After last week’s “breakthrough” with our method to sufficiently inhibit dopamine synthesis with 3IY it is time to start testing flies that express the optogenetic Chrimson channel in dopaminergic neurons from the PPM2 cluster.

ATR-Trial: Mix ATR directly with Sucrose / 3IY

Initially I stumbled across another problem, namely that the ATR, which is needed for the Chrimson channel to open, could not be applied in the same way as I did before. Usually, to prepare flies for JoyStick or T-Maze experiments, I would pipet 15µL of ATR onto their food. Here it was important to make sure to spread the ATR evenly across the surface since it has a bitter taste and flies would avoid consuming it if possible. This obviously would be problematic since then the basis of our experiment, optogenetic activation of the target neurons, could not be ensured.
Since for the 3IY treatment flies will not be kept in vials with the standard fly food, but vials with tissue paper soaked with sucrose, it was problematic that the tissue paper would simply soak up all the ATR in one spot. To battle this problem I tried mixing 20µL of ATR directly into the 3IY or sucrose solution. To confirm that this method still works I conducted a first trial only with control flies:

Flies that were kept in vials where the sucrose/3IY solution was not supplemented with ATR should not be affected by the light and should therefore not show any preference (CIs close to zero). Flies that could feed on ATR should avoid the light and show negative CIs, since the fly strain expresses the optogenetic channel in heat-sensing neurons and activation of these neurons would lead to an unpleasant sensation of heat.
The very low sample size is most likely the reason why both Negative control are not 0, but the fact that the group which was supplemented with ATR shows CIs close to -1 indicates that it’s okay to simply mix the ATR with the sucrose/3IY solution.

JoyStick-Results

After confirming the method to apply ATR we started JoyStick experiments with 5 groups:
Gr28bd+TrpA1+SUC+EtOH: Control without DA inhibition and no ATR (Negative CTRL)
Gr28bd+TrpA1+SUC+ATR: Control without DA inhibition and ATR (Positive CTRL without DA-inhibition)
Gr28bd+TrpA1+3IY+ATR: Control with DA inhibition and ATR (Positive CTRL without DA-inhibition)
PPM2+SUC+ATR: Experimental group without DA inhibition and ATR
PPM2+3IY+ATR: Experimental group with DA inhibition and ATR

For now the results look okay. CTRL groups with ATR already tend to avoid optogenetic activation, which is good. For all other groups a larger sample size (target = 50) is needed.