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Axonal Conduction Velocity Measurement
轴向传导速度测量

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Abstract

Action potential conduction velocity is the speed at which an action potential (AP) propagates along an axon. Measuring AP conduction velocity is instrumental in determining neuron health, function, and computational capability, as well as in determining short-term dynamics of neuronal communication and AP initiation (Ballo and Bucher, 2009; Bullock, 1951; Meeks and Mennerick, 2007; Rosenthal and Bezanilla, 2000; Städele and Stein, 2016; Swadlow and Waxman, 1976). Conduction velocity can be measured using extracellular recordings along the nerve through which the axon projects. Depending on the number of axons in the nerve, AP velocities of individual or many axons can be detected.
   This protocol outlines how to measure AP conduction velocity of (A) stimulated APs and (B) spontaneously generated APs by using two spatially distant extracellular electrodes. Although an invertebrate nervous system is used here, the principles of this technique are universal and can be easily adjusted to other nervous system preparations (including vertebrates).

Keywords: Nervous system(神经系统), Action potential(动作电位), Propagation(传播), Extracellular stimulation(细胞外刺激), Ectopic(异位), Nerve(神经), Spike timing(峰电位定时), Axon (轴突)

Background

Long-distance communication in the nervous system is mediated by APs that travel along axons. The ionic currents that flow across the axon membrane when an AP is generated (Hodgkin and Huxley, 1952) can be detected even outside of the neuron, using extracellular recording electrodes. AP conduction velocities in different neurons are quite variable and range from 200 meters per second (447 miles per hour) to less than 0.1 meters per second (0.2 miles per hour) (Kress et al., 2008; Kusano, 1966). In order to understand why there are differences in conduction velocity, the passive (membrane) properties of the axon need to be taken into account. Some axons propagate information more rapidly than others because of differences in properties that affect the time constant (e.g., resistance and capacitance) and the length constant (e.g., axon diameter, membrane permeability, and degree of myelination). Especially in unmyelinated axons, conduction velocity largely depends on the axon diameter, which in turn is also correlated with the amplitude of the extracellular AP (Stein and Pearson, 1971). Consequently, determining AP conduction velocity provides more than just information about signal movement and timing. It can also be used to characterize changes in intrinsic axon properties.

Materials and Reagents

Note: The materials and equipment listed refer to the equipment used in Städele and Stein (2016). 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 (http://backyardbrains.com).

  1. Petri dish (100 x 15 mm, Fisher Scientific, catalog number: FB0875713 ) lined with silicon elastomer (e.g., Sylgard 184, Sigma-Aldrich, catalog number: 761036 ; or Elastosil RT 601, Wacker Chemie, catalog number: 60063613 )
  2. Minutien pins (Fine Science Tools, catalog number: 26002-10 )
  3. Modeling clay (craft store)
  4. Syringe, filled with petroleum jelly (100% pure, pharmacy)
  5. Recording/stimulation electrodes
    Note: For details how to prepare the petroleum jelly filled syringe or recording/stimulating electrodes see our companion protocol ‘Extracellular axon stimulation’ by Städele, C., DeMaegd, M. and Stein, W.
  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 dehydrate, 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. Ruler or micrometer scale
  2. Stereomicroscope (e.g., Leica Microsystems, model: MS5 )
  3. Stimulator (A.M.P.I, model: Master 8 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 (Cambridge Electronic Design Limited, 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. Camera (e.g., AmScope, model: 3MP USB2.0 Microscope Digital Camera, catalog number MU300 )

Software

  1. ImageJ (National Insitute of Health) or a comparable software
  2. 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 Spike hound (http://spikehound.sourceforge.net)

Procedure

Figure 1 illustrates the experimental setup and electrode placement. In order to determine AP conduction velocity, it is important to keep in mind that there are differences in the procedure for measuring stimulated APs (Figure 1A) and spontaneously generated APs (Figure 1B). Especially in the latter case, it is crucial that APs are not initiated between the two recording sites, as this will result in wrong velocity measurements. APs can spread bi-directionally from their site of initiation, and if APs are initiated between the two recording sites, the measured difference in AP arrival times between recording sites is not representative of APs travelling the distance between the two (Figure 1C, and also step B2). Thus, in the following, detailed procedures are given for measuring conduction velocity of:
A. Stimulated APs by means of extracellular stimulation
B. Spontaneously initiated APs
In both cases, AP conduction velocity is measured as a function of the time the AP needs to travel from one site on an axon/nerve to another site (= ‘latency’). Conduction velocity can be calculated using:

With v as the conduction velocity,
∆x as the spatial distance between recording sites,
∆t as the time difference between recording sites (latency).


Figure 1. Representation of the electrode placement for measuring conduction velocity of stimulated APs (A) and spontaneously generated APs (B & C). A. Left: APs are initiated within the stimulation well and propagate along the axon (green arrows) towards the recording well. Note that APs propagate bi-directionally but are only detected at the recording site. Δx represents the distance between the stimulation and recording wells. Right: Extracellular recording of a nerve showing the latency (Δt) between the onset of the stimulation (stim. artifact) and the AP (gray box). B. Left: Correct electrode placement for measuring conduction velocity of spontaneously generated APs. Recording wells need to be placed in such a way that the AP is passing the wells without being initiated in-between the wells. Right: The latency in AP occurrence at the two recording sites reflects the time the AP needs to propagate from recording 1 to recording 2. C. Left: Incorrect electrode placement for measuring conduction velocity of spontaneously generated APs. Right: The latency between the two recording sites is small and it only reflects that the AP was initiated spatially closer to recording 1 than to recording 2.

  1. Measuring conduction velocity of stimulated APs
    The advantage of measuring conduction velocity of stimulated APs is that AP initiation is controlled by the experimenter. Thus, different AP frequencies can be tested, and APs can reliably be elicited in many different conditions (e.g., at different temperatures or in distinct neuromodulatory conditions). However, because the largest axons respond to the lowest stimulation amplitudes, measuring the velocities of small diameter axons may not be feasible with extracellular stimulation.
    1. Dissect the nerve of interest.
      Note: The dissected nerve should be long enough that the two wells constructed in step 2 are separate, and the entire length of the nerve contains the axon of interest. For a detailed protocol how to dissect the stomatogastric nervous system of C. borealis please review Gutierrez and Grashow (2009).
    2. Establish the extracellular recording and stimulation as described in companion protocol ‘Extracellular axon stimulation’ by Städele, C., DeMaegd, M. and Stein, W. For electrode placement see Figure 1A. 
    3. After determining the AP stimulation threshold, continuously stimulate the axon with a low frequency stimulation (1 to 2 Hz).
    4. Identify the AP on the extracellular recording (Figure 1A, right).
      Note: The stimulated AP will travel from the stimulation electrode to the recording site. From one stimulus pulse to the next, AP amplitude and shape should not change. Similarly, the latency between the stimulation onset and the AP occurrence should remain constant. If there is no consistency of AP amplitude or latency, then either no AP was elicited or the elicited AP does not pass the recording site, indicating that the axon might be damaged or might project through a different nerve.
    5. Measure conduction velocity as described in Data analysis.
    6. Variation of the experiment: In many axons, AP conduction velocity is frequency-dependent (Miller and Rinzel, 1981; Weidner et al., 2002) and/or history-dependent (Ballo et al., 2012; Kiernan et al., 1997). To test whether this is the case for the axon of interest, increase the stimulation frequency and observe whether conduction velocity changes as a function of frequency and/or number of APs. Instead of stimulating at a constant frequency, more biological relevant pulse sequences may be applied, such as burst-like changes in frequency (Ballo and Bucher, 2009).

  2. Measuring conduction velocity of spontaneously generated APs
    Conduction velocity measurements of spontaneously initiated APs does not require extracellular nerve stimulation. Many axons spontaneously generate APs, and accordingly, most nerve recordings show spontaneously occurring APs. By using spontaneously generated APs, the conduction velocities of small diameter axons can be assessed. However, the occurrence and frequency of the APs cannot be controlled by the experimenter. We are using the anterior gastric receptor neuron (AGR; Smarandache and Stein, 2007) of the stomatogastric nervous system of the Jonah crab (Cancer borealis) as an example. AGR spontaneously generates tonic spike activity (2 to 5 Hz) in its axon trunk that can be measured with extracellular electrodes (Daur et al., 2009; Städele and Stein, 2016).
    1. Dissect the nerve of interest. Then, instead of one stimulation and one recording well, both will be used as recording wells (Figure 1B).
    2. Connect the cable of the stimulation electrode to a second channel of the extracellular amplifier.
      Note: If the neuron of interest is bipolar or able to generate ectopic APs (e.g., Daur et al., 2009), make sure that the site where APs are initiated is not in between the two recording wells since the measured latencies will not reflect travel times between recording sites (Figure 1C). If necessary, this can be determined by adding a third recording well. The order at which the APs arrive at the recordings wells will be indicative of the initiation site. If either of the two outside wells records the AP first, the correct velocity can be measured using the difference in arrival times between the other recording wells. If the center well records the AP first, use longest latency between center and outside wells.
    3. Identify the AP of interest on the extracellular recordings.
      Note: The AP of interest should appear with a constant latency between both recording sites. Its shape and amplitude may differ between recording sites, but should be consistent for each site. If the AP of interest is not the largest unit on the extracellular recording, or difficult to identify for other reasons, see Notes.
    4. Measure conduction velocity as described in Data analysis.

Data analysis

  1. Measure the latency between AP occurrence at the two recording sites.
    1. For stimulated APs, measure the time difference between stimulus and recorded AP, as illustrated in Figure 1A.
      Note: Stimulation onset can be determined using the stimulus artifact, which spreads almost instantaneously through the Petri dish. In the recording software, measure the time between the peak of the stimulus artifact and the peak of the recorded action potential.
    2. For spontaneously elicited APs, measure the time difference between the peaks of the AP at recording 1 and recording 2, as illustrated in Figure 1B.
  2. Measure the distance between recording sites.
    Use a ruler or micrometer scale to measure the length of the nerve between the stimulation and recording wells. The shortest distance between both wells should be measured (well edge to well edge). There are different methods that can be used to measure distance. The simplest and least accurate method is using a ruler or micrometer. A more accurate method is the following:
    1. Put a size standard underneath the Petri dish (e.g., micrometer scale).
    2. Take a photo that shows the distance between the stimulation and recording well plus the size standard.
    3. Use ImageJ (National Insitute of Health) or a comparable software to measure the nerve distance between the two wells. Use the size standard to calibrate the distance measurement in the software.
  3. Calculate the conduction velocity using Equation 1.
    Note: For more accurate results, calculate, then average the conduction velocities of at least 10 APs.
  4. Use a paired t-test when comparing conduction velocities in two different conditions (e.g., before and after neuromodulator application) from the same preparation with a significance cut off value of P < 0.05.
  5. Use a repeat measures analysis of variance (ANOVA) when comparing multiple measurements of conduction velocity in different conditions (e.g., application of multiple neuromodulators, or measurements are multiple temperatures) from the same animal

Notes

  1. Using multisweeps and averages to increase measurement accuracy. To measure the latency between stimulus artifact and recording (or between recording 1 and recording 2), it is helpful to not only use single APs. Rather, multiple APs should be taken into account (Video 1). This will not only make the identification of time-correlated APs easier, but also allows to assess variability and changes in conduction velocity that may occur in different conditions. Time-correlated APs are most easily detected using a triggered sweep display (oscilloscope view, or ‘multisweep’, Video 2). In this view, multiple occurrences of the same AP are superimposed to show APs that occur at a fixed latency to the stimulation or the other recording, respectively. For this, an amplitude threshold is set in the software, through which the AP must rise to start a new sweep (‘triggering’). Consequently, only APs large enough to cross the threshold trigger new sweeps, and the latency of arrival of these APs at the recording site becomes obvious. If the threshold is set lower, APs of multiple axons may be detected. Depending on their conduction velocity, they may arrive at distinct, but fixed times at the recording. The APs of other axons will not be time locked to the trigger and hence occur randomly on the display. Using a triggered sweep display is particularly helpful to detect spontaneously generated APs, since it is difficult to determine time correlations between two recording sites using individual APs or without proper analysis.
    To determine the exact latency, it can be helpful to average several sweeps of triggered APs (Video 2). In this case, all randomly occurring APs will be small or absent, and only the time-locked APs show a clear peak that can easily be measured.

    Video 1. Video clip illustrating time-correlated APs using Spikerecorder software (Backyard Brains) for spontaneously generated APs. A similar procedure can be used for stimulated APs.

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    Video 2. Video clip illustrating spike-triggered multisweeps and averaging using Spike 2 software (CED) for spontaneously generated APs. A similar procedure can be used for stimulated APs.

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


  2. Noise issues. Electrical noise is a common problem associated with the recording of neuronal signals. The major source of electrical noise is pickup, which is caused by radiation from electrical devices and the recording equipment. This radiation produces currents in the electrodes and wires leading to the amplifiers in the recording system. Since most devices are powered by 60 Hz (US)/50 Hz (most other countries) alternating current, pickup appears as a distorted sine wave with a period of 16.7 (US)/20 (others) milliseconds. Noise may be reduced by using a grounded, metal enclosure (Faraday cage) around the preparation and the electrodes. The enclosure separates the source of the radiation from the electrodes. Grounding all recording and stimulation equipment to a single source will reduce noise. This can be done using a banana plug with a crocodile clamp on one side to connect the ground ports of the recording and stimulation equipment to the Faraday cage. For more information on the principles of Faraday cage function, see Chapman et al. (2015) and DeFreitas et al. (2012). The person operating the equipment might also be a source of noise, and he or she may need to be grounded as well. Additionally, shielded cables may be used to carry the signals from the electrodes to the amplifier.
    If the signal-to-noise ratio is still small, the issue might originate from a small signal rather than large noise. In this case, the recording well is likely leaky and should be remade.

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. Ballo, A. W. and Bucher, D. (2009). Complex intrinsic membrane properties and dopamine shape spiking activity in a motor axon. J Neurosci 29(16): 5062-5074.
  2. Ballo, A. W., Nadim, F. and Bucher, D. (2012). Dopamine modulation of Ih improves temporal fidelity of spike propagation in an unmyelinated axon. J Neurosci 32(15): 5106-5119.
  3. Bullock, T. H. (1951). Conduction and transmission of nerve impulses. Annu Rev Physiol 13: 261-280.
  4. Chapman, S. J., Hewett, D. P., and Trefethen, L. N. (2015). Mathematics of the Faraday cage. Siam Review 57(3): 398-417.
  5. Daur, N., Nadim, F. and Stein, W. (2009). Regulation of motor patterns by the central spike-initiation zone of a sensory neuron. Eur J Neurosci 30(5): 808-822.
  6. DeFreitas, J. M., Beck, T. W., and Stock, M. S. (2012). Comparison of methods for removing electromagnetic noise from electromyographic signals. Physiol Meas 33(2): 147-159.
  7. Gutierrez, G. J. and Grashow, R. G. (2009). Cancer borealis stomatogastric nervous system dissection. J Vis Exp (25).
  8. Hodgkin, A. L. and Huxley, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117(4): 500-544.
  9. Kiernan, M. C., Mogyoros, I., Hales, J. P., Gracies, J. M. and Burke, D. (1997). Excitability changes in human cutaneous afferents induced by prolonged repetitive axonal activity. J Physiol 500 ( Pt 1): 255-264.
  10. Kress, G. J., Dowling, M. J., Meeks, J. P. and Mennerick, S. (2008). High threshold, proximal initiation, and slow conduction velocity of action potentials in dentate granule neuron mossy fibers. J Neurophysiol 100(1): 281-291.
  11. Kusano, K. (1966). Electrical activity and structural correlates of giant nerve fibers in Kuruma shrimp (Penaeus japonicus). J Cell Physiol 68(3): 361-383.
  12. Meeks, J. P. and Mennerick, S. (2007). Action potential initiation and propagation in CA3 pyramidal axons. J Neurophysiol 97(5): 3460-3472.
  13. Miller, R. N. and Rinzel, J. (1981). The dependence of impulse propagation speed on firing frequency, dispersion, for the Hodgkin-Huxley model. Biophys J 34(2): 227-259.
  14. Rosenthal, J. J. and Bezanilla, F. (2000). Seasonal variation in conduction velocity of action potentials in squid giant axons. Biol Bull 199(2): 135-143.
  15. Smarandache, C. R. and Stein, W. (2007). Sensory-induced modification of two motor patterns in the crab, Cancer pagurus. J Exp Biol 210(Pt 16): 2912-2922.
  16. 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.
  17. Stein, R. B. and Pearson, K. G. (1971). Predicted amplitude and form of action potentials recorded from unmyelinated nerve fibres. J Theor Biol 32(3): 539-558.
  18. Swadlow, H. A. and Waxman, S. G. (1976). Variations in conduction velocity and excitability following single and multiple impulses of visual callosal axons in the rabbit. Exp Neurol 53(1): 128-150.
  19. Weidner, C., Schmelz, M., Schmidt, R., Hammarberg, B., Orstavik, K., Hilliges, M., Torebjork, H. E. and Handwerker, H. O. (2002). Neural signal processing: the underestimated contribution of peripheral human C-fibers. J Neurosci 22(15): 6704-6712.

简介

动作电位传导速度是动作电位(AP)沿着轴突传播的速度。测量AP传导速度有助于确定神经元的健康,功能和计算能力,以及确定神经元通信和AP启动的短期动力学(Ballo和Bucher,2009; Bullock,1951; Meeks and Mennerick,2007; Rosenthal和Bezanilla,2000;Städele和Stein,2016; Swadlow和Waxman,1976)。传导速度可以通过沿轴突投射的神经的细胞外记录来测量。根据神经中的轴突数量,可以检测个体或许多轴突的AP速度。
该协议概述了如何通过使用两个空间距离的细胞外电极来测量(A)刺激的AP和(B)自发产生的AP的AP传导速度。虽然这里使用无脊椎动物神经系统,但是这种技术的原理是普遍的,并且可以容易地调整到其他神经系统制剂(包括脊椎动物)。
【背景】神经系统中的长距离通讯是由沿轴突行进的AP介导的。当产生AP时流过轴突膜的离子电流(Hodgkin和Huxley,1952)可以使用细胞外记录电极在神经元外部检测。不同神经元中的AP传导速度是非常可变的,范围从每秒200米(447英里每小时)到小于0.1米每秒(每小时0.2英里)(Kress等人,2008; Kusano,1966)。为了理解为什么传导速度存在差异,需要考虑轴突的被动(膜)特性。由于影响时间常数(例如电阻和电容)和长度常数(例如,轴突直径,膜通透性和髓鞘形成程度)的性质差异,一些轴突可以比其他轴突更快地传播信息。特别是在无髓鞘轴突中,传导速度很大程度上取决于轴突直径,这反过来也与细胞外AP的幅度相关(Stein和Pearson,1971)。因此,确定AP传导速度不仅仅提供关于信号移动和定时的信息。它也可用于表征固有轴突属性的变化。

关键字:神经系统, 动作电位, 传播, 细胞外刺激, 异位, 神经, 峰电位定时, 轴突

材料和试剂

注意:所列材料和设备是指Städeleand Stein(2016)使用的设备。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( http://backyardbrains.com )。

  1. 用硅弹性体(例如,Sylgard 184,Sigma-Aldrich,目录号:761036)或Elastosil RT 601(Wacker Chemie)衬里的培养皿(100×15mm,Fisher Scientific,目录号:FB0875713) ,目录号:60063613)
  2. Minutien针(精细科学工具,目录号:26002-10)
  3. 造型粘土(工艺品店)
  4. 注射器,充满果冻(100%纯,药店)
  5. 记录/刺激电极
    注意:有关如何准备充气注射器或记录/刺激电极的详细信息,请参阅Städele,C.,DeMaegd,M。和Stein,W.的"伴侣协议"细胞外轴突刺激。 />
  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. 氯化钙脱水剂,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. 标尺或千分尺
  2. 立体显微镜(例如,Leica Microsystems,型号:MS5)
  3. 刺激器(A.M.P.I,型号:Master 8 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)
    低成本替代方案:Spikerbox(Backyard Brains,型号:Neuron SpikerBox)
  5. 数据采集板(剑桥电子设计有限公司,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。第二种低成本替代方案是Spikehound( http://spikehound。 sourceforge.net ),这也允许通过电脑声卡进行录制。 
  6. 相机(例如,,AmScope,型号:3MP USB2.0显微镜数码相机,产品目录号MU300)

软件

  1. ImageJ(国家卫生研究院)或类似软件
  2. 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Spike猎犬( http://spikehound。 sourceforge.net

程序

图1说明了实验装置和电极放置。为了确定AP传导速度,重要的是要记住,测量受激AP(图1A)和自发产生的AP(图1B)的过程存在差异。特别是在后一种情况下,至关重要的是AP将不会在两个记录站点之间启动,因为这将导致错误的速度测量。 AP可以从其启动位置双向传播,并且如果在两个记录站点之间启动AP,则记录站点之间的AP到达时间的测量差异不代表移动到两者之间的距离的AP(图1C,也是步骤B2)。因此,在下文中,给出了测量传导速度的详细步骤:
A.通过细胞外刺激刺激AP B.自发发起的受影响人士
在这两种情况下,AP传导速度作为AP需要从轴突/神经上的一个位点行进到另一个位点(="潜伏期")的时间的函数。传导速度可以用以下公式计算:

v 为传导速度,
作为记录站点之间的空间距离,Δx,
作为记录站点之间的时差(延迟),Δt

图1.用于测量受激AP(A)和自发产生的AP(B& C)的传导速度的电极放置的表示。A.左:AP在刺激井内启动并传播轴突(绿色箭头)朝向记录井。请注意,AP是双向传播的,但只在记录位点被检测到。 Δx表示刺激和记录井之间的距离。右:神经的细胞外记录显示刺激发作(刺激伪影)和AP(灰盒)之间的延迟(Δt)。 B.左:用于测量自发产生的AP的传导速度的正确的电极放置。记录井需要以这样一种方式进行放置,使得AP正在通过井而不在井之间启动。右:两个记录站点AP发生的延迟反映了AP需要从记录1传播到记录2的时间。C.左:用于测量自发生成的AP的传导速度的电极放置不正确。右:两个录音站点之间的延迟很小,只反映了AP在空间上比录音1更接近录音2。

  1. 测量刺激AP的传导速度
    测量受激AP的传导速度的优点是AP启动由实验者控制。因此,可以测试不同的AP频率,并且可以在许多不同的条件(例如,在不同的温度或不同的神经调节条件下)可靠地引出AP。然而,由于最大的轴突对最低的刺激幅度有反应,所以测量小直径轴突的速度可能与细胞外刺激是不可行的。
    1. 解剖感兴趣的神经。
      注意:解剖的神经应该足够长,使步骤2中构建的两个孔是分开的,并且神经的整个长度包含感兴趣的轴突。有关详细的方案如何解剖C. borealis的胃口胃神经系统,请查看Gutierrez和Grashow(2009)。
    2. 建立细胞外记录和刺激,如Städele,C.,DeMaegd,M.和Stein,W的伴随方案"细胞外轴突刺激"所述。对于电极放置,参见图1A。
    3. 确定AP刺激阈值后,用低频刺激(1〜2Hz)持续刺激轴突
    4. 识别细胞外记录上的AP(图1A,右图)。
      注意:刺激的AP将从刺激电极行进到记录位置。从一个刺激脉冲到下一个脉冲,AP幅度和形状不应该改变。类似地,刺激开始和AP发生之间的等待时间应保持不变。如果没有AP幅度或延迟的一致性,则不会引起AP或引发的AP不通过记录位点,表明轴突可能被损坏或可能通过不同的神经投射。 >
    5. 测量传导速度,如数据分析所述
    6. 实验的变化:在许多轴突中,AP传导速度是频率依赖性的(Miller和Rinzel,1981; Weidner等人,2002)和/或历史依赖性(Ballo等人,,2012; Kiernan等人,1997)。为了测试感兴趣的轴突是否是这种情况,增加刺激频率并观察传导速度是否随着AP的频率和/或数量的变化而变化。代替以恒定频率刺激,可以应用更多的生物相关脉冲序列,例如频率上的突发样变化(Ballo和Bucher,2009)。

  2. 测量自发生成的AP的传导速度
    自发发生的AP的传导速度测量不需要细胞外神经刺激。许多轴突自发产生AP,因此大多数神经记录显示出自发发生的AP。通过使用自发产生的AP,可以评估小直径轴突的传导速度。然而,实验者无法控制AP的发生和频率。作为例子,我们正在使用约纳蟹(胃癌)胃口胃神经系统的前胃部受体神经元(AGR; Smarandache和Stein,2007)。 AGR自发产生可以用细胞外电极测量的轴突干细胞(2〜5Hz)的强直刺激活性(Daur等人,2009;Städeleand Stein,2016)。
    1. 解剖感兴趣的神经。然后,代替一次刺激和一次记录,两者都将用作记录孔(图1B)。
    2. 将刺激电极的电缆连接到细胞外放大器的第二个通道。
      注意:如果感兴趣的神经元是双极的或能够产生异位AP(例如,Daur等,2009),请确保启动AP的位置不在两个记录孔之间,因为测量的延迟不会反映记录站点之间的旅行时间(图1C)。如有必要,可以通过添加第三记录井确定。 AP到达记录井的顺序将指示启动地点。如果两个外部井中的任一个首先记录了AP,则可以使用其他记录井之间的到达时间差来测量正确的速度。如果中心井首先记录AP,则在中心井和外部井之间使用最长的延迟。
    3. 识别细胞外记录上感兴趣的AP。
      注意:感兴趣的AP应在两个记录站点之间以一定的延迟出现。其形状和幅度在记录位置之间可能不同,但对于每个位置应该是一致的。如果感兴趣的AP不是细胞外记录上的最大单位,或者由于其他原因难以识别,请参见注释。
    4. 测量传导速度,如数据分析中所述。

数据分析

  1. 测量两个记录站点AP出现之间的延迟。
    1. 对于受刺激的AP,测量刺激和记录的AP之间的时间差,如图1A所示 注意:刺激发作可以使用刺激伪影确定,刺激伪影几乎瞬间通过培养皿传播。在录音软件中,测量刺激伪影的峰值与记录的动作电位的峰值之间的时间。
    2. 对于自发诱发的AP,测量记录1和记录2之间AP的峰值之间的时间差,如图1B所示。
  2. 测量记录位置之间的距离。
    使用标尺或千分尺来测量刺激和记录孔之间的神经长度。应测量两口井之间的最短距离(边缘到边缘)。有不同的方法可以用来测量距离。最简单和最不精确的方法是使用标尺或千分尺。一个更准确的方法是:
    1. 将尺寸标准放在陪替氏培养皿下方(例如,千分尺)。
    2. 拍摄照片,显示刺激和记录之间的距离加上尺寸标准。
    3. 使用ImageJ(国家卫生研究院)或类似的软件来测量两口井之间的神经距离。使用尺寸标准来校准软件中的距离测量。
  3. 使用公式1计算传导速度。
    注意:为了获得更准确的结果,请计算,然后平均至少10个AP的传导速度。
  4. 在比较两种不同条件(例如,神经调节剂应用前后)的传导速度时,使用配对的 em> P 0.05。
  5. 当比较来自同一动物的不同条件(例如,多个神经调节剂的应用或测量是多个温度)的传导速度的多次测量时,使用重复测量方差分析(ANOVA)

笔记

  1. 使用多扫描和平均值来提高测量精度。为了测量刺激伪像和记录(或记录1和记录2之间)之间的延迟,不仅使用单个AP是有帮助的。相反,应考虑多个接入点(视频1)。这不仅可以使时间相关AP的识别更容易,而且可以评估在不同条件下可能发生的传导速度的变化和变化。使用触发的扫描显示(示波器视图或"多扫描"视频2),最容易检测到时间相关的AP。在这种观点中,叠加了多次相同的AP,以显示分别以刺激或其他记录的固定延迟发生的AP。为此,在软件中设置幅度阈值,AP必须通过该阈值上升才能开始新的扫描("触发")。因此,只有足够超过阈值的AP才能触发新的扫描,并且这些AP到达记录站点的延迟变得明显。如果将阈值设定得较低,则可以检测多个轴突的AP。根据它们的传导速度,它们可以在记录时到达不同但固定的时间。其他轴突的AP不会被时间锁定到触发器上,因此在显示器上随机发生。使用触发的扫描显示器特别有助于检测自发生成的AP,因为使用单个AP或没有适当分析难以确定两个记录位点之间的时间相关性。
    为了确定准确的延迟,平均几次触发的AP(视频2)可能是有帮助的。在这种情况下,所有随机发生的AP将是小的或不存在的,并且只有时间锁定的AP显示可以容易地测量的清晰的峰。

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    视频1.使用Spikerecorder软件(后台脑)自动产生的AP的时间相关AP的视频剪辑。 类似的过程可用于受刺激的AP。
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    视频2.视频剪辑说明尖峰触发多扫描,并使用Spike 2软件(CED)对自发生成的AP进行平均。 类似的过程可用于受刺激的AP。
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  2. 噪音问题电噪声是与记录神经元信号相关的常见问题。电噪声的主要来源是拾取,这是由电气设备和记录设备的辐射引起的。该辐射在电极和导线中产生通向记录系统中的放大器的电流。由于大多数器件由60 Hz(US)/50 Hz(大多数其他国家)交流供电,因此拾音器将显示为16.7(US)/20(其他)毫秒的失真正弦波。通过在准备物和电极周围使用接地的金属外壳(法拉第笼)可以降低噪音。外壳将辐射源与电极分开。将所有记录和刺激设备接地到单个来源将降低噪音。这可以使用一侧的鳄鱼钳的香蕉插头将录音和刺激设备的接地端口连接到法拉第笼。有关法拉第笼功能的原理的更多信息,请参阅Chapman等人。 (2015)和DeFreitas等人。 (2012)。操作设备的人也可能是噪音源,他或她也可能需要接地。此外,屏蔽电缆可用于将信号从电极传送到放大器。
    如果信噪比仍然很小,则问题可能源于小信号而不是大噪声。在这种情况下,记录井很可能是泄漏的,应该进行重构。

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
    调整pH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

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. DeMaegd, M. L., Städele, C. and Stein, W. (2017). Axonal Conduction Velocity Measurement. Bio-protocol 7(5): e2152. DOI: 10.21769/BioProtoc.2152.
  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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