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.

Firstly, I did an experiment where Dop1R1 was knocked down via RNAi. The following shows the results:

For the next experiment, the controls groups were the same, however in the experimental group Amon was down regulated. These are the results:

Lastly, in the course the data for the larvae experiment was collected only under blue light, so I used the same crossings and tested them under red light to use as a control.

All figures show the results for preference tests (salt/pure) conducted under red and blue light for different gal4-lines.

MB145B:

SS01716:

SS02180:

The first figure shows the results for the preference test (salt/pure) conducted with amon-RNAi only under red light.

For the next experiment I conducted the same preference test but with the split-gal4 line MB143B under red and blue light.

The first experiment tested the activation of KCs with simultaneous ablation of pPAM which showed the following results:

Green: UAS-ChR2-XXL; LexAop-rpr x 58E02-LexA; H24-Gal4

Lightgrey: UAS-ChR2-XXL; LexAop-rpr x H24-Gal4

Darkgrey: UAS-ChR2-XXL x 58E02-LexA; H24-Gal4

The next experiment was conducted with the split-Gal4 line MB054B (DAN-f1/DAN-g1) and showed the following:

With the focus on dopaminergic neurons, I conducted two experiments with two different driver lines to see how the naive gustatory behavior in Drosophila larvae is affected.

The first cross I tested was R58E02-Gal4 x UAS-ChR2-XXL, which gave me the following results:

As seen in the figures, there was no effect under blue light.

For the second experiment I crossed TH-Gal4 x UAS-ChR2-XXL with these results:

With this experiment, there was an effect on gustatory behavior as there is a clear significant difference between the experimental group and the control group.