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Jul 2020
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In vivo Fluorescence Imaging of Extracellular ATP in the Mouse Cerebral Cortex with a Hybrid-type Optical Sensor
混合型光传感器对小鼠大脑皮层细胞外ATP的体内荧光成像   

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Abstract

Adenosine 5’-triphosphate (ATP) works as an extracellular signaling molecule for cells in the brain, such as neurons and glia. Cellular communication via release of ATP is involved in a range of processes required for normal brain functions, and aberrant communication is associated with brain disorders. To investigate the mechanisms underlying these cellular processes, various techniques have been developed for the measurement of extracellular ATP. To monitor the dynamics of extracellular ATP signaling with high spatiotemporal resolution, we recently developed a hybrid-type ATP optical sensor (ATPOS) that enables in vivo fluorescence imaging of extracellular ATP dynamics in the brain. ATPOS is synthesized by labeling an ATP-binding protein, Bacillus FoF1-ATP synthase ϵ subunit, with a small-molecular fluorescent dye Cy3. Injection of ATPOS into the cerebral cortex of living mice enables visualization of the wave-like propagation of extracellular ATP release in response to electrical stimulation. The protocol described here should be useful for visualizing ATP signaling in diverse processes involved in intercellular communication in the brain.

Keywords: ATP (ATP), Extracellular signaling molecule (细胞外信号分子), In vivo imaging (体内成象), Fluorescent sensor (荧光传感器), Brain (脑)

Background

Adenosine 5’-triphosphate (ATP), which is well known as the intracellular energy currency, works as an extracellular signaling molecule in various organs and tissues of the body (Zimmermann, 2016). In the brain, ATP is released from neurons and glia (Pankratov et al., 2006; Butt, 2011), and is involved in neurotransmission (Burnstock, 2007), neuron–glia interaction (Fields and Burnstock, 2006; Khakh and North, 2012), and regulation of blood flow (MacVicar and Newman, 2015). Release of ATP also occurs during pathological neuronal activity known as cortical spreading depression (CSD) (Schock et al., 2007; Heinrich et al., 2012), which is associated with migraine and cerebral ischemia (Lauritzen et al., 2011). Thus, extracellular ATP signaling is considered to play key roles not only in normal brain functions but also in brain disorders.


To better understand the roles of ATP signaling, it is important to visualize the spatiotemporal changes of extracellular ATP. Various techniques have been developed for monitoring extracellular ATP; for example, bioluminescence assays combined with microdialysis and electrophysiological methods using enzyme-coated electrodes can quantitate extracellular ATP in the brain of living animals (Gourine et al., 2005; Melani et al., 2005). However, these techniques provide little spatial information. In contrast to these non-imaging techniques, fluorescence imaging using optical sensors is a promising tool for monitoring the dynamics of a target molecule with high spatiotemporal resolution (Giepmans et al., 2006). To date, a variety of ATP sensors based on fluorescent proteins have been reported (Berg et al., 2009; Imamura et al., 2009; Tantama et al., 2013; Yaginuma et al., 2015; Arai et al., 2018; Lobas et al., 2019). However, their application in in vivo imaging of extracellular ATP remains difficult (Conley et al., 2017; Lobas et al., 2019) because they are not sensitive enough to detect ATP released into the extracellular space (Yegutkin, 2008). Moreover, these ATP sensors exhibit pH-dependent fluorescence changes due to the pH sensitivity of the fluorescent proteins used in the sensors (Tsien, 1998; Chudakov et al., 2010). This pH dependence is unfavorable for application in the brain, where the tissue pH fluctuates during neuronal activity (Chesler and Kaila, 1992; Magnotta et al., 2012).


We recently developed a hybrid-type ATP optical sensor, ATPOS, which consists of an ATP-binding protein, Bacillus FoF1-ATP synthase ϵ subunit, with a cysteine point mutation at glutamine-105 (ATPBP-Q105C) and a small-molecular fluorescent dye Cy3 bearing a cysteine-reactive maleimide group (Kitajima et al., 2020). ATPOS shows a large fluorescence response to ATP (~2 fold) with high affinity (Kd = ~150 nM), pH independence, and high selectivity, and is therefore suitable for fluorescence imaging of extracellular ATP dynamics (Kitajima et al., 2020). For application of ATPOS to extracellular ATP imaging in the brain, we used BoNT/C-Hc, a nontoxic subunit of Clostridium botulinum type C neurotoxin (Tsukamoto et al., 2005), through which ATPOS can be anchored to the surface of neuronal cell membranes. In addition, we adopted Alexa Fluor 488 (Alexa488) as a reference fluorophore, so that ATPOS can be used for dual-color ratiometric imaging. Accordingly, ATPOS is applied as a form of molecular complex with BoNT/C-Hc and Alexa488-labeled streptavidin (Figure 1). After delivery into the cerebral cortex of living mice, the ATPOS complex can aid visualization of wave-like extracellular ATP dynamics during CSD evoked by electrical stimulation (Kitajima et al., 2020). Here, we describe the protocols for the production of ATPOS and its application to fluorescence imaging of extracellular ATP in the mouse brain. These protocols will be useful for studying the roles of extracellular ATP signaling in various physiological and pathological processes in the brain.



Figure 1. Schematic illustration of the experimental workflow for the production of the ratiometric ATPOS complex. Procedure A: The ϵ subunit of FoF1-ATP synthase with a cysteine point mutation at glutamine-105 is expressed in bacterial cells. Procedure B: ATPOS is produced by labeling the ϵ subunit with Cy3 bearing a cysteine-reactive maleimide group. Procedure C: Biotin is covalently attached to ATPOS. Procedure D: BoNT/C-Hc is expressed in bacterial cells. Procedure E: Biotin is covalently attached to BoNT/C-Hc. Procedure G2: Biotinylated ATPOS, biotinylated BoNT/C-Hc, and Alexa488-labeled streptavidin are mixed at a molar ratio of 3:1:1 to assemble the ratiometric ATPOS complex via biotin-streptavidin linkage.

Materials and Reagents

  1. HiTrap TALON crude 1-ml column (BD Biosciences, catalog number: 29048565)

  2. TALON metal affinity resin (Clontech, catalog number: 635503)

  3. PD-10 desalting column (GE Healthcare, catalog number: 17085101)

  4. Empty disposable PD-10 column (GE Healthcare, catalog number: 17043501)

  5. Amicon Ultra-0.5 centrifugal filter unit (10 kDa cutoff) (Merck, catalog number: UFC5010)

  6. Amicon Ultra-0.5 centrifugal filter unit (30 kDa cutoff) (Merck, catalog number: UFC5030)

  7. Dental acrylic (GC, Fuji LUTE BC)

  8. Glass capillary (Harvard Apparatus, catalog number: 30-0053)

  9. 10-ml syringe (Terumo, catalog number: SS-10LZ)

  10. Plastic tube (Imamura, catalog number: 01605)

  11. Monopolar tungsten microelectrode (FHC, catalog number: UEWMGCSEKNNM)

  12. Escherichia coli BL21 (DE3)

  13. Wild-type mice (C57BL/6NCrSlc, male, postnatal 1-2 months old)

  14. Plasmid encoding ATPBP-Q105C (Kitajima et al., 2020)

  15. Plasmid encoding BoNT/C-Hc (Takikawa et al., 2014)

  16. Cy3 maleimide (GE Healthcare, catalog number: PA13131)

  17. Alexa488-labeled streptavidin (Thermo Scientific, catalog number: S11223)

  18. NHS-PEG4-biotin (Thermo Scientific, catalog number: A39259)

  19. Ampicillin (Nacalai Tesque, catalog number: 02739-32)

  20. Kanamycin (Nacalai Tesque, catalog number: 19839-44)

  21. Isopropyl-β-D-thiogalactopyranoside (IPTG) (Nacalai Tesque, catalog number: 19742-81)

  22. Lysozyme (Nacalai Tesque, catalog number: 19499-91)

  23. Triton X100 (Nacalai Tesque, catalog number: 12967-45)

  24. DNase I (Takara, catalog number: 2270A)

  25. Imidazole (Nacalai Tesque, catalog number: 19004-35)

  26. Medetomidine (Nippon Zenyaku, Domitor)

  27. Midazolam (Sandoz, Midazolam Injection 10 mg [SANDOZ])

  28. Butorphanol (Meiji Seika Pharma, Vetorphale)

  29. Mineral oil (Sigma, catalog number: M8410)

  30. Dimethyl sulfoxide (DMSO) (Nacalai Tesque, catalog number: 13408-64)

  31. Luria Bertani (LB) agar plates (see Recipes)

  32. 2× YT media (see Recipes)

  33. LB media (see Recipes)

  34. Phosphate-buffered saline (PBS) (see Recipes)

  35. 10× PBS (see Recipes)

  36. Artificial cerebrospinal fluid (aCSF) (see Recipes)

  37. HEPES-buffered saline (HBS) (see Recipes)

  38. Anesthetic solution (see Recipes)

Equipment

  1. 1-L flask

  2. French press (Aminco, model: FA-080B)

  3. Fast protein liquid chromatography system (GE Healthcare, model: AKTA purifier 10)

  4. Spectrophotometer (Eppendorf, model: Eppendorf BioPhotometer Plus)

  5. Heating pad (Bio Research Center, model: BWT-100A)

  6. Metal frame and frame holder (custom-made, Figures 2A and 2B)

  7. Dental drill (Nakanishi, model: ULTIMATE XL)

  8. Pipette puller (Sutter Instrument, model: P-97)

  9. Beveller (PRIME TECH, model: EZP-75)

  10. Wide-field microscope (Leica, model: M165FC)

  11. Objective lens (Leica, Plan APO 1.0×, catalog number: 10450028)

  12. Filter set (Leica, Filter set ET GFP3/CY3, catalog number: 10450612)

  13. High-speed scanning polychromatic light source (Hamamatsu Photonics, model: C7773)

  14. EM-CCD camera (TELEDYNE Photometrics, model: Evolve512)

  15. Stereotaxic manipulator (Luigs and Neumann, model: SM-5)

  16. Stimulus isolator (AMPI, model: ISO-Flex)

Software

  1. ImageJ/Fiji (NIH, version: 1.50e, https://imagej.net/Fiji)

  2. MetaMorph (Molecular Devices)

Procedure

  1. Expression and purification of ATPBP-Q105C

    1. Transform Escherichia coli BL21 (DE3) cells with the plasmid encoding ATPBP-Q105C (see Note 1) as follows. Add 100 ng plasmid to 30 μl competent cells in a microtube, gently mix, and incubate on ice for 5 min. Heat the cells at 37°C for 3 min, incubate on ice for 5 min, and add 300 μl LB media.

    2. Plate the cells on an LB agar plate containing 100 μg/ml ampicillin and incubate overnight at 37°C.

    3. Inoculate 5 ml 2× YT media containing 100 μg/ml ampicillin in a 50-ml tube with a single colony grown on the plate, and grow the culture at 37°C with shaking at 200 rpm for 6 h.

    4. Transfer 2 ml culture to a 1-L flask with 250 ml 2× YT media containing 100 μg/ml ampicillin, and grow the culture at 37°C with shaking at 200 rpm until the OD600 reaches 0.5-0.8.

    5. Add 1 ml 100 mM IPTG to the culture and incubate at 25°C with shaking at 160 rpm for 16 h.

    6. Collect the cells by centrifugation at 4°C for 15 min at 2,070 × g, and store the cell pellet at -80°C.

    7. Resuspend the cells in 12.5 ml PBS on ice. Add 1.3 ml PBS containing 10 mg/ml lysozyme and perform three freeze-thaw cycles. Add 150 μl 10% Triton X100, 150 μl 1 M MgCl2, and 9 μl 5 U/μl DNase I, and incubate at 4°C for 15 min.

    8. Centrifuge the cell lysate at 4°C for 30 min at 15,000 × g and collect the supernatant in a new tube.

    9. Equilibrate a HiTrap TALON crude 1-ml column with 5 ml PBS, and transfer the supernatant into the column.

    10. Apply 25 ml PBS containing 10 mM imidazole to the column for washing.

    11. Apply 20 ml PBS containing 150 mM imidazole to the column, and collect the eluate in 1 ml fractions in new tubes.

    12. Perform SDS-PAGE analysis to identify the fractions containing ATPBP-Q105C. Measure the protein concentration by the Bradford protein assay using BSA as a standard, and calculate the molar concentration of ATPBP-Q105C (15.9 kDa) (see Note 2).


  2. Fluorophore labeling

    1. Add 985 μl PBS to each of two new microtubes. Transfer 10 μl eluate containing 1 mM ATPBP-Q105C into each of the microtubes. Add 5 μl 10 mM Cy3 maleimide dissolved in DMSO to each microtube, gently mix, and incubate at 25°C for 45 min.

    2. Equilibrate a PD-10 desalting column with 25 ml PBS and transfer 2 ml mixture into the column.

    3. Apply 700 μl PBS to the column and discard the eluate.

    4. Apply 3 ml PBS to the column and collect the eluate containing the Cy3-labeled ATPBP-Q105C (ATPOS) in a new tube. Determine the concentration of ATPOS by the Bradford protein assay using BSA as a standard (see Note 3).


  3. Biotinylation of ATPOS

    1. Transfer 1,000 μl 6 μM ATPOS into a microtube containing 497 μl PBS. Add 3 μl 5 mM NHS-PEG4-biotin dissolved in DMSO, gently mix, and incubate at 25°C for 1 h.

    2. Equilibrate a PD-10 desalting column with 25 ml PBS and transfer 1.5 ml mixture into the column.

    3. Apply 1.2 ml PBS to the column and discard the eluate.

    4. Apply 3 ml PBS to the column and collect the eluate containing biotinylated ATPOS in a new tube.

    5. Transfer 500 μl eluate into an Amicon Ultra-0.5 centrifugal filter unit (10 kDa cutoff) and concentrate the solution by centrifugation at 4°C for 5 min at 14,000 × g. Apply the rest of the eluate to the centrifugal filter unit and repeat the centrifugation. Determine the concentration of biotinylated ATPOS by the Bradford protein assay using BSA as a standard (see Note 4); store at -80°C.


  4. Expression and purification of BoNT/C-Hc

    1. Transform Escherichia coli BL21 (DE3) cells with the plasmid encoding BoNT/C-Hc (see Note 1) as follows. Add 100 ng plasmid to 30 μl competent cells in a microtube, gently mix, and incubate on ice for 5 min. Heat the cells at 37°C for 3 min, and incubate on ice for 5 min. Add 100 μl LB media and incubate at 37°C for 20 min. Plate the cells on an LB agar plate containing 20 μg/ml kanamycin and incubate overnight at 37°C.

    2. Inoculate 5 ml LB media containing 20 μg/ml kanamycin in a 50-ml tube with a single colony grown on the plate, and grow the culture at 37°C with shaking at 200 rpm for 4 h.

    3. Transfer 2 ml culture into a 1-L flask with 250 ml 2× YT media containing 20 μg/ml kanamycin, and grow the culture at 37°C with shaking at 200 rpm until the OD600 reaches 0.5-1.0.

    4. Add 250 μl 100 mM IPTG to the culture and incubate at 25°C with shaking at 200 rpm for 24 h. Collect the cells by centrifugation at 4°C for 15 min at 2,070 × g and store the cell pellet at -80 °C.

    5. Resuspend the cells in 30 ml PBS on ice and lyse the cells under a pressure of 1,200 psi with a French press. Centrifuge the cell lysate at 4°C for 30 min at 15,000 × g and collect the supernatant in a new tube.

    6. Equilibrate 2 ml TALON metal affinity resin with 2 ml 10× PBS in a 50-ml tube, add the supernatant, and agitate the suspension on ice for 30 min.

    7. Transfer the suspension into an empty disposable PD-10 column and apply 50 ml PBS containing 5 mM imidazole to the column for washing.

    8. Apply 5 ml PBS containing 150 mM imidazole to the column and collect the eluate in 1 ml fractions in new tubes. Measure the protein concentration by the Bradford protein assay using BSA as a standard; determine the fraction containing the highest concentration of the protein (designated 1st fraction), the fraction containing the second highest concentration of the protein (designated 2nd fraction), and the fraction containing the third highest concentration of the protein (designated 3rd fraction) (see Note 5).

    9. Equilibrate a PD-10 desalting column with 25 ml PBS and transfer 2 ml 1st and 2nd fractions and 500 μl 3rd fraction into the column.

    10. Apply 500 μl 3rd fraction and 2.6 ml PBS to the column and collect 3.1 ml eluate containing BoNT/C-Hc in a new tube. Determine the concentration of BoNT/C-Hc by the Bradford protein assay using BSA as a standard and calculate the molar concentration of BoNT/C-Hc (51.6 kDa) (see Note 6).


  5. Biotinylation of BoNT/C-Hc

    1. Add 750 μl PBS to each of two new microtubes. Transfer 250 μl eluate containing 40 μM BoNT/C-Hc into each of the microtubes. Add 1 μl 200 mM NHS-PEG4-biotin dissolved in DMSO to each microtube, gently mix, and incubate on ice for 45 min.

    2. Equilibrate a PD-10 desalting column with 25 ml PBS and transfer 2 ml mixture into the column.

    3. Apply 500 μl PBS to the column and discard the eluate.

    4. Apply 3.1 ml PBS to the column and collect the eluate containing biotinylated BoNT/C-Hc in a new tube.

    5. Transfer 500 μl eluate into an Amicon Ultra-0.5 centrifugal filter unit (30 kDa cutoff) and concentrate the solution by centrifugation at 4°C for 5 min at 14,000 × g. Apply the rest of the eluate to the centrifugal filter unit in 500 μl aliquots and repeat the centrifugation. Determine the concentration of biotinylated BoNT/C-Hc by the Bradford protein assay using BSA as a standard (see Note 7); store at -80°C.


  6. Surgery

    1. Anesthetize a male C57BL/6 mouse by intraperitoneally injecting 0.1 ml/10 g body weight of anesthetic solution. Wait for 5-10 min and then assess the depth of anesthesia by tail pinch. Place the mouse under a wide-field microscope and maintain the rectal temperature at 37°C with a heating pad.

    2. Remove the scalp covering the skull with scissors, attach a custom-made metal frame to the exposed skull with dental acrylic (Figure 2A), and fix the metal frame with a custom-made frame holder (Figure 2B).

    3. Make a groove in the skull with a dental drill along the circumference of a 4-mm-diameter circle centered 3 mm posterior to the bregma and 3 mm lateral to the midline. Remove the bone flap within the circle with forceps, and leave the dura intact. Cover the surface of the exposed cortex with aCSF.



      Figure 2. Schematic illustration of the experimental set-up for the application of ATPOS to the mouse cerebral cortex. A. A custom-made metal frame is attached to the mouse skull. B. A custom-made frame holder. The metal frame is clamped at three positions by the upper nuts and the lower long nuts. C. ATPOS is pressure-injected into the cerebral cortex with a micropipet through the craniotomy by raising the cylinder containing mineral oil by 80 cm vertically from the pipet tip.


  7. Injection of ATPOS

    1. Prepare a glass micropipet as follows. Pull a glass capillary on a P-97 pipet puller and bevel the tip of the micropipet at an angle of 35 degrees relative to a grinding plate until the inner tip diameter reaches 10 μm.

    2. Prepare the ATPOS complex solution as follows. Transfer 3 μl 40 μM biotinylated ATPOS into a microtube containing 15.5 μl HBS, and add 1 μl 40 μM biotinylated BoNT/C-Hc. Add 0.5 μl 83 μM Alexa488-labeled streptavidin dissolved in PBS and gently mix.

    3. Load 20 μl ATPOS complex solution into the micropipet from its back end with a microloader pipet tip, and fill the rest of the inner space of the micropipet with 10 μl mineral oil. Attach the external cylinder of a 10-ml syringe to a 1.5-m-long plastic tube and add 5 ml mineral oil to the cylinder to fill the plastic tube. Connect the other end of the plastic tube to the back end of the micropipet (see Note 8).

    4. Fix the micropipette in a stereotaxic manipulator at an angle of 45 degrees relative to the brain surface and insert the pipette tip to a depth of 300 μm from the brain surface.

    5. Inject the ATPOS complex solution at 1 psi by raising the cylinder 80 cm vertically from the pipet tip for 20-30 min (Figure 2C).

    6. After the injection is finished, withdraw the micropipet from the brain.


  8. In vivo ATP imaging

    1. Find an appropriate imaging area by observing the fluorescence of ATPOS under 555-nm illumination, and focus on the brain surface by observing a brightfield image of the imaging area.

    2. Acquire Cy3 and Alexa488 fluorescence images with an EM-CCD camera at 2.5 Hz sequentially (200 ms duration for each fluorescence) by alternating 555-nm and 490-nm illumination for excitation of Cy3 and Alexa488, respectively (see Note 9).

    3. For drug application, load an appropriate volume of the drug dissolved in aCSF into a glass micropipet, and fill the rest of the inner space of the micropipet with mineral oil (see Note 10). Connect the micropipet with a plastic tube filled with mineral oil and inject the drug at 1 psi by raising the cylinder attached to the plastic tube 80 cm vertically from the pipet tip.

    4. To evoke CSD by electrical stimulation, fix a monopolar tungsten microelectrode in a stereotaxic manipulator at an angle of 45 degrees relative to the brain surface and place the tip of the electrode below the dura. Attach a ground wire to the custom-made metal frame for head fixation, and deliver a train of 100 μs pulses at 200 Hz lasting for 1 s with an intensity of 10 mA with a stimulus isolator.

Data analysis

  1. Open a series of acquired images using the ImageJ/Fiji software (ver. 1.50e), and split the images into a stack from the Cy3 fluorescence channel and a stack from the Alexa488 fluorescence channel (Figure 3A).

  2. Calculate the background intensity for each fluorescence channel as the mean pixel values in the cortical area that is not stained with the ATPOS complex, and subtract the background intensity from the stack of each fluorescence channel (Figure 3B).

  3. Process the stacks using a Gaussian spatial filter with a radius of 2 pixels (pixel size = 8 μm) (Figure 3C), and create a stack of ratiometric images by dividing the stack of the Cy3 fluorescence channel by that of the Alexa488 fluorescence channel (Figure 3D).

  4. Calculate the ratiometric fluorescence response of ATPOS (R/R0) as follows. Create an image showing the baseline of the fluorescence ratio (R0) by averaging the ratiometric images acquired before drug application or electrical stimulation (Figure 3E). Divide the stack of ratiometric images by the averaged image (Figure 3F) and plot a time-course of the mean fluorescence response in regions of interest (ROIs) selected in the cortical area stained with the ATPOS complex (Figure 4).



    Figure 3. Image processing with the ImageJ/Fiji software. A. Stacks of Cy3 (left) and Alexa488 (right) fluorescence channels obtained by splitting channels using menu commands (Image > Stacks > Tools > Deinterleave). B. Stacks of Cy3 (left) and Alexa488 (right) fluorescence channels obtained by subtraction of the background fluorescence using menu commands (Process > Math > Subtract). C. Stacks of Cy3 (left) and Alexa488 (right) fluorescence channels obtained by Gaussian-spatial filtering using menu commands (Process > Filters > Gaussian Blur). D. A stack of ratiometric images (left) obtained by division of the stack of the Cy3 fluorescence channel by that of the Alexa488 fluorescence channel (right) using menu commands (Process > Image Calculator). E. A ratiometric image (R0) (left) obtained by averaging frames acquired during the baseline period (right) using menu commands (Image > Stacks > Z Project). Enter frame numbers at the start and end of the baseline period in the ‘Start slice’ and ‘Stop slice’ boxes, respectively. In this example, the baseline period is 20 s (400 ms per frame × 50 frames). F. A stack of images (R/R0) (left) obtained by dividing the stack of ratiometric images by the averaged image (right) using menu commands (Process > Image Calculator).



    Figure 4. In vivo fluorescence imaging of extracellular ATP in the cerebral cortex. A-C. Visualization of the elevation of extracellular ATP levels after application of 10 mM ATP. A. The position of the micropipet for ATP injection (magenta) and the location of an ROI with a 15-pixel radius (blue). B. Representative images showing the ratiometric fluorescence response of ATPOS (R/R0) upon ATP injection. The extracellular ATP levels are increased around the micropipet. Time after the start of injection is presented above the images. Scale bar, 250 μm. C. A time-course of the ratiometric fluorescence response of ATPOS (R/R0) extracted from the ROI depicted in A. The magenta bar indicates ATP injection. D-F. Visualization of wave-like extracellular ATP dynamics during CSD evoked by electrical stimulation. D. The position of the stimulating electrode (yellow) and the location of an ROI with a 25-pixel radius (blue). E. Representative images showing the ratiometric fluorescence response of ATPOS (R/R0) upon electrical stimulation. A wave-like extracellular ATP release propagates from the stimulation site. Time after the initiation of electrical stimulation is presented above the images. Scale bar, 500 μm. F. Time-course of the ratiometric fluorescence response of ATPOS (R/R0) extracted from the ROI depicted in D.

Notes

  1. The bacterial expression construct is based on pET vectors in which the recombinant protein is expressed under the control of a T7 promotor. Therefore, one has to use a host strain bearing DE3 lysogen, such as BL21(DE3), with which IPTG induces the expression of T7 polymerase, leading to activation of the T7 promotor.

  2. The molar concentration of the purified ATPBP-Q105C is typically around 1 mM.

  3. The molar concentration of ATPOS is typically around 6 μM.

  4. The molar concentration of biotinylated ATPOS is typically around 40 μM.

  5. The protein concentration in the 1st, 2nd, and 3rd fraction is typically around 3 mg/ml, 2 mg/ml and 1 mg/ml, respectively.

  6. The molar concentration of BoNT/C-Hc is typically around 40 μM.

  7. The molar concentration of biotinylated BoNT/C-Hc is typically around 40 μM.

  8. It is important to prevent air bubbles getting into the micropipet or plastic tube.

  9. The obtained images have a dimension of 256 × 256 pixels, with a pixel size of 8 μm × 8 μm at the object plane.

  10. For application of ATP, load 20 μl 10 mM ATP dissolved in aCSF into a glass micropipet and fill the rest of the inner space of the micropipet with 10 μl mineral oil.

Recipes

  1. LB agar plates

    10 g tryptone

    5 g yeast extract

    5 g NaCl

    1 g glucose

    15 g agar

    Add Milli-Q H2O to 1 L

  2. 2× YT media

    16 g tryptone

    10 g yeast extract

    5 g NaCl

    Add Milli-Q H2O to 1 L

  3. LB media

    10 g tryptone

    5 g yeast extract

    5 g NaCl

    1 g glucose

    Add Milli-Q H2O to 1 L

  4. PBS (pH 7.4)

    8 g NaCl

    0.2 g KCl

    0.2 g KH2PO4

    2.9 g Na2HPO4·12H2O

    Add Milli-Q H2O to 1 L

  5. 10× PBS (pH 7.4)

    80 g NaCl

    2 g KCl

    2 g KH2PO4

    29 g Na2HPO4·12H2O

    Add Milli-Q H2O to 1 L

  6. aCSF

    7.3 g NaCl

    0.3 g KCl

    0.2 g NaH2PO4·2H2O

    2.2 g NaHCO3

    0.3 g CaCl2·2H2O

    0.2 g MgCl2·6H2O

    3.6 g glucose

    Add Milli-Q H2O to 1 L

  7. HBS

    2.4 g HEPES, 8.8 g NaCl, 0.2 g KCl. Add Milli-Q H2O to 1 L

  8. Anesthetic solution

    1.9 ml medetomidine (1 mg/ml)

    2.0 ml midazolam (5 mg/ml)

    2.5 ml butorphanol (5 mg/ml)

    Add 0.9% NaCl to 25 ml

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (18K14915 to H. Sekiya, 17H04764 and 18H04726 to D.A., 19K16251 to H. Sakamoto, 17K08584 to S.N., 25221304 to M.I., and 17H04029 and 19K22247 to K. H.), Japan Science and Technology Agency (PRESTO, JPMJPR17P1 to D.A.), and Takeda Science Foundation (to N.K.). This protocol was adapted from previous work (Kitajima et al., 2020).

Competing interests

The authors declare no conflicts of interest.

Ethics

All experimental procedures used in animal experiments have been approved by the Animal Welfare Committee of the University of Tokyo.

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简介

[摘要] 5'-三磷酸腺苷(ATP)是大脑中神经细胞和神经胶质细胞的一种细胞外信号分子。通过释放ATP进行的细胞通讯涉及正常大脑功能所需的一系列过程,而异常通讯与脑部疾病有关。为了研究这些细胞过程的基础机制,已开发出各种技术来测量细胞外ATP。Ť O监控外ATP信令以高时空分辨率的动态,我们最近开发出一种混合型ATP光学传感器(ATPOS)其能够在体内在脑细胞外ATP动态荧光成像。通过用小分子荧光染料Cy3标记ATP结合蛋白芽孢杆菌FoF 1 -ATP合酶ε亚基来合成ATPOS 。将ATPOS注射到活体小鼠的大脑皮层中,可以观察到响应电刺激的细胞外ATP释放的波状传播。这里描述的协议对于在大脑中细胞间通讯涉及的各种过程中可视化ATP信号应该是有用的。


[背景技术[ 0002 ]众所周知的细胞内能量货币腺苷5'-三磷酸(ATP)在人体各个器官和组织中作为细胞外信号分子(Zimmermann,2016)。在大脑中,ATP从神经元和神经胶质释放(Pankratov等,2006; Butt,2011),并参与神经传递(Burnstock,2007),神经元与神经胶质的相互作用(Fields和Burnstock,2006; Khkh和North, 2012年)和血流调节(MacVicar和Newman,2015年)。ATP的释放也发生在称为皮层扩散抑制(CSD)的病理神经元活动中(Schock等,2007; Heinrich等,2012),这与偏头痛和脑缺血有关(Lauritzen等,2011)。因此,认为细胞外ATP信号不仅在正常的脑功能中而且在脑疾病中均起关键作用。

牛逼Ø更好地理解ATP信号传导中的作用,以可视化的细胞外ATP的时空变化是很重要的。已经开发了用于监测细胞外ATP的各种技术。例如,生物发光测定小号结合微透析和使用酶电极涂覆可以QUANTI电生理学方法泰特在活的动物的脑细胞外ATP (Gourine等人,2005;梅拉尼。等人,2005) 。但是,这些技术提供的空间信息很少。相对于这些非成像技术,使用光学传感器荧光成像是一种用于监测目标分子的动态高时空分辨率的有前途的工具(Giepmans等人。,2006) 。迄今为止,基于荧光蛋白的各种ATP传感器已经报道(伯格等人,2009;今村等人。,2009; Tantama等人,2013;柳沼等人,2015;新井。等人,2018 ; Lobas等人,2019)。然而,由于它们灵敏度不足以检测释放到细胞外空间的ATP ,因此它们在细胞外ATP的体内成像中的应用仍然很困难(Conley等人,2017; Lobas等人,2019)。(Yegutkin,2008)。此外,由于用于传感器的荧光蛋白的pH敏感性,这些ATP传感器表现出pH依赖性的荧光变化(Tsien,1998; Chudakov等,2010)。这种pH依赖性不利于在大脑中应用,因为在该大脑中组织pH在神经元活动过程中会发生波动(Chesler和Kaila,1992; Magnotta等人,2012)。

我们最近开发了一种混合型ATP光学传感器ATPOS,该传感器由一个ATP结合蛋白,芽孢杆菌FoF 1 -ATP合酶ε亚基组成,在谷氨酰胺105(ATPBP-Q105C)和一个小分子上具有一个半胱氨酸点突变带有半胱氨酸反应性马来酰亚胺基团的荧光染料Cy3 (Kitajima et al。,2020)。ATPOS显示出对ATP的大荧光响应(〜2倍),具有高亲和力(K d = 〜150 nM ),pH独立性和高选择性,因此适用于细胞外ATP动力学的荧光成像(Kitajima et al。,2020 )。对于应用程序的ATPOS到外ATP成像在大脑中,我们使用的BoNT / C- Hc的,的无毒亚基肉毒梭菌C型神经毒素(冢本等人。,2005) ,通过它ATPOS可以被锚定到的神经元细胞的表面膜。此外,我们采用Alexa Fluor 488(Alexa488)作为参考荧光团,因此ATPOS可用于双色比例成像。因此,ATPOS以与BoNT / C - Hc和Alexa488标记的链霉亲和素的分子复合物形式使用(图1)。递送到活的小鼠的大脑皮层后,ATPOS络合物可以援助visualiz的通货膨胀波状外ATP动力学CSD期间诱发电刺激(北岛等人,2020) 。在这里,我们描述了ATPOS的生产协议及其在小鼠大脑中胞外ATP荧光成像中的应用。这些协议将是有用的用于研究荷兰国际集团的细胞外ATP信号转导的作用在大脑的各种生理和病理过程。





图1示意性图示了实验工作流程的所述生产的比例复合ATPOS。方法A:在细菌细胞中表达在谷氨酰胺-105处具有半胱氨酸点突变的FoF 1 - ATP合酶的ε亚基。程序B:通过用带有半胱氨酸反应性马来酰亚胺基团的Cy3标记ε亚基来生产ATPOS。程序C:将生物素共价连接至ATPOS。程序D:BoNT / C- Hc在细菌细胞中表达。程序E:生物素共价附于BoNT / C- Hc 。程序G2:以3:1:1的摩尔比混合生物素化的ATPOS,生物素化的BoNT / C - Hc和Alexa488标记的链霉亲和素,以通过生物素-链霉亲和素键合组装成比例的ATPOS复合物。

关键字:ATP, 细胞外信号分子, 体内成象, 荧光传感器, 脑



材料和试剂


1. HiTrap TALON粗1-ml色谱柱(BD Biosciences,目录号:29048565)     

2. TALON金属亲和树脂(Clontech ,目录号:635503)     

3. PD-10脱盐柱(GE Healthcare,目录号:17085101)     

4.空的一次性PD-10色谱柱(GE Healthcare,目录号:17043501)     

5. Amicon Ultra-0.5离心过滤器单元(截止至10 kDa )(Merck,目录号:UFC5010)     

6. Amicon Ultra-0.5离心过滤器单元(截留值30 kDa )(Merck,目录号:UFC5030)     

7.牙科用丙烯酸树脂(GC,Fuji LUTE BC)     

8.玻璃毛细管(哈佛仪器,目录号:30-0053)     

9. 10毫升注射器(Terumo,货号:SS-10LZ)     

10.塑料管(今村,货号:01605) 

11.单极钨微电极(FHC,目录号:UEWMGCSEKNNM) 

12.大肠杆菌BL21 (DE3) 

13.野生型小鼠(C57BL / 6NCrSlc,雄性,出生后1 - 2月龄) 

14.编码ATPBP-Q105C的质粒(Kitajima等,2020) 

15.编码BoN T / C - Hc的质粒(Takikawa等,2014) 

16. Cy3马来酰亚胺(GE Healthcare,目录号:PA13131) 

17. Alexa488标记的抗生蛋白链菌素(Thermo Scientific,目录号:S11223) 

18. NHS-PEG 4-生物素(Thermo Scientific,目录号:A39259) 

19.氨苄西林(Nacalai T esque ,目录号:02739-32) 

20.卡那霉素(Nacalai T esque ,目录号:19839-44) 

21.异丙基-β-D-硫代吡喃半乳糖苷(IPTG)(Nacalai T esque ,目录号:19742-81) 

22.溶菌酶(Nacalai T esque ,目录号:19499-91) 

23. Triton X100(Nacalai T esque ,目录号:12967-45) 

24. DNase I(宝酒,商品目录号:2270A) 

25.咪唑(Nacalai T esque ,目录号:19004-35) 

26.美托咪定(Nippon Zenyaku ,Domitor ) 

27.咪达唑仑(Sandoz,咪达唑仑注射液10毫克[SANDOZ]) 

28.布托啡诺(明治制衣制药公司,Vetorphale ) 

29.矿物油(西格玛,目录号:M8410) 

30.二甲基亚砜(DMSO)(Nacalai T esque ,目录号:13408-64) 

31. Luria Bertani(LB)琼脂板(请参阅食谱) 

32. 2 × YT媒体(请参阅食谱) 

33. LB介质(请参阅食谱) 

34.磷酸盐缓冲盐水(PBS)(请参阅食谱) 

35. 10 × PBS(请参阅食谱) 

36.人工脑脊液(aCSF )(请参阅食谱) 

37. HEPES缓冲盐水(HBS)(请参阅食谱) 

38.麻醉药(参见食谱) 



设备


1升烧瓶
法国媒体(阿明科,型号:FA-080B)
快速蛋白质液相色谱系统(GE Healthcare,型号:AKTA纯化仪10)
分光光度计(Eppendorf,型号:Eppendorf BioPhotometer Plus)
加热垫(生物研究中心,型号:BWT-100A)
金属框架和框架支架(定做,图小号2A和2B)
牙钻(中西,型号:ULTIMATE XL)
移液器拉拔器(Sutter仪器,型号:P-97)
贝弗勒(PRIME TECH,型号:EZP-75)
宽视野显微镜(Leica,型号:M165FC)
物镜(Leica,Plan APO 1.0 × ,目录号:10450028)
滤光片组(Leica,滤光片组ET GFP3 / CY3,货号:10450612)
高速扫描多色光源(滨松光电公司,型号:C7773)
EM-CCD相机(TELEDYNE光度学,型号:Evolve512)
立体定位操纵器(Luigs和Neumann,型号:SM-5)
刺激隔离器(AMPI,型号:ISO-Flex)


软件


ImageJ /斐济(NIH,版本:1.50e,https ://imagej.net/Fiji )
MetaMorph (分子设备)


程序


ATPBP-Q105C的表达和纯化
如下所述用编码ATPBP-Q105C的质粒转化大肠杆菌BL21(DE3)细胞(请参见注释1)。添加100纳克质粒〜30个μ升冰上感受态细胞中微管,轻轻混合,并孵育5分钟。加热该细胞在37 ℃下3分钟,在冰上孵育5分钟,并添加300个μ升LB培养基。
板上的细胞上的Ñ LB琼脂板含100 μ克/ ml氨苄青霉素和孵化过夜,在37 ℃。
接种5ml米升2 ×含有100 YT培养基μ微克/毫升氨苄青霉素的50毫升试管中具有生长在平板上的单菌落,并生长培养物在37 ℃下振荡培养,在200转6小时。
转移2毫升培养到1-L的烧瓶用250个升2 ×含有100 YT培养基μ微克/毫升氨苄青霉素,和生长培养在37 ℃下振荡培养,在200转,直到OD 600个达到0.5 - 0.8。
向培养物中加入1 ml 100 mM IPTG,并在25°C下以160 rpm摇动孵育16小时。
通过在2070 × g下于4°C离心15分钟收集细胞,并将细胞沉淀物储存于-80°C。
在冰上的12.5 ml PBS中重悬细胞。加入含有10 mg / ml溶菌酶的1.3 ml PBS,并执行三个冻融循环。添加150 μ升10%的Triton X100,150 μ升的1M的MgCl 2 ,和9 μ升5U / μ升DNA酶I,并孵育在4℃下15分钟。
将细胞裂解液在4°C下以15,000 × g离心30分钟,然后将上清液收集在新的试管中。
用5 ml PBS平衡HiTrap TALON粗1 ml色谱柱,并将上清液转移到色谱柱中。
将25 ml含10 mM咪唑的PBS应用于色谱柱以进行洗涤。
将含有150 mM咪唑的20 ml PBS应用于色谱柱,并在新管中以1 ml馏分收集洗脱液。
进行SDS-PAGE分析,以鉴定含有ATPBP-Q105C的馏分。测量由蛋白质浓度的Bradford蛋白测定使用BSA作为标准,并计算ATPBP-Q105C(15.9摩尔浓度kDa的)(见注2)。


荧光团标记
加入985 μ升PBS以每两个新的微管。转移10 μ升含1mM ATPBP-Q105C到每个微管的洗脱液。加入5 μ升10毫溶于DMSO的Cy3马来酰亚胺到每个微管,轻轻混合,并孵育在25℃下45分钟。
用25 ml PBS平衡PD-10脱盐柱,然后将2 ml混合物转移到柱中。
应用700 μ升PBS到COL UMN并丢弃洗脱液。
将3 ml PBS上样至色谱柱,并在新管中收集含有Cy3标记的ATPBP-Q105C(ATPOS)的洗脱液。确定由ATPOS的浓度的使用BSA作为标准Bradford蛋白测定(见注3)。


ATPOS的生物素化
转让1000 μ升6 μ中号ATPOS成微管含有497 μ升PBS。加入3 μ升5毫NHS-PEG 4 -生物素溶解在DMSO中,轻轻混合,并孵育在25℃下1个小时。
平衡的PD-10脱盐柱用25ml PBS和1.5转移米升混合物到列中。
应用1.2米升PBS到柱,弃去洗脱液。
应用3米升PBS到柱并收集含有在一个新的管生物素化ATPOS洗出液。
转移500 μ升洗脱液成的Amicon超离心0.5过滤器单元(10 kDa的截留)和精矿吃通过离心将该溶液在4℃下5分钟,在14000 ×克。将其余洗脱液加到离心过滤器上,然后重复离心。确定由生物素化的ATPOS的浓度的使用BSA作为标准Bradford蛋白测定(见注4); 储存在-80°C。


BoNT / C- Hc的表达与纯化
如下所述,用编码BoNT / C- Hc的质粒转化大肠杆菌BL21(DE3)细胞(见注1)。添加100纳克质粒〜30个μ升冰上感受态细胞中微管,轻轻混合,并孵育5分钟。将细胞在37 °C下加热3分钟,然后在冰上孵育5分钟。添加100 μ升LB培养基,孵育在37 ℃进行20分钟。板上的细胞上的Ñ LB琼脂含有板20 μ克/ ml卡那霉素,并培育过夜,在37 ℃。
接种5ml米升含有20 LB培养基μ克/ ml卡那霉素在50毫升试管中具有生长在平板上的单菌落,并生长培养物在37 ℃下振荡培养,在200转4小时。
转移2毫升培养成一个1升烧瓶中以250个升2 ×含有20 YT培养基μ微克/毫升卡那霉素,和生长培养在37 ℃下振荡培养,在200转,直到OD 600个达到0.5 - 1.0。
向培养物中添加250μl100 mM IPTG,并在25°C下以200 rpm摇动孵育24小时。收集通过离心将细胞在在2070 4℃,15分钟×克,并将细胞沉淀在存储- 80 ℃。
在冰上的30 ml PBS中重悬细胞,并用French press在1200 psi的压力下裂解细胞。将细胞裂解液在4°C下以15,000 × g离心30分钟,然后将上清液收集在新的试管中。
在50 ml试管中,用2 ml 10 × PBS平衡2 ml TALON金属亲和树脂,加入上清液,在冰上搅拌悬浮液30分钟。
将悬浮液转移到空的一次性PD-10色谱柱中,然后将50 ml含5 mM咪唑的PBS应用于色谱柱进行清洗。
将含有150 mM咪唑的5 ml PBS应用于色谱柱,并在新管中以1 ml馏分收集洗脱液。测量由蛋白质浓度的使用BSA作为标准Bradford蛋白测定; 确定FRA含有蛋白的最高浓度ction(指定1日分),含有该蛋白质的第二浓度最高的部分(表示为2次级分),以及含有该蛋白质的第三浓度最高的馏分(指定3次分数)(请参见注释5)。
平衡用25ml PBS的PD-10脱盐柱和传送2毫升1- ST和2次级分和500 μ升3 RD分数到列中。
应用500 μ升3次分数和2.6毫升的PBS于柱并收集3.1毫升洗脱液含有的BoNT / C- Hc的在新管中。确定的浓度的BoNT / C- Hc的通过所述使用BSA作为标准Bradford蛋白测定和计算的摩尔浓度的BoNT / C- Hc的(51.6 kDa的)(见注6)。


生物素化的的BoNT / C- Hc的
添加750 μ升PBS到每两个新的微管。转移250 μ升含有40洗脱液μ中号的BoNT / C- Hc的进入每个微管。加入1 μ升200毫NHS-PEG 4在冰上-生物素溶解在DMSO中至每个微管,轻轻混合,并孵育45分钟。
用25 ml PBS平衡PD-10脱盐柱,然后将2 ml混合物转移到柱中。
应用500微升PBS中,以该柱,弃去洗脱液。
将3.1 ml PBS应用于色谱柱,并在新管中收集含有生物素化的BoNT / C- Hc的洗脱液。
转移500微升洗脱液成的Amicon超离心0.5过滤器单元(30 kDa的截止)中并在4℃下以14000浓缩离心溶液5分钟×克。洗脱液的其余部分应用到离心过滤单元500个微升等分试样,并重复离心。确定生物素化的浓度的BoNT / C- Hc的通过所述使用BSA Bradford蛋白测定作为标准(参见编号OTE 7); 储存在-80°C。


手术
通过腹膜内注射0.1 ml / 10 g体重的麻醉溶液来麻醉雄性C57BL / 6小鼠。等待5 - 10分钟,然后通过尾捏评估麻醉深度。将鼠标放在大视野显微镜下,并用加热垫将直肠温度保持在37 °C。
用剪刀除去覆盖颅骨的头皮,用牙科丙烯酸树脂将定制的金属框架连接到裸露的颅骨上(图2A),并用定制的框架固定器固定金属框架(图2B)。
用牙钻在前颅骨后3毫米,中线外侧3毫米的4毫米直径圆的圆周上在颅骨上开一个凹槽。用镊子取下圆圈内的骨瓣,并保留硬脑膜。用aCSF覆盖裸露皮质的表面。




图2中的实验设置为示意图的ATPOS的应用到小鼠大脑皮层。答:定制的金属框架连接到鼠标头骨。B.定制框架支架。金属框架由上螺母和下长螺母夹紧在三个位置。C.用微型吸管通过开颅手术将装有矿物油的圆柱体从吸头尖端垂直向上抬高80厘米,通过开颅术将ATPOS压力注射到大脑皮层中。


注入ATPOS
如下准备玻璃微量移液器。上的P-97吸管牵拉拉玻璃毛细管和斜角的顶端微量吸以相对于研磨盘35度,直到内尖端直径达到10的角度μ米。
如下准备ATPOS综合解决方案。传输3 μ升40 μ中号生物素化ATPOS成微管含有15.5 μ升HBS,并添加1 μ升40 μ中号的生物素化的BoNT / C- Hc的。添加0.5 μ升83 μ中号Alexa488标记的链霉亲溶解在PBS中,轻轻混匀。
负载20 μ升ATPOS络合物溶液到微量移从与它的后端微加载吸管尖,并填充内S的其余部分的的速度微量吸用10 μ升矿物油。将10毫升注射器的外部气瓶连接到1.5米长的塑料管上,并向该气瓶中添加5毫升矿物油以填充塑料管。将塑料管的另一端连接到微量移液器的后端(请参见注8)。
固定在立体定位机械手微量以4的角度相对于脑表面5度并插入移液管尖端到300的深度μ米从大脑表面。
通过将量筒从移液器吸头垂直提升80 cm的位置20到30分钟,以1 psi的压力注入ATPOS复合溶液(图2C)。
注射完成后,从大脑中取出微量吸管。


体内ATP成像
通过在555 nm光照下观察ATPOS的荧光来找到合适的成像区域,并通过观察成像区域的明场图像将其聚焦在大脑表面上。
通过交替使用555 nm和490 nm的照明分别激发Cy3和Alexa488,用EM-CCD相机依次以2.5 Hz(每个荧光持续200 ms的时间)采集Cy3和Alexa488荧光图像(请参见注释9)。
对于药物应用,加载溶解在药适当体积的脑脊液到玻璃微量移,并填充的内部空间的其余部分微量吸用矿物油(见注10)。将微量移液管与装有矿物油的塑料管连接,并通过将连接到塑料管的量筒从移液管吸头垂直向上80厘米处以1 psi的压力注入药物。
要通过电刺激诱发CSD,请将单极钨微电极固定在相对于大脑表面成45度角的立体定向操纵器中,并将电极的尖端置于硬脑膜以下。将接地线连接到定制的金属框架上以进行头部固定,并使用激励隔离器以200 Hz的频率传递100μs的脉冲,持续1 s,强度为10 mA。


数据分析


打开序列o F,使用所获取的图像的ImageJ的/斐济软件(版本1.50e),并且图像分裂成从Cy3的荧光通道和从Alexa488的荧光通道(图3A)的堆叠的堆叠。
计算每个荧光通道的背景强度,作为皮层区域中未被ATPOS复合物染色的平均像素值,并从每个荧光通道的堆栈中减去背景强度(图3B)。
处理栈使用的高斯空间滤波器与2个像素(像素的大小= 8的半径μ米)(图3C),和创建的叠层比率通过该Alexa488的荧光信道的分割的Cy3荧光通道的堆栈中的图像(图3D)。
如下计算ATPOS的比例荧光响应(R / R 0 )。通过对在药物应用或电刺激之前获得的比例图像进行平均,来创建显示荧光比率(R 0 )基线的图像(图3E)。将比率图像的堆栈除以平均图像(图3F),并绘制在用ATPOS复合物染色的皮层区域中选定的感兴趣区域(ROI)中的平均荧光响应的时程图(图4)。




图3.图像处理用的ImageJ的/斐济软件。A.通过使用菜单命令(图像>堆栈>工具>解交织)拆分通道而获得的Cy3(左)和Alexa488(右)荧光通道的堆栈。B. Cy3的(左)和Alexa488的(右)的荧光通道的栈由减法获得的使用菜单命令(过程>数学>减法)背景荧光。C.使用菜单命令(“处理”>“滤镜”>“高斯模糊”)通过高斯空间滤波获得的Cy3(左)和Alexa488(右)荧光通道的堆栈。的D.堆栈比例由堆叠的分割而获得的图像(左)的通过的Cy3标记荧光通道的Alexa488的荧光信道使用(右)菜单命令(过程>图像计算器)。E.通过使用菜单命令(“图像”>“堆栈”>“ Z Project”)对在基线周期(右)期间获取的帧进行平均而获得的比例图像(R 0 )(左)。在“开始片”和“停止片”框中分别在基准时间段的开始和结束处输入帧号。在此示例中,基线周期为20 s(每帧400 ms × 50帧)。图像F.堆栈([R / [R 0 )(左)通过迪维获得丁的叠层比率通过使用菜单命令(过程>图像计算器)平均图像(右)图像。




图4.大脑皮层中细胞外ATP的体内荧光成像。A - C.施加10 mM ATP后细胞外ATP水平升高的可视化。A.用于ATP注射的微量吸管的位置(品红色)和半径为15像素的n ROI的位置(蓝色)。B.代表图像显示ATP注入时ATPOS的比例荧光响应(R / R 0 )。微吸管周围细胞外ATP水平升高。注射开始后的时间显示在图像上方。比例尺,250微米。C.从A中描绘的ROI提取的ATPOS的比例荧光响应(R / R 0 )随时间变化。洋红色条表示ATP注入。D - F .电刺激诱发的CSD期间,波状细胞外ATP动力学的可视化。D.刺激电极的位置(黄色)和半径为25个像素的n ROI的位置(蓝色)。E.代表性图像显示了在电刺激下ATPOS的比例荧光响应(R / R 0 )。波浪状的细胞外ATP释放从刺激部位传播。在图像上方显示了电刺激开始后的时间。比例尺,500微米。F.从D中描绘的ROI提取的ATPOS的比例荧光响应(R / R 0 )的时程。


笔记


Ť他细菌表达构建体基于的pET其中重组蛋白的控制下表达载体一个T7启动子。因此,人们必须使用一个宿主菌株轴承DE3溶素原,如BL21(DE3),通过该IPTG诱导的T7聚合酶的表达,导致活化的T7启动子。
纯化的ATPBP-Q105C的摩尔浓度通常约为1 mM。
ATPOS的摩尔浓度一般为约6 μ中号。
生物素化的ATPOS的摩尔浓度通常为大约40 μ中号。
在1中的蛋白质浓度ST ,2次和3次分数是通常大约3毫克/毫升,2毫克/毫升和1毫克/毫升分别。
的摩尔浓度的BoNT / C- Hc的通常为约40 μ中号。
生物素化的摩尔浓度的BoNT / C- Hc的通常为约40 μ中号。
重要的是要防止气泡进入微量移液管或塑料管。
Ť他获得的图像具有256个的尺寸× 256像素,与一个的8像素大小μ米× 8 μ米在物平面处。
对于ATP应用,负载20 μ升10毫ATP溶解于脑脊液到玻璃微量移并填充的内部空间的其余部分微量吸用10 μ升矿物油。


菜谱


LB琼脂板
10克胰蛋白try


5克酵母提取物


5克氯化钠


1克葡萄糖


15克琼脂


将Milli-Q H 2 O添加到1 L


2 × YT媒体
16克胰蛋白try


10克酵母提取物


5克氯化钠


将Milli-Q H 2 O添加到1 L


LB媒体
10克胰蛋白try


5克酵母提取物


5克氯化钠


1克葡萄糖


将Milli-Q H 2 O添加到1 L


PBS(pH 7.4)
8克氯化钠


0.2克KCl


0.2克KH 2 PO 4


2.9 g Na 2 HPO 4 ·12H 2 O


将Milli-Q H 2 O添加到1 L


10 × PBS(pH 7.4)
80克氯化钠


2克氯化钾


2克KH 2 PO 4


29 g Na 2 HPO 4 ·12H 2 O


将Milli-Q H 2 O添加到1 L


脑脊液
7.3克氯化钠


0.3克氯化钾


0.2 g NaH 2 PO 4 ·2H 2 O


2.2克碳酸氢钠3


0.3克CaCl 2 ·2H 2 O


0.2克MgCl 2 ·6H 2 O


3.6克葡萄糖


将Milli-Q H 2 O添加到1 L


哈佛商学院
2.4克HEPES,8.8克氯化钠,0.2克氯化钾。将Milli-Q H 2 O添加到1 L


麻醉药
1.9毫升美托咪定(1毫克/毫升)


2.0毫升咪达唑仑(5毫克/毫升)


2.5毫升布托啡诺(5毫克/毫升)


向25毫升中添加0.9%氯化钠


致谢


这项工作得到了日本教育,文化,体育,科学和技术部(MEXT)资助的科学研究补助金(KAKENHI)的支持(授予H. Sekiya的18K14915,授予DA的19H16251的17H04764和18H04726)。 H. Sakamoto,SN的17K08584,MI的25221304和KH的17H04029和19K22247),日本科学技术署(PRESTO,JP的JPMJPR17P1)和武田科学基金会(NK)。该协议改编自先前的工作(Kitajima等,2020)。


利益争夺


Ť他作者宣称没有冲突小号间的EST 。


伦理


一个在动物实验中使用LL实验过程已通过东京大学的动物福利委员会。


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Copyright Kitajima et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kitajima, N., Takikawa, K., Sekiya, H., Asanuma, D., Sakamoto, H., Namiki, S., lino, M. and Hirose, K. (2021). In vivo Fluorescence Imaging of Extracellular ATP in the Mouse Cerebral Cortex with a Hybrid-type Optical Sensor. Bio-protocol 11(11): e4046. DOI: 10.21769/BioProtoc.4046.
  2. Kitajima, N., Takikawa, K., Sekiya, H., Satoh, K., Asanuma, D., Sakamoto, H., Takahashi, S., Hanaoka, K., Urano, Y., Namiki, S., Iino, M. and Hirose, K. (2020). Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor. Elife 9: : e57544.
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