The overall goal of this procedure is to efficiently trap an image calcium transience in male C.Elegans head neurons upon exposure to pheromones. This method can help answer key questions in the neurobiology field, such as understanding sex specific neural circuit regulation. The main advantage of this technique, is that male C.Elegans can now be imaged efficiently in a micro-fluidic trap device.
Generally, individuals new to this method will struggle because loading animals can be difficult at first and biogenic pheromones don't elicit calcium transience in every animal. To begin assembly of the reservoirs, prepare three sets of tubing by inserting a needle into one end and a metal tube into the other. Then, for each reservoir, take a three way luer valve and attach a 30 milliliter syringe with its plunger removed, a three milliliter syringe and the tubing.
Fill each reservoir with the appropriate buffer, either stimulus S basal or S basal containing fluorescin. Then, remove any air bubbles from both the reservoir and tubing, by flicking and pumping the syringe. Now, fill a new three milliliter syringe and tubing with S basal and insert into the outlet port of the micro-fluidic device.
Gently apply a little pressure to the syringe until the buffer appears at the surface of the inlet holes. Next, connect the flow control, buffer and stimulus reservoir tubes to their appropriate inlet holes, ensuring that liquid drops are present on both the loading port hole and the tubing to be attached. Now, gently apply a little more pressure to the syringe until droplets appear in the worm loading port inlet, then insert a solid blocking pin into that port.
Next, remove the syringe from the outlet port and attach an outlet line connected to the house of vacuum. Now, inspect the device for any bubbles in the float channels in the video feed from the microscope. Click on live, to observe the live image and wait for bubbles to naturally dissipate before proceeding.
Next, turn on the fluorescent light source and use a GFP filter to confirm proper flow dynamics. Actuate the three wave valve and observe the fluorescent control stream appear in the video feed, when the valve is open. To begin, pick one worm onto an unseeded NGM agar plate and allow the worm to crawl away.
Then, float the plate with S basal. Draw up solution containing the worm into the syringe. Now, on the device, turn off the vacuum to stop the flow using the outlet luer valve.
Then, remove the solid pin that blocks the worm loading port. Insert the tubing containing worms into the loading port. Open the outlet luer valve and observe the live video feed.
Now, apply gentle pressure onto the syringe until the worm appears. Pull the worm back if it enters tail first, it must go in head first to be exposed to the stimulus. The user must pull the worm back before the tail fully enters the channel, otherwise, getting the worm out of the channel is extremely difficult and usually requires flushing the worm completely through the channel and loading a new worm.
Now, using pressure from the syringe, advance the worm through the channel and position the worm's head, so it is exposed to the buffer flow but not so far into the channel, that it is free to move about. A small amount of axial or rotational movement is expected in non-paralyzed animals. Although the addition of a paralytic to the buffers nearly eliminates this effect.
To decrease the likelihood of movement during trial, use older males which are more efficiently trapped, decrease the width or thickness of the worm loading port or increase the amount of time spent in the paralytic. Use the software to make recordings using blue light excitation. Set the exposure to 100 milliseconds, open the image acquisition window, set the time points to 300 with an interval at zero, then start the image acquisition.
After five seconds, apply the stimulus by changing the three way valve controlling the buffers. A 10 second stimulation is a good starting point and the length of stimulus can be adjusted as needed. After the stimulation, switch the flow back by pressing the button again and continue recording until the 30 second video is completed.
This allows the g camp fluorescence to return to base line. Now, let the preparation rest for 30 seconds before the next trial. Worms expressing g camp three in the no susceptive neuron, ASH, respond to one molar glycerol with visible changes in fluorescence within the ASH neuron, indicative of neural activity.
The neuron should remain in the area of analysis for the entire video, otherwise a movement artifact may occur. If so, move the analysis area for those time points and merge the intensity values to create a single trace. Males who are stimulated with the attractive biogenic pheromone ascaroside three during exposure, calcium transience were measured in the four CEM neurons.
From animal to animal and neuron to neuron, the traces varied in shape, sign and magnitude. Thus, it is very important to analyze many animals when studying pheromone responses. After watching this video, you should have a good understanding of how to load a male C elegans into a micro-fluidic device and analyze calcium transience in head neurons.
The same process can be used with hermaphrodites, to identify sec specific differences in neural responses. Once mastered, this technique can be done in half an hour for the first animal imaged. After the first recording, a new animal can be loaded in image in about 10 minutes.
Following this procedure, other methods like optogenetics can be combined with this protocol in order to further understand how neural circuits are regulated.
