Hi guys,

How are you?  Quick update: I did manage to get the speed reinforcement to work. Thus, I will start the screen this week!

This is the same experiment as I previously showed of Gr66a>Chrimson (ATR). The only difference is that the light was on for the whole experiment, so that the flies could see the light before the entered the arm. Previously the light switched on once the fly went into the arm. The phenotype is much stronger (there is some classical component in it). I was trying to reinforce left or right turns but it does not seem to work after a bit trying out. It makes sense ecologically I think, that the right or left turns are not coupled to the reinforcement systems. I also have been thinking about the CS-US relation bitter taste-turn directions does not make sense ecologically, but maybe if instead of bitter, I apply pain or heat …it could work. I was thinking of reinforcing orientation as well as a speed threshold, or any other variants. What do you think? I would appreciate some ideas. Since I want to make sure about what am I measuring: operant/place/classical…

Schematic of the Y-mazes closed loop.  A setup consisting of a rig with many Y-mazes is illuminated from below with a diffuser in between. Above a camera records from the behaving flies in the Y-mazes and track different parameters online over the time course of the experiment with a custom software. The initial paradigm detects whenever the fly enters the middle-vertical arm and consequently send the signal to the projector to illuminate that arm, which in turn reinforeces the fly behavior.

Below the validation tests at three different intensities (columns: intensities relative to projector max. output). I measured three different parameters for this validation (rows), for two conditions, with and without ATR (first and second graph). It seems like the paradigm affects both, number of entries and dwelling time within the arm. It seems like at the maximum intensity, even without ATR there might be an effect.

praktikumandmine

Here I attach the results in a pdf file from the students praktikum with an additional line I tested on my own meanwhile (Gr28bd and TrpA1 drivers together). They seem to work as a really good positive control btw, good for technique optimization.

For the students I tried out two different split drivers, the MB058B, which targets PPL1-a’2a2, and MB301B, which targets PAM-b2b’2. In addition the Gr5a driver, because it targets the “sugar” neurons. From the split drivers I wanted to see if I still get a validation from my initial model. MB301B seems to do quite what my model would predict but MB058B maybe not. Hopefully in a future screen I would be able to test many more and make a much more precise modelling.

linearmodelling

This is the markdown showing the protocol and results of the modelling for the choice in the T-maze. This is for calculating valence. Nevertheless, this needs to be confirmed with the results of more lines, it could be that it is overfitted, I would like to do in addition cross-validation. I´m actually doing crosses and finding new lines to have more lines to test.

Last week I continued the experiments for the optogenetics. These are my results:

and these are Lena’s results:

Compared to Lena’s results, my results are a bit too positive.

As a pilot for future experiments I tried to reproduce results from Keene et al., 2012. There, they substituted sugar by a laser to provoke PER after 24h of starvation (females, one week old). In TH-GAL4 they found 100% response when they pointed the laser to head or thorax. In tdc2-GAL4 indeed they found 50% of flies responding to heat but only when the laser was pointed to the thorax but not to the head.
Since we do not have this kind of laser, they used, I played around with an infrared light. Flies were fixed to hooks and a clamp as always. First, there was given a filter paper soaked with EVIAN-water (negative control), then there were 10s of heat (36-38°C), afterwards two positive controls: 30% sucrose on the legs and finally on the labellum.
In the figure, you can see that I am able to reproduce the methods with an infrared lamp instead of a laser. The TH-GAL4 flies crossed to TrpA respond much more to heat compared to the controls (see figure in blue and red). The proboscis extension appears already after a few seconds.
Unfortunately, I could not reproduce the results for tdc2-GAL4 (see figure in green). There was no response to heat found at all. (tdc2-GAL4 has to be ok, because I see fluorescence when crossed to GCaMP). Interestingly, the flies did not respond as strong to the 30% sucrose as expected. The starvation may have to be longer to increase motivation?
Next step will be to see a phenotype that can be seen by activating tdc2-GAL4. That may be PER after longer starvation or flight behavior.