Two-Choice Optogenetic Preference Assay#
In this protocol, you’ll use a simple maze to ask a simple question with a genetically precise tool: does activating a specific population of taste neurons with light cause a fly to approach or avoid that light?
You’ll be given a vial of flies that express the red light-gated ion channel CsChrimson in a particular population of gustatory receptor neurons: either Gr5a (sweet-sensing) or Gr66a (bitter-sensing). You won’t be told which you have. Using a two-choice assay, you’ll collect behavioral data, calculate a preference index, and use your results to figure out — and defend — which genotype you have.
This protocol follows Fu et al. (2023), “Using Drosophila Two-Choice Assay to Study Optogenetics in Hands-On Neurobiology Laboratory Activities”, published in the Journal of Undergraduate Neuroscience Education.
Supplies
3D-printed fly maze
2 testing tubes per maze (1 clear, 1 foil-wrapped)
Red LED light source
BNC Cable
Power cable
Lux meter app (on phone)
Timer
Small funnel
Ice and ice bucket
Vials of Gr5a>CsChrimson and Gr66a>CsChrimson flies
Setting up the fly maze#
The fly maze has a loading hole on top, with holes on each side for the testing. Two testing tubes — one clear, one wrapped in foil — insert into the two testing holes.
Insert the clear tube into one testing hole and the foil-wrapped tube into the other.
Set up your light#
Connect the red LED to the PowerLab Output using the BNC cable.
Connect the 5V power to the LED.
Configure LabChart to send 1 second pulses of red light every other second in Setup → Stimulator. This will allow you to control the pulse heights (2V-10V). More voltage will change LED intensity.
Position the red LED next to the clear tube. Use the lux meter to measure the light intensity at this distance.
Measure the output from your LED using a lightmeter on your phone.
Light intensity: ____________
Running the two-choice assay#
Place one of your vials of flies into ice until the flies are immobile.
Once the flies are immobile, using the funnel, tap flies from one vial into the top loading hole.
Wait one minute, or until you see any fly emerge into the testing side. Once you do, start the red LED pulse.
Wait one more minute for flies to move.
Count the clear-tube (red-light side) flies: Without moving the maze, quickly count how many flies are in the clear side. If you need to, you can take a picture!
Count the foil-tube (dark side) flies: Carefully remove the foil from the dark side and quickly count the flies within. Include any flies that you see within the loading space in this count as well, since they never made it to the red-light tube.
Record all three counts in Data Table 1.
Repeat with your other vial of flies.
Calculating the preference index#
For each vial, calculate a preference index (PI):
where \(N_{red}\) is the number of flies on the red-light side, \(N_{dark}\) is the number of flies on the dark side (foil tube + elevator hole), and \(N_{total}\) is the total number of flies tested.
PI ranges from −1 (every fly avoided the light) to +1 (every fly moved toward the light). A PI near 0 indicates no preference either way.
Guesses, please!
Based on your preference indices, do you think you have Gr5a>CsChrimson or Gr66a>CsChrimson flies?
Troubleshooting#
Observation |
Likely issue(s) |
Possible solution |
|---|---|---|
Flies aren’t moving at all after loading |
Flies are cold-stunned from handling, or starved too long |
Give flies a minute to recover before starting the timer; check starvation window |
Preference index is close to 0 for both genotypes |
Light intensity too low |
Recheck lux meter reading at 11 cm; note loading method used |
Flies escape during tube transfer |
Funnel not seated tightly, or flies not sufficiently tapped down |
Tap tube firmly on the bench before removing caps; work over a container |
Counted flies don’t match starting vial number |
Flies lost during transfer, or miscounted |
Recount; note any discrepancy in your data table rather than adjusting counts |