The overall goal of this procedure is to perform Ratiometric Calcium Imaging of the effects of hermaphrodite specific neuron, or HSN activity, on Caenorhabditis elegans behavior. This method can help answer key questions in the Behavioral Neuroscience field about how neuro circuit activity produces changes in animal behavior as animals alternate between behavioral states. This technique allows changes in intracellular calcium activity to be recorded with the high signal to noise ratio, while simultaneously tracking animal behavior and locomotion.

Though this method can provide insight into neuronal activity in C.elegans, it can also be applied to other transparent model systems including Drosophila larvae and Zebra fish. Demonstrating the procedure with me, will be Collin's Labs Students:Richard Kopcock, Pravat Dhakal, and Layla Nassar. To mount the animals, first apply OP50 bacterial food from a seeded nematode growth medium agar plate to the bottom of a platinum worm pick and use the pick to transfer approximately 20 late L four L-X 2, 000 for animals expressing the sensitive calcium reporter GCaMP5 and mCherry in HSN from the N-L three P promoter onto a seeded nematode growth medium plate.

Confirm the presence of the developing vulva under a stereo microscope as a clear dark spot surrounded by a white crescent and incubate the worms at 20 degrees Celsius for 24-40 hours. At the end of the incubation, apply OP50 to a new pick and transfer three worms to an unseeded nematode growth medium plate leaving a small portion of food on the new plate for the worms to feed on during the imaging. Next, use a flat rounded spatula to cut a 20 by 20 millimeter chunk from the unseeded plate and place the chunk, worm side down, onto one edge of a clean 24 by 60 millimeter number 1.5 coverslip.

Then, carefully slide the agar chunk into the center of the cover slip and place a 22 by 22 millimeter number one cover slip on top of the agar piece to reduce sticking and evaporation. To set up the imager, place the worms onto the stage of an inverted microscope and select the 20x subjective. Use the binocular eye pieces to select a worm for imaging and slide the infrared filter and place above the condenser.

Open an appropriate video analysis program and run the serial stage XY stage final communication script. Click on CsvWriter and select a folder for saving the recorded X and Y stage information. Then, click the green arrow head to initialize the digital acquisition devise.

In the infrared camera recording software under settings, select Toggle Camera Control Dialog. Then select the Custom Video Modes menu and select Mode 1 and Pixel Format Raw eight. Under Trigger Stroke, set the Trigger Input Line to three, the Polarity to high, and the Mode to 14.

Toggle Enable to stop the frame acquisition until the voltage trigger signal is received from the fluoresces camera. With the image viewing window open, click Record and select the folder for saving the image sequences. Select Buffered Recording Mode and save the images in JPEG format.

Then, click Start Recording to initialize the acquisition. Next, open the Capture tab in the Florescence Acquisition Software and set the exposure time to 10 milliseconds, the Binning to four X and the Image Depth to 16 bit. Select a centered camera Sub-Array of 512 pixels wide by 256 pixels high.

The program will automatically divide the wide image in half to generate the separate florescence channels. Click Show Output Trigger Options, and set all of the triggers to positive. Confirm that trigger one and trigger two are set to Exposure and trigger three is set to Programmable with a period of 25 milliseconds.

Then, under the sequence tab, select Time Lapse Under Scan Settings, set Field Delay one to 50 milliseconds to collect images at 20 hertz. Select save to Temporary Buffer. For Ratiometric Calcium Imaging, under the Sequence tab, click Start to begin recording and track the worm with the joystick keeping the cells and synapses of the interest in focus in the center of the field of view.

At the moment the florescence recording is started, triggers are sent to the waiting bright field camera and the stage controller which begin collecting synchronized images and stage position. Click the Stats button in the Histogram window to show pixel statistics for each channel. Adjust the LED power to ensure a maximum single pixel of mCherry florescence at the presynaptic terminus of greater than or equal to 8, 000 counts in the red channel giving approximately 12 bit dynamic range above background.

The Calcium Reporter Signals at the presynaptic terminus during resting or low calcium should be around 2, 500 counts in the green channel. Record the behavior until one egg laying active state is reached. Then, save a 10 minute subset containing 6, 000 frames before and 6, 001 frames after the first egg laying event.

Take care to keep the same subset of bright field images of worm behavior and time points of the X-Y stage position or the precise synchronization of data streams will be lost. For Image Segmentation, import the image sequences into an appropriate Ratiometric Quantization Software Program and right click on the time series in the sequence tab to set the time series to 20 frames per second and 1.25 micrometers per pixel. Under the Tools menu, select Ratio then select mCherry for Channel A and GCaMP5 for Channel B and click Calculate to subtract the background from each image sequence.

Select Apply a rainbow look up table to the ratio channel and use the mCherry Channel to generate an intensity modulated ratio channel to map the ratio color onto the brightness of the mCherry channel. Click Ratio to generate the two channels. To create on object finding protocol, drag the Find Objects using intensity tool under the measurements tab to the protocol pane and click the cog to set the window of mCherry intensity values and to Find Objects.

Select intensity values greater than or equal to two standard deviations above the background taking care that the cell body and presynaptic terminus is detected. Add any additional filters targeting the mCherry channel only as necessary to exclude undesired objects, like:head, tail, or gut florescence. Then, open the Measurements menu and select Make Measurement Item and choose all time points to execute image Quantization Analysis Protocol.

Simultaneous acquisition of GCaMP5 and mCherry florescence signals is used to generate a pixel by pixel ratio channel that compensates for changes in animal movement and focus. For each animal, changes in presynaptic intracellular HSN calcium can be correlated with behaviors evident in bright field imaging including egg release and changes in locomotion. Indeed, significant differences in worm speed before, during, and after the egg laying active state can be observed.

Zooming into one egg laying event, reveals a robust change in GCaMP5 florescence prior to the egg laying event. While the mCherry florescence remains relatively unchanged. In addition, measured changes in the GCaMP5:mCherry ratio clearly show an HSN calcium transient about 4 seconds prior to egg release.

The proceeds vulval muscle contraction evident of a clear slowing of worm locomotion that ends with egg release. Further, imaging and tracking of the worms as just demonstrated, allows visualization of the spacial organization of egg laying behavior from foraging, transient HSM calcium changes, and HSN activity that transitions into burst firing before locomotion to the egg laying location. Once mastered, this technique can be completed in an hour or two, if it's performed properly.

While attempting this procedure, it's important to remember to maintain focus on the cells of interest and their presynaptic terminals during the imaging steps. Following this procedure, other methods such as Optogenetic Activation, can be performed to answer additional questions about how the HSN responds to presynaptic stimulation. This technique has paved the way for researchers in the field of Neuroscience to explore egg laying circuit function in C.elegans.

After watching this video you should have a good understanding of how to perform Ratiometric Calcium Imaging of HSN in active Caenorhabditis elegans.