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Fluorescent Measurement of Synaptic Activity Using FM Dyes in Dissociated Hippocampal Cultured Neurons
使用FM染料对分离的海马培养神经元中的突触活动进行荧光测定   

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参见作者原研究论文

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Scientific Reports
Jul 2017

 

Abstract

Release and recycling of synaptic vesicles are essential for neurotransmission and synaptic plasticity. To gain mechanistic understanding of these processes, direct measurements of vesicle release and retrieval is indispensable. Styryl dyes like FM1-43 and FM4-64 have been widely used for this purpose and their loading and unloading are reliable measurements for synaptic vesicle release and retrieval in cultured neurons. This protocol describes in detail the procedure of using styryl dyes to label and measure synaptic vesicle uptake and release in cultured rat hippocampal neurons. We also include a brief description of hippocampal culture. In the end, we briefly discuss the commonality and difference among FM dye, pH-sensitive fluorescent proteins and quantum dots in terms of measuring synaptic vesicle behavior.

Keywords: FM1-43 (FM1-43), Synaptic transmission (突触传递), Fluorescence live imaging (荧光实时成像), Dissociated hippocampal culture (分离的海马培养物)

Background

Synaptic vesicles are indispensable for neurotransmission since they are the only organelle responsible for neurotransmitter release in chemical synapses. Their amount, release probability, fusion kinetics and recycling routes define synaptic transmission and neuronal communication. Various tools have been developed to probe synaptic vesicles, including electrophysiological recording of postsynaptic neurons, capacitance measurement of membrane trafficking, amperometry of oxidizable transmitters, electron microscope imaging of fixed synapses, and fluorescence imaging of vesicular labels in live neurons. Among all existing methods, the last is the only one that not only yields both spatial and temporal information about individual synapses but also provides high throughput (i.e., more data points from single synapses of different neurons). Various fluorescent probes based on different targeting and reporting mechanisms have been developed. Styryl dye (i.e., FM dyes including FM1-43, FM4-64, FM5-95), invented more than twenty years ago, remains a reliable and convenient tool. Due to its moderate affinity to lipid membrane and its lipid-sensitive emission, it can be readily loaded into recycled synaptic vesicles and released when those vesicles are exocytosed. Using more sensitive photodetectors like EMCCD, FM dyes can report single vesicle release events. Here, we provide a relatively complete description of FM-based imaging of synaptic vesicle release in primary cultures of rodent hippocampal neurons. In addition, we also discuss the commonality and the distinction between FM dyes and other fluorescent vesicle labels.

Materials and Reagents

  1. Pasteur pipette 9 in, cotton-plugged (Fisher Scientific, catalog number: 13-678-8B )
  2. Pasteur pipette 5.75 in (Fisher Scientific, catalog number: 13-678-20A )
  3. 24-well plates (Corning, Costar®, catalog number: 3524 )
  4. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 )
  5. Round 12 mm-Ø glass coverslips #0 (Thermo Fisher Scientific, special order, 0.085 mm thick)
  6. Aluminum foil (WebstaurantStore Food Service Equipment and Supply Company, Choice, catalog number: 12224X1HD )
  7. Platinum wires (Alfa Aesar, catalog number: 10286 )
  8. 24 x 40 coverslips (Fisher Scientific, special order), all 0.085 mm thick (i.e., size 0)
  9. Parafilm PM-996 (Bemis, catalog number: PM996 )
  10. 30-mm Ø Petri dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 153066 )
  11. 15 ml conical tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  12. 0.2 µm filter (Corning, catalog number: 431218 )
  13. 50 ml conical tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  14. 1.7 ml tubes (Corning, Axygen®, catalog number: MCT-175-C )
  15. Neonatal rat hippocampal neurons (P0-P3)
  16. Trypsin-EDTA (trypsin: 0.025%/EDTA: 0.01%) (Thermo Fisher Scientific, GibcoTM, catalog number: R001100 )
  17. Minimum Essential Medium (MEM, 1x) (Thermo Fisher Scientific, GibcoTM, catalog number: 51200038 )
  18. Glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 15023021 )
  19. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
  20. Transferrin (Sigma-Aldrich, catalog number: T1428 )
  21. L-glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
  22. Insulin (Sigma-Aldrich, catalog number: I5500 )
  23. Fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  24. Matrigel (Corning, catalog number: 354234 )
  25. 70% EtOH (Decon Labs, catalog number: 2401 )
  26. Ara-C (Cytarabine) (Sigma-Aldrich, catalog number: C1768 )
  27. B27 supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  28. Vacuum grease (Dow Corning, catalog number: 1597418 )
  29. FM1-43 (Biotium, catalog number: 70022 )
  30. 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX) (Abcam, catalog number: ab120046 )
  31. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5) (Abcam, catalog number: ab120003 )
  32. N-2-hydroxyethyl piperazine-n-2 ethanesulphonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034 )
  33. DNase (Sigma-Aldrich, catalog number: D5025 )
  34. 10 mM HCl (diluted with ddi water 1:100 from 1 N solution) (Sigma-Aldrich, catalog number: H9892 )
  35. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S641-212 )
  36. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
  37. Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: BP214 )
  38. Calcium chloride dihydrate (CaCl2·2H2O) (Acros Organics, catalog number: 207780010 )
  39. Matrigel stock solution (see Recipes)
  40. Hanks solution (see Recipes)
  41. Hanks + 20 (H + 20) (see Recipes)
  42. Dissociation solution (see Recipes)
  43. DNase solution (see Recipes)
  44. 2 mM Ara-C stock (see Recipes)
  45. Plating medium (see Recipes)
  46. Ara-C solution (see Recipes)
  47. Extracellular bath Tyrode’s saline (see Recipes)
  48. 90 K (High Potassium Tyrode’s) (see Recipes)

Equipment

  1. Bunsen burner (Sigma-Aldrich, catalog number: Z270326 )
  2. 250 ml beaker (Fisher Scientific, Fisherbrand, catalog number: FB100250 )
  3. Pyrex beaker (Corning, PYREX®, catalog number: 1000-100 )
  4. Inverted microscope (20x lens, Mercury arc lamp, filters for EGFP) (Nikon Instruments, model: Eclipse Ti-E )
  5. Electric field stimulation chamber (two platinum wires glued to the sides of RC-26G perfusion chamber) (Warner Instruments, model: RC-26G )
  6. PH-1 platform (Warner Instruments, model: PH-1 )
  7. Six Channel Perfusion Control Valve System (Warner Instruments, model: VC-6 )
  8. Inline heater (e.g., Warner Instruments, catalog number: 64-0104 )
  9. TC-344B Dual Channel Temperature Controller (Warner Instruments, model: TC-344B )
  10. Prior Lumen 200 Illuminator (Prior Scientific, model: Lumen 200 )
  11. Cell culture hood, CO2 incubator
  12. Set of dissection tools (Fine Science Tools)
  13. Stimulus Isolator SD9 (Grass Instruments, model: SD9 )
  14. Digidata 1440A (Molecular Devices, model: Digidata 1440A )
  15. Dissection microscope (Nikon Instruments)
  16. Centrifuge (Eppendorf, model: 5702 R )
  17. Computer with time-lapse imaging system
  18. Computer with Clampex software
  19. Andor iXon Ultra EMCCD (Andor)
  20. Vibration Isolation table (Newport)

Software

  1. Micro-Manager (Schneider et al., 2012) or Fiji (Schindelin et al., 2012)
  2. Clampex (Molecular Devices)
  3. Microsoft Excel (Microsoft)

Procedure

  1. Primary culture of rat hippocampal neurons
    Note: Preparation of neonatal neuronal culture is technically easier than embryonic culture.
    Primary cultures of dissociated postnatal rat hippocampal cells are prepared as previously described (Liu and Tsien, 1995). Briefly, rat hippocampi (CA1-CA3) are normally dissected from P0 to P1 Sprague-Dawley rats and dissociated into a single-cell suspension with a 10-min incubation in trypsin-EDTA (trypsin: 0.025%/EDTA: 0.01%, Thermo Fisher Scientific) followed by gentle trituration using three glass pipettes of decreasing diameters (~1 mm, 0.5 mm, and 0.2 mm), sequentially (Figure 1). Pipettes were obtained by fire-polishing the tips on a Bunsen burner. Soft borosilicate glass melts and shrinks easily if exposed to fire. For the best results, pipette tips are positioned vertically to the flame and are being twisted clockwise and counterclockwise for even heating and melting. Longer flaming makes the smaller diameter tips. It is also important to make sure that the smallest tips are not fully sealed, which sometimes happens. Ideally, the smallest diameter pipette lets cell suspension through slowly and ensures single cell dissociation.


    Figure 1. Example of fire-polished Pasteur pipette tips of different diameters used for dissociation of hippocampi

    1. Dissociated cells are recovered by centrifugation (200 x g, 5 min) at 4 °C and re-suspended in plating medium composed of Minimal Essential Medium (MEM, Thermo Fisher Scientific) with (in mM) 27 glucose, 2.4 NaHCO3, 0.00125 transferrin (Sigma-Aldrich), 2 L-glutamine (Thermo Fisher Scientific), 0.0043 insulin (Sigma-Aldrich) and 10%/vol fetal bovine serum (FBS, Gibco).
    2. 100 μl of cell suspension is seeded onto round 12 mm-Ø glass coverslips (200-300 cells/mm2) pre-coated with Matrigel (Corning) placed in 24-well plates (Fisher Scientific).
      Note: Use of Matrigel as a neuronal substrate vs. commonly used poly-L-lysine substantially improves cell culture quality. 100 μl of Matrigel is dispensed onto the surface of coverslip 1-24 h before use and is aspirated before the cell suspension is plated. Also, no antibiotics should be used in order to avoid unwanted effects on neuronal properties.
    3. Dissection tools and glassware used for cell culture are being cleaned with no detergents in order to prevent their detrimental impact on neuronal membranes. After use, tools are washed with deionized water; blood remains are removed with Kimwipes. For sterilization before the dissection, tools are incubated with 70% EtOH in a 250 ml beaker (Fisherbrand) for 30 min. To protect fine dissection tools from mechanical damage due to bumping to the bottom of the beaker–we cover the bottom with 2-3 Kimwipes. The coverslips we use are 12 mm #0 (Thermo Fisher Scientific, special order, 0.085 mm thick). Coverslips are autoclaved in a Pyrex beaker covered with 3 layers of aluminum foil for 30 min before use.
      Note: No any other special treatment of coverslips is needed.
    4. Cells are allowed to adhere to the substrate for 30-60 min before the addition of 1 ml plating medium. After 1-2 days in culture, an additional 1 ml medium containing (in mM) 27 glucose, 2.4 NaHCO3, 0.00125 transferrin, 0.5 L-glutamine, 0.002 Ara-C, 1%/vol B27 supplement (Thermo Fisher Scientific) and 5%/vol FBS is added. Ara-C (1 µM) in the culture media efficiently prevents astroglial proliferation.
    5. Experiments are performed between DIV 12 and 18 (when synaptic transmission is well established).

  2. Imaging setup
    1. For cell culture on coverslips, an inverted microscope is preferred due to the ability to use an oil-immersion objective with high N.A. high magnification. An XYZ motorized stage is preferred but not necessary.
    2. An RC-26G chamber (Warner Instruments) is modified with 2 platinum wires attached to the sides of the chamber for delivering electric field stimulation. The chamber is bottom-sealed with a 24 x 40 mm size 0 cover glass (0.085 mm thick) using vacuum grease and clamped on a PH-1 platform (Warner Instruments) placed on a microscope stage (Scientifica). We use a Nikon Eclipse Ti inverted microscope with a 20x Plan Apo VC objective (N.A. 0.75) for large areas or a 100x Apo VC objective (N.A. 1.40) for a detailed view of synaptic boutons.
      Note: The summative thickness of two #0 coverslips (one 24 x 40 coverslip, Fisher Scientific special order) which seals the bottom of the chamber and one 12 mm circular coverslip with cells) is equal to the thickness of one #1.5 standard coverslip (0.17 mm), for which most of the objective lenses are optimized and corrected. This eliminates the need to seal coverslips with cells directly to the bottom of the imaging chamber with vacuum grease.
    3. Solution exchange is achieved via gravity perfusion controlled by a VC-6 valve control system and a 6-channel manifold (Warner Instruments) with a constant rate of ~50 μl/sec which allows a complete exchange of the bath solution in the recording chamber within 30 sec.
    4. All experiments are performed at room temperature, but physiological temperature can also easily be achieved by using an inline heater (e.g., 64-0104, Warner Instruments) and a heated stage (e.g., PH-1 stage, Warner Instruments). Both can be set to 34-37 °C (e.g., controlled through TC-344B Dual Channel Temperature Controller, Warner Instruments). To avoid heating induced stage drift, preheating for at least 1 h is recommended. Autofocus such as Nikon Perfect Focus is also helpful.
    5. An RC-26G with attached stimulation wires is clamped in PH-1 and a home-made water-tight insert for the Scientifica stage.
      Note: It is advisable to have a leak-proof system for live imaging to minimize possible damage to the microscope. PH-1 is bolted to the stage insert. Laboratory Parafilm PM-996 is used as a seal.
    6. Excitation light sources can be an arc lamp or a laser launched either through a liquid light guide or a laser launcher box. We use a Prior Lumen 200 Illuminator connected to the microscope via a liquid light guide.
    7. Fluorescence excitation/dichroic/emission filter combination corresponding to selected FM dyes can be found online (e.g., http://www.chroma.com). Filters are either installed in the filter cubes inside the microscope housing, or separately within the light path. For example, FM1-43 imaging is done using a fluorescence filter set: Ex. 460/50; DIC: 495LP; Em: 510/25BP. All optical filters and dichroic mirrors are purchased from Chroma or Semrock.
    8. Image acquisition and synchronized perfusion are controlled via Micro-Manager and Clampex software. The acquisition settings including excitation power, fluorescence filter set (excitation, dichroic and emission filters), exposure time, camera gain and frame rate are all kept the same among different samples. Images are taken at 0.1 Hz rate. Baseline fluorescence is captured during 1 min before electrical/chemical stimulation/depolarization of neurons or for at least 3 frames.
    9. Imaging with Andor EMCCD. 1) Set Andor temperature to -80 °C; 2) Set Andor readout mode to 5 MHz (instead of 17 MHz); 3) Set Andor vertical speed to 0.5, instead of 3.3: these settings help to increase the dynamic range for imaging fluorescence intensity.

  3. Imaging procedure
    1. Synaptically mature primary rat hippocampal neurons (DIV 12-18) are incubated with 10 µM FM1-43 (i.e., SynaptoGreen C4, Biotium) for 0.5 h at 37 °C in a 5% CO2 incubator to load FM1-43 into synaptic vesicles through spontaneous exo-/endocytosis. For dye loading, 10 μl sterile 1 mM stock solution in ddiH2O mixed thoroughly with 990 μl of the conditioned plating medium from the neuronal culture will be added to and dispensed into a new well of the 24-well plate. Then, a coverslip with neurons is transferred to the well containing 10 μM FM1-43 by means of burner sterilized forceps.
    2. A more common loading procedure is acute loading by stimulation. For example, FM1-43 dye is loaded in the presence of high K+ saline, which promotes massive evoked exo-/endocytosis.
    3. For that, in a laminal flow culture hood, use burner sterilized forceps to transfer one coverslip of primary rat hippocampal culture from the 24-well plates to a 30-mm Ø Petri dish containing 3 ml of normal Tyrode’s solution. Bring the coverslips to the microscope. To prevent dye loss due to spontaneous neuronal activity, Glutamate receptor blockers (10 μM NBQX and 20 μM D-AP5) are added at least 1 min before imaging and are present in the bath throughout the whole imaging experiment.
    4. Transfer the coverslips to the imaging chamber preloaded with normal Tyrode’s solution with perfusion speed of 0.05 ml/sec.
    5. Stop perfusion by closing both inlet and outlet (i.e., vacuum), and remove ~90-95% solution from the chamber.
    6. Dropwise add 500 μl high K+ solution containing 10 µM FM1-43 (i.e., SynaptoGreen C4, Biotium) to the imaging chamber and mix it gently.
    7. Incubate the cells for 2 min with the dye and turn on the perfusion for 5-min washout of surface FM dyes with normal Tyrode’s solution. Healthy looking neurons with moderate FM1-43 labeling are identified at 20x magnification (as in Figure 2).
    Note: The two different loading procedures have their own pros and cons. If possible side effects due to membrane depolarization in high K+ are to be avoided it is advisable to use the first approach (30 min at 37 °C). Acute loading helps to achieve more directed plasma membrane targeting.


    Figure 2. Sample zoom-out images which show optimal cell density in DIC and overlay with FM1-43 fluorescence

    1. To induce synaptic vesicle release, either electric field stimulation or high K+ perfusion is used. The rate of evoked FM1-43 loss from presynaptic terminals is measured.
    2. The neuronal culture is exposed to electrical field stimulation at 10 Hz with 1 msec 70 V pulses generated by Grass SD9 stimulus isolator for 2 min or high-potassium (90 K) 1 min perfusions.
    3. Stimulation control is interfaced through Clampex software (Molecular Devices). An example of Clampex protocol that controls timing of electric field stimulation and switch between perfusion channels is shown in Figure 3.


      Figure 3. Example of Clampex protocol that can be used to control timing of electric field stimulation and to switch between solution perfusion channels. Sampling rate is set at 1 msec.

    4. SD9 is triggered with a 5 V TTL pulse from #4 digital output port on 1440A digidata (Molecular Devices). SD9 settings are shown in Figure 4. 70 V output pulses are delivered from positive and negative ports on SD9 to the platinum wires (Alfa Aesar 0.5 mm dia), super-glued to the sides of the RC-26G chamber. Stimulation conditions were optimized in whole-cell patch clamp recordings on current-clamped neurons and are sufficient to reliably evoke action potentials without obvious detrimental effects. In the example Clampex protocol (Figure 2), electric field stimulation is encoded as a 10 Hz, pulse train (Epoch B), which is 120,000 msec (120 sec = 2 min) long. Repetitive triggering of the stimulator through digital output #4 is encoded by the star (*) in digital bit pattern.


      Figure 4. Grass instruments SD9 stimulus isolator settings. Frequency–0, Delay–0, duration–10 x 0.1, Volts–7 x 10, output–mono, polarity–normal.

    5. Digital control of perfusion is achieved through a VC-6 valve controller. Individual perfusion channels are set to external trigger mode, and open or close in response to TTL pulse sent from the Digidata 1440A digital output.
    6. The Andor iXon Ultra EMCCD camera’s External Input/Output ‘Fire’ port is connected to the Start port on the Digidata 1440A, which allows it to trigger Clampex protocols via the Digitizer Start Input immediately when the imaging acquisition begins. The schematic diagram of controller/devices interconnections is depicted in Figure 5.


      Figure 5. Diagram of controller/devices interconnections

    7. Orientation of coverslips on the microscope is shown in Figure 6.
    8. Baseline fluorescence is captured for 1 min (or at least 3 frames) before electrical/chemical stimulation/depolarization of neurons.


      Figure 6. Diagram of coverslips’ orientation on the microscope

  4. Imaging analysis
    Imaging analysis in ImageJ/Fiji:
    1. Open ImageJ or Fiji (not Micro-Manager).
    2. File → Import image sequence.
    3. Image → Duplicate first (not a stack) and use it for ROI selection.
    4. If want to select/save/analyze a portion of image, e.g., area in focus: select it, right click, duplicate.
    5. If you want to smoothen the image: Process → Filters → Mean (e.g., average 4-5 pixel values).
    6. Process → FFT → Bandpass Filter, large structures–down to 40 px, small–down to 3, Suppress stripes: None, 5% tolerance of direction, Autoscale after filtering, Saturate image.
    7. Having duplicated the active image, open Image → Adjust → Threshold, Dark background-B&W.
    8. Set lower threshold level, click set, apply: the program assigns values in a range from 0 to 255 for the 8-bit image.
    9. Analyze → Analyze Particles: size 2-200 pixels (good range to cover synaptic structures). An example of threshold-defined and size-restricted ROIs is shown in Figure 7.


      Figure 7. Example of the threshold based selection of FM1-43 labeled ROIs. Image on the right depicts outlined ROIs determined based on the threshold and particle size.

    10. Image → Adjust → Brightness/Contrast: drag brightness to Max to be able to find background spots with almost no signal.
    11. Select 4 ROIs for background: One by one select background regions and click Add on the ROI manager.
    12. On the ROI manager, go to More → Save ROIs and save in the root folder, not in the stack.
    13. Go to original image stack, open ROI manager, uncheck, check back, show all.
    14. ROI manager, select More → Multi measure to get the intensity values for each ROI through the whole stack of images. Copy values to Excel.
      Notes:
      1. To correct for lateral drifting in a stack of images, have your stack open with the 1st frame active, to which you want to align subsequent frames, then go to: Plugins → Registration → StackReg → Translation. After correction, the new stack will have blank areas along the edges where drifting happened. To exclude those areas from analysis–just select the area you want to keep, right-click, duplicate and save the stack as a new image sequence in a new folder.
      2. StackReg is built into Fiji (if the Fiji installation does not have it, go to Help → Update, click ‘Manage update sites’, add http://sites.imagej.net/BIG-EPFL/ and it will be automatically installed at next update), but can be added to ImageJ as well. It requires TurboReg.
        http://bigwww.epfl.ch/thevenaz/turboreg/
        http://bigwww.epfl.ch/thevenaz/stackreg/
      3. MultiStackReg (available from http://bradbusse.net/downloads.html, requires StackReg and TurboReg) can be used to save the transformations and apply them to another stack. This is extremely useful if another channel that has a lower signal is imaged simultaneously and is difficult to automatically correct.

    Data analysis in Excel:
    1. Insert 1 column at the beginning of the data set to be able to calculate average background values (from the last 4 columns that represent background ROIs-regions of interest).
      Note: Columns of data contain fluorescence intensity values of individual ROIs over frame number, plotted in rows. Frame numbers can easily be translated into time points from the imaging frequency if need be. If the frequency is 0.1 Hz, interval between frames is 10 sec.
    2. Make a copy of the entire spreadsheet and subtract averaged background values (in column 1) from individual intensity values.
    3. Make a second copy of the spreadsheet with background values subtracted. Insert 2 rows on top of the data set. Calculate average values of the first 3 rows (baseline, Fmax) and last 3 rows (after 2-nd bout in 90 K, Fmin).
    4. Copy the spreadsheet one more time and normalize data as (F - Fmin)/(Fmax - Fmin) for individual ROI. Visual representation of Excel analysis can be found in Figure 8.


      Figure 8. Screenshots from Excel analysis. A. Background averaging column is added to the dataset, then a copy of the spreadsheet is made (con (2)). B. In the con (2) spreadsheet background is subtracted, frame number is translated to time, Fmax and Fmin values are calculated, graph panel is added for visual control of the FM1-43 fluorescence changes, then the spreadsheet is copied one more time (con (3)). C. In con (3) spreadsheet the data from con (2) is being normalized and presented as (F - Fmin)/(Fmax - Fmin) and application bars are being added to the graph.

    5. Calculate average values across multiple ROIs over time. An example of the resultant graph is shown in Figure 9. Sample images at baseline, end of electric field stimulation and end of second bout in 90 K+ are shown in Figure 10.
      Note: Different Fields of View (FOVs) contain different numbers of threshold-detected ROIs. To eliminate over-influence of FOVs with high ROI number on the averaged data, a set of randomly selected ROIs which is equal or smaller than the number of ROIs of the least populated FOV can be used for averaging. To randomize ROIs in excel–insert a new row on top of the ROI data set. In this new row, select cells, aligned to the columns with ROIs. Type in =rand(), then press Ctrl+Enter. Having the row of randomized values selected, go to Menu-Data-Sort-Expand the selection-Options-Sort left to right. Sort data by row1 (randomized values).


      Figure 9. Example of normalized FM1-43 fluorescence in response to 2 min 10 Hz electric field stimulation (green bar) or 1 min high potassium (90 K) chemical stimulation (red)


      Figure 10. Example images at baseline, end of electric field stimulation and end of second bout in 90 K+

Data analysis

Initial image analysis is done in ImageJ. Regions of interest (ROIs) are selected by using the same fluorescence intensity threshold across different samples. Average intensity from every ROI and average background intensities from four cell-free regions in every image stack are exported to Excel. The FM1-43 signal in every ROI is calculated as (F - Fmin)/(Fmax - Fmin), in which Fmax is the average of the first 3 frames at a baseline before stimulation, and Fmin is the average of the last 3 frames after multiple bouts with 90 K stimulation. All fluorescence intensity values are background subtracted.
To determine the minimum number of ROIs for FM1-43 destaining, a power analysis is performed using G*Power (Faul et al., 2007). An effect size of 25% is estimated with the error probability set to 0.05, power to 0.95 and an expected standard deviation of 40% is chosen based on FM destaining experiments performed in the lab. A sample size of 53 is needed to achieve significance with a two-tailed Student’s t-test. All image processing is performed in ImageJ. All experiments are performed in two to three different batches of cell cultures. All values presented are mean ± SEM. For calculating statistical significance, the Student’s t-test is used for 2-group comparison, and one-way analysis of variance (ANOVA) followed by the Tukey-Kramer method as post-hoc analysis is used for comparing three or more groups.

Notes

  1. Reproducibility and variability
    In our tests, we observed a moderate variability in FM loading and unloading in untreated control. This variation mostly originates from the variations of cell culture such as days in vitro and cell density. Therefore, reducing such variables can significantly improve experimental consistency. In addition, we notice that unhealthy neurons often have excessive FM staining, which we have used as a criterion to exclude damaged synapses.

  2. The comparison between FM and other synaptic vesicle labels
    Synaptic vesicles are so essential in neurobiology that many methodologies such as electrophysiology, amperometry, electron microscopy and fluorescence imaging have been developed over the past fifty years to study them. Fluorescence imaging of fluorophores that selectively target synaptic vesicles has the advantage of directness, ability to dynamically monitor synaptic events in real time with high throughput and high spatial resolution (Kavalali and Jorgensen, 2014). FM dyes are the most popular fluorescent labels (Gaffield and Betz, 2007). Other probes include fluorescently tagged antibodies recognizing vesicle luminal epitopes, vesicular proteins whose luminal domain is tagged with pH-sensitive fluorescent protein (Syn-pHFP) (Afuwape and Kavalali, 2016) and quantum dots (Qdots) with surface affinity to vesicular membranes (Zhang et al., 2009) or conjugated to antibodies recognizing vesicle luminal epitopes (Park et al., 2012). Below, we will make a concise comparison among those fluorescent labels in terms of operational difference.
    1. Targeting difference
      FMs are styryl dyes that randomly insert into cell membranes. With stimulation-induced vesicle release and compensatory retrieval, FM dyes are believed to end up mostly in recycled synaptic vesicles, partially in endosomes and lysosomes, and a little in Golgi and endoplasmic reticulum (ER). Qdots with vesicular membrane affinity and the vesicle-targeting antibodies conjugated to fluorophores including Qdots have similar targeting outcomes with a lower possibility of being targeted to Golgi and ER. Shortening loading time is the most effective way to reduce non-synaptic vesicle targeting. Differently, Syn-pHFPs are transgenically expressed and distributed to all cell membranes the original proteins reside in, which includes vesicles and the cell surface. Generally, it is believed that Syn-pHFPs label all synaptic vesicles, releasable and non-releasable.
    2. Signal difference
      Since FM dyes are prone to washout when surfaced, the signal reporting vesicle release is a decrease of their fluorescence. In contrast, Syn-pHFPs report exocytosis as an increase of fluorescence because exocytosis causes deacidification of the vesicular lumen, and their fluorescence decrease reports endocytosis and vesicle re-acidification.
    3. Normalization difference
      Different synaptic boutons contain different numbers of vesicles; therefore, normalization is important to obtain a measurement of population vesicle behavior. For FM dyes and other externally added probes, the most common and simplest way to normalize the response is to define the fluorescence intensity prior to stimulation as 100% with the intensity after exhaustive stimulation as 0. For genetically expressed Syn-pHFP, the most common normalization is to use a high concentration of NH4Cl solution to de-acidify all cytoplasmic compartments to completely unquench Syn-pHFP and achieve the maximum 100% fluorescence intensity. To determine minimal Syn-pHFP intensity (0%), cells are normally perfused with low pH-solution (e.g., pH 5.5 Tyrode’s). Clearly, both pH manipulations have a possible impact on synaptic vesicle behavior. Notably, our recent paper demonstrated that even a moderate concentration of NH4Cl (i.e., 5 mM) can change synaptic vesicle release and retrieval over an extended period of time (Lazarenko et al., 2017).

Recipes

  1. Matrigel stock solution
    1. Make Matrigel stock solution
      Thaw 10 ml bottle on ice in a cold room for 24-48 h, then aliquot in 1 ml aliquots and freeze. If you try to thaw it at a higher temperature, it will turn into sludge
    2. Make working Matrigel (i.e., what is called Matrigel in the protocol)
      1. Thaw 1 ml aliquot on ice for several hours and mix with 49 ml MEM
      2. Matrigel is thick and viscous, so you will probably need to flush the 1 ml aliquot with MEM to get the whole thing out
      3. Shake the MEM + Matrigel to mix and let it sit overnight in a cold room to dissolve completely before using
      4. Aliquot it into four 15 ml conical tubes with 12.5 ml in each
      5. Cover all tubes with aluminum foil to protect from light
      6. Do not use an aliquot older than one month, and do not filter Matrigel
  2. Hanks solution
    Hanks solution is directly made from Sigma-Aldrich H2837 powdered HBSS, or directly ordered from Invitrogen
    0.2 µm filter sterilize and aliquot in 50 ml tubes
  3. Hanks + 20 (H + 20)
    400 ml 1x Hanks + 100 ml fetal bovine serum (20% FBS)
    0.2 µm filter sterilized and aliquoted in 50 ml tubes
  4. Dissociation solution (500 ml)
    Hank’s salt solution +12 mM MgSO4·6H2O
  5. DNase solution
    Dissolve entire 375 kU in 5 ml sterilized Milli-Q water and aliquot in 40 µl aliquots
  6. 2 mM Ara-C stock (500 μl aliquots)
    1. 9.7 mg Ara-C (usually stored in refrigerator or cold room) to 20 ml of deionized water in a 50-ml conical tube
    2. Vortex well
    3. Filter (0.2 μm) into 50 ml conical tube
    4. Aliquot into 1.7 ml tubes, 500 μl each
    5. Store at -20 °C
  7. Plating medium
    500 ml MEM
    2.5 g glucose
    100 mg NaHCO3
    50 mg transferrin
    Mix well with stir bar on lab bench
    Note: You can also make 1 L solution and divide it for plating and Ara-C.
    50 ml FBS (~10%)
    5 ml 0.2 M L-glutamine
    1 ml insulin stock (12.5 mg/ml insulin in 10 mM HCl)
    0.2 µm filter sterilize and aliquot in 50 ml tubes in culture hood
  8. Ara-C solution
    500 ml MEM
    2.5 g glucose
    100 mg NaHCO3
    50 mg transferrin
    Mix well with stir bar on bench
    1.25 ml of 0.2 M L-glutamine
    10 ml B27 supplement
    500 µl Ara-C stock (2 mM, in Milli-Q water), for the final concentration 1 µM
    25 ml FBS (5%)
    0.2 µm filter sterilize and aliquot in 50 ml tubes
  9. Extracellular bath Tyrode’s saline
    150 mM NaCl
    4 mM KCl
    2 mM MgCl2
    2 mM CaCl2
    10 mM N-2 hydroxyethyl piperazine-n-2 ethanesulfphonic acid (HEPES)
    10 mM glucose
    pH 7.35
  10. 90 K (High Potassium Tyrode’s)
    64 mM NaCl
    90 mM KCl
    2 mM MgCl2
    2 mM CaCl2
    10 mM N-2 hydroxyethyl piperazine-n-2 ethanesulphonic acid (HEPES)
    10 mM glucose
    pH 7.35

Acknowledgments

The methods were adapted from (Lazarenko et al., 2017). Techniques were also adapted from all of the references cited. This work was supported by NIH Grant OD008761, NS094738 and DA025143 to Q.Z. We have no conflicts of interest or competing interests to declare.

References

  1. Afuwape, O. A. and Kavalali, E. T. (2016). Imaging synaptic vesicle exocytosis-endocytosis with pH-sensitive fluorescent proteins. Methods Mol Biol 1474: 187-200.
  2. Faul, F., Erdfelder, E., Lang, A. G. and Buchner, A. (2007). G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 39(2): 175-191.
  3. Gaffield, M. A. and Betz, W. J. (2006). Imaging synaptic vesicle exocytosis and endocytosis with FM dyes. Nat Protoc 1(6): 2916-2921.
  4. Kavalali, E. T. and Jorgensen, E. M. (2014). Visualizing presynaptic function. Nat Neurosci 17(1): 10-16.
  5. Lazarenko, R. M., DelBove, C. E., Strothman, C. E. and Zhang, Q. (2017). Ammonium chloride alters neuronal excitability and synaptic vesicle release. Sci Rep 7(1): 5061.
  6. Liu, G. and Tsien, R. W. (1995). Synaptic transmission at single visualized hippocampal boutons. Neuropharmacology 34(11): 1407-1421.
  7. Park, H., Li, Y. and Tsien, R. W. (2012). Influence of synaptic vesicle position on release probability and exocytotic fusion mode. Science 335(6074): 1362-1366.
  8. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  9. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  10. Zhang, Q., Li, Y. and Tsien, R. W. (2009). The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles. Science 323(5920): 1448-1453.

简介

突触小泡的释放和再循环对于神经传递和突触可塑性是至关重要的。 为了获得对这些过程的机械理解,直接测量囊泡释放和回收是必不可少的。 苯乙烯基染料如FM1-43和FM4-64已被广泛用于此目的,其装载和卸载是可靠的测量突触小泡释放和恢复培养的神经元。 该协议详细描述了使用苯乙烯基染料来标记和测量培养的大鼠海马神经元中的突触小泡摄取和释放的程序。 我们还包括对海马文化的简要描述。 最后,我们简要讨论FM染料,pH敏感荧光蛋白和量子点在测量突触小泡行为方面的共性和差异。
【背景】突触小泡是神经传递不可或缺的,因为它们是化学突触中负责神经递质释放的唯一细胞器。它们的数量,释放概率,融合动力学和再循环路线定义了突触传递和神经元交流。已经开发了多种用于探测突触囊泡的工具,包括突触后神经元的电生理学记录,膜运输的电容测量,可氧化的发射体的电流分析,固定突触的电子显微镜成像以及活神经元中的囊泡标记的荧光成像。在所有现有的方法中,最后一个是不仅产生关于个体突触的空间和时间信息,而且提供高吞吐量(即,来自不同神经元的单个突触的更多数据点)的唯一方法。已经开发了基于不同定向和报告机制的各种荧光探针。二十多年前发明的苯乙烯基染料(即FM染料,包括FM1-43,FM4-64,FM5-95)仍然是一种可靠而方便的工具。由于其对脂质膜的中等亲和力及其对脂质敏感的排放,可以容易地装载到再循环的突触囊泡中并且当这些囊泡被胞吐出时释放。使用更敏感的光电探测器如EMCCD,FM染料可以报告单个囊泡释放事件。在这里,我们提供了一个相对完整的基于FM的啮齿动物海马神经元原代培养的突触小泡释放成像的描述。此外,我们还讨论了FM染料和其他荧光泡标签的共性和区别。

关键字:FM1-43, 突触传递, 荧光实时成像, 分离的海马培养物

材料和试剂

  1. 巴斯德吸管9英寸,棉塞(Fisher Scientific,目录号:13-678-8B)
  2. 巴斯德吸管5.75英寸(Fisher Scientific,目录号:13-678-20A)
  3. 24孔板(Corning,Costar ®,产品目录号:3524)
  4. Kimwipes(KCWW,Kimberly-Clark,目录号:34155)
  5. 圆形12 mm-Ø玻璃盖玻片#0(Thermo Fisher Scientific,特殊订单,0.085 mm厚)
  6. 铝箔(WebstaurantStore食品服务设备和供应公司,选择,目录号:12224X1HD)
  7. 铂丝(阿法埃莎,目录号:10286)
  8. 24 x 40盖玻片(费希尔科学,特殊订单),全部0.085毫米厚( ,尺寸为0)
  9. Parafilm PM-996(Bemis,目录号:PM996)
  10. 30-mmØ培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:153066)
  11. 15ml锥形管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:339650)。
  12. 0.2μm过滤器(Corning,目录号:431218)
  13. 50ml锥形管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339652)
  14. 1.7毫升试管(Corning,Axygen,产品目录号:MCT-175-C)
  15. 新生大鼠海马神经元(P0-P3)
  16. 胰蛋白酶-EDTA(胰蛋白酶:0.025%/ EDTA:0.01%)(Thermo Fisher Scientific,Gibco TM,目录号:R001100)
  17. 最低必需培养基(MEM,1x)(Thermo Fisher Scientific,Gibco TM,目录号:51200038)
  18. 葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:15023021)
  19. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S5761)
  20. 转铁蛋白(Sigma-Aldrich,目录号:T1428)
  21. L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030081)
  22. 胰岛素(Sigma-Aldrich,目录号:I5500)
  23. 胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:26140079)
  24. Matrigel(Corning,目录号:354234)
  25. 70%EtOH(Decon Labs,目录号:2401)
  26. Ara-C(阿糖胞苷)(Sigma-Aldrich,目录号:C1768)
  27. B27补充物(Thermo Fisher Scientific,Gibco TM,目录号:17504044)。
  28. 真空脂(道康宁,目录号:1597418)
  29. FM1-43(Biotium,目录号:70022)
  30. 2,3-二氧代-6-硝基-1,2,3,4-四氢苯并[f]喹喔啉-7-磺酰胺二钠盐(NBQX)(Abcam,目录号:ab120046)
  31. D - ( - ) - 2-氨基-5-膦酰基戊酸(D-AP5)(Abcam,目录号:ab120003)
  32. N-2-羟乙基哌嗪-n-2乙磺酸(HEPES)(Sigma-Aldrich,目录号:H4034)
  33. DNase(Sigma-Aldrich,目录号:D5025)
  34. 10mM HCl(从1N溶液用1:100的二氯甲烷稀释)(Sigma-Aldrich,目录号:H9892)
  35. 氯化钠(NaCl)(Fisher Scientific,目录号:S641-212)
  36. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  37. 氯化镁六水合物(MgCl 2•6H 2 O)(Fisher Scientific,目录号:BP214)
  38. 氯化钙二水合物(CaCl 2•2H 2 O)(Acros Organics,目录号:207780010)
  39. 基质胶储备液(见食谱)
  40. 汉克斯解决方案(见食谱)
  41. 汉克斯+20(H + 20)(见食谱)
  42. 解离解决方案(见食谱)
  43. 脱氧核糖核酸解决方案(见食谱)
  44. 2 mM Ara-C储备(见食谱)
  45. 电镀介质(见食谱)
  46. Ara-C解决方案(见食谱)
  47. 细胞外浴蒂罗尔盐水(见食谱)
  48. 90 K(高钾台氏)(见食谱)

设备

  1. 本生灯(Sigma-Aldrich,目录号:Z270326)
  2. 250毫升烧杯(Fisher Scientific,Fisherbrand,目录号:FB100250)
  3. Pyrex烧杯(Corning,PYREX®,<目录号:1000-100)
  4. 倒置显微镜(20倍镜头,汞弧灯,绿色荧光蛋白过滤器)(尼康仪器,型号:Eclipse Ti-E)
  5. 电场刺激室(两根铂丝粘在RC-26G灌注室的两侧)(华纳仪器,型号:RC-26G)
  6. PH-1平台(华纳仪器,型号:PH-1)
  7. 六通道灌流控制阀系统(华纳仪器,型号:VC-6)
  8. 内联加热器(如,Warner Instruments,产品目录号:64-0104)
  9. TC-344B双通道温度控制器(华纳仪器,型号:TC-344B)
  10. 先前的流明200照明器(Prior Scientific,型号:流明200)
  11. 细胞培养罩,CO 2培养箱
  12. 一套解剖工具(精细科学工具)
  13. 刺激隔离器SD9(Grass Instruments,型号:SD9)
  14. Digidata 1440A(Molecular Devices,型号:Digidata 1440A)
  15. 解剖显微镜(尼康仪器)
  16. 离心机(Eppendorf,型号:5702 R)
  17. 计算机与延时成像系统
  18. 计算机与Clampex软件
  19. 安道尔iXon超EMCCD(安道尔)
  20. 隔振表(纽波特)

软件

  1. 微型经理(Schneider等人,2012年)或斐济(Schindelin等人,2012年)
  2. Clampex(分子设备)
  3. Microsoft Excel(Microsoft)

程序

  1. 大鼠海马神经元的原代培养 注意:新生儿神经元培养物的制备在技术上比胚胎培养更容易。
    如先前所述(Liu和Tsien,1995)制备解离的出生后大鼠海马细胞的原代培养物。简而言之,通常从P0至P1 Sprague-Dawley大鼠解剖大鼠海马(CA1-CA3),并在胰蛋白酶-EDTA(胰蛋白酶:0.025%/ EDTA:0.01%,Thermo)中孵育10分钟以解离成单细胞悬浮液Fisher Scientific),随后使用三个直径逐渐减小(〜1mm,0.5mm和0.2mm)的玻璃移液管轻轻研磨(图1)。移液器是通过在本生灯上进行抛光来获得的。如果暴露在火中,软硼硅酸盐玻璃容易熔化并收缩。为了获得最佳效果,移液枪头垂直于火焰定位,顺时针和逆时针扭转,以便加热和融化。更长的燃烧使得小直径的尖端。确保最小的尖端没有被完全密封也是很重要的,这有时会发生。理想情况下,最小直径的移液管让细胞悬浮缓慢,并确保单细胞解离。


    图1.用于海马分离的不同直径的火抛光的巴斯德移液管尖端的实例

    1. 通过在4℃下离心(200xg,5分钟)回收解离的细胞,并重悬于由最小必需培养基(MEM,Thermo Fisher Scientific)组成的平板培养基中,用(以mM计)的27葡萄糖,2.4NaHCO 3,0.00125转铁蛋白(Sigma-Aldrich),2L-谷氨酰胺(赛默飞世尔科技),0.0043胰岛素(Sigma-Aldrich)和10%/体积胎牛血清(FBS,Gibco) 。
    2. 将100μl细胞悬浮液接种到置于24孔板(Fisher Scientific)中的预涂有基质胶(Corning)的圆形12mm-Ø玻璃盖玻片(200-300个细胞/ mm 2)上。
      注意:使用Matrigel作为神经元底物与通常使用的聚-L-赖氨酸基本上改善细胞培养质量。在使用前1-24小时将100μl的Matrigel分配到盖玻片的表面上,并在细胞悬液电镀之前被吸出。而且,不应该使用抗生素以避免对神经元性质的不利影响。
    3. 用于细胞培养的解剖工具和玻璃器皿正在用不含洗涤剂的清洁剂来防止其对神经膜的不利影响。使用后,工具用去离子水洗涤; Kimwipes将血液保留下来。对于解剖前的消毒,将工具在250ml烧杯(Fisherbrand)中用70%EtOH孵育30分钟。为了防止由于碰到烧杯底部而造成机械损伤的精细解剖工具,我们用2-3根Kimwipes覆盖底部。我们使用的盖玻片是12mm#0(Thermo Fisher Scientific,特殊订单,0.085mm厚)。在使用之前,盖玻片在用3层铝箔覆盖的派热克斯(Pyrex)烧杯中高压灭菌30分钟。
      注:不需要其他特殊的盖玻片处理。
    4. 在加入1ml平板培养基之前,使细胞粘附于底物30-60分钟。在培养1-2天后,再加入含有(以mM计)27葡萄糖,2.4NaHCO 3,0.00125转铁蛋白,0.5L谷氨酰胺,0.002Ara-C,1%/体积B27的1ml培养基补充(Thermo Fisher Scientific)和5%/ vol FBS。 Ara-C(1μM)在培养基中有效地防止星形胶质细胞增殖。

    5. 在DIV 12和18之间进行实验(当突触传输已经建立时)
  2. 成像设置
    1. 对于盖玻片上的细胞培养,倒置显微镜是优选的,因为能够使用具有高N.A.高放大率的油浸物镜。
      XYZ电动舞台是首选,但没有必要
    2. 一个RC-26G室(华纳仪器公司)被修改了2个铂丝连接到房间的两侧,以提供电场刺激。使用真空油脂将该室用24×40mm大小的盖玻片(0.085mm厚)底部密封,并将其夹在放置在显微镜台(Scientifica)上的PH-1平台(Warner Instruments)上。我们使用Nikon Eclipse Ti倒置显微镜对20x Plan Apo VC物镜(N.A. 0.75)进行大面积测量,或者使用100x Apo VC物镜(N.A. 1.40)来获得突触boutons的详细视图。
      注意:两个#0盖玻片(一个24×40盖玻片,费舍尔科学特殊订单),密封腔室的底部和一个12毫米的圆形盖玻片的细胞总厚度等于一个#1.5的厚度标准的盖玻片(0.17毫米),大部分物镜都经过优化和校正。这样就不需要用真空脂将细胞直接密封到成像室的底部。
    3. 通过由VC-6阀门控制系统和6通道歧管(华纳仪器)控制的重力灌注实现溶液交换,恒定速率为约50μl/秒,这允许在记录室内完全交换浴液30秒。
    4. 所有的实验都是在室温下进行的,但是通过使用在线加热器(例如,64-0104,Warner Instruments)和加热阶段(例如 >,PH-1阶段,华纳仪器)。两者都可以设置为34-37°C(例如,,通过TC-344B双通道温度控制器,Warner Instruments控制)。为避免加热引起的阶段漂移,建议至少预热1小时。
      如尼康Perfect Focus等自动对焦也很有帮助
    5. 带有附加刺激线的RC-26G夹在PH-1和Scientifica舞台的自制防水插件中。
      注意:建议使用防泄漏系统进行实时成像,以尽可能减少对显微镜的损坏。 PH-1螺栓连接到舞台插入。实验室石蜡膜PM-996被用作密封件。
    6. 激发光源可以是通过液体光导或激光发射箱发射的弧光灯或激光。我们使用先前流明200照明器通过液体光导连接到显微镜。
    7. 对应于所选FM染料的荧光激发/二向色性/发射滤光片组合可以在网上找到( , http ://www.chroma.com )。滤光片安装在显微镜外壳内的滤光片立方体中,或者分开安装在光路中。例如,使用荧光滤波器组完成FM1-43成像:五十零分之四百六十零; DIC:495LP; Em:510 / 25BP。所有的滤光片和分色镜都是从Chroma或Semrock购买的。
    8. 图像采集和同步灌注通过Micro-Manager和Clampex软件进行控制。包括激发功率,荧光滤波器组(激发,二向色和发射滤波器),曝光时间,相机增益和帧速率的采集设置在不同样本之间保持相同。图像以0.1Hz的速率拍摄。基线荧光是在电刺激/化学刺激/神经元去极化之前1分钟或至少3帧捕获。
    9. Andor EMCCD成像。 1)将安道尔温度设定为-80℃; 2)将Andor读数模式设置为5 MHz(而不是17 MHz); 3)设置安道尔垂直速度为0.5,而不是3.3:这些设置有助于增加成像荧光强度的动态范围。

  3. 成像程序
    1. 将突触成熟的原代大鼠海马神经元(DIV12-18)与10μMFM1-43(即,SynaptoGreen C4,Biotium)在37℃在5%CO 2中孵育0.5小时, 2培养器通过自发外/内吞作用将FM1-43载入突触小泡。对于染料加样,用990μl来自神经元培养物的条件培养基充分混合的10μl无菌1mM的ddiH 2 O储备溶液将被加入并分配到24孔板的新孔中,好板。然后,将含有神经元的盖玻片通过燃烧器消毒镊子转移到含有10μMFM1-43的孔中。
    2. 更常见的加载程序是通过刺激的急性加载。例如,FM1-43染料在高K +生理盐水的存在下加载,其促进大量诱发的外/内吞作用。
    3. 为此,使用燃烧器消毒镊子将原生大鼠海马培养物的盖玻片从24孔板转移到含有3ml正常台氏液的30-mm Petri培养皿中。把盖玻片带到显微镜。为了防止由于自发性神经元活动引起的染料损失,谷氨酸受体阻断剂(10μMNBQX和20μMD-AP5)在成像前至少1分钟添加,并在整个成像实验中存在于浴中。
    4. 将盖玻片转移到预先装有正常台氏液的成像室,灌注速度为0.05毫升/秒。
    5. 通过关闭进口和出口(即,真空)停止灌注,并从腔室中取出〜90-95%的溶液。
    6. 逐滴加入含有10μMFM1-43(即,SynaptoGreen C4,Biotium)的500μl高K +溶液至成像室并轻轻混合。
    7. 用染料孵育细胞2分钟,用正常的Tyrode溶液打开表面FM染料5分钟冲洗的灌注。具有中等FM1-43标记的健康看上去的神经元在20倍放大倍数下被识别(如图2所示)。
    注:这两种不同的加载程序各有利弊。如果可能避免由于高K +中的膜去极化而引起的副作用,则建议使用第一种方法(37℃下30分钟)。急性负载有助于实现更多定向的质膜靶向。


    图2.显示DIC中最佳细胞密度和FM1-43荧光覆盖的样品缩小图像

    1. 为了诱导突触小泡释放,使用电场刺激或高K +灌注。测量从突触前末梢诱发的FM1-43损失的速率。
    2. 将神经元培养物暴露于10Hz的电场刺激下,使用Grass SD9刺激隔离器产生的1毫秒70V脉冲2分钟或高钾(90K)1分钟灌流。
    3. 通过Clampex软件(Molecular Devices)连接刺激控制。控制电场刺激的时间和灌注通道之间切换的Clampex协议的例子如图3所示。


      图3. Clampex协议示例,可用于控制电场刺激的时间和在溶液灌注通道之间切换。采样速率设置为1毫秒。

    4. SD9由1440A digidata(Molecular Devices)的#4数字输出端口的5 V TTL脉冲触发。 SD9的设置如图4所示.70 V输出脉冲从SD9的正负端口传输到铂丝(阿法埃塞0.5 mm直径)上,并与RC-26G腔体的两侧粘合。刺激条件在电流钳位神经元的全细胞膜片钳记录中被优化,并足以可靠地引起动作电位而没有明显的有害作用。在Clampex协议(图2)的例子中,电场刺激被编码为10Hz脉冲序列(Epoch B),其长度为120,000毫秒(120秒= 2分钟)。通过数字输出#4重复触发刺激器由星号(*)以数字位模式编码。


      图4.草乐器SD9刺激隔离器设置频率0,延迟0,持续时间10 x 0.1,电压-7 x 10,输出单声道,极性正常。

    5. 灌注的数字控制是通过一个VC-6阀门控制器来实现的。个别灌注通道设置为外部触发模式,并根据Digidata 1440A数字量输出发送的TTL脉冲打开或关闭。
    6. Andor iXon Ultra EMCCD摄像机的外部输入/输出“Fire”端口连接到Digidata 1440A上的Start(开始)端口,当成像采集开始时,该端口可立即通过Digitizer Start Input(数字化仪启动输入)触发Clampex协议。控制器/设备互连示意图如图5所示。


      图5.控制器/设备互连图

    7. 显微镜上的盖玻片的方向如图6所示。
    8. 基线荧光在神经元的电/化学刺激/去极化之前被捕获1分钟(或至少3帧)。


      图6.盖玻片在显微镜上的定位图

  4. 成像分析
    ImageJ / Fiji中的成像分析:
    1. 打开ImageJ或斐济(不是微管理器)。
    2. 文件→导入图像序列。
    3. 图像→首先复制(不是堆栈)并用于ROI选择。
    4. 如果想要选择/保存/分析图像的一部分,例如,焦点区域:选择它,右键单击,重复。
    5. 如果你想平滑图像:过程→过滤器→平均值(例如,平均4-5个像素值)。
    6. 过程→FFT→带通滤波器,大型结构 - 小至40 px,小至3,抑制条纹:无,方向容差为5%,滤波后自动缩放,图像饱和。
    7. 复制活动图像后,打开图像→调整→阈值,黑暗背景B&amp; W。
    8. 设置较低的阈值级别,单击设置,应用:程序为8位图像分配范围从0到255的值。
    9. 分析→分析粒子:大小2-200像素(覆盖突触结构的良好范围)。图7显示了一个阈值定义和大小受限的ROI示例。


      图7.基于阈值的FM1-43标记的ROI选择示例右侧的图片显示了根据阈值和粒度确定的概述ROI。

    10. 图像→调整→亮度/对比度:拖动亮度到最大,以找到几乎没有信号的背景斑点。
    11. 为背景选择4个ROI:逐个选择背景区域,然后在ROI管理器上单击添加。
    12. 在ROI管理器上,转至更多→保存ROI并保存到根文件夹中,而不是保存在堆栈中。
    13. 转到原始图像堆栈,打开ROI管理器,取消选中,查看,显示所有。
    14. 投资回报率管理器,选择更多→多重测量来获取整个图像堆栈中每个投资回报率的强度值。将值复制到Excel 。
      注意:
      1. 为了纠正一堆图像中的侧向漂移,在第一帧激活的情况下打开堆栈,然后进入:Plugins→Registration→StackReg→Translation。修正之后,新的堆叠沿着发生漂移的边缘将会有空白区域。要从分析中排除这些区域,只需选择要保留的区域,右键单击,复制并将堆栈作为新图像序列保存在新文件夹中即可。
      2. StackReg是建立在斐济(如果斐济的安装没有它,去帮助→更新,点击“管理更新网站”,添加 http://sites.imagej.net/BIG-EPFL/ 它会在下次更新时自动安装) ,但也可以添加到ImageJ中。它需要TurboReg。
        http://bigwww.epfl.ch/thevenaz/turboreg/
        http://bigwww.epfl.ch/thevenaz/stackreg/
      3. MultiStackReg(可从http://bradbusse.net/downloads.html获得,需要StackReg和TurboReg)可用于保存转换并将其应用到另一个堆栈。如果另一个具有较低信号的通道同时成像并且很难自动校正,这非常有用。

    在Excel中进行数据分析:
    1. 在数据集的开头插入1列,以便能够计算平均背景值(来自代表背景感兴趣区域(即感兴趣区域)的最后4列)。
      注意:数据列包含帧数中单个ROI的荧光强度值,按行绘制。如果需要,帧数可以很容易地从成像频率转换成时间点。如果频率是0.1赫兹,帧之间的间隔是10秒
    2. 制作整个电子表格的副本,并从个别强度值中减去平均背景值(在第1列)。
    3. 制作电子表格的第二个副本,并减去背景值。在数据集顶部插入2行。计算前3行(基线,F min
    4. 复制电子表格一次,并将数据规范化为单个ROI的(F-F min F min

      图8. Excel分析截图 A.将背景平均值列添加到数据集中,然后制作电子表格副本(con(2))。 B.在con(2)电子数据表中减去背景,将帧数转换为时间,F min和F min值被计算,图表面板被添加用于视觉控制的FM1-43荧光变化,然后电子表格复制一次(con(3))。 C.在con(3)电子数据表中,来自con(2)的数据被归一化并表示为(F_F min_min)/(F_max_F_min)分钟
    5. 计算多个投资回报率在一段时间内的平均值。结果图的一个例子如图9所示。图10中显示了在基线,电场刺激结束和第二次结束90 K +时的样品图像。
      注意:不同的视野(FOV)包含不同数量的阈值检测ROI。为了消除具有高ROI数量的FOV对平均数据的过度影响,可以使用随机选择的等于或小于最少填充的FOV的ROI数量的一组ROI进行平均。要在Excel中随机化ROI,请在ROI数据集顶部插入一个新行。在这个新行中,选择与具有ROI的列对齐的单元格。输入= rand(),然后按Ctrl + Enter。选择一行随机值后,进入菜单 - 数据分类 - 将选项 - 选项 - 从左向右排序。按行1(随机值)排序数据。


      图9.响应2分钟10Hz电场刺激(绿色条)或1分钟高钾(90K)化学刺激(红色)的归一化的FM1-43荧光的示例


      图10.在基线,电场刺激结束和第二回合结束时的示例图像90 K +

数据分析

初始图像分析在ImageJ中完成。感兴趣区域(ROI)通过在不同样品间使用相同的荧光强度阈值来选择。来自每个ROI的平均强度和来自每个图像堆栈中的四个无细胞区域的平均背景强度被输出到Excel。每个ROI中的FM1-43信号计算为(F-Fmin / F)/(Fmax-Fmin),其中F最大值 为了确定FM1-43脱色ROI的最小数目,使用G * Power(Faul等人,2007)进行功率分析。估计效应量为25%,误差概率设置为0.05,功率为0.95,根据实验中进行的FM脱色实验选择40%的预期标准偏差。需要53个样本量才能达到双尾学生 t - 测验的显着性。所有图像处理都在ImageJ中执行。所有的实验在两到三个不同批次的细胞培养中进行。所有数值均为平均值±SEM。为了计算统计显着性,使用Student's检验法进行2组比较,并且使用Tukey-Kramer方法的单向方差分析(ANOVA)作为事后分析比较三个或更多的组。

笔记

  1. 重复性和变异性
    在我们的测试中,我们观察到FM加载和卸载在未经处理的对照中适度的变化。这种差异主要来源于细胞培养的变化,如体外天和细胞密度。因此,减少这些变量可以显着提高实验的一致性。此外,我们注意到不健康的神经元通常具有过度的FM染色,这是我们用来排除受损突触的标准。

  2. 调频和其他突触小泡标签之间的比较
    突触囊泡在神经生物学中非常重要,以至于在过去的五十年中,已经开发出许多方法,如电生理学,电流分析法,电子显微镜和荧光成像等来研究它们。选择性靶向突触囊泡的荧光团的荧光成像具有直接性的优点,能够以高通量和高空间分辨率实时动态监测突触事件(Kavalali and Jorgensen,2014)。 FM染料是最受欢迎的荧光标签(Gaffield和Betz,2007)。其他探针包括识别囊泡腔表位的荧光标记的抗体,其具有pH敏感性荧光蛋白(Syn-pHFP)(Afuwape和Kavalali,2016)和具有表面亲和性的量子点(Qdots)标记的囊泡蛋白或者与识别囊泡腔表位的抗体缀合(Park等人,2012)。下面我们将就这些荧光标签在操作上的区别进行简要的比较。
    1. 针对性差异
      FMs是随机插入细胞膜的苯乙烯染料。随着刺激诱导囊泡释放和补偿性恢复,FM染料被认为主要在循环突触囊泡中,部分在内体和溶酶体中,而在高尔基体和内质网(ER)中则少量存在。具有囊泡膜亲和性的囊泡和缀合到包括Qdot的荧光团的囊泡靶向抗体具有相似的靶向结果,靶向高尔基体和ER的可能性较低。缩短加载时间是减少非突触小泡靶向的最有效方法。不同的是,Syn-pHFPs是转基因表达并分布到原始蛋白质所在的所有细胞膜,包括囊泡和细胞表面。一般认为,Syn-pHFPs标签的所有突触囊泡,可释放和不可释放。
    2. 信号差异
      由于FM染料在表面上易于洗脱,所以报告泡囊释放的信号是其荧光的降低。相反,Syn-pHFPs报道胞吐作为荧光的增加,因为胞吐引起囊泡腔的脱酸,并且它们的荧光减少报告胞吞作用和囊泡再酸化。
    3. 标准化差异
      不同的突触boutons包含不同数量的囊泡;因此,正态化对于获得群体囊泡行为的测量是重要的。对于FM染料和其他外部添加的探针,使响应正常化的最常见和最简单的方式是将刺激前的荧光强度定义为100%,其中强度刺激后的强度为0.对于遗传表达的Syn-pHFP,最常见的标准化是使用高浓度的NH 4 Cl溶液将所有胞质区室去酸化以完全去除Syn-pHFP,并达到最大100%的荧光强度。为了确定最小的Syn-pHFP强度(0%),细胞通常用低pH溶液(例如,pH 5.5 Tyrode's)灌注。显然,两种pH操作都可能对突触小泡行为产生影响。值得注意的是,我们最近的论文证明,即使是中等浓度的NH 4 Cl(即5mM)也可以在一段较长的时间内改变突触小泡的释放和回收(Lazarenko et。,2017)。

食谱

  1. 基质胶储备液
    1. 制造 Matrigel原液
      在冷室中将10毫升的瓶子在冰上解冻24-48小时,然后等分成1毫升的等分试样并冷冻。如果你试图在更高的温度下解冻,它会变成污泥
    2. 制作工作Matrigel (即,协议中称为Matrigel)
      1. 解冻1毫升冰块几小时,并与49毫升MEM
        混合
      2. 基质胶厚而且粘稠,所以你可能需要用MEM冲洗1毫升的等分试样,以获得整个事情
      3. 摇动MEM + Matrigel混合,让它在寒冷的房间里过夜,使用
        完全溶解
      4. 分成四个15毫升的锥形管,每个12.5毫升
      5. 用铝箔覆盖所有的管子,以免光照。
      6. 不要使用超过一个月的等分样品,也不要过滤Matrigel
  2. 汉克斯解决方案
    Hanks溶液直接由Sigma-Aldrich H2837粉末状HBSS制成,或直接从Invitrogen订购。
    0.2微米的过滤消毒和分装在50毫升管
  3. 汉克斯+20(H + 20)
    400毫升1x汉克斯+ 100毫升胎牛血清(20%胎牛血清)
    0.2微米过滤器灭菌,并分装在50毫升管
  4. 解离溶液(500毫升)
    Hank's盐溶液+12mM MgSO 4•6H 2 O
  5. DNase解决方案
    将整个375 kU溶解在5 ml无菌Milli-Q水中,分装成40μl等分试样
  6. 2mM Ara-C储液(500μl等分试样)
    1. 9.7毫克Ara-C(通常存放在冰箱或冷藏室)到50毫升锥形管中的20毫升去离子水
    2. 涡好
    3. 过滤(0.2微米)到50毫升锥形管
    4. 分装到1.7毫升管,每个500微升
    5. 在-20°C储存
  7. 电镀介质
    500毫升MEM
    2.5克葡萄糖
    100毫克NaHCO 3•/ 2 50毫克转铁蛋白
    在实验室工作台上用搅拌棒充分混合
    注意:您也可以制作1L的解决方案,并将其划分为镀层和Ara-C。
    50毫升FBS(〜10%)
    5毫升0.2 M L-谷氨酰胺
    1毫升胰岛素储备(12.5毫克/毫升胰岛素在10毫米氯化氢)
    0.2微米的过滤器消毒,并分装在50毫升管培养罩
  8. Ara-C解决方案
    500毫升MEM
    2.5克葡萄糖
    100毫克NaHCO 3•/ 2 50毫克转铁蛋白

    在搅拌棒上搅拌均匀 1.25ml的0.2M L-谷氨酰胺
    10毫升B27补充
    500μlAra-C储备液(2 mM,Milli-Q水),终浓度为1μM 25毫升FBS(5%)
    0.2微米的过滤消毒和分装在50毫升管
  9. 细胞外浴蒂罗德的生理盐水
    150 mM NaCl
    4 mM KCl
    2mM MgCl 2•/ 2 2mM CaCl 2 2/2 10毫摩尔N-2羟乙基哌嗪-n-2乙磺酸(HEPES)
    10 mM葡萄糖
    pH 7.35
  10. 90 K(高钾台氏)
    64 mM NaCl
    90 mM KCl
    2mM MgCl 2•/ 2 2mM CaCl 2 2/2 10毫摩尔N-2羟乙基哌嗪-n-2乙磺酸(HEPES)
    10 mM葡萄糖
    pH 7.35

致谢

这些方法改编自(Lazarenko等人,2017)。从所有引用的参考文献也改编了技术。这项工作得到了NIH Grant OD008761,NS094738和DA025143对Q.Z的支持。我们没有利益冲突或利益冲突申报。

参考

  1. Afuwape,O.A和Kavalali,E.T。(2016)。 用pH敏感的荧光蛋白成像突触小泡胞吐作用 - 内吞作用方法Mol Biol 1474:187-200。
  2. Faul,F.,Erdfelder,E.,Lang,A.G。和Buchner,A。(2007)。 G * Power 3:为社会,行为和生物医学科学提供灵活的统计能力分析程序。 Behav Res Methods 39(2):175-191。
  3. Gaffield,M.A。和Betz,W.J。(2006)。 用FM染料成像突触囊泡胞吐作用和内吞作用 Nat Protoc < 1(6):2916-2921。
  4. Kavalali,E.T。和Jorgensen,E.M。(2014)。 形象化突触前功能 Nat Neurosci 17(1) :10-16。
  5. Lazarenko,R.M.,DelBove,C.E.,Strothman,C.E。和Zhang,Q。(2017)。 氯化铵改变神经元的兴奋性和突触小泡的释放科学杂志< 7(1):5061。
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引用:Lazarenko, R. M., DelBove, C. E. and Zhang, Q. (2018). Fluorescent Measurement of Synaptic Activity Using FM Dyes in Dissociated Hippocampal Cultured Neurons. Bio-protocol 8(2): e2690. DOI: 10.21769/BioProtoc.2690.
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