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Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster
成年黑腹果蝇食窦肌肉9(CM9)的电生理记录   

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Abstract

The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows for the recording of both evoked (EPSPs) and spontaneous postsynaptic excitatory potentials (mEPSPs) at a mature glutamatergic synapse. Combined with CM9-specific genetic technologies, the CM9 NMJ provides a powerful experimental system to better understand the regulation of neurotransmitter release at a mature synapse.

Keywords: Drosophila(果蝇), Aging(衰老), Neuromuscular junction(神经肌肉接头), Neurotransmission(神经传递)

Background

A significant hurdle in defining changes in presynaptic function during aging has been due to the lack of a simple model system for performing the electrophysiological recordings necessary to thoroughly characterize the release of neurotransmitter from the presynaptic nerve terminal. Existing rodent models suffer from the significant cost issues associated with aging studies and the technical difficulty of using electrophysiological recordings on single defined nerve terminals with consistent release parameters. To overcome these obstacles, we have pioneered a model synaptic system in the adult Drosophila for analyzing the effects of age on presynaptic function during neurotransmission, the CM9 NMJ located on the fly proboscis (Rawson et al., 2012; Mahoney et al., 2014; Mahoney et al., 2016) (Figure 4A). Briefly, the presynaptic arbor of the CM9 motor neuron (MN) converges upon the 15 muscle fibres of the CM9 muscle to form 35 individual distinct innervations (Rawson et al., 2012). The CM9 MN has been shown to be necessary for the contraction of the CM9 muscle and is the only source of glutamatergic input for the CM9 muscle (Kimura et al., 1986; Gordon and Scott, 2009). Given the highly-conserved nature of the mechanisms underlying synaptic vesicle (SV) release between flies and mammals, and the resemblance to the central synapses found in the mammalian CNS, this makes the CM9 NMJ a powerful model for investigating presynaptic function.

Materials and Reagents

  1. Sterile disposable filter (0.22 μm pore size, aPES membrane 19.6 cm2 CA membrane) (such as Corning® 250 ml Vacuum Filter/Storage Bottle System, Corning, catalog number: 430767 )
  2. Borosilicate glass capillary with filament (OD 1.50 mm, ID 0.86 mm) (Sutter Instrument, catalog number: BF150-86-10 )
  3. Borosilicate glass capillary without filament (OD 1.50 mm, ID 0.86 mm) (Sutter Instrument, catalog number: B150-86-10 )
  4. Silver wire (A-M Systems, catalog number: 782000 )
  5. Diamond coated bench stone (such as DMT 8 in Dia-Sharp bench stone)
  6. Drawn out P200 tip
  7. 10 ml syringe (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: S7510-10 )
  8. Minutien pins (Fine Science Tools, catalog number: 26002-10 )
  9. Fine paint brush
  10. Flies of desired genotype and age
    Note: UAS constructs can be driven within the CM9 motor neuron via the use of the E49-Gal4 driver (E49-Gal4 from Ulrike Heberlein’s Gal4 collection).
  11. 50% Bleach
  12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G )
  13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653-250G )
  14. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 21115-250ML )
  15. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: 63069-500ML )
  16. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761-500G )
  17. Trehalose (Sigma-Aldrich, catalog number: T9531-25G )
  18. HEPES (Sigma-Aldrich, catalog number: H3375-25G )
  19. Sucrose (Sigma-Aldrich, catalog number: 84097-250G )
  20. Modified hemolymph like solution (HL3.1) (see Recipes)

Equipment

  1. Vannas spring scissors–2 mm cutting edge (Fine Science Tools, catalog number: 15000-03 )
  2. Tungsten Dissecting needle, 125 mm, Ultra fine (Roboz Surgical Instrument, catalog number: RS-6063 )
  3. Micro Dissecting needle holder, 5 ¼” (Roboz Surgical Instrument, catalog number: RS-6060 )
  4. Fine forceps (such as Dumont #5CO, Fine Science Tools, catalog number: 11295-20 and Dumont #3, Fine Science Tools, catalog number: 11231-30 )
  5. Dissecting stereoscopic zoom microscope (such as ZEISS, model: SteREO Discovery.V8 )
  6. Narrow Format Manipulator Systems (such as Sutter Instrument, model: ROE-200 )
  7. Horizontal Micropipette puller (such as programmable Flaming/Brown type micropipette puller, Sutter Instrument, model: P-97 ) with platinum filament
  8. Micro Forge electrode polisher (such as NARISHIGE, model: MF-900 )
  9. Upright confocal microscope (such as Olympus, model: BXS1WI ) with 10x and 40x water objectives
  10. Power lab 4/30 digital converter (ADInstruments, model: ML866 )
  11. Neuroprobe amplifier 1600 (A-M Systems, model: Model 1600 , catalog number: 680100)
  12. Stimulator (Digitimer, model: DS2A )
  13. Labchart 7 (ADInstruments)

Software

  1. Prism 6 (GraphPad software)
  2. Mini Analysis (comparable version 6.0.3, Synaptosoft Inc.)

Procedure

  1. Preparing silver chloride wire
    Place silver wire in a 50% Bleach/50% ddH2O solution so that at least ¾ of the wire is immersed in the liquid. Leave the wire in the bleach/water solution for ~5 min to allow for sufficient chloridization to occur.
  2. Preparing recording electrode–see JoVE video ‘Making patch pipettes and sharp electrodes with a programmable puller’ by Brown et al., 2008 for reference (see Notes).
    1. Insert a single silver-chloride wire into the electrode holder, ensuring that the end of the filament contacts the metal bottom of the holder.
    2. Carefully insert a glass capillary into the micropipette puller, and set puller program to create pipettes with adequate tip size and an internal resistance of 30 MΩ when filled with 3 M KCl. These parameters may be achieved by using the programmable P-97 Flaming/Brown puller (Figure 1B) (see Notes).


      Figure 1. Prepared electrodes. A. Representative suction electrode polished to ~12-15 μm I.D. B. Representative recording electrode pulled using the parameters indicated in Notes.

    3. Load the elongated tip syringe with 3 M KCl and dispense enough KCl into the glass capillary to sufficiently backfill the tip. Once the tip has sufficient KCl in place, fill the glass capillary with enough KCl to ensure the electrode is at least half full.
    4. Slide the silver-chloride wire into the glass capillary ensuring that it is immersed in the KCl.
    5. Insert recording electrode into the electrode micromanipulator that will be used for grounding (Figure 5.2).
  3. Preparing suction electrode
    1. Carefully insert a glass capillary tube, without filament, into the micropipette puller and set puller program to create pipettes with adequate tip size. Generally, the parameters used for creating the recording electrodes can also be used to make the suction electrode.
    2. Using a diamond coated bench stone (coarse side) break the tip sufficiently so that a flat opening of ~30 μm is created.
    3. Heat polish the electrode tip to reduce the I.D. to ~12-15 μm. Polishing is required to produce a sufficient taper to ensure that the CM9 MN does not come out of the electrode during recording and thus provides an adequate seal around the CM9 MN (Figure 1A).
    4. Once the suction electrode has been sufficiently polished, create a hole in the side of the electrode, about 1 cm down from the base of the tip, to allow for the placement of the silver wire. This is achieved by using the ‘fine’ side of the diamond coated bench stone to initially make a small hole. Once created, angle the suction electrode more acutely and continue scoring the glass. This will create a small gradient that will help to discourage bending of the silver wire required for stimulation (Figure 2).


      Figure 2. Representative image of the final suction electrode. Arrow indicates location of scored hole and gradient required to discourage bending of the silver wire required to stimulate the CM9 MN.

  4. Preparing fly for the experiment:
    1. Remove a single virgin female fly (see Notes) from food vial using a Pasteur pipette.
    2. Place the fly on ice until postural control is lost ~15/20 sec.
    3. Quickly transfer the fly to a small sylgard dissection surface and decapitate the head using a 30 G needle (Figure 3, right image).
    4. Place the back of the head on the dissection surface and pin the proboscis into an extended position (Figure 3, left image).
    5. Following successful pining (Figure 3, left image), cover the entire head in cold HL3 recording solution (see Recipes).


      Figure 3. Representative image showing fly before (right) and after decapitation (left). Dashed line indicates where on the fly to cut to successfully decapitate the head. Image on the left shows how to correctly pin out the head prior to dissecting the cuticle–black box indicates area of cuticle to be removed in order to expose the CM9 group of muscles.

    6. Dissect the anterior head cuticle containing the antennae from the preparation using a sharp tungsten needle to pierce the soft cuticle found underneath the faceplate and Vannas spring scissors to cut the cuticle. 5CO fine forceps are then used to remove any remaining tracheal air sacks that may remain after removing the faceplate. The use of 5CO forceps here to remove the remaining air sacks reduces the risk of damage to the nerve/muscles (Figure 3, left image and Figure 4B–black boxes).
    7. Re-pin the proboscis in a retracted position to increase the tension on the CM9 muscles (Figures 4B and 4C) (see Notes). The CM9 group of muscles run from the rostrum, identified by the brown/orange structure found at the end of the proboscis musculature located within the head cavity, to the bottom of the eye on either side of the head. Figure 4D indicates the translucency of the muscles and can be easily identified when compared to the more opaque fat bodies that remain within the head cavity post dissection.


      Figure 4. CM9 preparation. A. Diagram of Drosophila head indicating the approximate location of the CM9 muscle and motor neuron. B. Representative image of dissected fly head indicating the final positioning of the insect pins required to create sufficient tension on the muscles during recordings. C. Magnified image of CM9 muscles and MN (D) Dissected CM9 with * indicating the position of most cranial muscle fiber used for recordings.

    8. Move the dissection dish to the slide holder on the microscope stand (Figure 5.4). Once in position, place both the grounding wire (Figure 5.5) and stimulation grounding wire (Figure 5.7) into the HL3 bath to ensure the formation of a complete electrical circuit.
    9. Using the 10x objective (Figure 5.3), slowly lower the suction electrode (Figure 5.6) into the HL3 bath and continue motion of the electrode until it comes into the plane of view of the preparation. Generally, as long as care is taken when lowering the electrode, the manipulator setting can be left on 1.


      Figure 5. An overview of the rig setup. 1. Recording electrode head stage; 2. Recording. Electrode holder; 3. 10x/40x objective; 4. Microscope slide holder; 5. Grounding wire; 6. Suction electrode wire/holder; 7. Stimulator grounding wire.

    10. Once positioned, suck a loop of the CM9 MN (Figure 4C–Pharyngeal nerve) into the polished suction electrode and fill with modified HL3 to create an en passant configuration (Figure 6A). Sometimes it will be necessary to switch to the 40x objective for the purposes of sucking up the CM9 nerve as it is not always easily observed under the 10x objective.
      Note: There are 2 nerves that innervate the CM9 group of muscles but very little is known about the second smaller innervation and how it is involved in CM9 function. We do know that the second innervation is not a glutamatergic output due to the lack of positive vGLUT staining at the presynaptic nerve terminal. Care must be taken here to only suck up the CM9 MN and not the smaller innervation as this will impede the successful stimulation of the muscle.


      Figure 6. Representative mEPSP and EPSP traces from CM9 NMJ. A. Recording arrangement; B. Representative traces of an EPSP and mEPSP generated from a 7-day old animal.

  5. Intracellular recordings from CM9 muscle
    1. Slowly lower recording electrode into HL3 using coarse motion (setting 3/4) on the ROE-200 micromanipulator (Figure 5.1). Using the 10x objective, find the tip of the recording electrode and lower with caution until the electrode is in the plane of view of the preparation.
    2. Once the electrode is in the plane of view of the CM9 group of muscles finer motion is used to avoid impaling the muscle and breaking the electrode (setting 6/7 on ROE-200 micromanipulator). At this point, the objective should also be changed to 40x making sure to avoid hitting the electrodes already in position.
    3. Identify muscle to be recorded from, generally the most cranial CM9 muscle fiber accessible from the anterior position, and carefully impale the recording electrode into the muscle (Figure 4D).
  6. Stimulating the CM9 MN
    1. Stimulate the CM9 at 0.5-5 V for 300 μsec. The observance of a presynaptic based action potential is confirmed by the observance of a distinct voltage threshold for EPSP appearance (Figure 6B).
    2. Generally, animals 7-35 days old have EPSPs amplitudes of ~9 mV (± 0.36) and ~12 mv (± 0.55) for 42 days old animals. mEPSP amplitudes remain relatively constant throughout the lifespan of the fly (~1.13 mV [± 0.43]) (see Mahoney et al., 2014 for complete values for 7-60 days old virgin females).

Data analysis

  1. A Neuroprobe Amplifier Model 1600 is used in combination with a PowerLab 4/30 to amplify and digitize the data.
  2. LabChart 7 is used to record the data for at a rate of 1 Hz for 60 sec per fly taking care to record input resistance after the terminus of each 60 sec train.
  3. MiniAnalysis is used to analyze the amplitudes of both mEPSP and EPSPs (Figure 6B). From MiniAnalysis the values for EPSP and mEPSP amplitudes are tabulated in GraphPad Prism 6 from which appropriate statistical analysis is carried out to assess significance. Generally, all multiple comparisons are performed using a one-way ANOVA with a Bonferroni correction for multiple comparisons. All two-way comparisons are performed using a standard Student’s t-test.

Notes

  1. Fly husbandry: Virgin females are maintained at 25 °C and flipped every other day until needed for recording purposes. It is recommended that the flies used in the experiments do not grow in an over-populated environment (generally ~10-20 virgin females per vial).
  2. Muscle tension: To insure successful recordings, it is necessary to place sufficient tension on the CM9 group of muscles. This can be achieved by gently pushing the proboscis, via moving of the dissecting pin impaled proboscis, posteriorly towards the brain and sweeping the pin towards the base of the eye. Depending on which set of CM9 muscles are recorded from this will determine to which side the proboscis is repositioned.
  3. Electrode parameters: Heat–611, Pull–70, Vel–60, Del–130.
  4. A sharp recording electrode, similar to those used in this protocol, are used primarily to measure the electrical current passing through, or the voltage across, a neuronal/cellular membrane. These electrodes differ from classical whole cell patch pipettes in that the tip needs to be sufficiently sharp enough to penetrate the cellular membrane. On the other hand, the function of the suction electrode is to create a seal around the axon to allow for the detection of local circuit currents flowing around the axon as the action potential propagates, this can then be observed in the muscle via the use of the recording electrode. Depending on whether current clamp or voltage clamp mode is used this will determine the observance of either an EPSP (current clamp) or an EPSC (endplate postsynaptic current–voltage clamp).

Recipes

  1. Modified hemolymph like solution (HL3.1)

    Note: Remaining volume needed for 250 ml is made up of ddH2O. Both CaCl2 and MgCl2 are added to 10 ml HL3 prior to recording. Adjust pH to 7.31 before use.

References

  1. Brown A. L., Johnson, B. E., Goodman, M. B. (2008). Making patch-pipettes and sharp electrodes with a programmable puller. J Vis Exp (20): e939.
  2. Gordon, M. D. and Scott, K. (2009). Motor control in a Drosophila taste circuit. Neuron 61(3): 373-384.
  3. Kimura, K. I., Shimozawa, T. and Tanimura, T. (1986). Isolation of Drosophila mutants with abnormal proboscis extension reflex. J Exp Zool 239(3): 393-399.
  4. Mahoney, R.E., Azpurua, J. and Eaton, B.A. (2016). Insulin signaling controls neurotransmission via the 4eBP-dependent modification of the exocytotic machinery. Elife 5.
  5. Mahoney, R. E., Rawson, J. M. and Eaton, B. A. (2014). An age-dependent change in the set point of synaptic homeostasis. J Neurosci 34(6): 2111-2119.
  6. Rawson, J.M., Kreko, T., Davison, H., Mahoney, R., Bokov, A., Chang, L., Gelfond, J., Macleod, G. T. and Eaton, B. A. (2012). Effects of diet on synaptic vesicle release in dynactin complex mutants: a mechanism for improved vitality during motor disease. Aging Cell 11(3): 418-427.

简介

围绕哺乳动物突触前记录的复杂性是哺乳动物中枢神经系统(CNS)神经传递过程中突触前机制研究的重要障碍。 在这里,我们描述成人飞行神经肌肉接头(NMJ),西伯利亚肌肉9(CM9)NMJ,其允许记录诱发(EPSP)和自发性突触后兴奋性潜力(mEPSPs)在成熟的谷氨酸能突触。 结合CM9特异性遗传技术,CM9 NMJ提供了一个强大的实验系统,以更好地了解成熟突触神经递质释放的调节。
【背景】在老化过程中定义突触前功能变化的重要障碍是由于缺乏一个简单的模型系统,用于执行必要的电生理记录,以彻底地表征神经递质从突触前神经末梢的释放。现有的啮齿动物模型遭受与衰老研究相关的显着成本问题和在具有一致释放参数的单个定义的神经末梢上使用电生理记录的技术难度。为了克服这些障碍,我们在成年果蝇中开创了一种模型突触系统,用于分析年龄对神经传递过程中突触前功能的影响,CM9 NMJ位于飞翔的长鼻(Rawson等) ,2012; Mahoney等人,2014; Mahoney等人,2016)(图4A)。简而言之,CM9运动神经元(MN)的突触前心轴收敛于CM9肌肉的15个肌肉纤维,以形成35个独立的神经支配(Rawson等人,2012)。已显示CM9 MN对于CM9肌肉的收缩是必需的,并且是CM9肌肉的谷氨酸能输入的唯一来源(Kimura等人,1986; Gordon和Scott,2009)。鉴于苍蝇和哺乳动物之间突触小泡(SV)释放机制的高度保守性质以及哺乳动物CNS中发现的中枢突触的相似性,这使得CM9 NMJ成为调查突触前功能的有力模型。

关键字:果蝇, 衰老, 神经肌肉接头, 神经传递

材料和试剂

  1. 无菌一次性过滤器(0.22μm孔径,aPES膜19.6cm 2),CA膜)(例如Corning 250ml真空过滤器/储存瓶系统,Corning,目录号: 430767)
  2. 硼硅酸盐玻璃毛细管(外径1.50 mm,ID 0.86 mm)(Sutter Instrument,目录号:BF150-86-10)
  3. 无长丝的硼硅酸盐玻璃毛细管(OD 1.50 mm,ID 0.86 mm)(Sutter Instrument,目录号:B150-86-10)
  4. 银线(A-M系统,目录号:782000)
  5. 金刚石涂层台板石(如Dia-Sharp台阶上的DMT 8)
  6. 绘出P200提示
  7. 10ml注射器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:S7510-10)
  8. Minutien针(精细科学工具,目录号:26002-10)
  9. 精美油漆刷
  10. 所需基因型和年龄的苍蝇
    注意:通过使用E49-Gal4驱动程序(来自Ulrike Heberlein的Gal4系列的E49-Gal4),可以在CM9运动神经元内驱动UAS构建体。
  11. 50%漂白
  12. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G)
  13. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653-250G)
  14. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:21115-250ML)
  15. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:63069-500ML)
  16. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761-500G)
  17. 海藻糖(Sigma-Aldrich,目录号:T9531-25G)
  18. HEPES(Sigma-Aldrich,目录号:H3375-25G)
  19. 蔗糖(Sigma-Aldrich,目录号:84097-250G)
  20. 修饰的血淋巴样溶液(HL3.1)(见食谱)

设备

  1. 万纳斯弹簧剪刀-2毫米刀刃(精细科学工具,目录号:15000-03)
  2. 钨解剖针125毫米,超细(Roboz手术器械,目录号:RS-6063)
  3. 微型解剖针架5¼“(Roboz Surgical Instrument,目录号:RS-6060)
  4. 精镊子(如Dumont#5CO,Fine Science Tools,目录号:11295-20和Dumont#3,Fine Science Tools,目录号:11231-30)
  5. 解剖立体变焦显微镜(如ZEISS,型号:SteREO Discovery.V8)
  6. 窄格式机械手系统(如Sutter Instrument,型号:ROE-200)
  7. 水平式微量拔管器(如可编程火焰/棕色型微量吸管拔出器,Sutter Instrument,型号:P-97)
  8. 微锻电极抛光机(如NARISHIGE,型号:MF-900)
  9. 直立共焦显微镜(如奥林巴斯,型号:BXS1WI),具有10x和40x水位目标
  10. 电力实验室4/30数字转换器(AD仪器,型号:ML866)
  11. Neuroprobe放大器1600(A-M系统,型号:1600型,目录号:680100)
  12. 刺激器(Digitimer,型号:DS2A)
  13. Labchart 7(AD仪器)

软件

  1. Prism 6(GraphPad软件)
  2. 迷你分析(可比版本6.0.3,Synaptosoft Inc.)

程序

  1. 准备氯化银线
    将银线放在50%漂白/ 50%ddH 2 O溶液中,使至少3/4的线浸入液体中。将电线留在漂白/水溶液中约5分钟,以便发生足够的氯化
  2. 准备记录电极 - 参见2008年的Brown Joe视频“使用可编程拉杆进行贴片移液器和锋利的电极”。参考(见注释)。
    1. 将一根氯化银线插入电极支架,确保灯丝的端部接触支架的金属底部。
    2. 小心地将玻璃毛细管插入微量移液器拉拔器,并设置拉拔程序以填充3 M KCl时,产生足够的尖端尺寸和30MΩ内阻的移液器。这些参数可以通过使用可编程P-97 Flaming / Brown拉拔器来实现(图1B)(见注释)。


      图1.制备的电极。 A.代表性的吸电极抛光至〜12-15μmI.D. B.使用注释中所示的参数拉出代表性记录电极。

    3. 用3 M KCl装入细长尖端注射器,并将足够的氯化钾分配到玻璃毛细管中,以充分回填尖端。一旦尖端有足够的KCl就位,用足够的氯化钾填充玻璃毛细管,以确保电极至少半满。
    4. 将氯化银线滑入玻璃毛细管,确保浸入KCl中。
    5. 将记录电极插入将用于接地的电极显微操纵器(图5.2)。
  3. 准备吸电极
    1. 小心地将玻璃毛细管插入微量拔出器,并将拉丝器程序设置为具有足够的尖端尺寸的移液器。通常,用于创建记录电极的参数也可用于制造吸电极。
    2. 使用金刚石涂层台板(粗面)充分破裂尖端,使得产生〜30μm的平坦开口。
    3. 热抛光电极尖以减少I.D.至〜12-15μm。需要抛光才能产生足够的锥度,以确保CM9 MN在记录过程中不会从电极中脱出,从而在CM9 MN周围提供足够的密封(图1A)。
    4. 一旦吸入电极被充分抛光,在电极侧面形成一个距离电极底部约1厘米的孔,以便放置银线。这是通过使用金刚石涂层台阶的“细”侧来初始化一个小孔来实现的。一旦创建,吸引电极更锐利地倾斜,并继续打分玻璃。这将产生一个小的渐变,这将有助于阻止刺激所需的银丝的弯曲(图2)

      图2.最终吸引电极的代表性图像。箭头表示打击孔的位置和阻止刺激CM9 MN所需的银线弯曲所需的梯度。

  4. 为飞行准备实验:
    1. 使用巴斯德吸管从食物小瓶中取出一只处女雌蝇(见附注)。
    2. 将飞行物放在冰上,直至姿势控制失效〜15/20秒
    3. 快速将飞行物转移到一个小的Sylgard解剖表面,并使用30 G针头打死头部(图3,右图)。
    4. 将头背放在解剖面上,将长鼻针插入延伸位置(图3左图)。
    5. 在成功打浆(图3左图)后,在冷HL3记录解决方案中覆盖整个头部(见配方)。


      图3.代表性图像,显示飞前(右)和断头后(左)。 虚线表示在飞行中切割成功地斩首头。左侧的图像显示如何在剖开角质层之前正确地排出头部黑色框表示要移除的角质层的区域,以暴露CM9组肌肉。

    6. 使用尖锐的钨针将准备好的包含天线的头部头皮角质层解剖,以穿透面板下方的软角质层和Vannas弹簧剪刀切割角质层。然后使用5CO细镊子去除可能在移除面板后残留的任何剩余气管气囊。在这里使用5CO镊子去除剩余的空气袋可减少损伤神经/肌肉的风险(图3,左图和图4B-黑盒)。
    7. 将长鼻针重新插入收缩位置,以增加CM9肌肉的张力(图4B和4C)(见注)。 CM9组肌肉从位于头腔内的长鼻肌肉结束处发现的棕色/橙色结构识别,在头部两侧的眼睛底部。图4D显示肌肉的半透明度,与残留在头腔内解剖后的更不透明的脂肪体相比较,可以很容易地识别。


      图4. CM9准备。 A.表示CM9肌肉和运动神经元的大致位置的果蝇的图。 B.解剖的飞头的代表性图像,指示在记录期间在肌肉上产生足够张力所需的昆虫针的最终定位。 C. CM9肌肉和MN的放大图像(D)解剖CM9与*表示用于记录的大多数颅肌纤维的位置。

    8. 将解剖盘移至显微镜架上的支架上(图5.4)。一旦就位,将接地线(图5.5)和刺激接地线(图5.7)放入HL3浴中,以确保形成完整的电路。
    9. 使用10x物镜(图5.3),将吸电极(图5.6)缓慢降低到HL3浴中,并继续电极的运动,直到其进入准备平面。通常,只要注意降低电极时,可以将机械手设置保持为1.


      图5.钻机设置概述。 1。记录电极头阶段; 2.录音。电极座3.10x / 40x目标;显微镜滑块架;接地线吸电极线/支架; 7.刺激器接地线。

    10. 一旦定位,将CM9MN(图4C-咽神经)的环吸入抛光的吸引电极中,并用修改的HL3填充以产生通过结构(图6A)。有时,为了吸收CM9神经,有必要改用40倍的目标,因为在10x目标下并不总是很容易观察到。
      注意:有两个神经支配着CM9组的肌肉,但是对于第二个较小的神经支配以及如何参与CM9功能却知之甚少。我们知道,由于在突触前神经末梢缺乏阳性vGLUT染色,第二次神经支配不是谷氨酸能输出。必须注意,只能吸收CM9 MN,而不是较小的神经支配,因为这将阻碍成功的肌肉刺激。


      图6. CM9 NMJ的代表性mEPSP和EPSP迹线。 :一种。录音安排; B.由7天龄的动物产生的EPSP和mEPSP的代表性痕迹。

  5. 来自CM9肌肉的细胞内记录
    1. 在ROE-200显微操作器上使用粗略运动(设置3/4)将记录电极缓慢降低为HL3(图5.1)。使用10x物镜,找到记录电极的尖端,并小心,直到电极处于制备平面。
    2. 一旦电极处于CM9组肌肉细胞运动的平面,就可以避免刺激肌肉和破坏电极(设置在ROE-200显微操作器上的6/7)。在这一点上,目标也应该改为40倍,确保避免击中已经就位的电极。
    3. 确定肌肉记录,一般是从前位置可以获得的最颅骨CM9肌肉纤维,并将记录电极小心地刺入肌肉(图4D)。
  6. 刺激CM9 MN
    1. 在0.5-5 V下刺激CM9 300微秒。通过遵守EPSP外观的不同电压阈值来确认遵守基于突触前的动作电位(图6B)。
    2. 一般来说,7-35天的动物对42天龄的动物的EPSPs幅度为〜9mV(±0.36)和〜12mv(±0.55)。 mEPSP幅度在整个飞行寿命期间保持相对恒定(约1.13mV [±0.43])(参见Mahoney等人,2014年为7-60天龄的女性女性的完整值)。 />

数据分析

  1. 1600型的Neuroprobe放大器与PowerLab 4/30结合使用,用于放大和数字化数据。
  2. LabChart 7用于以1 Hz的速率记录数据,每次飞行60秒,请注意在每60秒火车终点后记录输入电阻。
  3. MiniAnalysis用于分析mEPSP和EPSPs的幅度(图6B)。从MiniAnalysis中,EPSP和mEPSP幅度的值在GraphPad Prism 6中列出,从中进行适当的统计分析以评估显着性。通常,使用具有Bonferroni校正的单因素方差分析进行所有多重比较以进行多次比较。所有的双向比较都是使用标准学生的测试进行的。

笔记

  1. 养殖业:维珍女性维持在25°C,每隔一天翻转一次,直到需要进行记录。建议实验中使用的苍蝇不会在人口稠密的环境中生长(通常每瓶约10-20个女性)。
  2. 肌肉紧张:为了确保成功录音,必须在CM9肌肉组上施加足够的张力。这可以通过轻轻推动长鼻,通过将解剖的针刺入的长鼻子向后推向大脑并将针朝向眼睛的基部扫过来实现。根据哪一组CM9肌肉记录,这将决定哪一侧长鼻子重新定位。
  3. 电极参数:Heat-611,Pull-70,Vel-60,Del-130。
  4. 与本协议中使用的那些类似的尖锐记录电极主要用于测量通过的电流或跨越神经元/细胞膜的电压。这些电极与传统的全细胞贴片移液管不同,因为尖端需要足够锐利以穿透细胞膜。另一方面,吸引电极的功能是在轴突周围产生密封,以便当动作电位传播时检测在轴突周围流动的局部电路电流,然后可以通过使用记录电极。根据是否使用电流钳位或电压钳模式,这将决定是否遵守EPSP(电流钳)或EPSC(终板突触后电流电压钳位)。

食谱

  1. 修饰的血淋巴样溶液(HL3.1)

    注意:250毫升所需的剩余体积由ddH 2 O组成。添加了CaCl 2 和MgCl 2 在记录之前至10毫升HL3。使用前请将pH调至7.31。

参考

  1. Brown AL,Johnson,BE,Goodman,MB(2008)。  使用可编程拉杆制作贴片移液器和锋利电极。 (20):e939。
  2. Gordon,MD和Scott,K.(2009)。电动机控制在果蝇味道电路。 神经元 61(3):373-384。
  3. Kimura,KI,Shimozawa,T.and Tanimura,T。(1986)。分离具有异常长鼻反射的果蝇突变体。 J Exp Zool 239(3):393-399。
  4. Mahoney,R.E.,Azpurua,J.and Eaton,B.A。 (2016)。胰岛素信号通过4eBP-依赖性的细胞增殖机制的修改。 5.
  5. Mahoney,RE,Rawson,JM和Eaton,BA(2014)。  突触内稳态设定点的年龄依赖性变化。 Neuros ci 34(6):2111-2119。
  6. Rawson,JM,Kreko,T.,Davison,H.,Mahoney,R.,Bokov,A.,Chang,L.,Gelfond,J.,Macleod,GT and Eaton,BA(2012)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/22268717”target =“_ blank”>饮食对dynactin复合突变体突触小泡释放的影响:改善活力的机制在运动疾病期间。老年细胞 11(3):418-427。
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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: © 2017 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. Eaton, B. A. and Mahoney, R. E. (2017). Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster. Bio-protocol 7(14): e2401. DOI: 10.21769/BioProtoc.2401.
  2. Mahoney, R. E., Rawson, J. M. and Eaton, B. A. (2014). An age-dependent change in the set point of synaptic homeostasis. J Neurosci 34(6): 2111-2119.
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