Flexible Valence Coding by Dopaminergic PPM2 Neurons in Drosophila

Dopaminergic systems are involved in various physiological processes including motivation and valuation. Studies on Drosophila melanogaster imply that certain insect dopaminergic neurons (DANs) are central for the mediation of valuation, as activity in these neurons can substitute for teaching signals in classical conditioning. This view may oversimplify the complex circuitry of the fly’s dopaminergic system. We focus on an understudied population of DANs and their involvement in valence coding. Using a combination of optogenetics and different operant self-stimulating paradigms, we investigated whether flies expressing an optogenetic channel in DANs of the posterior protocerebral posterior medial cluster (PPM2) would approach or avoid optogenetic stimulation. Flies’ choice was not consistent over the course of our experiments: Initially, animals avoided the stimulating light but this avoidance weakened over time and shifted to mild approach behavior in the final stages. Pharmacologically impairing DA synthesis abolished both effects. Actively exposing flies to the stimulating light aimed to identify whether the valence shift was due to accumulating neural signaling/dopamine release during the experiments. However, exposing flies to light before testing did not induce any preference changes in one-minute choice, hinting that the behavioral change does not occur from prolonged signaling in these neurons. The observation that the very same dopaminergic neurons can mediate both, avoidance and approach behavior in our self stimulating experiments, challenges a central, dopamine-mediated valuation system. Additional findings from flies that express the optogenetic channel in heat-sensing neurons further weaken the claim of centralized dopaminergic punishment neurons, as heat-punishment was independent of dopamine.

TH-C-AD; TH-D-DBD x UAS-CD8::GFP Stainings

Green: GFP
Magenta: BRP

First T-Maze trial with previous light exposure

Final results of yellow light T-Maze experiments with dopamine depletion using 3IY

In previous experiments we tested flies expressing the optogenetic CsChrimson channel in PPM2 neurons. We observed mild avoidance at 1 minute, which decreased over the time course of 10 minutes, even leading to positive choice indices. To verify whether the observed effect was instructed by dopaminergic signaling, I depleted flies of dopamine using the competitive tyrosine-hydroxylase inhibtor 3-Iodo-L-Tyrosine, and tested flies again in red and yellow light T-Maze, for 1 and 10 minutes. The figure below shows the results for the yellow light T-Maze. Although we can see the same trend from avoidance in the beginning to mild approach after 10 minutes, this effect was not significant. For the 3IY treated experimental groups, we can assume that the general effect mediated by these neurons seems to be absent, when animals are depleted of dopamine.

Although I could not observe a significant effect here, I plan to follow up these experiments with yet another set of T-Maze experiments. This time I plan to actively activate the neurons prior to testing. I hypothesize, that prolonged activation of the neurons might affect their valence for the animal and therefore expect animals which experienced this activation to show differences in 1 minute testing.

Yellow T-Maze Results

I performed the first set of T-Maze experiments, which included 3IY treated flies, with red light. In this experiment I could nicely reproduce earlier results in PPM2 flies only treated with ATR. Flies initially showed weak avoidance of optogenetic stimulation, and developed a weak approach-behavior over the time course of ten minutes. In the 3IY treated flies I found similar avoidance after 1 minute of testing but, interestingly, flies kept the same level of avoidance also for a choice time of 10 minutes. This indicates that initial avoidance might be independent of dopamine, but prolonged or repeated release of the neurotransmitter from PPM2 neurons might lead to circuit changes, weakening avoidance behavior, potentially even changing it to approach.

The new set of experiments uses yellow light instead of red light. This experiments are especially interesting, as I observed stronger effects for the experimental group for yellow light.

There are two main points to discuss about the data.
First, the negative control (Gr28bd+SUC+EtOH) … I was hoping that I solved the problem with the avoidance in flies that were not treated with ATR. These flies should not avoid optogenetic stimulation since without the chromophore, there should be no, or at least very weak, activation of the CsChrimson channel. Even if the sample size of 5 is rather low, it is a bit worrying that when these flies were tested for 10 minutes ((Gr28bd+SUC+EtOH (B)), they show avoidance comparable to control flies that were treated with ATR and tested for 1 minute ((Gr28bd+SUC/3IY+ATR (A)).

On the other hand, the experimental groups look pretty good. For now, I was not only able to reproduce results from the first 1 vs. 10 minute T-Maze testing with yellow light, it also seems that 3IY-treated PPM2 flies show the same phenotype as when tested in red light.

For the next few weeks I will have to increase sample sizes, aiming for 30 for each of the experimental groups. I will only include a few control groups treated with ATR, as the effect size here seems to allow for a lower N. Presumably, I will include more untreated control flies, to see whether the avoidance will persist or if the current results simply arise from the low sample size.

Update 28/11/25

Proceeding with T-Maze experiments

After pausing T-Maze experiments because of the issues with my negative control, I should now be abled to proceed.

Since it’s been a bit more than 2 months since the last results, this was the state of the experiments back then:

Now I added the following results:

Leading to an updated version of all results:

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.

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.

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.

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.