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Jul 2013

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Fluorescent Measurement of Synaptic Activity Using SynaptopHluorin in Isolated Hippocampal Neurons
采用SynaptopHluorin在分离海马神经元中进行突触活性荧光测量   

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Abstract

This protocol comprises the entire process of fluorescent measurement of vesicle recycling using the probe SynaptopHluorin, a pH-dependent GFP variant whose fluorescence increases at the synapse upon vesicle release due to fluorescence quenching in acidic vesicles. This technique provides a genetic tool to monitor synaptic vesicle recycling in real time in cultured hippocampal neurons.

Materials and Reagents

  1. Embryonic hippocampal neurons (E18)
  2. SynaptopHluorin
    Note: pHluorin-based fusion-proteins in which ecliptic pHluorin was fused to an intraluminal loop of synaptotagmin (Fernandez-Alfonso and Ryan, 2006; Wienisch and Klingauf, 2006).
  3. 0.03% trypsin (-20 °C) (Sigma-Aldrich)
  4. 0.1% (w/v) poly-L-lysine (-20 °C) (Peptides International)
  5. Neurobasal medium (2-8 °C) (Life Technologies, Gibco®)
  6. B-27 (-20 °C) (Life Technologies, Gibco®)
  7. Glutamine (final concentration: 0.5 mM, -20 °C) (Life Technologies, Gibco®)
  8. Glutamate (final concentration: 0.025 mM) (Sigma-Aldrich)
  9. Fetal bovine serum (FBS) (-20 °C) (Life Technologies, Gibco®)
  10. Fluo-4 AM calcium indicator (-20 °C) (Life Technologies, Molecular Probes®, catalog number: F14201 )
  11. DL-2-Amino-5-phosphonopentanoic acid (APV) (final concentration: 50 µM, solution in 4 °C) (Sigma-Aldrich, catalog number: A5282 )
  12. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (final concentration: 10 µM , -20 °C) (Sigma-Aldrich, catalog number: C127 )
  13. Bafilomycin A1 (final concentration: 5 µM, 20 °C) (Calbiochem®, catalog number: 196000 )
  14. Vacuum grease (Dow-Corning, for mounting the coverslip)
  15. HEPES-buffered saline (HBS) extracellular solution (4 °C) (see Recipes)
  16. 2x HeBS (see Recipes)

Equipment

  1. Pasteur pipette
  2. No. 1, 12-mm diameter glass coverslips (Warner Instruments)
    Note: No.1 glass coverslips are washed with HCl and ethanol, then autoclaved. Autoclaved glass coverslips are treated in 0.1% (w/v) poly-L-lysine for overnight. poly-L-lysine is aspirated on the next day, and glass coverslips are washed with sterile water three times.
  3. Inverted microscope (63x oil immersion lens, Mercury arc lamp, filters for EGFP) (ZEISS)
  4. Computer with time-lapse imaging system
  5. Electric field stimulation chamber (two parallel platinum wire bath electrodes) (Warner Instruments)
  6. Perfusion pump (Ismatec® REGLO Digital 2-Ch Var-Speed Pump; 0.005 to 59 ml/min) (Cole-Parmer, catalog number: WU-78016-40 )

Software

  1. Axiovision LE software
  2. Microsoft Excel

Procedure

  1. Primary cultures of rat hippocampal neurons
    Primary rat hippocampal neurons are prepared from rat fetuses (Sprague-Dawley, day 18 of gestation; Harlan, Indianapolis, IN) as described previously (Krueger et al., 2003; Kaech and Banker, 2006; Li et al., 2008a, b; Beaudoin et al., 2012; Park et al., 2014). After isolation of hippocampi from prenatal brains using autoclaved tools, they are incubated for 12 min with 0.03% trypsin. The hippocampal neurons are dissociated by trituration through a sterilized polished Pasteur pipette. The neurons are seeded onto plates containing medium with 5% FBS. After 3 h incubation, cells are maintained in neurobasal medium supplemented with B-27, glutamine, and antibiotics. Neurons are grown at 37 °C in a 5% CO2 and 20% O2 humidified incubator. Neurons are transfected with 2.5 M calcium phosphate at day 5 after plating with an efficiency of 1-5%. Briefly, transfection cocktail solutions are prepared as follows and incubated at room temperature for 20 min: 15 μl H2O, 1.8 μl 2.5 M of CaCl2, 1.5 μg of cDNA, and 15 μl of 2x HeBS (for a well of a 12 well plate with 500 μl media). Cocktail solution is added into the cell culture medium already present in the well and incubated for 30 min. Then this transfection medium is replaced with conditioned media after washing three times.
    1. Embryonic hippocampal neurons (E18) are dissected then incubated for 12 min with 0.03% trypsin.
    2. The hippocampal cells are dissociated by trituration through a polished Pasteur pipette.
    3. Neurons are plated on 0.1% (w/v) poly-L-lysine -coated 12-mm diameter coverslips at a density of 5 x 104 cells /each well in a 12 well plate; this is relatively low density to be able to view neuronal processes.
    4. Cells are cultured in Neurobasal medium, supplemented with 1x B-27 (it is ordered as 50x, but final is 1x 10 ml in 500 ml Neurobasal medium), 0.5 mM glutamine, 25 μM glutamate, and 5% FBS.
    5. 3 h after plating, the medium is replaced with serum-free Neurobasal supplemented with B-27 and 0.5 mM glutamine.
    6. Cultures typically contain few glial cells, start to form synapses after 6 days in vitro, and are typically used for synaptic studies 16 to 21 days after plating.

  2. Expressing SynaptopHluorin in cultured hippocampal neurons
    The calcium phosphate (Jiang and Chen, 2006) method is used to transfect hippocampal neurons on DIV 4 or 5 with SynaptopHluorin with 1.5 µg DNA per well.
    Transfection efficiency is ~1-5% (see Figure 1). Transfection is performed at DIV 5. Neurons are imaged at DIV 16-21.


    Figure 1. Example calcium phosphate transfection of a healthy neuron expressing a GFP-labeled mitochondrial protein. Shown are the phase contrast A, green fluorescent B and merged images C. Bar=20 μm

  3. Preparing stimulation
    1. The coverslips are mounted in the electric field stimulation chamber with vacuum grease (see Figure 2).
    2. 50 μl HBS is immediately added to cover the neurons and platinum wires. The seal should be sufficiently tight to prevent leaks.
    3. The chamber is perfused at a rate of 2 ml/min at room temperature with HBS solution with10 µM CNQX, 50 µM APV to prevent recurrent activity.
    4. All experiments are performed at room temperature but physiological temperatures can be used.


    Figure 2. Image of a recording chamber. Recording chamber with inserted coverslip A, and a diagram of orientation of a coverslip on a microscope B.

  4. Stimulation
    1. Healthy SynaptopHluorin expressing-neurons are located using the excitation and emission filters for EGFP.
      Note: It is important to choose synapses that do not have very high levels of resting fluorescence because bright synapses tend to represent axons of unhealthy cells or cells highly overexpressing SynaptopHluorin molecules on the cell surface.
    2. Before stimulation, it is important to fix the exposure time and set up time-lapse image program on the computer.
      Notes:
      1. Exposure time should be long enough to have significant pixel intensity and short enough to limit significant photo bleaching. In our experiments, we use exposure times no longer than 100 ms.
      2. Bleaching should be minimized so that there is no need to correct for bleaching during analysis. If bleaching is obvious, controls should be performed without stimulation to estimate bleaching rate to correct mathematically the final data for bleaching.
    3. At least three images must be obtained before stimulation to have sufficient data points to assure stable baseline fluorescence (Figure 3). The time course of fluorescent SynaptopHluorin responses is then obtained from time-lapse images taken every 3 sec (Figure 3).
    4. Following baseline imaging, neurons are stimulated with 100 stimuli at 5 Hz (50 mA, 1 ms pulses) for 20 sec (Figure 3). This should deplete the readily releasable pool and begin to use more slowly releasing pools. Brief stimuli work better for hippocampal neurons. We suggest starting at a low current pulse and gradually increasing the current until the optimum stimulus effect is obtained. Wait at least one minute before repeating stimuli since synapses need to recover after each stimulus. Also, mature neurons form the most complete synaptic connections so if there is little response it is helpful to use older cultures.
      Notes:
      1. In our experience, 16-21 day-old hippocampal neurons plated at a density 5 x 104 cells/a well of a 12 well plate are the best for the experiment.
      2. Another method to the check the maturity of the cells is to use fluo-4 AM calcium indicator. Neurons are loaded with 5 μM fluo-4AM (final) The stock should be prepared per manufacturers’ instructions and incubated at 37 °C for 30 min in the dark. Fluorescent axonal varicosities correspond to synaptic boutons along hippocampal cell axons detected in phase images of the cultured neurons (Komai et al., 2006). Then neurons are stimulated (50 mA, 1 ms) at 5 Hz for 20 sec. The time course of fluorescent responses of indicator is measured from the time-lapse images taken every 3 sec. If the neurons have a robust response to fluo-4 AM, this predicts future success using SynaptopHluorin.
    5. During stimulation using SynaptopHluorin, images are obtained during the stimulus and after the stimulus for a total of 120 sec.
    6. After 5 min at rest neurons can be stimulated again at 5 Hz for 20 sec in the presence of 5 μM bafilomycin A1 in HBS. Neurons should sit at least 30 sec to allow bafilomycin A1 diffusion into the cell. After bafilomycin exposure, fluorescence will come to a plateau even after stimulation ends and this peak fluorescence value will be used as the absolute maximum of fluorescence intensity.


    Figure 3. Example plots of change in synaptopHluorin fluorescence over time. Images of fluorescent puncta were obtained for two cells (a control and a cell overexpressing Bcl-xL) that were stimulated with 100 stimuli at 5 Hz. Shown are the change in fluorescence values relative to baseline for 5 sample synaptopHluorin puncta for each cell before, during and after stimulation. The time course of recovery to baseline can be measured by fitting curves to the recovery portion of the data.

  5. Image analysis
    1. In General, an axon expressing SynaptopHluorin with high baseline fluorescence is not able to produce significant fluorescence increases upon stimulation. These neurons have an abnormally high surface expression of SynaptopHluorin; therefore one should avoid using these synapses. It is important to choose the right size regions of interest (ROIs). SynaptopHluorin diffuses within the plasma membrane of an axon away from the presynaptic bouton upon stimulation which increases the area of fluorescence (Li and Murthy, 2001). If the ROI is too small, it will not include this lateral movement, therefore you will not see the appropriate change in fluorescence. Because the average diameter of a bouton is about 1-2 µm and the lateral movement of SynaptopHlourin is an equal distance from a central point (Li and Murthy, 2001) we suggest the ideal size of an ROI for an individual bouton is about 4 µm2 (Granseth et al., 2006). The mean fluorescence signal of each ROI of a fixed size for all images is measured (see Figure 4). Image J can also be used for analysis. All fluorescent signals are background subtracted using an ROI off of the cell of similar size.


      Figure 4. Demonstration of synaptopHluorin puncta. Examples are shown of three different times of fluorescent measurement, indicated by yellow arrows. Examples of ROIs (red boxes) are shown on the top row and these images are repeated without the red boxes in the second row for clarity. Bar=5 μm

    2. Fluorescence values for baseline values are obtained by averaging 3 images at rest before the stimulus period. The time course of fluorescent responses of SynaptopHluorin are measured from time-lapse images taken every 3 sec. Images are analyzed using Axiovision LE software (or Image J) and Microsoft Excel.
    3. Fluorescence changes of puncta in response to a stimulus (∆F)=F-F0 (F=actual fluorescence of each ROI, F0: The initial fluorescence intensity which is the fluorescence before stimulus). Normalize fluorescence change of SynaptopHluorin puncta ∆F/F0 in order to see the differences before, during and after electrical stimulation.
      Notes:
      1. If the images move laterally during the time course, this will have to be corrected or the time course will not be accurate. The actual baseline fluorescence value varies in an individual synapse by about 10% between the first and second sets of stimuli. The absolute (non-normalized) resting (baseline) fluorescence may be measured from unquenched SynaptopHluorin molecules on the plasma membrane. The amount of unquenched SynaptopHluorin fluorescence may differ at any synapse, especially between different neurons. For this reason, measuring the absolute baseline fluorescence may better represent the actual data. These data should be shown in the published manuscript along with the normalized data.
      2. How do you prevent bias in selecting synapses? Do analysis randomly, make at least 3 different cultures (make the same standard culture each time), do control and experimental groups blinded to the experimental condition. Check all the images to make sure they did not move and are very stable. Do not choose bright synapses; set up the right size of the ROI and randomly choose boutons to do analysis. Try to average all records, the more analysis you do, the better representation of the average behavior of synapses and the less possibility of bias by a selection procedure.
      3. What kinds of artifacts do you see? Sometimes you see a change in fluorescence during the stimulation that does not recover after stimulation ceases. This may be caused by abnormally high surface expression of SynaptopHluorin or can occur in unhealthy cells. Some synapses never appear to respond. This may represent a bouton whose fluorescence sits below the detection threshold. There are different modes of vesicle retrieval whose relative prominence depends on the type of synapse. In our hippocampal images it was difficult to discern these different modes using SynaptopHluorin. SynaptopHluorin molecules are mobile and the lateral spread of fluorescence from the site of exocytosis to other parts of the bouton can cause artifacts in analysis. Investigators might want to consider using synaptophysin-pHluorin, which has lower mobility than SynaptopHluorin. In addition, interactions between closely apposed fluorescent puncta can perturb the fluorescence levels during stimulation. One should omit from analysis any puncta displaying these artifacts.
      4. Because SynaptopHluorin molecules are mobile, sometimes we can see that diffusion of SynaptopHluorin out of a small ROI causes a fast decline of the signal. This constitutes an artifact and is not related to endocytosis. To overcome it, choose a slightly larger ROI area to prevent significant diffusional loss of synaptopHluorin.

Recipes

  1. HEPES-buffered saline (HBS) extracellular solution
    119 mM NaCl
    2.5 mM KCl
    2 mM CaCl2
    2 mM MgCl2
    25 mM HEPES
    30 mM glucose
    Buffered to pH 7.4
    Warmed to room temperature before use
  2. 2x HeBS
    274 mM NaCl
    10 mM KCl
    1.4 mM Na2HPO4.7H2O
    15 mM glucose
    42 mM HEPES

Acknowledgments

The methods were adapted from (Li et al., 2013) Techniques were also adapted from all of the references cited. The authors wish to thank Miesenbock, G. (Miesenbock et al., 1998) for providing the synaptopHluorin construct for these studies. This work was supported by grant NIH NS064967 to EAJ.

References

  1. Beaudoin, G. M., 3rd, Lee, S. H., Singh, D., Yuan, Y., Ng, Y. G., Reichardt, L. F. and Arikkath, J. (2012). Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7(9): 1741-1754.
  2. Fernandez-Alfonso, T. and Ryan, T. A. (2006). The efficiency of the synaptic vesicle cycle at central nervous system synapses. Trends Cell Biol 16(8): 413-420.
  3. Granseth, B., Odermatt, B., Royle, S. J. and Lagnado, L. (2006). Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51(6): 773-786.
  4. Jiang, M. and Chen, G. (2006). High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat Protoc 1(2): 695-700.
  5. Kaech, S. and Banker, G. (2006). Culturing hippocampal neurons. Nat Protoc 1(5): 2406-2415.
  6. Komai, S., Licznerski, P., Cetin, A., Waters, J., Denk, W., Brecht, M. and Osten, P. (2006). Postsynaptic excitability is necessary for strengthening of cortical sensory responses during experience-dependent development. Nat Neurosci 9(9): 1125-1133.
  7.  Krueger, S. R., Kolar, A. and Fitzsimonds, R. M. (2003). The presynaptic release apparatus is functional in the absence of dendritic contact and highly mobile within isolated axons. Neuron 40(5): 945-957.
  8. Li, H., Alavian, K. N., Lazrove, E., Mehta, N., Jones, A., Zhang, P., Licznerski, P., Graham, M., Uo, T., Guo, J., Rahner, C., Duman, R. S., Morrison, R. S. and Jonas, E. A. (2013). A Bcl-xL-Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis. Nat Cell Biol 15(7): 773-785.
  9. Li, H., Chen, Y., Jones, A. F., Sanger, R. H., Collis, L. P., Flannery, R., McNay, E. C., Yu, T., Schwarzenbacher, R., Bossy, B., Bossy-Wetzel, E., Bennett, M. V., Pypaert, M., Hickman, J. A., Smith, P. J., Hardwick, J. M. and Jonas, E. A. (2008a). Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons. Proc Natl Acad Sci U S A 105(6): 2169-2174.
  10. Li, H., Chen, Y., Jones, A. F., Sanger, R. H., Collis, L. P., Flannery, R., McNay, E. C., Yu, T., Schwarzenbacher, R., Bossy, B., Bossy-Wetzel, E., Bennett, M. V., Pypaert, M., Hickman, J. A., Smith, P. J., Hardwick, J. M. and Jonas, E. A. (2008b). Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons. Proc Natl Acad Sci U S A 105(6): 2169-2174.
  11. Li, Z. and Murthy, V. N. (2001). Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses. Neuron 31(4): 593-605.
  12. Miesenbock, G., De Angelis, D. A. and Rothman, J. E. (1998). Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394(6689): 192-195.
  13. Park, H. A., Licznerski, P., Alavian, K. N., Shanabrough, M. and Jonas, E. A. (2014). Bcl-xL Is Necessary for Neurite Outgrowth in Hippocampal Neurons. Antioxid Redox Signal.
  14. Wienisch, M. and Klingauf, J. (2006). Vesicular proteins exocytosed and subsequently retrieved by compensatory endocytosis are nonidentical. Nat Neurosci 9(8): 1019-1027.

简介

该协议包括使用探针SynaptopHluorin的荧光测量的荧光测量的整个过程,所述探针是pH依赖性GFP变体,其荧光在囊泡释放时由于酸性囊泡中的荧光猝灭而在突触上增加。 这种技术提供了一种遗传工具来监测突触小泡实时回收培养的海马神经元。

材料和试剂

  1. 胚胎海马神经元(E18)
  2. SynaptopHluorin
    注意:基于pHluorin的融合蛋白,其中黄体pHluorin融合到突触结合蛋白的腔内环(Fernandez-Alfonso和Ryan,2006; Wienisch和Klingauf,2006)
  3. 0.03%胰蛋白酶(-20℃)(Sigma-Aldrich)
  4. 0.1%(w/v)聚-L-赖氨酸(-20℃)(Peptides International)
  5. 神经基础培养基(2-8℃)(Life Technologies,Gibco )
  6. B-27(-20℃)(Life Technologies,Gibco )
  7. 谷氨酰胺(终浓度:0.5mM,-20℃)(Life Technologies,Gibco )
  8. 谷氨酸(终浓度:0.025mM)(Sigma-Aldrich)
  9. 胎牛血清(FBS)(-20℃)(Life Technologies,Gibco )
  10. Fluo-4AM钙指示剂(-20℃)(Life Technologies,Molecular Probes ,目录号:F14201)
  11. DL-2-氨基-5-膦酰基戊酸(APV)(终浓度:50μM,4℃溶液)(Sigma-Aldrich,目录号:A5282)
  12. 6-氰基-7-硝基喹喔啉-2,3-二酮(CNQX)(终浓度:10μM,-20℃)(Sigma-Aldrich,目录号:C127)
  13. Bafilomycin A1(终浓度:5μM,20℃)(Calbiochem ,目录号:196000)
  14. 真空润滑脂(Dow-Corning,用于安装盖玻片)
  15. HEPES缓冲盐水(HBS)细胞外溶液(4℃)(参见配方)
  16. 2x HeBS(请参阅配方)

设备

  1. 巴斯德移液器
  2. 1号,12mm直径的玻璃盖玻片(Warner Instruments)
    注意:1号玻璃盖玻片用HCl和乙醇洗涤,然后高压灭菌。 将高压灭菌的玻璃盖玻片在0.1%(w/v)聚-L-赖氨酸中处理过夜。 聚-L-赖氨酸在第二天吸出,玻璃盖玻片用无菌水洗涤三次。
  3. 倒置显微镜(63x油浸镜头,汞弧灯,EGFP过滤器)(ZEISS)
  4. 带延时成像系统的计算机
  5. 电场刺激室(两个平行的铂丝电极电极)(Warner Instruments)
  6. 灌注泵(Ismatec REGLO Digital 2-Ch Var-Speed Pump; 0.005至59ml/min)(Cole-Parmer,目录号:WU-78016-40)

软件

  1. Axiovision LE软件
  2. Microsoft Excel

程序

  1. 大鼠海马神经元的原代培养物
    如先前所述(Krueger等人,2003; Kaech和Banker,2006; Li等人)从大鼠胎儿(Sprague-Dawley,妊娠的第18天; Harlan,Indianapolis,IN)制备原代大鼠海马神经元; 等,2008a,b; Beaudoin等人,2012; Park等人,2014)。使用高压灭菌工具从产前脑中分离海马后,将它们与0.03%胰蛋白酶孵育12分钟。海马神经元通过研磨通过无菌抛光的巴斯德吸管分离。将神经元接种在含有含5%FBS的培养基的平板上。孵育3小时后,将细胞维持在补充有B-27,谷氨酰胺和抗生素的神经基础培养基中。神经元在37℃下在5%CO 2和20%O 2加湿培养箱中生长。在铺板后第5天用2.5M磷酸钙转染神经元,效率为1-5%。简言之,如下制备转染混合物溶液,并在室温下温育20分钟:将15μlH 2 O,1.8μl2.5M CaCl 2,1.5μgcDNA ,和15μl的2x HeBS(对于具有500μl培养基的12孔板的孔)。将鸡尾酒溶液加入到已经存在于孔中的细胞培养基中并孵育30分钟。然后在洗涤三次后用条件培养基替换该转染培养基
    1. 解剖胚胎海马神经元(E18),然后与0.03%胰蛋白酶孵育12分钟。
    2. 通过抛光的巴斯德吸管研磨使海马细胞解离。
    3. 将神经元接种在0.1%(w/v)聚-L-赖氨酸包被的12-mm上 直径的盖玻片,密度为5×10 4个细胞/每个孔 孔板;这是相对低的密度能够看到神经元 处理
    4. 细胞在补充的Neurobasal培养基中培养 与1x B-27(它被排序为50x,但最后是1×10毫升在500毫升 Neurobasal培养基),0.5mM谷氨酰胺,25μM谷氨酸和5%FBS。
    5. 铺板后3小时,用补充有B-27和0.5mM谷氨酰胺的无血清Neurobasal替换培养基。
    6. 培养物通常包含少量胶质细胞,开始形成突触 6天后在体外,并且通常用于突触研究16至   21天后。

  2. 在培养的海马神经元中表达SynaptopHluorin 磷酸钙(Jiang和Chen,2006)方法用于用每孔1.5μgDNA的SynaptopHluorin转染DIV 4或5上的海马神经元。
    转染效率约1-5%(见图1)。转染在DIV 5进行。神经元在DIV 16-21成像

    图1。 表达GFP-标记的线粒体蛋白的健康神经元的磷酸钙转染实例显示相位对比A,绿色荧光B和合并的图像C. Bar = 20μm

  3. 准备刺激
    1. 盖玻片用真空油脂安装在电场刺激室中(见图2)。
    2. 立即加入50μlHBS以覆盖神经元和铂 电线。密封件应足够紧以防止泄漏。
    3. 在室温下以2ml/min的速率灌注室 HBS溶液,含有10μMCNQX,50μMAPV以预防复发活性。
    4. 所有实验在室温下进行,但可以使用生理温度。


    图2。 记录室的图像。带有插入盖玻片A的记录室,以及显微镜B上盖玻片的方向图。

  4. 刺激
    1. 使用EGFP的激发和发射滤光片定位健康的表达SynaptopHluorin的神经元。
      注意:重要的是选择没有很高的突触 休息荧光的水平,因为明亮的突触倾向于代表 不健康细胞的轴突或高度过表达SynaptopHluorin的细胞   分子在细胞表面。
    2. 在刺激之前,重要的是固定曝光时间并在计算机上设置延时图像程序。
      注意:
      1. 曝光时间应足够长,以具有重要的像素 强度和短到足以限制显着的光漂白。 在我们的 实验中,我们使用曝光时间不超过100毫秒。
      2. 漂白应该最小化,以便没有必要纠正 漂白。 如果漂白是明显的,控制应该 在没有刺激的情况下执行以估计漂白率来校正 数学上用于漂白的最终数据
    3. 至少三个 图像必须在刺激之前获得足够的数据 点以确保稳定的基线荧光(图3)。 时间 然后从中获得荧光SynaptopHluorin反应的过程 每3秒拍摄一次延时图像(图3)。
    4. 以下 基线成像,用5Hz下的100个刺激刺激神经元(50 mA,1ms脉冲)20秒(图3)。 这应该耗尽 可释放池,并开始使用更缓慢释放的池。 简要 刺激对海马神经元工作更好。 我们建议从a开始 低电流脉冲并逐渐增加电流直到最佳   获得刺激效果。 等待至少一分钟,然后重复 刺激,因为突触需要在每次刺激后恢复。 还有,成熟   神经元形成最完整的突触连接,所以如果有 一点反应是有帮助的使用老的文化。
      注意:
      1. 根据我们的经验,16-21天的海马神经元在a 密度5×10 4 细胞/12孔板的孔是 实验。
      2. 检查成熟度的另一种方法 细胞是使用fluo-4 AM钙指示剂。 神经元加载5 μMfluo-4AM(最终) 并在37℃在黑暗中孵育30分钟。 荧光灯 轴突静脉曲张对应于沿海马的突触boutons 在培养的神经元的相图像中检测到的细胞轴突(Komai et et al。,2006)。 然后在5Hz下刺激神经元(50mA,1ms)20 秒。 测量指示剂的荧光响应的时间过程 从每3秒拍摄的延时图像。 如果神经元有一个 对fluo-4 AM的强烈响应,这预示着未来的成功使用 SynaptopHluorin。
    5. 在使用SynaptopHluorin的刺激期间, 在刺激期间和刺激之后获得图像 共120秒。
    6. 静息5分钟后,可刺激神经元 在5μMbafilomycin A1存在下在HBS中再次在5Hz下进行20秒。 神经元应该至少坐30秒以允许巴佛洛霉素A1扩散 进入细胞。 bafilomycin暴露后,荧光会来到a 甚至在刺激结束后的高原和这个峰值荧光值 将被用作荧光强度的绝对最大值。


    图3. 突触点荧光随时间变化的实例图。显示了5个样品的荧光值相对于基线的变化。对于5个样品,荧光斑点的荧光斑点的图像是针对在5Hz下用100个刺激物刺激的两个细胞(对照和过表达Bcl-xL的细胞)获得的。每个细胞在刺激之前,期间和之后。恢复到基线的时间过程可以通过拟合曲线到数据的恢复部分来测量
  5. 图像分析
    1. 一般来说,表达具有高基线的SynaptopHluorin的轴突 荧光不能产生显着的荧光增加 刺激。这些神经元具有异常高的表面 表达突触蛋白因此应该避免使用这些 突触。重要的是选择合适大小的感兴趣区域 (ROI)。 SynaptopHluorin扩散在轴突的质膜内 远离突触前bouton刺激增加 荧光面积(Li和Murthy,2001)。如果ROI太小,它 不会包括这种横向运动,因此你不会看到 适当的荧光变化。因为a的平均直径 bouton约1-2微米,SynaptopHlourin的横向运动  与中心点的距离相等(Li和Murthy,2001) 个体bouton的ROI的理想尺寸为约4μm2(Granseth等人,2006)。每个ROI的平均荧光信号 测量所有图像的固定尺寸(参见图4)。图像J也可以 用于分析。所有荧光信号减去背景 使用与相似大小的单元格相关的ROI

      图4。 SynaptopHluorin斑点的证明。 示例显示了三个不同时间的荧光测量,由黄色箭头指示。 ROI(红色框)的示例显示在顶行,并且为了清楚起见,在第二行中重复这些图像而没有红色框。 Bar =5μm

    2. 荧光值  对于基线值通过平均3个静止图像来获得 刺激期。荧光反应的时间过程 SynaptopHluorin是从每3秒拍摄的延时图像测量的。 使用Axiovision LE软件(或Image J)和 Microsoft Excel。
    3. 响应a的斑点的荧光变化 刺激(ΔF)= F-F 0(F =每个ROI的实际荧光,F 0):初始 荧光强度,其是刺激前的荧光)。 按顺序标准化SynaptopHluorin斑点的荧光变化ΔF/F O 以查看电刺激之前,期间和之后的差异 注意:
      1. 如果图像在时间过程中横向移动,则会有 或者时间过程将不准确。实际上 基线荧光值在个体突触中变化约10%  在第一和第二组刺激之间。绝对 (非标准化)静息(基线)荧光 未淬灭的SynaptopHluorin分子在质膜上。数量 的未淬灭的SynaptopHluorin荧光可以在任何突触, 特别是在不同神经元之间。为此,测量 绝对基线荧光可更好地表示实际数据。 这些数据应与出版的手稿一起显示 归一化数据。
      2. 如何防止选择突触的偏见? 做随机分析,做至少3个不同的文化(使同样 标准培养每次),对照组和实验组盲法 到实验条件。 检查所有图像以确保它们 没有移动,非常稳定。 不要选择明亮的突触; 建立 正确的ROI大小和随机选择boutons做分析。 尝试平均所有记录,你做的分析越多,越好 表示突触的平均行为和较少 通过选择过程的偏差的可能性。
      3. 什么类型 你看到的工件? 有时你会看到荧光的变化 刺激后不恢复的刺激停止。 这可能   由SynaptopHluorin的异常高表面表达引起 可能发生在不健康的细胞中。一些突触从来没有出现响应。 这可以表示荧光位于检测之下的bouton 阈。有不同的囊泡检索模式  突出取决于突触的类型。在我们的海马图像  使用SynaptopHluorin难以辨别这些不同的模式。 SynaptopHluorin分子是移动的和横向扩散 荧光从胞吐位点到bouton的其他部分 可能会导致分析中的工件。调查员可能想考虑 使用突触泡蛋白-pHluorin,其具有较低的迁移率 SynaptopHluorin。此外,互动之间紧密相连 荧光斑点可以扰乱荧光水平 刺激。应该从分析中省略显示这些的点 工件。
      4. 因为SynaptopHluorin分子是移动的, 有时我们可以看到SynaptopHluorin扩散出来的小 ROI导致信号的快速下降。这构成了一个工件 并且与胞吞作用无关。要克服它,选择一个 较大的ROI面积以防止显着的扩散损失 synaptopHluorin。

食谱

  1. HEPES缓冲盐水(HBS)细胞外溶液
    119 mM NaCl
    2.5mM KCl
    2mM CaCl 2 2 / 2mM MgCl 2/
    25 mM HEPES
    30mM葡萄糖 缓冲至pH 7.4
    使用前温度升至室温
  2. 2x HeBS
    274 mM NaCl 10 mM KCl
    1.4mM Na 2 HPO 4 SubO 2 .7H 2 O 2。 15mM葡萄糖 42 mM HEPES

致谢

方法改编自(Li等人,2013)。技术也改编自所有引用的参考文献。 作者希望感谢Miesenbock,G(Miesenbock等人,1998)为这些研究提供synaptopHluorin构建体。 这项工作得到了NIH NS064967授予EAJ的支持。

参考文献

  1. Beaudoin,G.M.,3rd,Lee,S.H.,Singh,D.,Yuan,Y.,Ng,Y.G.,Reichardt,L.F.and Arikkath,J。(2012)。 从早期出生后的小鼠海马和皮质培养锥体神经元。 Nat Protoc 7(9):1741-1754。
  2. Fernandez-Alfonso,T.and Ryan,T.A。(2006)。 突触囊泡循环在中枢神经系统突触的效率。趋势Cell Biol 16(8):413-420。
  3. Granseth,B.,Odermatt,B.,Royle,S.J.and Lagnado,L。(2006)。 网格蛋白介导的胞吞作用是海马突触小泡检索的主要机制。 Neuron 51(6):773-786。
  4. Jiang,M。和Chen,G。(2006)。在低密度神经元培养中的高Ca 2 + 磷酸转染效率。 Nat Protoc 1(2): 695-700。
  5. Kaech,S。和Banker,G。(2006)。 培养海马神经元。 Nat Protoc 1(5) :2406-2415。
  6. Komai,S.,Licznerski,P.,Cetin,A.,Waters,J.,Denk,W.,Brecht,M.and Osten,P。(2006)。 突触后兴奋性是加强经验依赖性发展期间的皮质感觉反应所必需的。 Nat Neurosci 9(9):1125-1133。
  7.   Krueger,S.R.,Kolar,A。和Fitzsimonds,R.M。(2003)。 突触前释放装置在没有树突接触的情况下起作用,并且在分离的轴突内具有高度移动性。 a> Neuron 40(5):945-957。
  8. Li,H.,Alavian,KN,Lazrove,E.,Mehta,N.,Jones,A.,Zhang,P.,Licznerski,P.,Graham,M.,Uo,T.,Guo,J.,Rahner ,C.,Duman,RS,Morrison,RSand Jonas,EA(2013)。 Bcl-xL-Drp1复合物调节内吞过程中的突触小泡膜动力学。 Nat Cell Biol 15(7):773-785
  9. Li,H.,Chen,Y.,Jones,AF,Sanger,RH,Collis,LP,Flannery,R.,McNay,EC,Yu,T.,Schwarzenbacher,R.,Bossy,B.,Bossy-Wetzel, E.,Bennett,MV,Pypaert,M.,Hickman,JA,Smith,PJ,Hardwick, M.和Jonas,E.A。(2008a)。 Bcl-xL在培养的海马神经元中诱导Drp1依赖性突触形成。 Proc Natl Acad Sci USA 105(6):2169-2174。
  10. Li,H.,Chen,Y.,Jones,AF,Sanger,RH,Collis,LP,Flannery,R.,McNay,EC,Yu,T.,Schwarzenbacher,R.,Bossy,B.,Bossy-Wetzel, E.,Bennett,MV,Pypaert,M.,Hickman,JA,Smith,PJ,Hardwick,JMand Jonas,EA(2008b)。 Bcl-xL在培养的海马神经元中诱导Drp1依赖性突触形成。 Proc Natl Acad Sci USA 105(6):2169-2174。
  11. Li,Z.and Murthy,V.N。(2001)。 在海马突触可视化突触小泡的突触后吞噬。 神经元 31(4):593-605。
  12. Miesenbock,G.,De Angelis,D.A。和Rothman,J.E。(1998)。 用pH敏感的绿色荧光蛋白显现分泌和突触传递。自然 394(6689):192-195。
  13. Park,H.A.,Licznerski,P.,Alavian,K.N.,Shanabrough,M.and Jonas,E.A。(2014)。 Bcl-xL是海马神经元中神经突生长所必需的。 抗氧化反应信号。
  14. Wienisch,M。和Klingauf,J。(2006)。 通过补偿性内吞作用被胞吐和随后检索的囊泡蛋白是不同种的。 Nat Neurosci 9(8):1019-1027。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, H., Park, H. and Jonas, E. A. (2014). Fluorescent Measurement of Synaptic Activity Using SynaptopHluorin in Isolated Hippocampal Neurons. Bio-protocol 4(23): e1304. DOI: 10.21769/BioProtoc.1304.
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