Fussl was crossed with either Stinger (ctrl) or a UAS-TNT line to block the synaptric transmission of the Fussl positive neurons. A third construct was used but did not yield any data due to difficulties with their flight performance. The Fussl-Stinger along with Fussl-TNT flies do also show difficulties in flying. These differences will be assessed.

The experiment was done as a pilot experiment before doing a larger scale.

The data is a bit inconsistent but shows a positive and reassuring numerical difference. The control is a bit lower than expected, compared to WTB flies (showing usually a PI 0f 0.6). The flies have a slightly different background than wtb flies and have pale orange eyes (still no apparent impairments in vision). Further experiments will be conducted before proceeding with a larger sample size of the flies.

Positive Control: Gr28bd-G4, TrpA1-G4

Parameters: Light: intensity (500 Lux side, 1000 Lux bottom); frequency = 20Hz; Delay = 1 ms; Duration = 9.9 ms; volts = 6.4

Red lines: completed

mb025b: not selected against tubby

Results of the T-maze screen analysis, both individual and combined.

Yellow 1 (Positive Control): Gr28bd-G4, TrpA1-G4

Parameters: Light: intensity (500 Lux side, 1000 Lux bottom); frequency = 20Hz; Delay = 1 ms; Duration = 9.9 ms; volts = 6.4

Yellow 1 (Positive Control):  Gr28bd-G4, TrpA1-G4

Light: intensity (500 Lux side, 1000 Lux bottom), frequency (20Hz)

Not confocal immages (going to the confocal this week)

The construct I am analyzing is the FoxP-IsoB_Gal4, so with the Gal4 inserted in the 8th exon

Larvae

Adult VNC

Some traces from this week just so that you have an idea how do they look like. To me they are not the optimal traces I expected. But one can see some signal there. I will start the screen hoping to get enough good traces without too much work.

what do you think is the best quality control for accepting a trace for the analysis or not. I was thinking the 3D mapping gives a good hint but without quantification.

In addition I was writting to Andi Straw to solve the issue of running two cameras in the same computers, but now answer

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.

Since we have problems with Madeleine’s project, I will make crosses to test that the flies are ok before using them for PKC RNAi experiments.

the putative elavGal$;tubGal80ts flies will be crossed to w; AKAP/Cyo flies. Then the son ElavGal4/Y, tubGal80ts/+ will be crossed to w.

Only non Cyo male flies should get white eyes, if not, the stock is not clean.

In parallel, I will also start crossing nsybGal4 into the tubGal80ts line (2 to 4 crosses necessary depending on the eye color of the heterozygotes).