Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Our research uses optogenetic tools to study how neural circuits in Drosophila melanogaster govern behaviors, like thermotaxis and gestation. By controlling specific neurons with light, we aim to understand how these circuits drive sensory responses and influence decision making with high precision. Optogenetics is a valuable technology in neuroscience, offering precise control of neural activity using light.

In Drosophila melanogaster, tools like CsChrimson and GtACR2 enable targeted activation and inhibition of neurons. These tools allow researchers to manipulate neural circuits, study behavior, and explore sensor responses with high specificity. Our protocol offers a simple, cost-effective and reproducible approach to optogenetic manipulation in Drosophila.

It uses commercially available materials, making it accessible in labs and classrooms with limited resources. The methods are also highly adaptable, enabling the study of attractive and avoidance behaviors with minimal technical challenges. To begin, obtain male and female Drosophila flies, expressing GtACR2 in heating cells.

Align two steel plates on separate hotplates so their edges meet. Place a plastic sheet protector on top and secure it with tape to minimize movement. Now, position a piece of white paper on the sheet protector to reduce background noise signals, and place a clear plastic cover on top of the white paper.

Then cut a hole in the bottom of a polystyrene foam box to accommodate the camera and blue light approximately 12 centimeters above the experimental surface. Position the camera and the blue light to minimize glare, while ensuring activation. Set the camera to record with a one-second time lapse, narrow field, and a resolution of 4, 000 by 3, 000 pixels.

Adjust the hotplate settings to maintain a surface temperature of 25 plus or minus one degree Celsius and 31 plus or minus one degree Celsius on the respective steel plates. Monitor the temperatures of the steel plates using a surface temperature probe before and after each trial. Next, place the plastic cover on the 25-degree-Celsius steel plate.

Now, using a fly aspirator, gently release a single fly under the cover. Position the box above the experimental area to create dim light below 10 lux, and let the fly acclimate for one minute. After the acclimation period, lift the box and quickly adjust the plastic cover so the center of the cover aligns with the steel plate boundary.

Initiate the trial, turn on the camera and blue light at 20 kilolux. Capture the activity of the flies for two minutes. After two minutes, turn off the camera and light.

Dispose of the flies using the aspirator. Anesthetize starved male and female flies expressing CsChrimson in sweet-taste receptor neurons on ice. Apply seven to 10 small dots of glue on a glass slide.

Position one fly ventral side up on each glue dot, ensuring the thorax and the wings contact the glue to minimize movement. Fan the wings out to each side to increase the adhesive surface area. Place wet paper towels in a humidity box and transfer the slides to the box.

After two hours of recovery, place the slide under the microscope. Use a syringe to deliver a droplet of water to satiate the flies, preventing thirst-induced proboscis extensions. Manually hold a red laser pointer and shine red light at the proboscis or head of a single fly at 700 lux.

Observe the proboscis extension response through the microscope within a 30-second window. After testing the light-induced proboscis extension, examine the response to 4%sucrose. Expel a droplet of sucrose at the end of the syringe needle and bring it close to the fly's proboscis.

First, assemble the fly maze for the experiment. In dark or low-light conditions, place 10 male and 10 female CsChrimson-expressing flies into the loading tube and connect it to the holding chamber. Tilt the elevator and gently tap the tube to move the flies into the holding chamber.

After transferring the flies to the holding chamber, use the elevator to lower them between the loading tube and testing tube holes. Then remove the loading tube. Position the fly maze approximately 13 centimeters away from the 1, 000-milliampere red light source without turning the light on.

Lower the elevator until the holding chamber aligns with the testing tube holes, allowing the flies to move freely between the foil-wrapped and uncovered testing tubes. Simultaneously, turn on the red light at approximately 40 kilolux to activate CsChrimson. Allow the flies to choose between the red light-exposed tube and the shaded tube for a minute.

After one minute, raise the elevator between the loading tube and testing tube holes. Remove the flies from each tube. Count them and record the numbers.

Clean the fly elevator and fly maze using distilled water after each trial. In the blue-light, optogenetic, thermotactic, positional preference assay, flies avoided the 31-degrees-Celsius side under the control conditions, while in blue light with ATR supplementation, flies exhibited no preference between 25 and 31 degrees Celsius, indicating HC neuron inhibition via GtACR2 activation. Under control conditions, flies showed minimal proboscis extension response, while red light activation with ATR supplementation resulted in significant proboscis extension, demonstrating the activation of sweet-sensing neurons by CsChrimson.

In the red-light, optogenetic, fly maze assay, control groups showed no preference, while red light activation with ATR supplementation caused flies to avoid the uncovered tube, indicating bitter-sensing neuron activation by CsChrimson.