Calcium Imaging in Individual Neurons Regulating Egg-Laying Behavior in Caenorhabditis elegans

1. Strains, Culture Media, and Mounting of Animals

1. Grow C. elegans worms at 20 °C on standard 60-mm Nematode Growth Medium (NGM) agar plates seeded with OP50 E. coli bacterial food.
2. Prepare two plasmids for each cell-specific promoter of interest: one, driving expression of GCaMP5 to record intracellular Ca²⁺, and the second, driving expression of mCherry to allow for ratiometric quantitation of GCaMP5 fluorescence changes and simplify object finding and measurement.
   NOTE: GCaMP5:mCherry ratiometric imaging corrects for fluctuations in GCaMP5 fluorescence that result from changes in focus and animal movement, not actual changes in intracellular Ca²⁺.
3. Inject GCaMP5- and mCherry-expressing plasmids along with the pL15EK visible rescue marker plasmid into the gonads of LX1832 lite-1(ce314), lin-15(n765ts) X mutant animals and recover non-Muv lin-15(+) animals expressing GCaMP5 and mCherry from a high-copy transgene. Use the lite-1 mutant background to reduce blue-light avoidance behavior.
4. Integrate transgenes to chromosomes to reduce mosaicism and simplify the generation of compound mutant strains.
   NOTE: The LX2004 strain described here carries an integrated high-copy transgene that expresses GCaMP5 and mCherry in the HSNs from the nlp-3 promoter (see Table of Materials). The nlp-3 promoter was found to drive strong expression in the HSNs of late L4 and adult animals without causing significant defects in HSN development or egg-laying behavior compared to other promoters tested (e.g.,tph-1, egl-6, and unc-86). This strain and others that express GCaMP5 and mCherry in the ventral cord (VC) motor neurons, vulval muscles, and uv1 neuroendocrine cells are available from the Caenorhabditis Genetics Center, and details of their construction have been described.
5. Apply OP50 bacterial food from a seeded NGM agar plate to the bottom of a platinum worm pick and use it to transfer ~ 20 late L4 LX2004 animals that express GCaMP5 and mCherry in the HSNs from the nlp-3 promoter. Ensure that the developing vulva appears in a stereomicroscope as a clear dark spot surrounded by a white crescent. Incubate animals at 20 °C for 24-40 h.
6. After the incubation, apply OP50 to the pick and transfer ~ 3 of the worms to an unseeded NGM plate, leaving a small amount of food behind for the worms to feed upon during imaging. Ensure sufficient food, as too little bacterial food will encourage the worms to wander away from the center of the plate, while too much food will increase background fluorescence and cause hypoxia.
7. Use a flat rounded spatula to cut a ~ 20 mm x 20 mm chunk from the plate carrying the worms and transfer the chunk face-down onto the center of a clean 24 mm x 60 mm #1.5 coverslip (Figure 1). Start the application from one side to keep bubbles from being trapped under the coverslip and interfering with imaging. Apply a 22 mm x 22 mm #1 coverslip to the top of the chunk to reduce sticking and evaporation.

2. Hardware and Instrumentation Setup

1. Place the staged, transgenic worms onto the stage of an inverted microscope with a ≥ 0.7 Numerical Aperture Plan-Apochromat 20x objective, motorized XY stage controllable with a joystick, an image splitter, and fluorescence filters for simultaneous GCaMP5 and mCherry excitation and emission, cameras for fluorescence and infrared brightfield imaging, and a triggerable Light Emitting Diode (LED) illumination system (Figure 2A).
   ​NOTE: An 80/20 beam-splitter sends 80% of the image signal to the fluorescence camera and 20% to the brightfield camera. An upright microscope can also be used, but the NGM chunk should be placed in between a glass slide and the large coverslip in this case.
   1. Use the binocular eyepieces to select a worm for imaging. When an animal is selected, slide the infrared filter in place above the condenser.
2. Transistor-transistor logic (TTL) triggers
   1. Attach coaxial cables from each of the three TTL trigger output lines on the fluorescence camera. Connect the first output to the BNC input #3 of the LED illumination system.
   2. Connect the second output to a BNC 'banana' adapter with green and brown wires from the 8-pin GPIO connector running to GPIO input #3 (pin 4) and ground (pin 5) of the infrared camera, respectively.
   3. Connect the third output to a BNC 'banana' adapter with jumper wires running to the #8 digital input and ground in the digital acquisition device (Figure 2B).
3. Digital acquisition device (DAQ)
   1. Connect the DAQ microcontroller board (see Table of Materials) to the PC via a USB cable. Update the firmware with the standard Firmata protocol (see Table of Materials) and configure the USB port to communicate at 57600 baud.
4. LEDs
   1. Run the LED Controller software (see Table of Materials). Switch the Trigger Mode from 'Continuous' to 'Gated' and select Trigger Channel '3' for both the 470 and 590 nm LEDs.
   2. Turn on and enter LED power for each LED (e.g., set the 470 nm LED to 20% and the 590 nm LED to 40%).
5. Run the serial-stage communication script 'XY-stage-final' in Bonsai (see Table of Materials). Click on the grey 'CsvWriter' node and select a folder to save the recorded X and Y stage information (Figure 3). Press the green arrowhead in the toolbar to initialize the DAQ.
   NOTE: The script will start recording the X and Y stage position when the fluorescence camera sends a TTL signal. This script outputs four columns of data: frame number, X position (µm), Y position (µm), and the interval between frames (s).
6. In the infrared camera recording software (see Table of Materials), under 'Custom Video Modes,' select "Mode 1" (2 x 2 binning; 1,024 x 1,024 pixels), and "Pixel Format Raw 8". Under 'Trigger / Strobe', set the trigger input line (GPIO) to "3", the polarity to "High", and the 'Mode' to "14". Toggle 'Enable' to stop frame acquisition until a TTL trigger signal is received.
   1. Leaving that window open, click the red "Record" button on the main camera view toolbar. Select the folder for image sequences to be saved. Select 'Buffered' Recording mode and save 'Images' in JPEG format. Click "Start Recording" to initialize the acquisition.
7. Fluorescence camera and image splitter
   NOTE: GCaMP5 and mCherry fluorescence channels must be collected simultaneously to ensure proper image registration for ratiometric quantitation. An image splitter allows two-channel acquisition of the GCaMP5 and mCherry fluorescence onto one sensor.
   1. In the 'Capture' tab of the image acquisition software (see Table of Materials), set exposure time to 10 ms, binning to 4x, and image depth to 16-bit. Select a centered camera subarray of 512 pixels wide by 256 pixels high. Click 'Show Output Trigger Options' and set all Triggers to 'Positive.'
      ​NOTE: The DAQ can occasionally miss TTL triggers if they are 10 ms or less.
   2. Ensure that 'Trigger 1' and 'Trigger 2' are set to 'Exposure' while 'Trigger 3' is set to 'Programmable' with a 'Period' of 25 ms. Under the 'Sequence' tab, select 'Time Lapse' with a 'Field Delay1' of 50 ms to collect images at 20 Hz. Select 'Save to Temporary Buffer.'
8. PMT gain and laser intensity during three channel confocal imaging
   1. Set the PMT gain so the background fluorescence is at a level just above the minimum (black level). Increase 561 nm (green) laser power until a single saturated 12-bit or 16-bit pixel is observed at the presynaptic terminus in the mCherry channel.
   2. Adjust 488 nm (blue) laser intensity so that GCaMP5 fluorescence is just visible at the presynaptic terminus above the background. This low setting prevents the saturation of the GCaMP5 pixels when the fluorescence increases in response to strong Ca²⁺ transients. Open the confocal pinhole all the way to maximize light capture.

3. Ratiometric Ca²⁺ Imaging and Behavior Recording

1. Under the 'Sequence' tab in the fluorescence acquisition software, click "Start" to begin recording. Track the worm with the joystick, keeping the cell(s) and synapses of interest in focus and in the center of the field of view (FOV). Click the 'Stats' button in the histogram window to show pixel statistics of each channel.
2. Adjust the LED power to ensure a maximum single-pixel mCherry fluorescence at the presynaptic terminus of ≥ 8,000 counts (~ 4,000 photoelectrons), giving a ~ 12-bit dynamic range above the background (~ 100 photoelectrons). GCaMP5 signals at the presynaptic terminus during resting (low) Ca²⁺ should be around ~ 2,500 counts – just visible above background.
3. Record until one egg-laying active state is reached; typically, this occurs every 20-30 min in a wild-type worm. Save a 10-min subset (12,001 frames), 6,000 frames before and after the first egg-laying event (frame 6,001). Be sure to keep the same subset of brightfield images of worm behavior and timepoints of XY stage position, or the precise synchronization of data streams will be lost.
4. Use ImageJ and the BioFormats plugin (see Table of Materials) to convert image sequences to Image Cytometry Standard (.ics) format so that it can be imported into the Ratiometric Quantitation software.