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
PPM2 flies avoid the elevator
A: Red Light, 1min (N = 25-30)
B: Yellow Light, 1min (N = 26-30)
C: Red Light, 10 min (N = 18-30)
D: Yellow Light, 10min (N = 16-30)
Comparison of 1 and 10 minute T-Maze for a DAN-PPM2 driver line.
To investigate whether there are subsets of dopaminergic neurons that are responsible for “reward” or “punishment” in the absence of stimuli, different transgenic Drosophila lines were screened for approach or avoidance of neuronal activation. The different lines expressed the optogenetic “Chrimson” channel in different sets of dopaminergic neurons and were tested in different operant self-stimulating experiments (T-Maze (red light), T-Maze (yellow light), Y-Maze and JoyStick). Except for two, none of the tested lines displayed consistent preference or avoidance in all four different experiments. Both lines (TH-D’ and TH-D1) drive expression in the same DAN clusters including PPL1_FB, PPM2 and PPM3.
Since there are now more refined Gal4 driver lines, enabling expression of the optogenetic channel in only subsets of neurons compared to the original driver lines, T-Maze and JoyStick experiments were repeated with these new lines.
Rescreening in JoyStick and T-Maze showed that out of the three tested lines only one line displayed (tendencies for) light-avoidance as the original driver line. This was the line driving expression in the PPM2 cluster. (JoyStick data from other lines not shown, PPM3 could not be tested in T-Maze due to technical limitations). Interestingly in the JoyStick, this line displayed a shift in preference that was similar to the shift observed in the original TH-D’ driver line.
As mentioned above, JoyStick experiments had shown that after initial avoidance of the light (represented by negative PIs), in the last training periods flies would show PIs close to zero. In the T-Maze experiments flies are usually tested for only 1 minute. To find out whether fly preference would show a same development when choice time was extended, I conducted a set of T-Maze experiments, with a choice time of 10 minutes.
These results suggest that in the flies expressing the Chrimson channel in PPM2 DANs, prolonged choice time leads to a shift in “valence”, as also observed in the broadly expressing driver line TH-D’. This shift was only observed under yellow light.
Comparing control groups in yellow and red light, there seem to be differences in the activation of the Chrimson channel under red and yellow light. Whether this can be explained by different penetration capabilities due to the different wave lengths will be subject to further research.
Comparison PPM2 1 min and 10 min T-Maze
Results for yellow light. (A) Represents 1 minute testing (B) represents 10 minute testing.
Sample sizes:
Gr28bd+TrpA1: (A) –> 26; (B) –> 16
Gr28bd+TrpA1(Co): (A) –> 26; (B) –> 16
PPM2: (A) –> 30; (B) –> 30
Results for red light. (A) Represents 1 minute testing (B) represents 10 minute testing.
Sample sizes:
Gr28bd+TrpA1: (A) –> 27; (B) –> 17
Gr28bd+TrpA1(Co): (A) –> 25; (B) –> 17
PPM2: (A) –> 30; (B) –> 25