测量中使用FM标签的神经元的胞外分泌

Biology

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Summary

囊泡释放动力学的能力来衡量,可以帮助提供洞察到一些神经递质的基础。在这里,我们采用实时成像与红色荧光染料FM 4-64标记,以测量在海马神经元的文化突触前囊泡释放率的囊泡。

Cite this Article

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Newton, J., Murthy, V. Measuring Exocytosis in Neurons Using FM Labeling. J. Vis. Exp. (1), e117, doi:10.3791/117 (2006).

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Abstract

囊泡释放动力学的能力来衡量,可以帮助提供洞察到一些神经递质的基础。在这里,我们使用实时成像与FM染料标记,以监测囊泡突触前囊泡释放率。 FM4 - 64是一个红色的荧光双亲苯乙烯染料嵌入到突触小泡的膜,内吞作用是刺激。亲脂相互作用导致的染料,大大提高了荧光,从而散发出耀眼的的信号时,囊泡和的名义,在细胞外液时相关。经过清洗的步骤是用来帮助去除质膜的内外部染料,其余的FM是集中在囊泡,然后被驱逐又一轮的电刺激诱导的胞外分泌时。囊泡释放率是测量荧光的产生减少。由于调频染料可以适用于外部和瞬时,它是一个有用的工具,用于确定在神经元文化的胞外分泌率,尤其是在比较之间转突触和相邻的控制boutons率。

Protocol

细胞:大鼠(或鼠标)海马(14-28天)在体外神经元文化。

刺激:电器,通过交付两个铂电极; 70-90毫伏

显微镜的60X石油镜头:一个倒置的CCD荧光显微镜。

软件:Slidebook(智能成像创新,圣莫尼卡,加利福尼亚)

进一步详情请参阅页的补充方法

  1. 暖的HEPES缓冲液(HBS)至室温。添加谷氨酸受体拮抗剂APV(终浓度为50微米)和CNQX(10μM决赛)。每个FM实验通常需要20-30毫升哈佛商学院。对于工作的10微米调频4-64(分子探针)的浓度,在哈佛商学院的稀释原液1:1000(与拮抗剂添加)。每个实验2毫升调频soln。用铝箔覆盖的解决方案,以防止光线照射。

  2. 安装在盖玻片含有的神经元在电极上腔。检查室媒体室顶部的水平,并完全覆盖的电极。删除任何多余的溶液从底部的盖玻片。新增石油镜头(如果使用油镜头)。

  3. 成立灌注仪器。先用几毫升以下的每个给定的顺序(加入未来之前,让每一个流彻底):水,乙醇,水,那么哈佛商学院。接下来添加哈佛商学院(保存在今后的步骤手工添加几毫升),并关闭灌注。在盖玻片边缘设置灌注口两侧的吸力。这是最好的,如果灌注插座达到进入会议厅,其中作为吸应休息在最高层。虽然加入哈佛商学院(灌注或手工),打开的吸力,并调整其位置,使其保持在合适的水平完全覆盖电极媒体。

  4. 看该地区的盖玻片上,你想的形象,并尝试大致把它重点。优化区域含有许多突触,但不应该如此密集的单独的进程变得难以区分。应该有没有最小的细胞体,多余的膜(如星形胶质细胞的团块),或其他非特异性材料(如皮棉)。 FM染料可能非特异性坚持这些。

  5. 使用“工作组”过滤立方体(激发绿灯,并收集红远红排放)。 Slideview“捕捉偏好”提供10Hz的图像后,发病900 AP的设置0(通常为300至900的AP使用此加载刺激)。在图像窗口中,选择“荧光与红色的”过滤器设置,改变曝光时间为100 ms。关闭任何预先存在的timelapse设置。不要采取任何图像,直到您准备开始刺激。

  6. 确保吸力/打开。快速添加FM soln(2毫升)室在另一端的吸。在图像窗口中立即按好了图像,并开始刺激。刺激后等待30-45秒,然后迅速洗出调频加入哈佛商学院(我用吸管的手,但是这也可以通过灌注完成)〜2毫升。

  7. 通过灌注〜10分钟,洗净,与哈佛商学院的调频。流速应该是1-1.5毫升/分钟。在此清洗步骤,改变刺激的偏好,以便刺激发病开始@ 10图像(通常为900至1200的AP @ 10赫兹这脱色刺激)。入洗约7分钟,仔细检查的喜好(刺激呈现在图像10)。现在使用小焦点的窗口中选择一个次区域和重点。确保刺激的设置已被更改,然后再进行下一步。以一个单一的形象,看是否洗净完成(个别突触应明确punctated)。如果不增加额外0.5mL/min的流速,再等2分钟。

  8. 一旦清洗完成后,进行必要的调整曝光时间仍收到良好的,明确的信号(通常在50-100毫秒。曝光时间应尽量减少由于快速漂白的FM)。然后设置timelapse喜好采取每5秒38图像。检查的灌注,使有足够的哈佛商学院5分钟,并留下灌注流动。从timelapse开始脱色。

  9. destain后,改变刺激的设置,交付后的图像0 1200 AP @ 10HZ(通常为1200至2000年的AP使用这最后一步)。关闭的timelapse设置,并采取单一的形象,开始刺激。灌注仍然应该流淌这一步,将有助于消除任何剩余的水泡调频染料,以确定基线/“总释放的”荧光。刺激结束后,关闭的刺激设置,将成为关注的焦点地区。关闭灌注和吸,然后采取两个最终的基准图像。无论是单个图像或Z - Stack的图像(0.5微米步骤),可采取。 Z轴堆叠有用的情况下,在实验过程中有一个在焦平面转变。

  10. 调频实验已经完成。如果立即这样做更多的实验,镜头和分庭应清洗之间的每一次试验,但灌注仪器不需要直到最后的实验后清洗。完全与水,再用乙醇冲洗设备。

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Discussion

300动作电位(AP)的是足以诱发囊泡循环至少有一个全面,装载了300接入点或更是经常给标签的囊泡回收池刺激。虽然更加激烈负荷刺激,如900的AP,可能会允许必须标明的其他囊泡的名义,这也增加了染料可以嵌入外膜的时间,从而导致更多的非特异性染色。我已经找到了清洗步骤是至关重​​要的,以确保正确的脱色。重要的是要灌注流速为每分钟1至1.5毫升,一般为10 + 2分钟。 〜7分钟后洗净,监察染料去除的程度,可以采取单一图像。如果有必要,流量和或洗时间可略有增加。对于我们的实验,它是不可取的洗着手,因为这增加了通过自发的胞吐事件可能发生甚至处于静止状态时,细胞失去囊泡标签的可能性大于10-12分钟。对于脱色步骤,900至1200的AP管理,释放所有标记的​​囊泡。此外,另有1200至2000年的AP往往给获得“释放的荧光”的基准值,用于脱色数据正常化。

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Materials

Name Company Catalog Number Comments
APV Sigma-Aldrich A5282
FM 4-64 Molecular Probes, Life Technologies T-3166
CNQX disodium salt Sigma-Aldrich C-239

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References

  1. Betz, W. J., Bewick, G. S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science. 255, 200-203 (1992).

Comments

4 Comments

  1. The data you can see on the screen look af if you see a lot of photobleaching. Is this true?
    How do you correct for this?

    Reply
    Posted by: Anonymous
    September 20, 2008 - 2:20 PM
  2. As in all fluorescence imaging experiments, bleaching can be corrected for in several ways.
    First, we use a sequence of frames where no stimulation was applied to obtain the bleaching rate
    (make sure to subtract a background region from all the frames). We then fit an exponential
    to this control background-subtracted data.
    Bleach(i) = Bleach(0)*exp(-i/tau) Then, we go back to the original data (minus the background) and apply the exponential correction.  F_corrected(i) = F(i)/exp(-i/tau)

    There are many descriptions of bleach correction on the web (just google).
    A nice description of bleach correction was recently described in:
    http://www.jneurosci.org/cgi/content/full/²5/19/4766/DC1 (see second supplementary material)
    A technical article on general calibration can be found in Journal of Microscopy, ²16:15-²4 (²004)
     

    Reply
    Posted by: Anonymous
    September 25, 2008 - 10:49 AM
  3. Thanks for this nice protocol.
    I started working with this technique and found your video really helpful.
    One question i would like to ask is related to the reproducability of this kind of experiments. Can you do repeated loading / unloadings on the same set of synapses?

    E.g., in my first experiments i use 600Ap/²0Hz stimuli for loading and destaining of the synapses, respectively. Image acquisation is done with with 1 Hz and a total of 70 images (²0 images before/after stimulation).

    While i get nice punctae with corresponding destaining curves in the first loading/unloading cycle, i found it vitually impossible to do a second round of stimulations at the same set of synapses. After the second loading the whole neurites become very bright, no punctae are identifiable and there is also no clear destaining, apart of photo bleaching.

    Can it be that the dye is damaging the synapses? (I use neutral density filters with ~5% transmission and select the exposure times as short as possible).

    Any comment or help on this is highly appreciated!

    Best,

    Paul

    Reply
    Posted by: Anonymous
    September 17, 2009 - 6:12 AM
  4. Paul,

    It is not uncommon to have difficulty for repeated staining. It is often related to one of two things:

    (i) Too much background: it is possible that you are ending up with too much surface stain with repeated loading and you are swamping the actual intravesicular signal. You can test this by skipping the labeling step in the first round - just stimulate as many times to mimic stainign and destaining, but don't add FM dye. Then, try the second round staining.

    (ii) The preparation becoming unhealthy either due to excessive electrical stimulation or phototoxicity. You can test this by skipping stimulation the first round and just have dye present for the same time, wash it out. Then, wait and do the second round as usual. To test phototoxicity, you can do staining and destaining in the first round, but just don't image at all. Then, start imaging only for the second staining.

    In the end, I am afraid that you may have to try several combinations of things.

    Best,

    -venki murthy

    Reply
    Posted by: Anonymous
    September 22, 2009 - 12:31 PM

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