ND -log6 filter with an ND -log5 filter (or any other ND log filter, as required).
    5. Wait at least 30 sec in the dark, and then apply an additional 5 sec light pulse, using the pulse generator.
    6. Repeat steps 5d-e, while you gradually increase the light intensity using the other ND log filters (the last pulse should be generated with no filter at all).
      *It is important to begin with dim lights and gradually increase the light intensity since this would keep the fly in a relative dark adapted state suitable for the subsequent more intense stimulus.


    Figure 6. Typical ERG trace recorded from wild type fly in response to intense orange light. The upper filled bar represents the light monitor (LM); the down-pointing upper arrow indicates the “on” transient and the bottom up-pointing arrow indicates the “off” transient, both arising from the second order lamina neurons; the right up-pointing arrow indicates a slow component arising from the pigment (glia) cells; the slowly rising corneal negative peak amplitude also originates from the pigment cells.

  6. PDA protocol:
    1. Use white-eyed fly; place an orange filter in front of the Xenon lamp and apply two/three 5 sec light pulses, at 5-10 sec dark intervals using the pulse generator.
    2. Wait at least 30 sec in the dark, place a blue filter in front of the Xenon lamp, and apply two/three 5 sec light pulses, at 5-10 sec dark intervals.
    3. Wait at least 30 sec in the dark, place an orange filter in front of the Xenon lamp, and apply two 5 sec light pulses, at 5-10 dark intervals.


    Figure 7. Induction and suppression of a PDA. An intense blue light pulse, which converted ~80% of the rhodopsin photopigment to its metarhodopsin state resulted in a saturated prolonged corneal negative response that continued in the dark, owing to maximal activation of the TRP and TRPL channels, making R1-6 cells non responsive. Two additional intense blue lights elicited smaller responses (without the ON and OFF transients) that originated from the R7 and R8 cells, in which a PDA was not induced. The following red light, which photoconverted metarhodopsin back to rhodopsin suppressed the PDA after the light was turned off. Intense orange light pulses were applied before and after the PDA. LM: light monitor.

  7. Analyze traces using a data analysis program (see analysis example in Figure 8).


    Figure 8. An example of ERG intensity response analysis of Cpn1% Drosophila strain. Cpn1% strain is a Calphotin-RNAi induced transgenic fly, in which the endogenous Ca2+ buffer protein, calphotin (CPN) is reduced to 1% of normal, in R1-6 photoreceptors (Weiss et al., 2012). A comparison of ERG responses to orange light pulses among three fly strains: A. Superposition of 3 sample traces showing the ERG responses to maximal intensity orange light pulses of wild type (WT, control), Cpn1% and Cpn1%/Rh1:CalX rescue flies (transgenic Cpn1% flies, which also over-express a Ca2+ pump that extrudes Ca2+ from the photoreceptor cell). B. Intensity-response curves of ERG responses of the above three fly strains. The different curves were measured from WT, Cpn1% and Cpn1%/Rh1:CalX flies (mean ± S.E.M, n=4-8).

Notes

  1. Raising flies: Standard Drosophila protocol dictates that flies are raised on a standard corn starch substrate, supplemented with yeast extracts and at 24 °C. It is recommended that the flies used in the experiments do not grow in an over-populated environment.
    For intensity response and PDA protocols, use flies that were raised in the dark, for at least 24 h (preferably 48-72 h). In addition, all steps of the fly preparation must be performed in the dark, using red lights only.
    For optimal ERG recording, use flies 1-3 days old.
  2. In cases where it is important to maintain the integrity of the eye, it is possible to place a dot of Redux cream on the top of the eye, and insert the electrode into the cream only, without piercing the eye.

Recipes

  1. Ringer's solution
    Ingredient
    Con. mM
    NaCl
    130
    KCl
    2
    MgCl2
    5
    CaCl2
    2
    HEPES (pKa=7.5 @ 25 °C)
    10
    Mix the ingredients according to the order of their appearance in the table above
    Adjust pH to 7.15, using HCl
    Filter through a sterile disposable filter, and stored at 4 °C (Indefinite shelf life if stored properly)

Acknowledgments

We thank Anatoly Shapochnikov for constructing the Wax Filament Heater. The construction of the ERG protocol was supported by the Israel Science Foundation (ISF).

References

  1. Kohn, E. and Minke, B. (2011). Methods for studying Drosophila TRP channels. TRP Channels. M. X. Zhu. Boca Raton (FL).
  2. Minke, B. (1982). Light-induced reduction in excitation efficiency in the trp mutant of Drosophila. J Gen Physiol 79(3): 361-385.
  3. Minke, B. (2012). The history of the prolonged depolarizing afterpotential (PDA) and its role in genetic dissection of Drosophila phototransduction. J Neurogenet 26(2): 106-117.
  4. Weiss, S., Kohn, E., Dadon, D., Katz, B., Peters, M., Lebendiker, M., Kosloff, M., Colley, N. J. and Minke, B. (2012). Compartmentalization and Ca2+ buffering are essential for prevention of light-induced retinal degeneration. J Neurosci 32(42): 14696-14708.

简介

光转导是将光转化为中枢神经系统使用的电信号的过程。无脊椎动物光转导是由磷酸肌醇信号级联介导的过程,其特征在于磷脂酶C(PLC)作为效应酶和瞬时受体电位(TRP)通道作为其靶标。使用无脊椎动物光感受器的巨大优势是制备的简单性,光刺激的容易性,关键分子组分的强大表达,最重要的是应用分子遗传学功能的能力。这最后一个特征主要归因于果蝇Melanogaster作为首选的动物模型。
视网膜电图(ERG)是从整个眼睛进行的细胞外电压记录,其反映了由于光刺激而引起的视网膜的总电活动。果蝇ERG光响应是稳健和容易获得的,因此使其成为识别由于突变导致的光反应缺陷的方便方法。延长的去极化后电位(PDA)是一种有用的ERG现象,可以在强烈的蓝光下从白眼苍蝇记录。由于光响应终止的失败,由于色素视紫红质的大量光转化引起的称为元视紫质的暗稳定状态。不同于轻度重合的ERG记录,其在光刺激停止后迅速下降到暗基线,在光偏移之后,PDA响应持续很长时间(小时)。然而,通过应用强烈的橙色光刺激,通过将视紫红质素转换回视紫红质,可以随时将该反应抑制到黑暗的基线(参见图7; Minke,2012)。 PDA已被用作筛选视觉缺陷突变体的重要工具(Minke,2012)。

关键字:果蝇的感光, 视网膜电图(ERG), 响应, 果蝇突变体, 体内电记录

材料和试剂

  1. 无菌一次性过滤器(0.2μm孔径,aPES膜,75mm直径)[例如NalgeneRapid-Flow无菌一次性过滤器单元(Thermo Fisher Scientific)], br />
  2. 1硼硅酸盐玻璃毛细管(OD 1.0mm,ID 0.58mm)(Harvard Apparatus)
  3. 3银丝(直径1mm,长度10cm)[例如Electrode AG/AGCL 1mm DIA(WPI)]
  4. 1ml/2ml注射器+ 1ml/2ml具有细长末端的注射器[例如MEDI-PLUS 1ml无针(KDL Medical Product Company)]
  5. 使用PVDF膜[例如Millex-GV注射器驱动过滤器单元(Merck Millipore Corporation)]的注射器过滤器(0.22μm孔径,适用于4mm注射器尖端)]
  6. 白眼的果蝇蝇(可在Bloomington Drosophila Stock Center购买)
    注意:红眼果蝇也适合于ERG记录,但在这些苍蝇中不能诱导PDA。
  7. 由石蜡和蜂蜡的混合物组成的自制低温熔融蜡
  8. 用于心电图的Redux霜剂[例如Redux ElectrolyteCrème(Parker Laboratories)]
  9. 林格的解决方案(参见配方)

设备

  1. 水平微量移液器拉出器[例如可编程的Flaming/Brown型微量移液器拉出器(Sutter Instrument Company,型号:P-97)]。也可以使用较便宜的拉片(例如Narishige垂直拉片(NARISHIGE Group,型号:PP-830)]
  2. 具有注射器的脉冲气流麻醉系统[例如睡眠系统(Inject + Matic Sleeper)]连接到CO 2容器
  3. 精细镊子(如Biologie镊子编号)和精细油漆刷(如Rekab油漆刷系列40,第3号)
  4. 飞行架[如Alnico Shallow Pot Magnet(Eclipse Magnetics)](图1,#2,图2,#2和视频1)
  5. 精细镊子[如镊子(Biologie,型号:5号)]和精细漆刷[如油漆刷(Rekab,型号:40系列,3号)]
  6. 自制蜡丝加热器+由铂铱(0.25 mm直径)灯丝和支架组成的烙铁(图3)
  7. 解剖立体缩放显微镜[尼康立体显微镜(尼康公司,型号:SMZ-2)]
  8. (例如KG3和RG620(SCHOTT North America,型号:KG3和RG620))的"冷"照明器(例如KL 1500 LCD(SCHOTT North America,型号:KL 1500 LCD) br />
  9. 具有电信号处理器(如Clampex)和数据分析程序(如Clampfit)的计算机
  10. 具有至少4种不同放大设置的立体显微镜(例如具有6,12,25和50放大倍数的Wild M5(Leica Microsystems)](参见图1,#3和视频1)
  11. 振动隔离表
  12. 黑色法拉第笼覆盖在黑布(图1,#6和视频1)
  13. 开/关磁体块以提供飞行架的方便放置[例如完整的磁性基座(Eclipse Magnetics,型号:E905)](图2,#5和视频1)
  14. 4个三轴粗机械微操纵器(例如三轴紧凑型粗微型操纵器(Tritech Research,Narishige,型号:M-2)](图1,#4,图2,#3和视频1)
  15. 1个三轴,精细,非常稳定的机械显微操纵器,用于保持记录电极[例如Leitz Mechanical Micromanipulator(Leica Microsystems)](图1,#8)
  16. 用于光监测的光检测器光电晶体管(固定在一个微操纵器上)(图1,#5,图2,#4和视频1)。
  17. 快门系统[例如LS2 2 mm单稳态快门(Uniblitz,Vincent Associates ?)] +快门驱动器[例如VCM-D1 Single Channel Uni- stable(Uniblitz,Vincent Associates )]
  18. 高压无臭氧75 W氙灯(工作在50 W)+ Schott KG3热过滤器+ 2聚光镜+光纤(直径3mm,长1.3米),将光线导入法拉第笼进行光线刺激
  19. 灯电源(如PTI LPS-220)
  20. 数据采集??系统[例如Digidata 1550数字化仪(Molecular Devices,型号:Digidata 1550数字化仪)]
  21. 2个玻璃电极夹适用于毛细管O.D. 1毫米(图1,#1,图2,#1和视频1)
  22. 微电极前置放大器系统,具有头级阻抗测试仪,至少x10放大[如细胞外前置放大器电流泵系统(Dagan,型号:2400A)](图1,#7和视频1)
  23. 脉冲发生器[如Master 8和Master 9(A.M.P.I,型号:Master 8和Master 9)]
  24. 中性密度(ND)滤光片[诸如ND滤光片(Balzer)和包括红色(SCHOTT North America,型号:RG610),Orange(SCHOTT North America,型号:OG590)和Blue(SCHOTT North America,型号:BG63) ]


    图1. ERG设置概述:#1电极夹,#2飞行架,#3立体显微镜,#4微型操纵器,#5光检测器光电晶体管,#6 - 法拉第笼,#7 - 放大器前级,#8 - Leica-Leitz机械微型操纵器


    图2. ERG设置的放大视图:#1电极夹,#2飞行架,#3-微操纵器,#4光探测器探头,#5磁铁块


    图3.用于固定活飞的自制仪器:铂铱(0.25 mm直径)灯丝电气连接到具有受控电流输出的电源。灯丝通过绝缘塑料连接到铜棒支架(加热器和烙铁)

    视频1. ERG集的一般概述
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程序

由ERG记录的光响应的主要组分是(1)细胞外记录的感光体电位,(2)在光脉冲开始和结束时的"开"和"关"瞬态,由二阶椎板神经元,和(3)色素(神经胶质)细胞的缓慢反应。感光体电位是由光诱导的瞬态受体电位(TRP)和TRP样(TRPL)通道开口产生的对光的生理反应。响应于光的强步进,ERG的感光器的分量由初始角膜负快速瞬变组成,其由于光适应而下降到较低的稳态相。光感受器细胞的去极化触发角膜阳性"开"瞬态的诱导,通过标记反转在光感受器轴突和层的大单极神经元之间的突触。颜色细胞的响应分别表现为角膜负的缓慢上升和光照后的ERG的缓慢衰减。这些缓慢的组分起因于通过经由TRP和TRPL通道的感光细胞的K sup + +外流引起的胞外K sup +的增加,其使感光器周围的色素细胞去极化细胞(参见图6; Minke,1982)。

  1. 准备电极。
    1. 将两根氯化银细丝插入电极 固定器,确保灯丝的端部与之接触 金属底座。
    2. 小心地将玻璃毛细管插入 ?微量移液器,和设置拉出程序来创建移液器 足够的尖端尺寸(约1μm),锥形(约4mm)形状 ?和电阻5-10MΩ。这些参数可以通过使用来实现 可编程P-97火焰/棕色拉拔器上有三个加热循环(注意 ?这些设置在较便宜的拖拉器中不是必需的)
    3. 加载 细长的注射器与林格的解决方案,使用额外的 注射器,通过注射器过滤器过滤溶液,就像这样。 使用细长的,用林格氏溶液填充每个玻璃毛细管 注射器(确保毛细管至少半满)。
    4. 将氯化银灯丝(连接到电极夹)滑入玻璃毛细管
    5. 用记录吸管插入一个电极夹 电极微操纵器将用于接地(接地 电极,如图2中#1所示),并插入另一个电极 支架与记录吸管进入前置放大器头阶段 (有源电极,如图1,#7和视频1所示)。
  2. 确保工作集正确连接并打开(如图1,图3和视频1所示)。
  3. 准备实蝇的实验:以下所有程序应在深红色照明下进行,以保持苍蝇在黑暗适应条件下。为获得深红色照明,将红色滤光片插入"冷"照明器和氙气灯中
    1. 打开"冷"照明器,在飞行固定期间提供红光。
    2. 麻醉使用飞行睡眠注射器在小瓶中飞行
    3. 将苍蝇从小瓶中倒出,放在睡眠容器上,同时向下按CO 2 sub 发光踏板。
    4. 选择一个飞行的实验,并覆盖其余的培养皿。
    5. 确保飞行在正确的方向 - 躺在它的一边 它的背向手将被用来固定飞的飞 (如图4所示)。
    6. 将一滴蜡放在烙铁上。
    7. 用镊子将翅膀从翅膀中提起,然后使用烙铁将翅膀固定到飞镖架上(见图4)。
    8. 使用烙铁和蜡滴,固定飞毛的以下部件(见图4):
      1. 后背:用蜡将大部分飞行连接到支架表面。
      2.  腿:将烙铁的尖端放到接合处 点的腿,等待飞行带来它的腿到那一点,和 融化蜡以将所有的腿一起覆盖 *小心不要覆盖 ?胸部和腹部的苍蝇,那里的苍蝇气管 开口位于,以防止呼吸。
      3.  头部:小心地在头部和背部之间,颈部区域放置一滴低温熔融蜡。
        *注意不要使头部过热,而要遮盖面朝上的眼睛 用蜡固定头部到后面。过热的头不会 ?杀死苍蝇,但会消除光反应。
      4. 躯干可选:使用一小滴蜡,将躯干连接到躯干的上端。
        *确保飞头,胸部和腿正确固定,并且在实验过程中无法移动。
        *即使这些器官的微小运动也会导致大量的电子假象。


      图4.用蜡正确固定在飞行架上的活飞机

    9. 将一滴Redux霜剂放在苍蝇体的下端。
    10. 将支架放在法拉第笼中,放在磁铁块上(当处于 ON模式),并确保飞行距离大约0.5厘米 光导末端。
    11. 看看最小的放大 的立体显微镜以粗略地定位记录电极 在飞蝇的上方,地面电极在飞行的上面 躯干/上背,使用微操纵器
    12. 逐渐 放大立体显微镜的放大倍率(通常x 25是 充分放大),确保飞仍在中心 的视野(如图5所示)。
    13. 使用显微操纵器(如图5所示)将接地电极插入苍蝇的上躯干/背部。
    14. 优选地,将记录有源电极插入外部 蝇眼的周边(组织有点的区域 sturdier),使用显微操纵器。
      * ERG振幅是 在所有角膜区域大致相似。因此,确切的位置 ?角膜上的记录电极并不重要。


      图5.在高(左)和低(右)放大倍率下放置在眼睛和躯干中的电极飞行

  4. 关闭法拉第笼,确保其完全变暗。
    在执行记录之前,最好将放大器DC偏移调整到0,但这并不重要,因为实验目的是测量相对于黑暗基线的ERG振幅。
    *确保设备和接地的所有金属部件之间具有良好的电气连接。这对于消除50/60Hz实验室电的拾取是重要的。
  5. ERG强度响应协议:
    1. 开始记录实验,使用电信号处理器计算机程序。
    2. 在氙灯的前面放置一个橙色过滤器,以及ND -log6过滤器
    3. 在黑暗中等待至少30秒,然后使用脉冲发生器施加5秒光脉冲。
    4. 用ND -log5过滤器(或任何其他ND日志过滤器,根据需要)替换ND -log6过滤器。
    5. 在黑暗中等待至少30秒,然后使用脉冲发生器施加另外5秒的光脉冲。
    6. 重复步骤5d-e,同时逐渐增加光线 强度使用其他ND日志过滤器(最后一个脉冲应该是 生成没有过滤器)。
      *从昏暗开始很重要 ?灯光并逐渐增加光强度,因为这将保持 ?飞行在相对暗适应状态适合后续 更强烈的刺激。


    图6.从中记录的典型ERG跟踪 野生型蝇响应强烈的橙色光。 上方的填充栏 表示光监视器(LM);向下的上箭头 表示"开"瞬态,底部向上箭头表示 "off"瞬态,二者都来自二级椎板神经元; 右上箭头表示由于所产生的慢分量 颜料(胶质)细胞;缓慢上升的角膜负峰值振幅 也来自颜料细胞
  6. PDA协议:
    1. 使用白眼蝇;在氙灯前放置橙色过滤器 并应用两个/三个5秒光脉冲,以5-10秒的黑暗间隔使用 ?脉冲发生器
    2. 在黑暗中等待至少30秒,放置a 蓝色滤光片前面的氙灯,并应用两/三5秒光 脉冲,间隔5-10秒。
    3. 等待至少30秒 黑暗,在氙灯前放置一个橙色过滤器,并应用两个5 ?秒光脉冲,以5-10暗间隔


    图7.感应和 抑制PDA。强烈的蓝光脉冲,将?80% 的视紫红质色素到其逆转视蛋白状态导致a 饱和长期角膜阴性反应继续 暗,由于TRP和TRPL通道的最大激活,使得 R1-6细胞无反应。两个额外的强烈的蓝色光引起 较小的响应(没有ON和OFF瞬变) 来自R7和R8细胞,其中不诱导PDA。下列 红光,其将光敏视紫红质转化回视紫红质 在灯关闭后抑制PDA。强烈的橙色光 在PDA之前和之后施加脉冲。 LM:光监视器
  7. 使用数据分析程序分析跟踪(参见图8中的分析示例)。


    图8.Cpn 1%果蝇 菌株的ERG强度响应分析的实例: Cpn < %菌株是Calphotin-RNAi诱导的转基因蝇,其中在R1-6光感受器中内源性Ca 2+超级缓冲蛋白(calphotin,CPN)降低至正常的1% Weiss等人,2012)。在三种蝇株中对橙色光脉冲的ERG反应的比较:A.显示对野生型(WT,对照),Cpn 1%的最大强度橙色光脉冲的ERG反应的3个样品迹线的叠加和Cpn 1%/Rh1:CalX营养苍蝇(转基因Cpn <1%),其也过表达挤出的Ca 2+泵来自感光细胞的Ca 2+)。 B.上述三种蝇株的ERG反应的强度 - 反应曲线。从WT,Cpn <1%和Cpn <1> /Rh1:CalX苍蝇(平均值±S.E.M,n = 4-8)测量不同的曲线。

笔记

  1. 饲养蝇:标准的果蝇方案规定在标准玉米淀粉底物上补充酵母提取物并在24℃下培养果蝇。建议实验中使用的苍蝇不会在人口过多的环境中生长。
    对于强度反应和PDA方案,使用在黑暗中培养的苍蝇至少24小时(优选48-72小时)。此外,飞行准备的所有步骤必须在黑暗中进行,只使用红灯 要获得最佳ERG记录,请使用1-3天龄的苍蝇。
  2. 在重要的是保持眼睛的完整性的情况下,可以在眼睛的顶部放置一个Redux奶油点,并且只将电极插入奶油,而不刺入眼睛。

食谱

  1. 林格的解决方案
    成分
    Con。 mM
    NaCl
    130
    KCl
    2
    MgCl 2
    5
    CaCl <2>
    2
    HEPES(pKa = 7.5 @ 25℃)
    10
    根据其在上表中的出现顺序混合各成分
    使用HCl
    将pH调节至7.15 通过无菌一次性过滤器过滤,并储存在4°C(如果储存适当,保存期限不确定)

致谢

我们感谢Anatoly Shapochnikov建造蜡丝加热器。 ERG协议的建设得到了以色列科学基金会(ISF)的支持。

参考文献

  1. Kohn,E.和Minke,B。(2011)。 研究果蝇 TRP通道的方法。 TRP通道。摘要:博卡拉顿(FL)。
  2. Minke,B。(1982)。 光诱导的 trp 突变体中激发效率的降低> Drosophila 。 J Gen Physiol 79(3):361-385。
  3. Minke,B。(2012)。 长期去极化后电位(PDA)的历史及其在遗传切除中的作用 phototransduction。 J Neurogenet 26(2):106-117。
  4. Weiss,S.,Kohn,E.,Dadon,D.,Katz,B.,Peters,M.,Lebendiker,M.,Kosloff,M.,Colley,N.J.and Minke, 隔室和Ca 2 + 缓冲是预防光诱导的视网膜变性 J Neurosci 32(42):14696-14708。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rhodes-Mordov, E., Samra, H. and Minke, B. (2015). Electroretinogram (ERG) Recordings from Drosophila. Bio-protocol 5(21): e1636. DOI: 10.21769/BioProtoc.1636.
  2. Weiss, S., Kohn, E., Dadon, D., Katz, B., Peters, M., Lebendiker, M., Kosloff, M., Colley, N. J. and Minke, B. (2012). Compartmentalization and Ca2+ buffering are essential for prevention of light-induced retinal degeneration. J Neurosci 32(42): 14696-14708.
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