The overall goal of this methodology is to analyze navigational behavior of drosophila larva in response to simultaneously optogenetic stimulation of its olfactory neurons. This method enables the comprehensive dissection of olfactory circuit function and complements studies on how olfactory circuit function translates into behavior response. The main advantage of this technique is its specificity and temporal control by using a light instead of traditional odor gradients to activate individual olfactory neurons.
To begin the experiment, build a light deprived behavior arena. Construct a box that is 89 by 61 by 66 centimeters, made of black-colored plexiglass acrylic sheets, three millimeters thick. Place the box on a tabletop in the behavior room.
Next, mount a monochrome USB 3.0 CCD camera fitted with an IR Longpass 830 nanometer filter and an eight millimeter F1.4 C mount lens on the center of the ceiling of the black box. Place two infrared LED strips on the tabletop to illuminate the larvae in the dark arena. Then, obtain a 22 centimeter by 22 centimeter square aluminum plate to build the LED platform.
In the center of the plate, use a metal cutter to cut a hole that is large enough to fit around the CCD camera. Cover the metal plate with red LED strip lights. Solder the LED light strip wires in series, and feed the strip wires into an optocoupler relay controlled by a Raspberry Pi 2B microprocessor.
Next, mount the LED platform around the CCD camera. Then install and configure an Ubuntu MATE Raspbian Jessie Linux-based operating system on the Raspberry Pi processor before connecting the optocoupler relay to the LED strips. Attach a power supply to power the LED strips and the optocoupler.
Ensure there is homogenous irradiance at various points around the CCD camera in the behavior arena. Ensure the absolute irradiance at the surface of the arena with the help of a spectrometer and determine it to be approximately 1.3 watts per meter squared throughout the surface of the arena. Maintain the flies on standard fly food at 25 degrees Celsius, 50 to 60%relative humidity in a 12-hour, 12-hour light/dark cycle.
In order to express CsChrimson in a single pair of ORNs, cross the virgin females from a UAS-IVS-CsChrimson line to males from an ORX-GAL4 line. After male and female flies in a cross have been allowed to mate and lay eggs for 48 hours, transfer the adults to a fresh vial. On the surface of the food vial containing eggs, add 400 microliters of a mixture containing 400 micromolar all-trans-retinal, or ATR, dissolved in dimethyl sulphoxide, or DMSO, in 89 millimolar sucrose dissolved in distilled water.
Once ATR is added to the food vials containing eggs, incubate the vials in the dark for an additional 72 hours. After incubation, extract third instar larvae from the surface of fly food by floating them using a high-density 15%sucrose solution. Using a P1000 micro-pipette, separate the larvae floating on the surface of the sucrose solution into a 1, 000 milliliter glass beaker.
Wash larvae four times by exchanging 800 milliliters of fresh distilled water in the glass beaker each time. After the final wash, allow the larvae to rest for 10 minutes before subjecting them to the behavior assay. For the behavior assay, maintain a consistent temperature and humidity in the behavior room.
Prepare larval crawling medium by pouring 150 milliliters of melted 1.5%agarose into a 22 centimeter by 22 centimeter square Petri dish. Allow the agarose to solidify and cool for one to two hours in the Petri dishes before using them in the behavior assay. After the agarose has solidified, transfer no more than 20 of the prepped third instar larvae to the center of the Petri dish and cover the Petri dish with its lid.
Place the Petri dish in the behavior arena under the CCD camera. Turn on the 850 nanometer infrared LED light source to visualize larvae in the video. Start the CCD camera to record larval movement.
Finally, move on to data processing and analysis. When ORN 7a expressing CsChrimson was stimulated by light, there was a significant decrease in run length compared to control animals. Conversely, when ORN 42a expressing CsChrimson was stimulated by light, there was a significant increase in run length compared to control animals.
We found that for ORN 42a, different temporal patterns of light stimulation elicited different behavioral responses. While we have demonstrated this method for the first order olfactory receptor neurons, our method also permits a variety of future applications, such as measuring the impact of downstream neurons such as projection neurons and local neurons on larval behavior. We expect this technique to pave the way for a comprehensive dissection of larval olfactory circuit function.
