The 1st cross produced eggs, but they did not hatch or become pupae. In all of the 10 vials, eggs were observed, but no pupae. On the other hand, the 2nd cross did not produce any eggs. (The first trial was kept up for 10 days. After 10 days, a new trial began from the same stock. ) Control and the other two crosses produced eggs and hatched new flies.

The same crosses were repeated to ensure the lethality. In the second trial, ‘T4/T5xWTB’ cross produced eggs and pupae. But there is still the possibility that the T4/T5xWTB is also a lethal crossing. ‘T4/T5xTNTE’ cross again produced only eggs but no pupae. The other two crosses produced pupae again and became dark, but haven’t hatched yet. The second attempt is 10 days old.

Control groups fed with ATR (Gr28bdTrpA1_Suc_ATR and Gr28bdTrpA1_Suc_ATR) look fine and for now the avoidance by activating heat sensing neurons does not seem to depend on dopamine. The negative control (Gr28bdTrpA1_Suc_EtOH) shows stronger avoidance than we would expect. Also PIs of PPM2 lies do not follow the same pattern as we previosly observed… My current hypothesis is that some sort of mutation led to the Chrimson channel being more sensitive to light, even without ATR. Since we rear the PPM2 flies in light until they receive the ATR this might affect their behavior.

Luisa’s results for yellow light:

After the 10th day, cross 2 and the control group hatched new flies. On the 13th day, all of the groups have hatched flies. None of the crosses seems to be lethal.

Confirmation of 3IY+ATR

After figuring our how to apply 3IY to the flies and confirming that we can simply mix in the ATR with the sucrose to apply it, I stumbled upon another problem: when 3IY and ATR are used together the tissue paper will go from yellow to orange:

Since we cannot know if this affects the action of 3IY I conducted a final trial in the open field, measuring locomotion of WTB flies after treating them with 3IY and ATR for 48h.

N_{WTB_3IY_ATR} = 16 ; N_{WTB_3IY_EtOH} = 13; N_{WTB_SUC_EtOH} = 8

It seems, that ATR does not affect the action of 3IY and we can proceed with our experiments.

T-Maze results

These are the first results from the set of T-Maze experiments with 3IY treatment. I used red light (1600 Lux) with a decision time of one minute. P-values above plots indicate results of Wilcoxon’s test.

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

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.