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Jun 2016

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Extracellular Axon Stimulation
细胞外轴突刺激   

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Abstract

This is a detailed protocol explaining how to perform extracellular axon stimulations as described in Städele and Stein, 2016. The ability to stimulate and record action potentials is essential to electrophysiological examinations of neuronal function. Extracellular stimulation of axons traveling in fiber bundles (nerves) is a classical technique in brain research and a fundamental tool in neurophysiology (Abbas and Miller, 2004; Barry, 2015; Basser and Roth, 2000; Cogan, 2008). It allows for activating action potentials in individual or multiple axons, controlling their firing frequency, provides information about the speed of neuronal communication, and neuron health and function.

Keywords: Action potentials (动作电位), Electrophysiology (电生理学), Neuron (神经元), Threshold (阈值), Artifact (伪影), Anode (阳极), Cathode (阴极)

Background

Extracellular axon stimulation elicits action potentials (APs) without the need of introducing electrodes into neurons. This protocol describes cathodal stimulation, which uses the fact that the membrane potential of a neuron at rest is negative while the extracellular surrounding is positive in comparison. Two electrodes are needed: (1) a stimulation electrode (cathode) placed in close proximity to the axon, and (2) a reference electrode (anode) placed in the bath. When activated, the stimulation electrode adds electrons and thus negative charge to the outside of the axon. This makes the outside of the axon less positive and, as a consequence, decreases the potential difference between inside and outside of the neuron. The result is a local depolarization inside the axon. If sufficient in magnitude, this elicits an AP. The elicited AP originates close to the stimulation electrode and propagates bi-directionally along the axon.

The threshold current needed to elicit APs depends on several parameters, including (1) axon diameter (thicker axons are depolarized first), (2) the distance between stimulation electrode and axon, and (3) stimulation amplitude and duration. The duration must be limited to less than the duration of an AP to prevent the neuronal membrane from becoming refractory. Thus, short current pulses at threshold amplitude are typically used to elicit individual APs. Since thicker axons are recruited at lower stimulus amplitudes, extracellular stimulation works best if the axon of interest is the one with the largest diameter in the nerve. If smaller axons are targeted, larger stimulus amplitudes are required, which may activate larger axons first, in addition to the smaller axons of interest.

Materials and Reagents

Note: The materials and equipment listed refer to the equipment used in Städele and Stein (2016). The principles of retrograde extracellular axon stimulations are universal and the procedures can be easily adapted to other preparations. To reduce costs, comparable materials, equipment and software may be used that serve the same functions. For the general public or a teaching classroom, we suggest utilizing equipment from Backyard Brains (https://backyardbrains.com).

  1. Syringe, filled with petroleum jelly for preparing extracellular recording and stimulation wells
    For preparing syringes, the following materials will be needed:
    1. Petroleum jelly (100% pure, pharmacy)
    2. 100 ml glass beaker (for melting petroleum jelly)
    3. 5 ml Luer lock syringe (e.g., BD, catalog number: 309603 )
    4. Injection needle (20 G x 1.5”, e.g., Santa Cruz Biotechnology, catalog number: sc-359535 )
    5. Sand paper (80 to 100 grit, hardware store)
  2. Recording/stimulation electrodes
    For preparing electrodes, the following materials will be needed:
    1. Stainless steel wire, uncoated (A-M Systems, catalog number: 794800 )
      Low-cost alternative: Minutien pins (see below) or sewing pins
    2. Electrical wire, red and black PVC insulated (Southwire, model: 22 gauge stranded, catalog number: 57572444 , hardware store)
    3. Wire stripper (hardware store)
    4. Needle-nose pliers (hardware store)
    5. Heat shrink tubing (hardware store)
    6. Tin solder, 3/32 in. (Forney, catalog number: 38109 )
  3. Petri dish lined with silicon elastomer (e.g., Sylgard 184, Sigma-Aldrich, catalog number: 761036 ; or Elastosil RT 601, Wacker Chemie, catalog number: 60063613 )
  4. Minutien pins (Fine Science Tools, catalog number: 26002-10 )
  5. Modeling clay (craft store)
  6. Dissected nervous system
    Note: We are using adult Jonah crabs (Cancer borealis), purchased from The Fresh Lobster Company (Gloucester, MA).
  7. Physiological saline (see Recipes)
    The recipe for C. borealis saline can be found in Table 1
    1. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
    2. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M9272 )
    3. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
    4. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    5. Trizma base (Sigma-Aldrich, catalog number: T1503 )
    6. Maleic acid (Sigma-Aldrich, catalog number: M0375 )

Equipment

  1. Heating plate (Thermo Fisher Scientific, Thermo ScientificTM, model: NuovaTM Stirring HotPlates, catalog number: SP18425Q )
  2. Stereomicroscope (e.g., Leica Microsystems, model: MS5 )
  3. Stimulator (A.M.P.I, model: Master8 Pulse Stimulator )
    Low cost alternative: Pulse Pal V2 (Sanworks, catalog number: 1102 )
  4. Amplifier (A-M Systems, model: Differential AC Amplifier 1700 , catalog number: 690000)
    Low cost alternative: Spikerbox (Backyard Brains, model: Neuron SpikerBox )
  5. Data acquisition board (CED, model: Power 1401-3A )
    Low-cost alternative: by using the BYB Spike Recorder, data can be digitized by using the microphone jack and soundcard on a computer/laptop. A second low-cost alternative is Spikehound (http://spikehound.sourceforge.net), which also allows recording through the computer soundcard
  6. Soldering station (Apex Tool, Weller, model: Station 50/60W 120 V WES51 , catalog number: WES51)
  7. Voltmeter (FLIR Systems, Extech, model: EX330 , catalog number: 203489911)
  8. Forceps (e.g., Fine Science Tools, model: Dumont #5, catalog number: 11251-10 )

Software

  1. Recording software (Spike2 version 7.12, Cambridge Electronic Design Limited)
    Low cost alternative: BYB Spike Recorder (freeware, available on https://backyardbrains.com/products/spikerecorder) or Spikehound (http://spikehound.sourceforge.net)

Procedure

Figure 1 illustrates the experimental setup and electrode placement. To stimulate an axon, a section of the nerve of interest will be electrically isolated from the rest of the nervous system by using a petroleum jelly well. The success of the stimulation will be monitored by extracellularly recording the elicited AP on a spatially distant part of the axon.


Figure 1. Schematic of the experimental setup and electrode placement. Illustrated is a Petri dish that contains the nerve. The blue cylinders represent petroleum jelly wells, the black lines illustrate electrode placement and their connection to stimulation and recording equipment.

  1. Preparing petroleum jelly syringes
    1. Transfer petroleum jelly to a glass beaker.
    2. Place glass beaker on heating plate and warm petroleum jelly up until it is fully melted.
      Note: The melting point of petroleum jelly is between 45 °C and 60 °C. Avoid boiling! Boiling will create air bubbles.
    3. Take the 5 ml Luer lock syringe and fill it with the melted petroleum jelly by pulling the jelly up through the tip.
      Note: Wear gloves to protect yourself from burns.
    4. Let the filled syringe sit at room temperature until petroleum jelly is solidified.
      Note: To prevent air bubbles, place the filled syringe upwards (with the tip towards the ceiling) until the petroleum jelly solidifies.
    5. Cut off the tip of a 20-gauge injection needle using the wire-cutting pliers.
      Note: Use sand paper to smoothen the sharp edges.
    6. Bend the front part of the injection needle by an angle of ~70° (Figure 2).
    7. Add the needle to the syringe.


      Figure 2. Petroleum jelly filled syringe used for preparing the extracellular stimulation/recording wells

  2. Preparing stimulation and recording electrodes
    Note: Repeat the following steps to create all necessary electrodes including the stimulating, recording, and grounding electrodes. The stimulating and recording electrodes require both a red and black wire, while the ground only needs a single wire.
    1. Cut the electrical wires to the desired length.
    2. Use the wire stripper to remove 5 to 10 mm of the insulation on each side of each red/black wire.
    3. Take the pre-heated soldering iron and coat the stripped part of the cable with tin-solder (Figure 3A).
    4. Take needle-nose pliers and bend the tin-solder covered wire part into a loop (Figure 3B).
    5. Pass the stainless steel wire through the loop.
      Note: Instead of stainless steel wire, minutien or sewing pins can be used as well.
    6. Fill the loop with tin-solder to attach the stainless steel wire to the black/red wire (Figure 3C).
    7. Use a voltmeter to check if the stainless steel wire has the proper electrical contact with the wire.
    8. Solder a suitable plug onto the other side of the cable to attach it to the stimulator/amplifier.
    9. Use heat shrink tubing to insulate the blank solder joints.
    10. In addition to stimulation and recording electrodes, a single ground electrode is necessary to reduce recording noise and stimulation artifact. Repeat the above steps using a black wire to build a single electrode.


      Figure 3. Preparation of extracellular stimulation/recording electrode. A. Electrical wire with insulated and tin-solder coated front part B; Same wire, bent into a loop; C. Stainless steel wire attached to loop with tin-solder.

  3. Dissect the nerve of interest
    Note: For a detailed protocol how to dissect the stomatogastric nervous system of C. borealis please review (Gutierrez and Grashow, 2009).
  4. Transfer the nervous system to a silicone lined Petri dish.
    Note: The silicon elastomer should coat the bottom ~5 mm of the Petri dish.
    Place the nervous system in the middle of the dish.
    1. Fill the Petri dish with physiological saline (see Table 1 for C. borealis saline).
    2. Use minutien pins to mount the nervous system by either directly pinning through the end of nerves or by pinning through connective tissue still attached to the nervous system.
      Note: Do not pin through the section of nerve being recorded as this will damage axons.
  5. Secure the dish from displacement by attaching it to the work surface with modeling clay (Figure 4).
  6. Place the ground electrode into the bath. Use the modelling clay to prevent the ground electrode from displacement. Connect the ground wire to the amplifier ground.


    Figure 4. Petri dish with isolated stomatogastric nervous system. The photo shows the placement of the stimulation and recording wells along a stomatogastric nerve, saline inflow and outflow, and ground wire. The Petri dish is attached to the work surface with modelling clay. For extracellular stimulation and recording, no vibration isolation laboratory tables are needed. For better visualization, nerves have been retraced (gray and pink lines). 

  7. Prepare the extracellular stimulation/recording wells.
    1. With the use of a stereomicroscope make two concentric circles at two locations on the nerve of interest with the petroleum jelly filled syringe as shown in Figure 5. One well will serve as stimulation well, the other will serve as recording well. Add layers of petroleum jelly until the wells extend above the saline level (Figures 5A and 5B).
      Note: The well isolates a small section of the nerve from the rest of the bath as petroleum jelly is nonconductive.
    2. Ensure that the wells are tight and not leaky.
      Note: Add one or two drops of saline to the inside of the well. If the level of saline does not decrease, the well is sufficiently tight and isolates the nerve section from the rest of the dish (Figure 5C).


      Figure 5. Petroleum jelly well preparation. A. An initial concentric circle was formed around a small section of the nerve of interest. B. Finished well. Petroleum jelly layers were added until the top most layer extended out of the saline. C. Leak test. Saline was added inside the well to test if it is sufficiently isolating the nerve section from the rest of the bath.

  8. Place stimulation electrodes.
    1. Connect the stimulation cable with the stimulator (red wire = cathode, black wire = anode).
    2. Use forceps to place the steel wire (cathode) inside the stimulation well (Figure 6). Firmly press the wire into the silicone.
      Note: Avoid puncturing nerve or well. Place the end of the electrode deep enough into the silicone so the wire will not become dislodged at any point during the remainder of the experiment.
    3. Use forceps to place the anode outside of the well, but still close to it.
      Note: Wires must not touch each other since this will create a short circuit and axons will not be stimulated.
    4. Secure the wires using modelling clay.


      Figure 6. Arrangement of stimulation wires in the petroleum jelly well

  9. Repeat step 8 for the recording wires.
  10. Turn the extracellular amplifier on and check if spontaneous APs are visible on the extracellular recording.
    Notes:
    1. For C. borealis, the amplifier filter settings should: amplification: 10,000; high pass filter: low cut-off 100 Hz; low pass filter: high cut-off 500 Hz.
    2. Detected AP waveforms do not directly reflect membrane potential changes. APs are detected using a differential recording between the inside and the outside of the recording well.
  11. Stimulate the nerve extracellularly.
    Initially, set the stimulation parameters on the Master8 to 1 ms stimulation pulse duration, 1 Hz stimulation frequency, and 0 nA/Volt. After the stimulation threshold has been determined, more physiological parameters may be used.
    1. Use the extracellular recording to first identify the stimulus artifact (Figures 7A and 7B).
      Note: Stimulation artifacts can be easily distinguished from APs because the size of the artifact increases linearly with stimulus amplitude. If the stimulation artifact is too large, the polarity of the cables should be reversed. The stimulation artifact will be smallest when the cathode (and not the anode) is placed inside the stimulation well.


      Figure 7. Adjustment of the extracellular stimulation threshold. Shown are extracellular recordings of a nerve in the stomatogastric nervous system during subthreshold (A, B), threshold (C) and suprathreshold stimulation (D). A. During subthreshold stimulation with low stimulus amplitude only the stimulation artifact can be observed, but no AP. The artifact occurs simultaneously with the stimulus pulse. B. Increasing stimulation amplitude below threshold will not elicit APs, but increase the stimulation artifact amplitude. C. Increasing stimulus amplitude to threshold elicits an AP. Its waveform appears with a fixed time delay after the stimulation artifact (gray box). The axon with the largest diameter in a nerve will be recruited first. Note that the AP waveform does not represent the different phases of an AP, but rather the potential difference between the inside and outside of the petroleum jelly well as the AP enters and leaves the well. D. Increasing the stimulation amplitude above threshold recruits additional, smaller diameter axons. Note that the shape and amplitude of the detected waveform changes when other axons are recruited (compound AP). For better visualization, the artifact amplitude was cropped (indicated by *).

    2. Determine the stimulation threshold by slowly increasing the stimulation amplitude until an AP becomes visible (Figure 7C).
      Note: The AP should appear with a time delay after the stimulation artifact. APs can be distinguished from the artifact because they occur only after stimulus threshold is reached, and their time of occurrence and amplitude does not increase linearly with the stimulus amplitude. Note that if stimulus amplitude is further increased, APs from other axons may be recruited, which will change the size and shape of the detected signal (compound APs during suprathreshold stimulation, see Figure 7D and Video 1).
    3. If the recording software has a trigger function, use it to control the sweeps of the extracellular recording to permit a fast time base, and a comparison of stimulus artifact and AP shapes.

      Video 1. Video clip illustrating subthreshold, threshold and suprathreshold stimulation

Data analysis

Data analysis will differ depending on the experiment, but the following steps should be kept in mind while designing an experiment using extracellular axon stimulation.

  1. To ensure that APs are elicited, it is essential to differentiate between stimulus artifact and arriving APs at the recording site. Elicited APs arrive with a constant delay after the stimulus pulse, and are independent of stimulus amplitude (Figure 7C). In contrast, the stimulation artifacts scale with stimulus amplitude and arrive almost instantaneously (Figures 7A and 7B).
  2. At subthreshold stimulation amplitudes, only the stimulation artifact is visible (Figure 7A). To test whether the recorded signal is indeed the artifact, the time between stimulus onset and signal arrival at the recording site can be measured, using vertical cursors. For signal arrival, the first obvious peak of the signal can be used. Further, the amplitude of the arriving signal should be measured between signal minimum and maximum, using horizontal cursors. At least 10 consecutive stimuli should be averaged to determine mean arrival time and amplitude.
  3. Increase and decrease stimulus amplitude slightly, and measure arrival time and amplitude of the recorded signal again. If the arrival time occurs at the same time as the stimulus pulse and amplitude scales with stimulus amplitude (Figure 7B), the recorded signal is the stimulation artifact. Measure at least 10 consecutive signals at each stimulus amplitude.
    Note: A t-test can be used to compare measurements at different stimulus amplitudes (significance cut off value of P < 0.05).
  4. Increase stimulus amplitude further until a second, delayed signal arrives at the recording site (threshold stimulation, Figure 7C). If this is the AP, it should occur suddenly when stimulus amplitude is raised (no slow build-up). Measure arrival times and amplitudes for at least 10 consecutive APs.
    Note: APs are unaffected by changes of stimulus amplitude. Confirm that the recorded signal is indeed the AP by slightly increasing the stimulus amplitude before measuring arrival times and amplitudes again. Arrival times of APs depend on the distance between stimulation and recording sites.
  5. Increasing the stimulus amplitude further (suprathreshold, Figure 7D) may recruit additional APs, whose delay and amplitudes can be measured using the same analysis.

Notes

  1. Prior to attempting axon stimulation, record spontaneous AP activity of at least 10 consecutive APs. There may be need to further reduce noise by grounding the stimulator to the amplifier and by utilizing a faraday cage.
  2. Instead of steel, silver chloride may be used for the ground wire. This is particularly important if additional intracellular recordings are planned and/or a drift of the recorded membrane potential must be avoided.
  3. If the stimulation artifact is large and has a long duration, the anode and cathode of the stimulator may be inverted. In this case, the polarity of the stimulation electrodes needs to be reversed, because the anode causes the extracellular space to become more positive and, as a consequence, the neuron hyperpolarizes locally. Since this hyperpolarization is surrounded by areas which, relatively, are depolarized, APs can still be elicited (anodic stimulation). However, APs stimulated by anodic stimulation have a higher threshold, causing the stimulus artifact to be larger.
  4. If no extracellular recording of the stimulated nerve is feasible, the success of the stimulation can be assessed by monitoring changes in activity of postsynaptic neurons, circuits, muscles, or behavior. In this case, higher stimulus frequencies are recommended to achieve clear postsynaptic responses.

Recipes

  1. Cancer borealis physiological saline recipe (see Table 1)

    Table 1. Cancer borealis physiological saline recipe

    Note: Adjust pH to 7.4-7.6 with Trizma base and maleic acid.

Acknowledgments

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG STE 937/9-1), National Science Foundation (NSF IOS 1354932), Illinois State University, and the German Academic Exchange Service.

References

  1. Abbas, P. J. and Miller, C. A. (2004). Biophysics and physiology. In: F.-G. Zeng and R. R. Fay. (Eds). Cochlear Implants: Auditory prostheses and electric hearing. Springer, 20: 149-212.
  2. Barry, J. M. (2015). Axonal activity in vivo: technical considerations and implications for the exploration of neural circuits in freely moving animals. Front Neurosci 9: 153.
  3. Basser, P. J. and Roth, B. J. (2000). New currents in electrical stimulation of excitable tissues. Annu Rev Biomed Eng 2: 377-397.
  4. Cogan, S. F. (2008). Neural stimulation and recording electrodes. Annu Rev Biomed Eng 10: 275-309.
  5. Gutierrez, G. J. and Grashow, R. G. (2009). Cancer borealis stomatogastric nervous system dissection. J Vis Exp(25)
  6. Städele, C. and Stein, W. (2016). The site of spontaneous ectopic spike initiation facilitates signal integration in a sensory neuron. J Neurosci 36(25): 6718-6731.

简介

这是一个详细的协议,说明如何执行细胞外轴突刺激,如Städele和Stein,2016所述。刺激和记录动作电位的能力对神经元功能的电生理检查至关重要。 在纤维束(神经)中行进的轴突的细胞外刺激是脑研究中的一种经典技术,也是神经生理学的基础工具(Abbas和Miller,2004; Barry,2015; Basser和Roth,2000; Cogan,2008)。 它允许在单个或多个轴突中激活动作电位,控制其发射频率,提供关于神经元通信速度以及神经元健康和功能的信息。
【背景】细胞外轴突刺激引起动作电位(AP),而不需要将电极引入神经元。该方案描述了阴极刺激,其使用静息神经元的膜电位为负,而细胞外周围为正的事实。需要两个电极:(1)放置在轴突附近的刺激电极(阴极)和(2)置于浴中的参比电极(阳极)。当激活时,刺激电极向轴突的外部添加电子,从而增加负电荷。这使得轴突的外侧不太积极,因此减小神经元内外的潜在差异。结果是轴突内部局部去极化。如果数量足够,这会引起AP。引发的AP起始于靠近刺激电极并沿着轴突双向传播。
引起AP所需的阈值电流取决于几个参数,包括(1)轴突直径(较粗略的轴突首先被去极化),(2)刺激电极和轴突之间的距离,以及(3)刺激幅度和持续时间。持续时间必须限制在少于AP的持续时间以防止神经元膜变得难治性。因此,通常使用阈值幅度的短电流脉冲来引出各个AP。由于在较低的刺激振幅下招募较厚的轴突,所以如果感兴趣的轴突是神经中最大直径的轴突,则细胞外刺激效果最好。如果靶向较小的轴突,则需要较大的刺激振幅,除了感兴趣的较小轴突之外,还可以首先激活较大的轴突。

关键字:动作电位, 电生理学, 神经元, 阈值, 伪影, 阳极, 阴极

材料和试剂

注意:所列材料和设备是指Städeleand Stein(2016)使用的设备。逆行细胞外轴突刺激的原理是普遍的,并且程序可以容易地适应于其它制剂。为了降低成本,可以使用具有相同功能的可比较材料,设备和软件。对于公众或教学教室,我们建议使用后院大脑的设备( https://backyardbrains.com )。

  1. 注射器,充满石油用于制备细胞外记录和刺激井
    对于准备注射器,需要以下材料:
    1. 石油果冻(100%纯,药店)
    2. 100毫升玻璃烧杯(用于熔化凡士林)
    3. 5ml鲁尔锁定注射器(例如,BD,目录号:309603)
    4. 注射针(20G×1.5",例如,Santa Cruz Biotechnology,目录号:sc-359535)
    5. 沙纸(80至100粒,五金店)
  2. 记录/刺激电极
    对于制备电极,将需要以下材料:
    1. 不锈钢丝,未涂层(A-M系统,目录号:794800)
      低成本替代方案:Minutien针脚(见下文)或缝纫针脚
    2. 电线,红色和黑色PVC绝缘(Southwire,型号:22号,目录号:57572444,五金店)
    3. 剥线钳(五金店)
    4. 针尖钳(五金店)
    5. 热缩管(五金店)
    6. 锡焊,3/32英寸(Forney,目录号:38109)
  3. 用硅弹性体(例如,Sylgard 184,Sigma-Aldrich,目录号:761036;或Elastosil RT 601,Wacker Chemie,目录号:60063613)衬里的培养皿
  4. Minutien针(精细科学工具,目录号:26002-10)
  5. 造型粘土(工艺品店)
  6. 解剖神经系统
    注意:我们正在使用从The Fresh Lobster Company(格洛斯特,马萨诸塞州)购买的成年约拿蟹(Cancer borealis)。
  7. 生理盐水(见食谱)
    C的食谱。北欧盐水可以在表1中找到
    1. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9625)
    2. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M9272)
    3. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C7902)
    4. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
    5. Trizma碱(Sigma-Aldrich,目录号:T1503)
    6. 马来酸(Sigma-Aldrich,目录号:M0375)

设备

  1. 加热板(Thermo Fisher Scientific,Thermo Scientific TM,型号:Nuova TM 搅拌HotPlates,目录号:SP18425Q)
  2. 立体显微镜(例如,,Leica Microsystems,型号:MS5)
  3. 刺激器(A.M.P.I,型号:Master8 Pulse Stimulator)
    低成本替代方案:Pulse Pal V2(Sanworks,目录号:1102)
  4. 放大器(A-M系统,型号:差分AC放大器1700,目录号:690000)
    低成本替代品:Spikerbox(Backyard Brains,型号:Neuron SpikerBox)
  5. 数据采集板(CED,型号:Power 1401-3A)
    低成本的替代方案:通过使用BYB Spike Recorder,可以使用计算机/笔记本电脑上的麦克风插孔和声卡对数据进行数字化。第二种低成本的替代方案是Spikehound( http://spikehound.sourceforge.net ),这也允许通过计算机声卡记录
  6. 焊接站(Apex工具,Weller,型号:Station 50/60W 120 V WES51,目录号:WES51)
  7. 电压表(FLIR Systems,Extech,型号:EX330,目录号:203489911)
  8. 镊子(例如,,Fine Science Tools,型号:Dumont#5,目录号:11251-10)

软件

  1. 录音软件(Spike2版本7.12,剑桥电子设计有限公司)
    低成本替代方案:BYB Spike Recorder(免费软体,可在 https://backyardbrains .com/products/spikerecorder )或Spikehound( http://spikehound.sourceforge。网)

程序

图1说明了实验装置和电极放置。为了刺激轴突,通过使用凡士林,一部分感兴趣的神经将与神经系统的其余部分电隔离。刺激的成功将通过在轴突的空间上远离的部分细胞外记录所引起的AP来监测。


图1.实验装置和电极放置示意图。说明了含有神经的培养皿。蓝色圆筒代表石油井,黑线表示电极放置及其与刺激和记录设备的连接。

  1. 制备凡士林注射器
    1. 将果冻转移到玻璃烧杯中。
    2. 将玻璃烧杯放在加热板上并加热加热,直至其充分熔化。
      注意:凡士林的熔点在45°C和60°C之间。避免沸腾!沸腾会产生气泡。
    3. 取5毫升鲁尔锁定注射器,并将熔化的果冻通过尖端拉出,将其填满。
      注意:戴上手套以防止灼伤。
    4. 让填充的注射器坐在室温下,直到果冻固化。
      注意:为防止气泡,请将注满的注射器向上(尖端朝向天花板)放置,直至果冻固化。
    5. 使用切线钳切断20针注射针尖。
      注意:使用砂纸来平滑锋利的边缘。
    6. 将注射针的前部弯曲约70°的角度(图2)。
    7. 将针头插入注射器。


      图2.用于制备细胞外刺激/记录孔的石油冻胶注射器

  2. 准备刺激和记录电极
    注意:重复以下步骤以创建包括刺激,记录和接地电极在内的所有必要的电极。刺激和记录电极需要红色和黑色线,而地面只需要一根线。
    1. 将电线切割成所需长度。
    2. 使用剥线钳去除每根红/黑线两侧绝缘层的5〜10毫米
    3. 使用预热烙铁,并用锡焊料涂覆电缆的剥离部分(图3A)。
    4. 用针尖钳将锡焊包线部分弯曲成环(图3B)
    5. 将不锈钢丝穿过环路。
      注意:不用不锈钢丝,也可以使用细节或缝纫针。
    6. 用锡焊填充回路以将不锈钢丝连接到黑/红线(图3C)。
    7. 使用电压表检查不锈钢线是否与导线正确接触。
    8. 将合适的插头焊接到电缆的另一侧以将其连接到刺激器/放大器。
    9. 使用热缩管将空白焊点绝缘。
    10. 除了刺激和记录电极之外,还需要单个接地电极来减少记录噪声和刺激伪影。使用黑色线重复上述步骤以构建单个电极。


      图3.细胞外刺激/记录电极的制备。 A.带有绝缘和锡焊锡涂层前面部分的电线B;相同的线,弯曲成环; C.不锈钢丝与锡焊接在一起。

  3. 解剖感兴趣的神经
    注意:对于详细的方案,如何解剖C. borealis的口腔胃肠神经系统请参阅(Gutierrez and Grashow,2009)。
  4. 将神经系统转移到硅胶衬里的培养皿上 注意:硅弹性体应涂覆培养皿的底部〜5毫米。
    把神经系统放在盘子的中间。
    1. 用生理盐水填充陪替氏培养皿(见表1,见北极生理盐水)。
    2. 使用细节针来安装神经系统,通过直接固定在神经末端或通过钉扎仍然附着到神经系统的结缔组织。
      注意:不要穿过被记录的神经部分,因为这会损伤轴突。
  5. 通过用造型粘土将其固定在工件表面上(图4)将盘从位移中固定
  6. 将接地电极放入浴中。使用造型粘土防止接地电极发生位移。将接地线连接到放大器地。


    图4.具有孤立口腔胃神经系统的培养皿。 照片显示刺激和记录孔沿口胃神经,盐水流入和流出以及接地线的位置。培养皿用造型粘土附着在工作面上。对于细胞外刺激和记录,不需要隔离实验台。为了更好的可视化,神经被回溯(灰色和粉色线)。 

  7. 准备细胞外刺激/记录井。
    1. 使用立体显微镜,在图5所示的充气注射器上,在感兴趣的神经的两个位置上形成两个同心圆。一个孔将用作刺激,另一个将用作记录。加入石油胶层,直到孔延伸到盐水位以上(图5A和5B)。
      注意:由于凡士林不导电,所以将井的一小部分神经与其余的浴隔离。
    2. 确保井紧密而不漏水。
      注意:在井内添加一滴或两滴盐水。如果生理盐水的水平不下降,则井足够紧密,并将神经部分与其余部分隔开(图5C)。


      图5.石油凝胶井制备。A.在感兴趣的神经的一小部分周围形成初始同心圆。成绩好加入石油冻胶层,直到最上面的层从盐水中延伸出来。 C.泄漏试验。将盐水加入孔内以测试是否将神经部分与其余浴室充分隔离
  8. 放置刺激电极。
    1. 用刺激器连接刺激电缆(红线=阴极,黑线=阳极)。
    2. 使用镊子将钢丝(阴极)放置在刺激井内(图6)。将线牢固地插入硅胶中。
      注意:避免穿刺神经或良好。将电极的末端足够深地放入硅胶中,以便在实验的剩余时间内,导线不会在任何时候脱落。
    3. 使用镊子将阳极放置在井外,但仍然靠近。
      注意:电线不能互相接触,因为这将导致短路,轴突不会被刺激。
    4. 使用造型粘土固定电线。


      图6.刺激丝在石油井中的布置

  9. 对记录线重复步骤8。
  10. 转动细胞外放大器,并检查自发性AP是否在细胞外记录上可见。
    注意:
    1. 对于C. borealis,放大器滤波器设置应该:放大:10,000;高通滤波器:低截止频率100 Hz;低通滤波器:高截止频率500 Hz。
    2. 检测到的AP波形不直接反映膜电位变化。使用记录井的内部和外部之间的差分记录来检测AP。
  11. 刺激神经细胞外。
    最初,将Master8上的刺激参数设置为1 ms刺激脉冲持续时间,1 Hz刺激频率和0 nA/Volt。在确定刺激阈值之后,可以使用更多的生理参数。
    1. 使用细胞外记录首先识别刺激伪影(图7A和7B) 注意:刺激伪影可以很容易地与AP区分开,因为人造物的大小随着刺激振幅线性增加。如果刺激伪影太大,则电缆的极性应相反。当阴极(而不是阳极)放置在刺激井内部时,刺激伪影将最小。


      图7.细胞外刺激阈值的调整。 显示亚阈值(A,B),阈值(C)和上阈值刺激(D)期间口腔胃神经系统神经的细胞外记录。 A.在亚阈值刺激与低刺激幅度下只有刺激伪影可以观察到,但没有AP。伪像与刺激脉冲同时发生。 B.增加刺激幅度低于阈值不会引起AP,但增加刺激伪影幅度。将刺激振幅增加到阈值引起AP。其波形在刺激伪影(灰盒)后出现固定的时间延迟。首先招募直径最大的神经轴突。注意,AP波形不表示AP的不同相位,而是AP的进入和离开井之后的石油井内外的潜在差异。 D.增加超过阈值的刺激振幅招募额外的较小直径的轴突。注意,当招募其他轴突时,检测到的波形的形状和幅度变化(复合AP)。为了更好的可视化,伪影幅度被裁剪(用*表示)。

    2. 通过缓慢增加刺激幅度直到AP变得可见来确定刺激阈值(图7C) 注意:AP应在刺激伪影之后出现时间延迟。 AP可以与伪像区分开来,因为它们仅在达到刺激阈值之后发生,并且它们的发生时间和幅度不随刺激振幅线性增加。请注意,如果刺激幅度进一步增加,可能会招募来自其他轴突的AP,这将改变检测到的信号的大小和形状(复位AP在超阈值刺激期间,参见图7D和视频1)。 >
    3. 如果记录软件具有触发功能,可以使用它来控制细胞外记录的扫描,以允许快速的时间基准,以及刺激伪影和AP形状的比较。

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      要播放视频,您需要安装较新版本的Adobe Flash Player。

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数据分析

数据分析将根据实验而有所不同,但在设计使用细胞外轴突刺激的实验时,应牢记以下步骤。

  1. 为了确保AP被引出,必须区分刺激伪影和记录站点的到达AP。激发的AP在刺激脉冲之后以恒定的延迟到达,并且与刺激振幅无关(图7C)。相比之下,刺激伪影随着刺激振幅而缩放,几乎瞬间到达(图7A和7B)。
  2. 在亚阈值刺激振幅下,只有刺激伪影是可见的(图7A)。为了测试记录的信号是否确实是人造物,可以使用垂直光标测量刺激开始与记录位置的信号到达之间的时间。对于信号到达,可以使用信号的第一个明显的峰值。此外,应使用水平光标在信号最小值和最大值之间测量到达信号的幅度。平均至少要连续10次刺激,以确定平均到达时间和幅度
  3. 稍微增加和减少刺激振幅,并再次测量记录信号的到达时间和幅度。如果到达时间发生在刺激脉冲和振幅与刺激振幅(图7B)相比的同时,记录的信号是刺激伪影。在每个刺激振幅下测量至少10个连续的信号。
    注意:可以使用t检验来比较不同刺激幅度下的测量值(显着性切断值P <0.05)。
  4. 进一步增加刺激振幅,直到第二个延迟信号到达记录位置(阈值刺激,图7C)。如果这是AP,当刺激振幅提高时(不会缓慢增加),应该突然发生。测量至少10个连续AP的到达时间和振幅。
    注意:AP不受刺激振幅变化的影响。通过在测量到达时间和幅度之前稍微增加刺激振幅,确认记录的信号确实是AP。 AP的到达时间取决于刺激和记录位点之间的距离。
  5. 进一步增加刺激幅度(阈值,图7D)可以招募额外的AP,其延迟和幅度可以使用相同的分析来测量。

笔记

  1. 在尝试轴突刺激之前,记录至少10个连续AP的自发性AP活动。可能需要通过将刺激器接地到放大器并利用法拉第笼来进一步降低噪音
  2. 代替钢,氯化银可用于接地线。如果计划额外的细胞内记录和/或必须避免记录的膜电位的漂移,这是特别重要的。
  3. 如果刺激伪影较大并且持续时间较长,则刺激器的阳极和阴极可能反转。在这种情况下,刺激电极的极性需要反转,因为阳极导致细胞外空间变得更积极,因此神经元在局部极化。由于这种超极化被相对地被去极化的区域包围,AP仍然可以被引发(阳极刺激)。然而,通过阳极刺激刺激的AP具有更高的阈值,导致刺激伪影更大
  4. 如果没有细胞外记录的刺激神经是可行的,刺激的成功可以通过监测突触后神经元,电路,肌肉或行为的活动的变化来评估。在这种情况下,建议使用较高的刺激频率来实现清晰的突触后反应。

食谱

  1. 癌症北极生理盐水配方(见表1)

    表1. 生理盐水配方

    注意:使用Trizma碱和马来酸将pH调节至7.4-7.6。

致谢

这项工作得到了德意志民主共和国(DFG STE 937/9-1),国家科学基金会(NSF IOS 1354932),伊利诺伊州立大学和德国学术交流处的资助。

参考文献

  1. Abbas,PJ和Miller,CA(2004)。  生物物理学和生理学在:F.-G.曾和R. R. Fay。 (Eds)。耳蜗植入物:听觉假肢和电听力。 Springer ,20:149-212。
  2. Barry,JM(2015)。 Axonal activity 在体内:技术考虑和对自由移动动物神经回路探索的意义。前线Neurosci 9:153。
  3. Basser,PJ和Roth,BJ(2000)。  新电刺激可激活组织的电流。 Annu Rev Biomed Eng 2:377-397。
  4. Cogan,SF(2008)。  神经刺激和记录电极 Annu Rev Biomed Eng 10:275-309。
  5. Gutierrez,GJ和Grashow,RG(2009)。  < (胃癌)口腔胃神经系统解剖。 J Vis Exp (25)
  6. Städele,C.和Stein,W.(2016)。  自发性异位癫痫发作的部位促进感觉神经元中的信号整合。 Neurosci 36(25):6718-6731。
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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. Städele, C., DeMaegd, M. L. and Stein, W. (2017). Extracellular Axon Stimulation. Bio-protocol 7(5): e2151. DOI: 10.21769/BioProtoc.2151.
  2. Städele, C. and Stein, W. (2016). The site of spontaneous ectopic spike initiation facilitates signal integration in a sensory neuron. J Neurosci 36(25): 6718-6731.
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