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Measuring Caenorhabditis elegans Sleep during the Transition to Adulthood Using a Microfluidics-based System
使用基于微流体的系统测量处于过渡到成年期的秀丽隐杆线虫的睡眠   

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Abstract

C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski and Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al., 2008; Singh et al., 2011). During lethargus, animals cycle between periods of immobility (sleep bouts) and periods of active locomotion (motion bouts). Sleep bouts resemble sleep in other species based on behavioral criteria, including cessation of feeding and locomotion, increased arousal threshold for response to sensory stimulation, rapid reversibility, and homeostatic response to sleep loss. Several assays have been developed to study sleep in C. elegans (Belfer et al., 2013; Bringmann, 2011; Nelson et al., 2013; Raizen et al., 2008). Here, we contribute a detailed protocol for assessment of C. elegans sleep during lethargus, which has been used successfully by many research groups, incorporating simple microfluidic chambers, a low cost camera with lighting system, and computational analysis based on image subtraction. We note that this system could be easily adapted to assess sleep in any small animal.

Keywords: C. elegans sleep(秀丽隐杆线虫的睡眠), Lethargus(休眠), Microfluidic chambers(微流体室), Image subtraction(图像相减法), Total sleep(总睡眠), Lethargus duration(休眠持续时间), Average bout duration(每次休眠平均持续时间), Quiescence(静止)

Background

C. elegans sleep is often assessed based on the cessation of locomotion, which is a common characteristic of sleep across the animal kingdom. Due to the intermittent nature of sleep bouts during C. elegans developmental sleep, computer vision is generally used to track the activity of C. elegans during lethargus. Animals are constrained to a single focal plane to keep them in focus. Because C. elegans can be exhausted in liquid by prolonged swimming (Ghosh and Emmons, 2008), C. elegans sleep researchers rely almost exclusively on assay formats that enforce crawling, not swimming. Also, because food may dramatically alter behavior, C. elegans sleep studies are usually undertaken in the presence of bacterial food. There are two major assay formats: animals confined to larger spaces and tracked across multiple developmental stages (Belfer et al., 2013; Nelson et al., 2013; Raizen et al., 2008) or animals confined to small spaces and tracked for shorter time intervals (Bringmann, 2011; Singh et al., 2011). The original report of C. elegans sleep utilized the first assay format, which permitted detection of sleep during each larval lethargus as animals crawled on the surface of a culture dish. This required one camera per animal, which limited throughput (Raizen et al., 2008). More recent work generally uses small chambers arranged in tight groupings, which allows simultaneous tracking of multiple animals with one camera. Various small-chamber formats are available. An automated polydimethylsiloxane (PDMS) chamber system is available with 60 chambers, but this format requires high levels of consistency in chamber loading with media and seeding bacteria, which is best achieved with robotic systems (Nelson et al., 2013). As these systems are not widely available to academic labs, most groups use other assay formats. Very small chambers constructed from agarose hydrogels have also been used to constrain locomotion of L1 larvae and it is possible that these could be reformatted for use with older C. elegans larval stages (Bringmann, 2011). But, agarose hydrogels are not easily reusable and most C. elegans sleep research focuses on the last lethargus, during the transition to adulthood. Here, we describe in detail a tractable PDMS-based, small chamber assay system, which allows simultaneous tracking of up to 10 L4 to adult animals. This is a variation of an earlier 6-chamber format assay (Singh et al., 2011). The assay is based on reusable PDMS chips and requires a minimal space to track animals in the last larval lethargus. The small chamber assay format described here requires reagents and equipment that are readily available in most C. elegans laboratories and has been adapted by several groups beyond our own.

Materials and Reagents

  1. 1.5 ml Eppendorf tube
  2. Glass slides (Fisher Scientific, catalog number: 12-550-343 )
  3. Tape (e.g., FisherbrandTM 0.75 in. colored label tapes, Fisher Scientific, catalog number: 15-901-20A for white tapes)
  4. 100 mm Petri dishes (e.g., Sigma-Aldrich, catalog number: P5856 )
  5. Cover slip, 25 x 25 mm, glass (VWR, catalog number: 48368084 )
  6. 15 ml Falcon tubes (Corning, catalog number: 352095 )
  7. Parafilm (Bemis, catalog number: PM999 )
  8. FisherbrandTM Pasteur pipette (Fisher Scientific, catalog number: 22-183624 ) with dropper bulb
  9. Double sided tape (e.g., Scotch permanent double sided tape, Staples, catalog number: 504829 )
  10. C. elegans strains: standard wild type laboratory strain, N2. Additional strains are also available at the Caenorhabditis Genetics Center (CGC, http://cbs.umn.edu/cgc)
  11. Escherichia coli OP50 strain (available at the CGC)
  12. Antibiotics-treated OP50 (made from E. coli, preparation procedure provided below)
  13. Sylgard® 184 silicone elastomer kit (Dow Corning, catalog number: BCBI10824 )
  14. Ethanol
  15. LB agar (BD, catalog number: 240110 )
  16. Kanamycin (Sigma-Aldrich, catalog number: K1876 )
  17. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-212 )
  18. BactoTM peptone (BD, BactoTM, catalog number: 211677 )
  19. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: BP213-1 )
  20. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: BP510-250 )
  21. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  22. Potassium phosphate buffer (pH 6)
  23. BactoTM agar (BD, BactoTM, catalog number: 214010 )
  24. Liquid nematode growth media (NGM) for re-suspending antibiotic-treated OP50 (see Recipes)
  25. 2% agar for sealing chambers (see Recipes)

Equipment

  1. Microfluidic chamber chip (design and instructions provided)
  2. Template mask/mold: http://www1.simtech.a-star.edu.sg/smfhttp://www.flowjem.com
  3. Oven
  4. Spectrometer (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 Spectrophotometers , catalog number: ND-2000C)
  5. Heating block (e.g., Benchmark two-block digital dry bath, Benchmark, catalog number: BSH1002 )
  6. Centrifuge for 50 ml conical tubes (e.g., Eppendorf, model: 5810 R )
  7. Tabletop centrifuge for 1.5 ml Eppendorf tubes (e.g., Eppendorf, model: 5424 )
  8. Shaker (e.g., Eppendorf, New BrunswickTM, model: Innova® 44 , catalog number: M1282-0000)
  9. Dissection scope (e.g., Zeiss, model: SteREO Discovery.V8 ) or other light source
  10. Camera with time-lapse imaging software or custom script (at least 2MP, e.g., Zeiss AxioCam ICc3 [Zeiss, model: AxioCam ICc3 ] with ZEN software or less expensive cameras with custom imaging acquisition software you write or obtain)
  11. Computer for image acquisition and analysis

Software

  1. MATLAB (MathWorks, Inc.)
  2. Open source software: Python 2.7.3 or up (but not Python 3), Numpy 1.6.2 or up, Scipy-0.10.1 or up, and Matplotlib-1.1.1 or up

Procedure

  1. Making microfluidic chips
    Each chamber on the microfluidic chip is designed to contain one animal, which allows unambiguous tracking of movement by an individual animal. We consistently hand-loaded chips containing 10 individual assay chambers. Chambers were designed to provide sufficient space for locomotion, and to promote crawling. Swimming leads to exhaustion-induced sleep and, thereby, confounds developmental sleep assessment. For assessing sleep after larval stage 4 (L4) during the L4/adult lethargus, a chamber height of 30 μm confines animals to a single focal plane and promotes crawling (Figure 1). Additionally, each chamber contains numerous hexagonal posts of 50 μm in diameter. These are closely spaced to promote crawling, with a minimum gap of 130 μm, based on previous work (Lockery et al., 2008). We provide an AutoCAD file for a 10-chamber microfluidic chip in the Supplement. Chamber designs must be altered if a different size animal or a different developmental stage is to be examined. For example, if an earlier developmental stage is preferred, a shorter chamber height and smaller gaps between posts should be used to promote animal crawling.


    Figure 1. The 10-chamber PDMS chip. Scale bar = 2 mm.

    1. Order template mask/mold for microfluidic chip. Any commercial sources for custom mold and chip fabrication are appropriate, e.g., http://www1.simtech.a-star.edu.sg/smf, http://www.flowjem.com.
    2. Create microfluidic chips from the mold using a Sylgard® 184 silicone elastomer (PDMS) kit with 1:10 ratio of cross-linker/curing agent A to siloxane B. PDMS should be poured to roughly 4 mm thick over the mold. Cure the PDMS in an oven at 55 °C for 18 h. Peel away the PDMS from the mold and trim to create chips with chambers centered in a roughly 14 x 16 mm rectangle chip.
    3. New PDMS chips are hydrophobic, which makes it hard to fill chambers with liquid. Soaking the chips in OP50 liquid culture (from step B2, below) at room temperature overnight, followed by washing with water and ethanol renders the PDMS less hydrophobic. To completely de-gas chips before loading animals, chips are left to dry on the bench for a week at room temperature in an unsealed container.

  2. Preparing antibiotics-treated OP50
    1. Streak out OP50 E. coli onto an LB agar plate from the glycerol stock (see https://www.addgene.org/plasmid-protocols/bacterial-plates/). Culture at 37 °C overnight.
    2. The next day, inoculate a single colony into 100 ml liquid LB (https://www.addgene.org/plasmid-protocols/inoculate-bacterial-culture/). Grow OP50 culture at 30 °C for 18 h at 220 rpm or 37 °C for 12 h at 220 rpm.
    3. Remove 300 μl from the overnight liquid LB culture and add 600 μl LB. Determine bacteria concentration by optical density using a spectrometer at 600 nm. Concentrate the culture to a final OD600 of 10 by spinning, removing excess liquid, and re-suspending the bacterial pellet. Add kanamycin to the re-suspended bacteria to yield a final concentration of 25 ng/ml of antibiotic; this will prevent OP50 growth. Store the concentrated OP50 + kanamycin culture at 4 °C.
      Note: The bacteria have to be completely static for a sleep assay, else bacterial metabolic products lead to C. elegans stress and less sleep. Therefore, the concentrated OP50 + kanamycin culture has to age for at least a week before setting up a sleep assay. Then, it should be used within five weeks.

  3. Loading animals to the chambers
    1. Before setting up a sleep assay, turn on a heating block to 90 °C and use it to melt 2% agar for use in step D2.
    2. Transfer 200 μl of OP50 + kanamycin culture into a 1.5 ml Eppendorf tube. Centrifuge at 845 x g for 4 min to pellet the bacteria. Remove the supernatant and resuspend the pellet with 300 μl liquid NGM. This creates ‘movie food’ (final OD600 of 6.7) that animals eat while in the chamber. Movie food must be prepared fresh for each assay and used within a few hours.
    3. Pick early- to mid-L4 larval stage animals (Figure 2) to new plates for each genotype/treatment to be tested. Morphological characteristics of the vulval developmental staging can be found in (Mok et al., 2015). Animals from L4.2 to L4.5 described in Figure 1 of this article are usually loaded into chambers for the sleep assessment.


      Figure 2. Early- and mid-L4 larval stage animals. A. Early L4 animal with a white crescent and arrow points to a triangle vulval lumen that is corresponding to L4.2 stage in Figure 1 of the reference (Mok et al., 2015). B. Mid-L4 animal with a white crescent and arrow points to a crown shaped vulval lumen that is corresponding to L4.5 stage in Figure 1 of the reference (Mok et al., 2015). Scale bars = 100 μm.

    4. Place a glass slide on the dissection scope stage and center the chip on the slide. Press the adhesive side of lab tape firmly onto the chip to clean both the top (side with chambers) and the bottom (flat side). Place the chip with the top chamber-side facing up and open (Figure 3A).
    5. Take 5 μl of movie food and place 1 drop (approximately 0.5 μl) on each of the 10 chambers (Figure 3B). Movie food drops should not touch each other. Pick one animal into each drop. One chamber can be left empty as a blank control (Figure 3C). Try to avoid bringing solid OP50 from the plate to the movie food, as bacterial growth in the chamber is detrimental to animals. Once all the animals are loaded to the chambers, place the cover glass onto the chip (Figure 3E). Practice loading of chips is suggested, which increases ability to trap one animal per chamber, with no big air bubbles inside the chamber.
      Notes:  
      1. The amount of movie food on each chamber when the cover slip is dropped in step C5 is critical for success. Animals will move across the chip surface with too much liquid, resulting in two or more animals in the same chamber and loss of data from both. Conversely, too little liquid results in large air bubbles that expand over time and interfere with motion detection by image subtraction. We find that in dry seasons especially during winter, even small air bubbles will expand quickly over time. To prevent data loss, we recommend a modification after step C5. After sealing the cover slip and chip with agar, tape the slide inside a 100 mm Petri dish, and fill the Petri dish with ddH2O up to the level of the cover slip. Make sure there is no water above the cover slip. Use double sided tape to secure the Petri dish onto the imaging platform.
      2. Movie food droplets can evaporate while animals are being loaded. In initial trials, we recommend distributing 10 μl of movie food onto the 10 chambers, yielding 1 μl/chamber. And, in initial trails, load only 5 animals onto each chip. Also, it may be convenient to move animals from plates to an extra large drop of liquid NGM near the chambers, followed by distribution of animals into drops atop chambers. Remove the extra large drop before proceeding to step C5.
    6. Press a single cover slip down firmly onto the chip. Use molten 2% agar (from the 90 °C heating block) to create a ring of agar that completely seals the junction between the chip and cover glass (Figure 3F). This prevents liquid evaporation and air infiltration into the chambers.
      Notes:
      1. Regrettably, it is common for inexperienced practitioners to have multiple animals trapped in the same chamber or to have air bubbles in chambers. To maximize the number of usable chambers with single animals and to prevent air bubbles, we recommend spreading the movie food droplet on each chamber with your pick so it covers the entire chamber surface, but does not contact the movie food droplet atop the next chamber (Figure 3D). If the chip is very hydrophobic, plasma cleaning will render the PDMS chip temporarily hydrophilic. Or, incubate chip in OP50 overnight again (step A3).
      2. The velocity of cover slip addition is critical. Too fast results in creating bubbles; too slow results in animals shifting to other chambers. Practicing without animals is recommended.


        Figure 3. Procedures to load animals to the chambers. A. Cleaned chip ready for the assay. B. Distribution of food droplets into each chamber. C. Early- to mid-L4 animals loaded into each droplet, leaving two as blank controls. D. Spreading of movie food across each chamber surface aid before adding the coverslip will decrease the odds of having two animals trapped in the same chamber. E. Coverslip is now in place, which traps each animal into a single chamber. Scale bars = 1 mm. F. The cover glass-chip-slide sandwich assembled for imaging. Scale bar = 5 mm.

  4. Acquiring images at set frequency
    1. After animals are loaded to the chambers and sealed with cover glass, tape the coverslip/chip assembly to the imaging platform so the edges of the chamber are parallel to the edges of the camera image and the chambers with animals are centered in the camera image. Carefully center the chambers to provide the most uniform illumination and consistent results.
    2. Set up the computer/camera system to record activity for an extended length of time (usually 12 h) to catch each lethargus entry and exit, as well as sleep bout number and duration. Animals must be brought into sharp focus for maximum detection of movement. See notes below for camera setups. Image capture frequency is determined by experimental design and is dependent on appropriate camera settings and computer storage/processing available. We normally acquire one image every 10 sec for 12 h. This allows easy discrimination between control/wild type and mutant animals with sleep defects. The sleep defects observed include changes in total time in sleep bouts, changes in bout duration or frequency, and altered lethargus duration. These can be calculated with the analysis strategy described below. Postural changes in sleep have been reported; it should be possible to detect these using other image analysis programs (Ghosh and Emmons, 2008; Iwanir et al., 2013).
    3. After images are captured, the cover slip removed, the agar and animals are discarded. The chip should be washed with water for reuse. Chips can be used indefinitely, unless damaged. We have not been able to recover individual animals from chambers.
    Notes:
    1. Any dissection scope mounted with a 2MP or higher camera equipped with time-lapse image acquisition software can be used to acquire the images. Or, simply suspend a camera and lens that can image an area slightly larger than the chambers, above a reflected illumination glass stage. We have used a 50 mm fixed focal length 10 megapixel lens with manual focus and iris (F-stop: 2.0, Filter: 30.5, Pitch: 0.5, Graftek Imaging), combined with Allied Vision Technologies Guppy Pro F-503 color CCD camera (Edmund Optics). These were combined with a Schneider C-mount Extension Tube Kit (B & H Photo) to achieve the desired field of view, as described in our previous on-line article (Huang, 2013). Any camera and lens system that can yield a high resolution, high contrast image of animals in the chamber region can be used, as shown in Figure 3.
    2. Black and white images are strongly recommended. We use a variety of cameras and software for image acquisition. The software should provide JPG images as output.
    3. Lighting must be consistent and stable. Set the lighting to give best contrast between the animals and the background to maximize detection of motion. The images in Figure 4 are a good illustration of this. Also bright illumination will minimize the impact of changes in room illumination. Once lighting levels are determined that give consistent and good results, use the lighting level for all future experiments.
    4. Camera noise and other noise should be determined for each experimental set-up, based on image analysis of chambers run without animals, using experimental levels of illumination. See below for details.

  5. Analyzing images 
    1. Introduction to image analysis: the first step of analysis is image subtraction. The algorithm calculates pixel difference between adjacent images for each chamber and then subtracts camera noise for each pixel (Singh et al., 2011). The output file from this analysis contains a list for each chamber of pixel differences for each consecutive pair of images. Below is a step-by-step guide to use the Hart laboratory image subtraction program in MATLAB. Go to https://github.com/Huiyan-Huang and download all the files from the image subtraction branch. Before running this analysis, the camera noise has to be determined. Please refer to the note section below to set the variable tolerance (tol) for the image subtraction script. Tolerance has to be adjusted for each camera using the experimental levels of illumination.
      1. Consolidate JPG images from one chip into a single folder. The image names must increment sequentially (e.g., test0001.jpg, test0002.jpg, …). In this example, the image folder name is called yyyy.mm.dd.N2.12h.
        Note: If your movies are in video format, they must be saved as individual JPG images.
      2. Open MATLAB on the same computer. Within MATLAB, open the WormGUI_10 folder from the ‘Current Folder’ window in the MATLAB interface. Open WormGUI_new.m file in MATLAB. Click the green ‘Run’ button above the MATLAB editor window. If WormGUI_new.m is not found in the current folder or on the MATLAB path, a message window will pop up. Select ‘Add to Path’ (Figure 4A) and the WormGUI_new window will appear (Figure 4B).
      3. The WormGUI window needs to know where the image files to be analyzed are stored on the computer. Copy the path name (e.g., C:\Users\Hart_E5430\Desktop\Sleep movies\yyyy.mm.dd.N2.12h) for the folder containing your image files and paste it in the window that says ‘First.image.file.name’ in the WormGUI. Add the name of your first image file to the path name (e.g., C:\Users\Hart_E5430\Desktop\Sleep movies\yyyy.mm.dd.N2.12h\test0001.jpg). Do the same for the path and name of your last image file in the window below that says ‘Last.image.file.name’ (Figure 4C) (e.g., C:\Users\Hart_E5430\Desktop\Sleep movies\yyyy.mm.dd.N2.12h\test4320.jpg)
      4. Select the chamber to be analyzed using the buttons to the left. Click ‘Mark Region’ to open the first image. Draw a box around the first chamber to mark it as Region 1. Close the popped up image. Do the same for the next 9 chambers. The boxes you draw here do not need to be equal in size. But, for sleep analysis, the entire chamber must be contained within the demarcated region to avoid loss of experimental data (Figure 4D).
      5. Click the ‘Run’ button in the WormGUI to start the analysis. When the message window shows ‘Processing – Please wait’, do not close the WormGUI window (Figure 4E). Keep an eye on the MATLAB command window, if error messages occur, close the WormGUI window and repeat from step E1a.
      6. When the message in the WormGUI changes to ‘FINISHED PROCESSING’ (Figure 4F), fill in the window below that says ‘Output.file.name’ with a name for your result file with a .txt extension (e.g., yyyy.mm.dd.N2.12h.txt). Click the ‘Save’ button (Figure 4G) and close the window when the message says ‘Finished Saving’ (Figure 4H).
        g. The results file will be in the MATLAB current folder. We recommend creating a new results folder on your computer (e.g., C:\Users\Hart_E5430\Desktop\Sleep movies\Results) and copying the results file into it. Note the path of this new results file for the analysis in the next step (e.g., C:\Users\Hart_E5430\Desktop\Sleep movies\yyyy.mm.dd.N2.12h.txt).
      Note: Value of the variable ‘tol’ is determined empirically for each camera/illumination intensity and is equal to the maximum pixel difference between adjacent images when no motion occurs in the chamber. We recommend taking a series of images (e.g., every 10 sec for 20 min) for an empty chip and running the image subtraction analysis and sleep metrics calculation (below). In our image acquisition systems, tol is usually between 25 and 50. Values of tol that are too low or high will mask sleep or motion, respectively. ‘tol’ should be iteratively determined and set to the lowest value that gives no motion for empty chambers. To initially set the value for tol, open MATLAB. Within MATLAB, open the WormGUI_10 folder from the ‘Current Folder’ window in the MATLAB interface. Open WormActivity.m file in MATLAB. In the MATLAB ‘Editor’ window, change the value for tol in line 18 (Figure 5A). Click the ‘Save’ button on the top. Using the same value for tol, open RunWorms.m and change the value for tol in line 22 (Figure 5B). Save the changes.


      Figure 4. Image subtraction analysis. A. Possible message when running the WormGUI_new script; B. The WormGUI_new pop-up window; C. Path for the first and last images filled into the windows; D. The pop-up window for selecting each chamber as a region; E. Message for processing image subtraction; F. Message for finished processing; G. Name for result file entered; H. Message for finished saving the result file.


      Figure 5. Set the value for viable tol. A. Set the tol in WormActivity.m; B. Set the tol in RunWorms.m.

    2. Here, image subtraction values are used to determine a rolling average for sleep across time, an analysis strategy first suggested by Raizen et al. in 2008. Each image subtraction event is assigned a value of either 1 (animal did not move, pixel difference zero or less) or 0 (animal moved, pixel value non-zero). The image subtraction result file is first converted to a list of 0’s and 1’s for individual animals. Then, lethargus duration, total sleep, average bout duration, and other metrics are calculated. Comparison of the total sleep of mutant animals to wild type control animals is not sufficient to rule out changes in sleep. We suggest, at a minimum, reporting lethargus duration and bout duration in the analysis as well, as some mutant strains with aberrantly short sleep bout duration have normal total sleep quantity during L4/adult lethargus due to increases in lethargus duration. Bout number and frequency are also important metrics, but they can be derived from total sleep, average bout duration and lethargus duration. It should be noted that detailed descriptions of changes in sleep and motion bout timing and duration within lethargus are possible and may be important in future studies.
      1. First, we define the metrics.
        1. Lethargus entry is defined as fractional Quiescence (fQ) stays above 0.1 for at least 20 min. fQ is calculated as the rolling average for 60 frames, i.e., 10 min. Lethargus entry is further adjusted to be the first frame of the first bout from the 60 frames used to calculate fQ.
        2. Lethargus exit is defined as fQ stays below 0.1 for at least 20 min. Lethargus exit is further adjusted to be the last frame of the last bout from the 60 frames used to calculate fQ.
        3. Lethargus duration is the duration between lethargus entry and exit.
        4. Total sleep is the total time spent in sleep between lethargus entry and exit. Note that behavioral lethargus may extend past the time of vulval eversion in some mutant strains.
        5. Average bout duration is the average bout duration across all of lethargus, between lethargus entry and exit.
        6. Bout number is the total number of bouts between lethargus entry and exit. Note that bout frequency decreases later in lethargus.
      2. Below is a step-by-step guide to use the Hart lab sleep metrics calculation script, run in Python.
        1. First, install the following free software: Python 2.7.3 or up (but not Python 3), Numpy 1.6.2 or up, Scipy-0.10.1 or up, and Matplotlib-1.1.1 or up.
        2. Once Python is installed on your computer, go to https://github.com/Huiyan-Huang and download the TenChamberSingleFileV2.py file from the sleep metrics calculation branch. Copy the file to your ‘C:\Users\Documents\Results’ folder from the image subtraction analysis. This script calculates sleep metrics for images taken every 10 sec. If a different image frequency is used, please contact us for modified instructions.
        3. To run the analysis, right click ‘TenChamberSingleFileV1.py’, select ‘Edit with IDLE’ (Figure 6A). The Python script will be opened. Find the line with path=r’C:\Users\...\...\...txt’. Substitute ‘C:\ Users\...\...\...txt’ with the path for your result file from the above image subtraction algorithm (e.g., path=r’C:\Users\Hart_E5430\Desktop\Sleep movies\Results\ yyyy.mm.dd.N2.12h.txt’).
          Note: It is important that you do not accidentally delete any part of the quotation mark (‘’) and the ‘r’ in front of the quotation mark. Also make sure the line beneath says ‘ChamberN=10’.
        4. In the top menu bar, choose ‘File’, and click ‘Save’ (Figure 6B). Then, choose ‘Run’, and click ‘Run Module’ (Figure 6C). A Python shell will popup. Once you see two ‘>>>’ (Figure 6D), the analysis is done.
        5. Now, in the same path as your .txt file, there will be two new files. One shows you the fractional quiescence over frames (Figure 7) and the other contains the detailed analysis for each chamber (Table 1).


          Figure 6. Calculating sleep metrics. A. Opening the Python script to calculate the sleep metrics; B. Saving the changes made for the analysis; C. Running the Python module to calculate the sleep metrics; D. Python shell has now finished processing.


          Figure 7. Fractional quiescence (fQ) plots for animals in each chamber. Each point represents the rolling average of surrounding 60 frames (10 min), when motion is assigned 0 and inactivity is assigned 1. Chamber 5 and chamber 10 did not contain animals, but did contain air bubbles which changed in size while imaging was occurring, which is reported as some movement in the graphs. The x-axis is frame number and the y-axis shows fQ. The red dotted line marks the 0.1 fQ threshold that is used to call entry and exit of lethargus. Here, we show the fQ plots for L4 to adult animals. For fQ plots of animals at other developmental stages, please refer to Figure 1 in a previous report of C. elegans sleep (Raizen et al., 2008).

          Table 1. Corresponding sleep metrics for N2 wild type animals shown in Figure 6
          name
          entry/h
          exit/h
          duration/h
          Corrected totalQ/min
          tAPD/s
          t#
          1
          3.4
          7.0
          3.5
          50
          25
          124
          2
          3.9
          6.5
          2.6
          45
          24
          113
          3
          4.9
          7.0
          2.1
          28
          21
          82
          4
          4.3
          6.7
          2.4
          37
          25
          90
          5
          0.0
          12.0
          12.0
          701
          948
          45
          6
          3.7
          6.9
          3.2
          56
          22
          153
          7
          3.0
          6.3
          3.3
          59
          21
          168
          8
          3.5
          6.7
          3.2
          51
          23
          136
          9
          3.3
          6.0
          2.7
          52
          22
          143
          10
          0.0
          12.0
          12.0
          701
          992
          43

Data analysis

Table 1 provides an example of sleep analysis results. We normally compare the total sleep (Table 1, ‘Corrected totalQ/min’ column) between mutant and wild type/control animals. Some mutant animals also differ in lethargus duration (Table 1, ‘duration/h’ column) and average bout duration (Table 1, ‘tAPD/s’ column). Ideally, control animals and experimental animals should be loaded on the same chip or other strategies should be employed to allow direct comparison between different genotypes, different trials and/or different days of data collection. A minimum of 10 animals for each genotype should be examined; larger numbers for experimental and control animals will be required if differences between genotypes are small or behavior is variable for a given genotype. Statistic analysis should be done with comparable number of animals run for both wild type/control and genotype of your interest. A Student’s t-test can be used to compare the results, if both samples fall in a normal Student’s t-distribution. Other analysis should be employed if the results follow other distributions.
Data from chambers with two or more animals must be excluded, as image subtraction does not track individual animals, just movement within the chamber. Data from chambers with big air bubbles that do not disappear before animals enter lethargus must be excluded as well, as bubble movement cannot be discriminated from animal movement by image subtraction. Meanwhile, animals that do not recover to normal levels of active locomotion within 30 min of chamber loading are excluded from analysis, as these are animals that were trapped or died during recording. This can be determined by rapidly reviewing the images the next day, before analysis.

Notes

  1. Temperature impacts sleep during lethargus. Studies should be done with consistent temperature control.
  2. We recommend using previously defined C. elegans strains for calibration. N2 and egl-4 loss of function are commonly used.
  3. If illumination, image size, noise, and other parameters are set appropriately, total sleep for 10 to 15 N2 wild type animals at 22 degrees Celsius should average 50 to 65 min during L4/adult lethargus.

Recipes

  1. Liquid nematode growth media (NGM) for re-suspending antibiotics-treated OP50
    0.3 g NaCl
    0.25 g peptone
    97.5 ml ddH2O
    100 μl 1 M MgSO4
    100 μl 1 M CaCl2
    100 μl 5 mg/ml cholesterol
    2.5 ml 1 M potassium phosphate buffer (pH 6)
    Filter sterilize and aliquot into 15 ml Falcon tubes
    Take out one tube at a time and aliquot into 1.5 ml tubes
    Note: The 15 ml aliquot can be sealed with Parafilm and stored at 4 °C for years, if not contaminated. The 1.5 ml aliquot can be stored at room temperature for months.
  2. 2% agar for sealing chambers
    2 g agar
    100 ml ddH2O
    Microwave to dissolve/melt agar. Aliquot 2% agar into small quantities and store at 4 °C
    Note: As long as nothing is growing in the agar, it should be good to use for years.

Acknowledgments

This work was supported by the National Institutes of Health NIH R01 NS055813 (A.C.H.) and Postdoctoral Fellowship in Translational Neuroscience (H.H.). This is a modification of a protocol reported previously (Singh et al., 2011). All experimental protocols were approved by appropriate institutional review boards. We have no conflict of interest.

References

  1. Belfer, S. J., Chuang, H. S., Freedman, B. L., Yuan, J., Norton, M., Bau, H. H. and Raizen, D. M. (2013). Caenorhabditis-in-drop array for monitoring C. elegans quiescent behavior. Sleep 36(5): 689-698G.
  2. Bringmann, H. (2011). Agarose hydrogel microcompartments for imaging sleep- and wake-like behavior and nervous system development in Caenorhabditis elegans larvae. J Neurosci Methods 201(1): 78-88.
  3. Ghosh, R. and Emmons, S. W. (2008). Episodic swimming behavior in the nematode C. elegans. J Exp Biol 211(Pt 23): 3703-3711.
  4. Huang, H., Zhu, C. T. and Hart, A. (2013). How to measure lethargus quiescence without monopolizing the dissection microscopes in your lab. The Worm Breeder’s Gazette 19(3).
  5. Iwanir, S., Tramm, N., Nagy, S., Wright, C., Ish, D., and Biron, D. (2013). The microarchitecture of C. elegans behavior during lethargus: Homeostatic bout dynamics, a typical body posture, and regulation by a central neuron. Sleep 36(3): 385-395.
  6. Lockery, S. R., Lawton, K. J., Doll, J. C., Faumont, S., Coulthard, S. M., Thiele, T. R., Chronis, N., McCormick, K. E., Goodman, M. B. and Pruitt, B. L. (2008). Artificial dirt: microfluidic substrates for nematode neurobiology and behavior. J Neurophysiol 99(6): 3136-3143.
  7. Mok, D. Z. L., Sternberg, P. W., and Inoue, T. (2015). Morphologically defined sub-stages of C. elegans vulval development in the fourth larval stage. BMC Dev Biol 15:26.
  8. Nelson, M. D., Trojanowski, N. F., George-Raizen, J. B., Smith, C. J., Yu, C. C., Fang-Yen, C. and Raizen, D. M. (2013). The neuropeptide NLP-22 regulates a sleep-like state in Caenorhabditis elegans. Nat Commun 4: 2846.
  9. Raizen, D. M., Zimmerman, J. E., Maycock, M. H., Ta, U. D., You, Y. J., Sundaram, M. V. and Pack, A. I. (2008). Lethargus is a Caenorhabditis elegans sleep-like state. Nature 451(7178): 569-572.
  10. Singh, K., Chao, M. Y., Somers, G. A., Komatsu, H., Corkins, M. E., Larkins-Ford, J., Tucey, T., Dionne, H. M., Walsh, M. B., Beaumont, E. K., Hart, D. P., Lockery, S. R. and Hart, A. C. (2011). C. elegans Notch signaling regulates adult chemosensory response and larval molting quiescence. Curr Biol 21(10): 825-834.
  11. Singh, K., Ju, J. Y., Walsh, M. B., DiIorio, M. A. and Hart, A. C. (2014). Deep conservation of genes required for both Drosophila melanogaster and Caenorhabditis elegans sleep includes a role for dopaminergic signaling. Sleep 37(9): 1439-1451.
  12. Trojanowski, N. F. and Raizen, D. M. (2016). Call it Worm Sleep. Trends Neurosci 39(2): 54-62.

简介

C。发展过程中的线虫睡眠受到动物界保护的基因和细胞机制的限制(Singh等人,2014; Trojanowski和Raizen,2016)。 C。线虫发育睡眠通常在成年过渡期间进行评估,2.6h时间间隔称为lethargus(Raizen等人,2008; Singh等人, 2011)。在嗜睡期间,动物在不动的时期(睡眠开始)和积极运动时期(运动发作)之间循环。睡眠状态基于行为标准,包括停止进食和运动,增加唤醒阈值以响应感觉刺激,快速可逆性和对睡眠损失的稳​​态反应,类似于其他物种的睡眠。已经开发了几种用于在C中研究睡眠的测定法。 (Belfer等人,2013; Bringmann,2011; Nelson等人,2013; Raizen等人, 2008)。在这里,我们提供了一个详细的评估方案。线虫在lethargus期间睡眠,许多研究组已经成功使用,结合简单的微流体室,具有照明系统的低成本照相机和基于图像减法的计算分析。我们注意到,这个系统可以很容易地适应于评估任何小动物的睡眠。

背景 C。睡眠通常根据运动停止进行评估,这是整个动物界睡眠的常见特征。由于C期间睡眠发作的间歇性。线虫发育睡眠,计算机视觉通常用于跟踪C的活动。线虫在lethargus期间。动物被约束到单个焦平面以保持它们的焦点。因为C.线虫可以通过长时间的游泳用尽液体(Ghosh和Emmons,2008),C。线虫睡眠研究人员几乎完全依赖于强化爬行而不是游泳的检测方式。另外,因为食物可能会大大改变行为。线虫睡眠研究通常在细菌食物的存在下进行。有两种主要的测定形式:动物被限制在较大的空间并且跨越多个发育阶段进行跟踪(Belfer等人,2013; Nelson等人,2013; Raizen 幼虫阶段(Bringmann,2011)。但是,琼脂糖水凝胶是不容易重复利用的,而且大部分是C。线虫睡眠研究着重于在成年过渡期间的最后一次嗜睡。在这里,我们详细描述了一种易于处理的基于PDMS的小室测定系统,其允许同时跟踪到成年动物的10L4。这是早期6室格式测定的一种变化(Singh等人,2011)。该测定是基于可重复使用的PDMS芯片,并且需要最小的空间来跟踪最近的幼虫lethargus中的动物。这里描述的小室测定形式需要在大多数C中容易获得的试剂和设备。 elegans 实验室,并已经被我们自己以外的几个团体改编。

关键字:秀丽隐杆线虫的睡眠, 休眠, 微流体室, 图像相减法, 总睡眠, 休眠持续时间, 每次休眠平均持续时间, 静止

材料和试剂

  1. 1.5 ml Eppendorf管
  2. 玻璃片(Fisher Scientific,目录号:12-550-343)
  3. Tape(,Fisherbrand TM> 0.75 in。彩色标签带,Fisher Scientific,目录号:15-901-20A,用于白色胶带)
  4. 100毫升培养皿(例如,Sigma-Aldrich,目录号:P5856)
  5. 玻璃盖(25 x 25 mm),玻璃(VWR,目录号:48368084)
  6. 15 ml Falcon管(Corning,目录号:352095)
  7. 石蜡膜(Bemis,目录号:PM999)
  8. Fisherbrand TM 巴斯德移液器(Fisher Scientific,目录号:22-183624),带滴管灯泡
  9. 双面胶带(例如,苏格兰永久性双面胶带,订书钉,目录号:504829)
  10. C。线虫菌株:标准野生型实验室菌株N2。遗传学中心(CGC,)也可获得额外的菌株http://cbs.umn.edu/cgc
  11. 大肠杆菌OP50菌株(可在CGC获得)
  12. 抗生素治疗的OP50(由大肠杆菌制成,其制备方法如下)
  13. Sylgard ®硅橡胶套件(Dow Corning,目录号:BCBI10824)
  14. 乙醇
  15. LB琼脂(BD,目录号:240110)
  16. 卡那霉素(Sigma-Aldrich,目录号:K1876)
  17. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358-212)
  18. Bacto TM 蛋白胨(BD,Bacto TM,目录号:211677)
  19. 硫酸镁七水合物(MgSO 4·7H 2 O)(Fisher Scientific,目录号:BP213-1)
  20. 氯化钙脱水(CaCl 2·2H 2 O)(Fisher Scientific,目录号:BP510-250)
  21. 胆固醇(Sigma-Aldrich,目录号:C8667)
  22. 磷酸钾缓冲液(pH 6)
  23. Bacto TM琼脂(BD,Bacto TM,目录号:214010)
  24. 液体线虫生长培养基(NGM)用于重新悬浮抗生素治疗的OP50(参见食谱)
  25. 2%琼脂密封腔(见配方)

设备

  1. 微流控腔芯片(提供设计和说明)
  2. 模板面具/模具: http://www1.simtech.a -star.edu.sg/smf http://www.flowjem .com
  3. 烤箱
  4. 光谱仪(例如,Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000分光光度计,目录号:ND-2000C) />
  5. 加热块(例如,基准两块数字干浴,基准,目录号:BSH1002)
  6. 用于50ml锥形管的离心机(例如,Eppendorf,型号:5810R)
  7. 用于1.5ml Eppendorf管的台式离心机(例如,Eppendorf,型号:5424)
  8. Shaker(例如,Eppendorf,New Brunswick TM ,型号:Innova 44,目录号:M1282-0000)
  9. 解剖范围(例如,蔡司,型号:SteREO Discovery.V8)或其他光源
  10. 具有延时成像软件或自定义脚本(至少2MP,例如的Zeiss AxioCam ICc3 [Zeiss,型号:AxioCam ICc3])与ZEN软件或具有定制成像采集软件的便宜的相机相机或获取)
  11. 用于图像采集和分析的计算机

软件

  1. MATLAB(MathWorks,Inc.)
  2. 开源软件:Python 2.7.3或以上(但不是Python 3),Numpy 1.6.2或更高版本,Scipy-0.10.1或更高版本,以及Matplotlib-1.1.1或以上版本

程序

  1. 制造微流控芯片
    微流体芯片上的每个腔室被设计成包含一个动物,其允许单个动物的运动的明确跟踪。我们一直手动装载包含10个单独测定室的芯片。房间设计为提供足够的空间,以促进爬行。游泳导致疲劳诱发的睡眠,从而混淆了发育性睡眠评估。为了在L4 /成年嗜睡期间评估幼虫期4(L4)之后的睡眠,室高度为30μm,将动物限于单个焦平面并促进爬行(图1)。另外,每个室包含许多直径为50μm的六角柱。根据以前的工作,这些紧密间隔以促进爬行,最小间隙为130μm(Lockery等人,2008)。我们在补充。如果要检查不同大小的动物或不同的发育阶段,必须改变腔室设计。例如,如果较早的发育阶段是优选的,则应使用更短的室高度和柱之间较小的间隙来促进动物爬行。


    图1. 10室PDMS芯片。 比例尺= 2 mm。

    1. 订购模板面膜/模具用于微流控芯片。任何用于定制模具和芯片制造的商业来源都是适当的,例如, http://www1.simtech.a-star.edu.sg/smf http://www.flowjem.com
    2. 使用具有1:10比例的交联剂/固化剂A与硅氧烷B的Sylgard 184硅氧烷弹性体(PDMS)试剂盒从模具中制备微流控芯片。将PDMS倒入约4毫米厚在模具上。在55℃的烘箱中固化PDMS 18小时。从模具中剥离PDMS并修剪以创建以大约14×16mm矩形芯片为中心的腔室的芯片。
    3. 新的PDMS芯片是疏水的,这使得难以用液体填充室。将芯片浸泡在OP50液体培养物中(来自步骤B2,下面)在室温下过夜,然后用水和乙醇洗涤,使PDMS疏水性较差。在装载动物之前,要将完全脱气的碎屑在室温下放置在不含盖的容器中的板凳上干燥一周。

  2. 准备抗生素治疗OP50
    1. 连线OP50 E。 (参见 https://www.addgene.org/plasmid-protocols/bacterial-plates/)。文化在37°C过夜。
    2. 第二天,将单个菌落接种到100ml液体LB中( https://www.addgene.org/plasmid-protocols/inoculate-bacterial-culture/)。在220 rpm或37℃下,以220 rpm在30℃下培养OP50培养18小时,持续18小时
    3. 从过夜液体LB培养物中取出300μl,加入600μlLB。使用600 nm的光谱仪,通过光密度确定细菌浓度。通过纺丝将培养物浓缩至最终OD 600以上,除去多余的液体,并重新悬浮细菌颗粒。向再悬浮的细菌中加入卡那霉素,得到最终浓度为25ng/ml的抗生素;这将阻止OP50的增长。将浓缩的OP50 +卡那霉素培养物在4℃下储存 注意:细菌必须对于睡眠测定是完全静止的,否则细菌代谢产物导致C。电影 压力和睡眠较少。因此,浓缩的OP50 +卡那霉素培养物必须在进行睡眠测定之前至少一周。然后,应在五周内使用。

  3. 装载动物到房间
    1. 在设置睡眠测定之前,将加热块打开至90°C,并使用它来熔化2%琼脂以用于步骤D2。
    2. 将200μlOP50 +卡那霉素培养物转移到1.5 ml Eppendorf管中。以845×g离心4分钟以沉淀细菌。取出上清液,并用300μl液体NGM重悬细胞沉淀。这产生了动物在房间里吃的"电影食物"(最终OD <600> 6.7)。电影食物必须为每种测定新鲜准备,并在几小时内使用。
    3. 选择早期到中期L4幼虫阶段动物(图2)到每个待测试的基因型/治疗的新平板。外阴发育分期的形态特征可以在(Mok等人,2015年)中找到。本文图1中描述的L4.2至L4.5的动物通常装入室内进行睡眠评估

      图2.早期和中期L4幼虫阶段动物。 A.具有白色新月形和箭头的早期L4动物指向三角形阴道腔,其对应于图1中的L4.2阶段参考文献(Mok等人,2015)。 B.具有白色新月形和箭头的中L4动物指向相当于参考图1中的L4.5阶段的冠状外阴腔(Mok等人,2015)。比例尺=100μm
    4. 将玻片放在解剖范围台上,并将芯片放在幻灯片上。将实验胶带的粘合剂侧面牢固地压在芯片上,以清洁顶部(带腔室的侧面)和底部(平坦侧面)。放置芯片,使顶部腔侧朝上并打开(图3A)。
    5. 取5μl电影食物,并在10个房间的每一个上放置1滴(约0.5μl)(图3B)。电影食物滴不应相互接触。在每一滴中挑一只动物。一个室可以作为空白对照留空(图3C)。尽量避免将固体OP50从板上带到电影食品中,因为室内的细菌生长对动物是不利的。一旦所有的动物都被装载到室中,将盖玻片放在芯片上(图3E)。建议实践加载芯片,这增加了每个腔室捕获一只动物的能力,室内没有大的气泡。
      注意:  
      1. 当步骤C5中盖子掉落时,每个房间上的电影食物的数量是成功的关键。动物将以太多的液体移动穿过芯片表面,导致两个或更多个动物在同一个室中,并且两者的数据丢失。相反,太少的液体导致大的气泡随时间膨胀并且通过图像减去而干扰运动检测。我们发现在冬季特别是冬季的干燥季节,即使小气泡也会随着时间的推移而迅速扩大。为了防止数据丢失,我们建议在步骤C5之后进行修改。用琼脂密封盖板和芯片后,将载玻片带到100 mm培养皿内,并用ddH >直到盖子的水平。确保盖子上方没有水。使用双面胶带将培养皿固定在成像平台上。
      2. 电影食物小滴可以在动物装载时蒸发。在初步试验中,我们建议您将10μl电影食物分配到10个小室,产生1μl/室。而且,在初始步骤中,每只芯片上只装载5只动物。此外,将动物从板移动到室附近的超大液滴NGM可能是方便的,随后将动物分配到小室顶部的滴液中。在继续步骤C5之前,移除额外的大滴。
    6. 将单个盖子滑落到芯片上。使用熔融的2%琼脂(来自90°C加热块)以产生完全密封芯片和玻璃盖之间的接合处的琼脂环(图3F)。这样可以防止液体蒸发和空气渗入室内 注意:
      1. 令人遗憾的是,没有经验的从业者常常将多只动物藏在同一个室内或在室内产生气泡。为了最大化使用单个动物的可用室数量并防止气泡,我们建议您在每个室内将电影食品液滴放在您的选择位置,以覆盖整个房间表面,但不会接触下一个房间顶部的电影食品液滴(图3D)。如果芯片非常疏水,等离子体清洁将使PDMS芯片暂时具有亲水性。或者,再次在OP50中孵化芯片(步骤A3)。
      2. 覆盖滑移加速的速度至关重要。太快造成气泡;动物太慢会转移到其他房间。建议无动物进行练习。


        图3.将动物装载到室内的步骤。 A.清洁的芯片准备好进行测定。 B.每个室上食物液滴的分布。 C.早期到中间L4动物装入每个液滴,留下两个作为空白对照。 D.在添加盖玻片之前,电影食物在每个房间表面上的传播将减少两只动物被困在同一个房间内的可能性。 E.盖片现在已经到位了,它将每只动物陷入一个单一的房间。比例尺= 1 mm。组装盖玻片芯片 - 滑块三明治成像。比例尺= 5毫米。

  4. 以设定的频率获取图像
    1. 将动物装载到房间并用盖玻璃密封后,将盖玻片/芯片组件胶带到成像平台,使得室的边缘平行于照相机图像的边缘,并且具有动物的室在照相机图像中居中。仔细中心室以提供最均匀的照明和一致的结果。
    2. 设置电脑/摄像机系统,以记录长时间的活动(通常为12小时)以捕获每个lethargus进入和退出,以及睡眠次数和持续时间。必须将动物置于尖锐的焦点以最大程度地检测运动。有关相机设置,请参阅下面的说明。图像捕获频率由实验设计确定,并且取决于适当的相机设置和可用的计算机存储/处理。我们通常每10秒获取一个图像12小时。这使得控制/野生型和具有睡眠缺陷的突变动物之间容易区分。观察到的睡眠缺陷包括睡眠时间总体变化,发作持续时间或频率变化,以及改变的嗜睡持续时间。这些可以用下面描述的分析策略计算。报告了睡眠姿势的变化;应该可以使用其他图像分析程序来检测这些图像(Ghosh和Emmons,2008; Iwanir等人,2013)。
    3. 拍摄图像后,取下盖子,弃掉琼脂和动物。该芯片应用水清洗再使用。芯片可以无限期使用,除非损坏。我们无法从室内收回个别动物。
    注意:
    1. 使用配备有延时图像采集软件的2MP或更高摄像头安装的任何解剖范围可用于获取图像。或者,简单地暂停相机和镜头,可以在反射的照明玻璃舞台上面对可能对稍微大于室的区域进行成像的照相机和镜头。我们使用了50mm固定焦距的10万像素镜头,手动对焦和光圈(F-stop:2.0,Filter:30.5,Pitch:0.5,Graftek Imaging),与Allied Vision Technologies Guppy Pro F-503彩色CCD相机Edmund Optics)。这些与施耐德C-Mount延伸管套件(B& H照片)结合,以实现所需的视野,如我们以前的在线文章(Huang,2013)所述。可以使用任何可以在室区域产生动物的高分辨率,高对比度图像的相机和透镜系统,如图3所示。
    2. 强烈建议使用黑白图像。我们使用各种相机和软件进行图像采集。该软件应提供JPG图像作为输出。
    3. 照明必须一致和稳定。设置照明以使动物和背景之间达到最佳对比度,以最大程度地发现运动。图4中的图像很好地说明了这一点。此外,明亮的照明将最小化房间照明变化的影响。一旦确定了提供一致和良好结果的照明级别,请使用照明级别进行所有未来的实验。
    4. 对于每个实验装置,应使用实验照明水平,基于对没有动物运行的室的图像分析来确定相机噪声和其他噪声。详情请见下文。

  5. 分析图片 
    1. 图像分析简介:分析的第一步是图像减法。该算法计算每个室的相邻图像之间的像素差异,然后减去每个像素的相机噪声(Singh等人,2011)。该分析的输出文件包含每个连续图像对的每个像素区域列表。以下是在MATLAB中使用Hart实验室图像减法程序的分步指南。转到 https://github.com/Huiyan-Huang 并下载所有文件从图像减法分支。在进行此分析之前,必须确定相机的噪音。请参阅下面的注释部分设置图像减法脚本的变量容差(tol)。必须使用实验照明水平对每台摄像机进行公差调整。
      1. 将JPG图像从一个芯片整合到一个文件夹中。图像名称必须按顺序递增(例如,,test0001.jpg,test0002.jpg,...)。在此示例中,映像文件夹名称称为yyyy.mm.dd.N 2 .12h。
        注意:如果您的电影是视频格式,则必须将其保存为单独的JPG图像。

      2. 在同一台电脑上打开MATLAB。在MATLAB中,从MATLAB界面的"当前文件夹"窗口打开WormGUI_10文件夹。在MATLAB中打开WormGUI_new.m文件。单击MATLAB编辑器窗口上方的绿色"运行"按钮。如果在当前文件夹或MATLAB路径中找不到WormGUI_new.m,将弹出一个消息窗口。选择"添加到路径"(图4A),并显示WormGUI_new窗口(图4B)。
      3. WormGUI窗口需要知道要分析的图像文件在计算机上的存储位置。复制包含您的文件夹的路径名称( eg 。,C:\ Users \ Hart_E5430 \ Desktop \ Sleep movies \ yyyy.mm.dd.N 2 .12h)图像文件并粘贴到WormGUI中的"First.image.file.name"窗口中。将您的第一个图像文件的名称添加到路径名称( eg 。,C:\ Users \ Hart_E5430 \ Desktop \ Sleep movies \ yyyy.mm.dd.N 2 .12h \ test0001.jpg)。对于"Last.image.file.name"(图4C)(例如。,C:\ Users \ Hart_E5430 \")的窗口中的最后一个图像文件的路径和名称,桌面\睡眠电影\ yyyy.mm.dd.N 2 .12h \ test4320.jpg)
      4. 使用左侧的按钮选择要分析的室。点击"标记区域"打开第一个图像。在第一个房间周围绘制一个盒子,将其标记为区域1.关闭弹出的图像。在接下来的9个房间做同样的事情。你在这里绘制的盒子不需要大小相同。但是,对于睡眠分析,整个房间必须包含在划定的区域内,以避免实验数据的丢失(图4D)。
      5. 单击WormGUI中的"运行"按钮开始分析。当消息窗口显示"处理 - 请稍候"时,不要关闭WormGUI窗口(图4E)。关注MATLAB命令窗口,如果发生错误消息,请关闭WormGUI窗口并重复步骤E1a。
      6. 当WormGUI中的消息更改为"完成处理"(图4F)时,请在下面的窗口中填写"Output.file.name",其结果文件的名称为.txt扩展名(例如< em> .yyyy.mm.dd.N 2 .12h.txt)。单击"保存"按钮(图4G),并在消息说"完成保存"(图4H)时关闭窗口。
        G。结果文件将在MATLAB当前文件夹中。我们建议您在计算机上创建一个新的结果文件夹(例如,,C:\ Users \ Hart_E5430 \ Desktop \ Sleep movies \ Results),并将结果文件复制到其中。请注意下一步中分析的新结果文件的路径(例如,C:\ Users \ Hart_E5430 \ Desktop \ Sleep movies \ yyyy.mm.dd.N 2 < /sub>.12h.txt)。
      注意:变量'tol'的值根据每个摄像机/照明强度经验确定,并且等于在室中没有运动时相邻图像之间的最大像素差。我们建议为空芯片拍摄一系列图像( 例如 ,每10秒20分钟一次),并运行图像减法分析和睡眠指标计算(下文)。在我们的图像采集系统中,tol通常在25到50之间。tol的太低或过高的值将分别掩盖睡眠或运动。应该迭代确定'tol',并将其设置为不给空房间运动的最低值。要初始设置tol的值,打开MATLAB。在MATLAB中,从MATLAB界面的"当前文件夹"窗口打开WormGUI_10文件夹。在MATLAB中打开WormActivity.m文件。在MATLAB"编辑器"窗口中,更改第18行中tol的值(图5A)。点击顶部的"保存"按钮。对于tol使用相同的值,打开RunWorms.m并更改第22行中tol的值(图5B)。保存更改。


      图4.图像减法分析。 A.运行WormGUI_new脚本时可能出现的消息; WormGUI_new弹出窗口; C.将第一张和最后一张图像填入窗户的路径; D.选择每个房间作为区域的弹出窗口; E.消息处理图像减法; F.完成处理消息; G.输入结果文件的名称; H.完成保存结果文件的消息。


      图5.设置可行tol的值。 A.在WormActivity.m中设置tol; B.在RunWorms.m中设置tol。

    2. 这里,图像相减值用于确定时间上的睡眠的滚动平均值,Raizen等人首先提出的分析策略。每个图像减法事件被赋予1(动物没有移动,像素差为零或更小)或0(动物移动,像素值非零)的值。图像减法结果文件首先转换为个体动物的0和1的列表。然后,计算lethargus持续时间,总睡眠,平均持续时间和其他指标。突变动物与野生型对照动物的总睡眠比较不足以排除睡眠的变化。我们建议,至少在分析中报告嗜睡持续时间和持续时间,因为一些具有异常短睡眠持续时间的突变株,由于lethargus持续时间的增加,在L4 /成人嗜睡期间具有正常的总睡眠数量。数字和频率也是重要的指标,但它们可以从总睡眠,平均持续时间和嗜睡持续时间得出。应该注意的是,在睡眠中的变化和运动时间和睡眠期间的详细描述是可能的,并且在将来的研究中可能是重要的。
      1. 首先,我们定义指标。
        1. Lethargus条目定义为分数静止(fQ)保持在0.1以上至少20分钟。 fQ计算为60帧的滚动平均值,即10分钟。 Lethargus条目进一步调整为从用于计算fQ的60帧开始的第一个帧的第一帧。
        2. Lethargus出口定义为fQ保持在0.1以下至少20分钟。 Lethargus出口进一步调整为最后一帧的最后一帧,用于计算fQ的60帧。
        3. 紫薇持续时间是进入和退出的时间。
        4. 睡眠总睡眠时间是入睡时间。请注意,在某些突变株中,行为学上的嗜睡可能会延伸到外阴外翻的时期。
        5. 平均回合持续时间是所有lethargus之间的平均持续时间,在lethargus进入和退出之间。
        6. 布鲁斯数字是lethargus进入和退出之间的总和数。请注意,发作频率在夜间降低。
      2. 以下是使用Hart实验室睡眠指标计算脚本(以Python运行)的分步指南。
        1. 首先,安装以下免费软件:Python 2.7.3或以上(但不是Python 3),Numpy 1.6.2或更高版本,Scipy-0.10.1或更高版本,以及Matplotlib-1.1.1或更高版本。
        2. 一旦您的计算机上安装了Python,请转到https://github.com/Huiyan-Huang并从睡眠指标计算分支下载TenChamberSingleFileV2.py文件。从图像减法分析将文件复制到您的"C:\ Users \ Documents \ Results"文件夹。该脚本计算每10秒拍摄的图像的睡眠度量。如果使用不同的图像频率,请与我们联系以获得修改的说明。
        3. 要运行分析,右键单击"TenChamberSingleFileV1.py",选择"使用IDLE编辑"(图6A)。 Python脚本将被打开。找到path = r'C:\ Users \ ... \ ... \ ... txt的行。使用上述图像减法算法( eg 。,path = r'C)中的结果文件的路径替换"C:\ Users \ ... \ ... \ ... txt" \ Users \ Hart_E5430 \ Desktop \ Sleep movies \ Results \ yyyy.mm.dd.N 2 .12h.txt')。
          注意:重要的是不要意外删除引号('')和引号前面的'r'的任何部分。还要确保下面的行表示"ChamberN = 10"。
        4. 在顶部菜单栏中,选择"文件",然后单击"保存"(图6B)。然后选择"运行",然后单击"运行模块"(图6C)。将弹出一个Python shell。一旦看到两个'>>>'(图6D),就进行分析。
        5. 现在,在与.txt文件相同的路径中,将有两个新文件。一个显示了帧上的分数静止(图7),另一个包含每个室的详细分析(表1)。


          图6.计算睡眠指标。 A.打开Python脚本以计算睡眠指标; B.保存对分析所做的更改; C.运行Python模块来计算睡眠指标; D. Python shell现已完成处理。


          图7.每个室中动物的分数静态(fQ)曲线。每个点表示周围60帧(10分钟)的滚动平均值,当运动被分配为0并且不活动被分配1.室5并且腔室10不包含动物,但确实包含在成像期间尺寸变化的气泡,其被报告为图中的一些运动。 x轴是帧号,y轴表示fQ。红色虚线表示0.1 fQ阈值,用于调用lethargus的进入和退出。在这里,我们显示L4成年动物的fQ图。对于其他发育阶段的动物的fQ图,请参见上一份报告中的图1。 elegans 睡眠(Raizen等人,2008)。

          表1.图6所示的N2野生动物的相应睡眠指标
          名称
          条目/h
          退出/h
          持续时间/ h
          已更正总计/分钟
          tAPD/s
          t#
          1
          3.4
          7.0
          3.5
          50
          25
          124
          2
          3.9
          6.5
          2.6
          45
          24
          113
          3
          4.9
          7.0
          2.1
          28
          21
          82
          4
          4.3
          6.7
          2.4
          37
          25
          90
          5
          0.0
          12.0
          12.0
          701
          948
          45
          6
          3.7
          6.9
          3.2
          56
          22
          153
          7
          3.0
          6.3
          3.3
          59
          21
          168
          8
          3.5
          6.7
          3.2
          51
          23
          136
          9
          3.3
          6.0
          2.7
          52
          22
          143
          10
          0.0
          12.0
          12.0
          701
          992
          43

数据分析

表1提供了睡眠分析结果的示例。我们通常比较突变体和野生型/对照动物之间的总睡眠(表1,"校正的总Q/min"列)。一些突变动物的嗜睡持续时间也不同(表1,"持续时间/小时"列)和平均持续时间(表1,'tAPD/s'栏)。理想情况下,对照动物和实验动物应该加载在相同的芯片上,或者应采用其他策略,以允许不同基因型,不同试验和/或不同天数据收集之间的直接比较。应检查每种基因型至少10只动物;如果基因型之间的差异很小或给定基因型的行为是可变的,则需要较大的实验和对照动物数量。应该使用与您感兴趣的野生型/对照型和基因型相似数量的动物进行统计学分析。如果两个样本均属于正常的Student's - 分布,则可以使用Student's t -test来比较结果。如果结果遵循其他分布,则应采用其他分析。
必须排除具有两个或更多个动物的房间的数据,因为图像减法不跟踪单个动物,只是在房间内的移动。必须排除动物进入夜蛾之前不消失的气泡数据,因为气泡运动不能通过图像减法与动物运动区别开来。同时,在室内装载30分钟内未恢复正常活跃运动水平的动物将被排除在分析之外,因为它们是在记录期间被捕获或死亡的动物。这可以通过在分析前快速查看第二天的图像来确定。

笔记

  1. 温度影响睡眠期间的睡眠。研究应该在温度控制一致的情况下进行
  2. 我们建议使用先前定义的 C。线虫菌株用于校准。 N2和 egl-4通常使用功能损失。
  3. 如果适当地设定照明,图像大小,噪音等参数,则在L4 /成人嗜睡期间,在22摄氏度的10〜15NN野生型动物的总睡眠平均值应为50〜65分钟。

食谱

  1. 液体线虫生长培养基(NGM)用于再悬浮抗生素治疗的OP50
    0.3克NaCl
    0.25克蛋白胨
    97.5ml ddH 2 O
    100μl1M MgSO 4
    100μl1M CaCl 2
    100μl5 mg/ml胆固醇
    2.5ml 1M磷酸钾缓冲液(pH6)
    过滤消毒,并分成15ml Falcon管 一次取出一根管,并分装成1.5 ml管 注意:15ml等分试样可以用Parafilm密封,并在4℃下储存多年,如果没有被污染。 1.5ml等分试样可以在室温下储存数月。
  2. 2%琼脂密封腔
    2 g琼脂
    100ml ddH 2 O 微波溶解/熔化琼脂。等分2%琼脂少量储存在4°C
    注意:只要琼脂中没有增长,多年来应该是好的。

致谢

这项工作得到了国立卫生研究院NIH R01 NS055813(A.C.H.)和翻译神经科学博士后研究员(H.H.)的支持。这是以前报告的协议的修改(Singh等人,2011)。所有实验方案均经适当的机构审查委员会批准。我们没有利益冲突。

参考文献

  1. Belfer,SJ,Chuang,HS,Freedman,BL,Yuan,J.,Norton,M.,Bau,HH和Raizen,DM(2013)。  Caenorhabditis -in-drop数组用于监视C。电影行为静止行为。 睡眠 36(5):689-698G。
  2. Bringmann,H。(2011)。琼脂糖水凝胶微室在秀丽隐杆线虫幼虫中成像睡眠和唤醒样行为和神经系统发育。 Neurosci Methods 201(1):78-88。
  3. Ghosh R.和Emmons,SW(2008)。  线虫游泳中的游泳行为。 elegans 。 J Exp Biol 211(Pt 23):3703-3711。
  4. Huang,H.,Zhu,CT and Hart,A.(2013)。  如何测量lethargus静止,而不垄断实验室中的解剖显微镜。 > Worm Breeder's Gazette 19(3)。
  5. Iwanir,S.,Tramm,N.,Nagy,S.,Wright,C.,Ish,D.,and Biron,D。(2013)。  C的微架构。线虫在lethargus期间的行为:静止反应动力学,典型的身体姿势,以及中枢神经元的调节。 36(3):385-395。
  6. Lockery,SR,Lawton,KJ,Doll,JC,Faumont,S.,Coulthard,SM,Thiele,TR,Chronis,N.,McCormick,KE,Goodman,MB和Pruitt,BL(2008) ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/18337372"target ="_ blank">人造污垢:用于线虫神经生物学和行为的微流体底物。 > J Neurophysiol 99(6):3136-3143。
  7. Mok,DZL,Sternberg,PW和Inoue,T。(2015)。  形态学定义的子阶段。线虫第四个幼虫期的外阴发育。 BMC Dev Biol 15:26。
  8. Nelson,MD,Trojanowski,NF,George-Raizen,JB,Smith,CJ,Yu,CC,Fang-Yen,C.和Raizen,DM(2013)。< a class ="ke-insertfile"href = http://www.ncbi.nlm.nih.gov/pubmed/24301180"target ="_ blank">神经肽NLP-22调节秀丽隐杆线虫中的睡眠状态。 Nat Commun 4:2846.
  9. AI(2008)。  Lethargus是秀丽秀丽秀丽秀丽状态的睡眠状态 451(7178):569-572。
  10. Singh,K.,Chao,MY,Somers,GA,Komatsu,H.,Corkins,ME,Larkins-Ford,J.,Tucey,T.,Dionne,HM,Walsh,MB,Beaumont,EK,Hart, Lockery,SR and Hart,AC(2011)。  < em> C。线虫 Notch信号调节成年化学感觉反应和幼虫蜕皮静止。 21卷(10):825-834。
  11. Singh,K.,Ju,JY,Walsh,MB,DiIorio,MA和Hart,AC(2014)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov/pubmed/25142568"target ="_ blank">深层保护黑腹果蝇和秀丽隐杆线虫所需的基因包括多巴胺能信号的作用。 睡眠 37(9):1439-1451。
  12. Trojanowski,NF和Raizen,DM(2016)。  通话它蠕虫睡眠。 趋势Neurosci 39(2):54-62。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Huang, H., Singh, K. and Hart, A. C. (2017). Measuring Caenorhabditis elegans Sleep during the Transition to Adulthood Using a Microfluidics-based System. Bio-protocol 7(6): e2174. DOI: 10.21769/BioProtoc.2174.
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