Behavioral genetics typically includes experiments to optogenetically or chemogenetically activate or inhibit neuron(s) of interest. Here is a general list of best practices when approaching these experiments.

Behavioral Genetics Strategies#

Activation Strategies#

• CsChrimson: red-shifted channelrhodopsin best targeted with 590-700nm light between 20-90 μW/mm². Can be tagged with various fluorophores, we have CyRFP3 and mVenus in house

Inhibition Strategies#

• GTACR: anion channelrhodopsin best targeted with ~520nm light with ~2 mW/mm². Can be tagged with various fluorophores, we have EYFP in house.
  • caveats: all experiments done in darkness (panels activate ACR). Required light intensity often prompts significant control effects. Dependent on local chloride dynamics as well
• HfACR/ RubyACR: red-shifted anion channelrhodopsin best targeted with ~660nm light with ~20 μW/mm². Can be tagged with various fluorophores, we have EYFP in house.
  • caveats: newer reagent, Sophia/Victoria beginning to use
• Shibire: temperature-sensitive gene that, when heated, the gene’s product (dynamin orthologue) is disrupted, leading to a halt in synaptic vescle recycling and inhibiting synaptic transmission. Permissive temperature is 19-22 deg C, and restrictive temperature is 29-31 deg C.
  • caveats: temperature can be challenging to control perfectly accurately
• Kir: inwardly rectifying potassium channel.
  • caveats: permanently silences neurons, potential developmental effects/compensation
• Tetanus toxin light chain (TeTxLC): permanently blocks synaptic transmission, silencing neurons.
  • caveats: permanently silences neurons, potential developmental effects/compensation

As a reminder, there are specific versions of each of these genetic strategies, be conscientious when selecting a variant. For example, different variants of GTACR have different properties.

Creating a Well-Controlled Experiment#

Split line/ GAL4 line Selection#

Before beginning experiments, you should examine your line for confounding cells. You should assume any labeled cell will be activated with your strategy. In an ideal world, we have perfect lines that only label our cell of interest. The next best thing is to have 2 separate lines labeling your cell of interest with non-overlapping distractor cells.

Control Fly Selection#

There are many control fly options available. The current control structure for behavioral genetics in the lab (Fall 2025) is as follows (mainly for optogenetic experiments):

Split line control:

Split GAL4 line 1 x UAS GFP

Split GAL4 line 2 x UAS GFP

Effector control:

empty-split x UAS effector

Experimental flies

Split GAL4 line 1 x UAS effector

Split GAL4 line 2 x UAS effector

** note: prior studies from the lab used ISOD1 flies, but more recently these flies have been behaving so poorly the lab has shifted away from using them as they are poor behavioral controls

Running Your Experiment#

• Fly Rearing: If using a channelrhodopsin, flies must be reared on ATR food (found in the cold room, covered in foil), and kept on ATR food in darkness prior to experiments
• Plan to stain a small number of flies for your intended marker to ensure the effector/split combo is working as intended
• You should pilot multiple light intensities to find what bests creates and effect in experimental flies while mitigating effects (light effect, heat effect) in control flies
  • these effects include more activity while light is on (heat), slowing to light onset (startle effect to light), bouts of walking triggered by light onset (light)
• In general, shorter optogenetic stimulation is preferred given the possibility of depolarization block (CsChrimson) or other effects beyond the expected.
• Behavior flies (head closed) often have better behavior. Past experiments have used a general threshold of running at least 10mm/s for most running bouts as a cutoff.

See Notes on Getting Robust Behavior, Controlling enclosure temperature and humidity, for other information