Light-induced activation of Chrimson does not induce HSP70 expression

In the previous experiment, no detectable HSP70 signal was observed following optogenetic activation of heat-sensing neurons for 5 or 15 min (first experiment). To investigate whether longer stimulation could trigger HSP70 expression, the light-exposure duration was extended. Additional control groups were included to determine if the absence of HSP70 could be explained by the presence or absence of all-trans-retinal (ATR), the ethanol used as its solvent or the flickering light exposure itself.

As a positive control, wild-type Berlin (WTB) flies were again exposed to 37 °C for 1 h to induce a heat shock response. The optogenetic groups (genotype ♂ Gr28bd; TrpA1 × ♀ norpA; 20× UAS-Chrimson) received ATR-containing food and were exposed to flickering red light for 15, 30 and 60 min. One group reveived ATR-containing food but was not exposed to light (ATR, 0 min). This control was used to assess whether ATR feeding alone could influence HSP70 expression. Another group reveived ethanol without ATR and was exposed to light for 60 min (ethanol, 60 min). Since ethanol was used as the solvent for ATR, this group showed if ethanol itself could affect HSP70 expression during light exposure. As an absolute negativ control for HSP70 expression under these conditions, a group only received water and was not exposed to light (water, 60 min).

HSP70 expression was analyzed by Western blotting, using the same methods as in the previous studies. It was detectable only in the WTB positive control following real heat shock. No HSP70 signal was detected in any of the other groups, including the ATR-fed flies exposed to light for up to 60 min and all negative control groups.

These results show that not even 60 min of optogenetic stimulation triggers HSP70 expression, which leads to the assumption that optogenetic activation of heat sensitive neurons generally does not lead to any HSP70 expression under the tested conditions. The negative controls also provided no evidence of detectable HSP70 expression associated with ATR feeding alone, ethanol feeding alone or flickering light exposure in the absence of ATR.

To rule out the possibility that the genetic modification required for Chrimson expression affects the flies ability to express HSP70, flies of the same genoytpe will be exposed to a real heat shock at 37 °C for 1 h to determine if they can induce HSP70 expression in response to actual heat shock.

Testing optogenetic activation of heat-sensing neurons as a stimulus for HSP70 expression

In other animals, such as C. elegans, activation of specific sensory neurons can induce the expression of heatshock genes in other tissues, suggesting that neuronal activity may trigger a systemic heatshock response. However, it’s not clear if similar mechanism exists in Drosophila melanogaster.

For the following experiment, flies of the genotype ♂Gr28bd; TrpA1 x ♀norpA; 20* UAS chrimson were used for optogenetic activation of heat-sensing neurons. They inherit both the heat-sensitive ion channels and the light-activated channel Chrimson under UAS control. When fed with all-trans-retinal (ATR), Chrimson becomes functional in neurons. Upon light exposure, Chrimson activates these neurons, causing them to fire as the flies were experiencing extreme heat, even though the actual temperature remains normal.

For practical reasons, only female flies were tested. The flies were fed ATR-containing food for two days before testing. For optogenetic stimulation, the flies were exposed to red flickering light for 0 min as negative control, 5 min or 15 min. Following light exposure, the flies were allowed to recover for 1 h at 25 °C before being frozen in liquid nitrogen. As positive control, WTB flies were heated for 1 h at 37 °C and processed in the same way.

The results were analyzed by Western blotting. HSP70 was detected using a rat anti-HSP70 (1:3000) primary antibody and a donkey anti-rat (1:10000) secondary antibody with fluorescence detection at 700 nm (red). Actin was used as loading control and detected using a rabbit anti-actin (1:10000) primary antibody and a goat anti-rabbit (1:10000) secondary antibody at 800 nm (green). HSP70 signal intensity was quanified and normalized to the corresponding actin signal.

HSP70 was detected only in the positive control and was not detectable in any of the optogenetic stimulation groups. This shows that short light exposure to activate heat-sensing neurons does not cause HSP70 production under this conditions. The duration or intensity of the optogenetic stimulation may have been insufficient to trigger a measurable heat-shock response. Therefore, the same genotype of flies will be tested using 1 h of light exposure, corresponding to the duration of the actual heat-shock treatment. Additional control groups will also be included to rule out other possible HSP70-expressing effects (following experiment).

Miray- JoyStick with 0273-Gal4 (Red Light 500 Lux)

Ömer – Yellow Light T-Maze: 0273-Gal4 x NorpA;20xUAS-Chrimson

Update: 10/08/26

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.

First T-Maze trial with previous light exposure

Since we observe a behavioral shift in TH-C-AD;TH-D-DBD flies over the time course of ten minutes, I hypothesized that this might be either due to an excess neuronal stimulation during this ten minutes (threshold) or that flies do learn something about the contingency between their own behavior and the resulting stimulation. To test these hypotheses, I stimulated flies for 5 minutes with the same light stimulus usually used for the experiment. During this 5 minutes, flies had no control over the stimulation therefore excluding a learned association of behavior and outcome. If the behavioral switch was merely due to excess stimulation, this 5 minutes of uncontrollable exposure should also affect behavior.

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:

Almost there

In this set of JoyStick experiments, I tested flies expressing the CsChrimson channel in OA-VuMa octopaminergic neurons, which are the Drosophila counterparts to the honeybee OA-VUMmx1 neurons. There are still a few flies left, but for now activation of these neurons does not seem to mediate any strong innate valence. There may be a slight tendency for approach in the alter periods but it might also be by chance, since we can already observe positive values in the pretest.