T-Maze results dopamine inhibition
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.
NWTB_3IY_ATR = 16 ; NWTB_3IY_EtOH = 13; NWTB_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.
First results from optgenetic experiments with PPM2 flies after inhibiting dopamine synthesis with 3IY
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.
The never ending story has ended
After 7 attempts to figure out the treatment with the dopamine-synthesis inhibitor 3IY we finally managed to get it right.
Trial 6 was a minor set back since I used the same method that had worked before but prepared 8 mL instead of 2 mL. 3IY is not really soluble so I thought that the first “fraction” of the solution might not contain enough of the inhibitor to sufficiently deplete dopamine. I tested WTB flies with the first fraction of the 8 mL preparation.
I did not observe differences between groups. N(3IY)= 14; N(SUC) = 12
Since I cannot prepare the 3IY for each vial I am going to use (~ 8 per testing day) separately and put them on the vortex for 30 minutes each I tried if shortly vortexing and stirring afterwards would enough. Additionally, I tried tissue and filter paper again since the previous tissue paper trial might have had the same problem that 3IY was not present in high enough amounts in the first fraction.
N= 14 for each group; FP = fitler paper; TP = tissure paper
Since this method seems to work, I can now prepare enough vials at the same time and start with the actual experiments. I only need to figure out how to apply the ATR….
JoyStick results with 650 Lux yellow light
We were having some issues with our Negative Control for the JoyStick. Without ATR the flies should not avoid optogenetic activation since the channel should not be functional, yet still they did. We lowered the light intensity from 800 Lux to 650 to see whether the light was to strong and would activate the Chrimson channel, even without ATR.
Alisa’s experiments
Alisa+Daniel experiments
Final 3-Iodo-L-Tyrosine trial before testing PPM2 flies.
I confirmed the action of the dopamine synthesis inhibitor 3-Iodo-L-tyrosine (3IY) in a previous trial, where treated flies showed decreased walking speed and distance when tested in an open field. In this trial I tested the flies “fresh out of the tube” so that they were able to feed on 3IY right to the point where they were tested. For the main experiment however, flies will be transferred from the food 2-5 hours before the experiment. To confirm, that the effect of 3IY treatment will persist also after this time span I moved flies from 3IY supplemented vials to fresh, empty vials at least 4 hours before measuring walking behavior in the open field. The results confirm, that the effect of 3IY is still present after this time.
3-Iodo-L-tyrosine Trial 3
For this trial I prepared 2mL of 3IY (10mg/ml in 5% Sucrose) 30 minutes before applying it to 8 pieces of filter paper stacked in the glass vial. I pipetted the whole volume of 2mL into the vial and discarded the excess liquid.
Trying to fix the Negativ Control for JoyStick Experiments
Recent results revealed problems with our negative control (Gr28bd-Gal4; TrpA1-Gal4+ EtOH) treated with EtOH. As the food for these flies was not substituted with ATR but only with Ethanol (ATR is dissolved in EtOH), the Chrimson channel should be non-functional in this group and the flies should therefore not avoid light. However, we observed negative PIs for this group. Since some activation of the Chrimson channel could be possible even without ATR, I tried to lower the used light intensity to values where the positve control would still avoid light, but the intensity would be too low for the Chrimson channel to activate when no ATR is present.
It seems that 650 Lux might still be too high. Therefore I will conduct another trial with 500 Lux.
JoyStick results for re-testing PPM2 flies
It’s not yet confirmed, that the 3IY treatment is working
Results from different dopamine depletion trials with 3-Iodo-Tyrosine (3IY)
To see whether the initial preference but also the preference shift, observed for flies expressing the optogenetic Chrimson-Channel in neurons from the PPM2 cluster (tested for preference in JoyStick and T-Maze), does depend on dopamine signaling, I want to repeat these experiments with dopamine depleted flies. For this I plan to use 3-Iodo-Tyrosine, which should reduce dopamine production. To verify the effect 3IY I conducted two sets of experiments, testing locomotion and geotaxis. Successfully depleting dopamine should decrease locomotion and performance in geotaxis assays. At this point both experiments have the same outcome, that the tested method to apply 3IY does not work.
Locomotion in open field arena
Geotaxis in Benzer Counter-Current
