Christine DamrauView Profile
Tested the third cross. Now, it is around 15n each group. After a control experiment I preferred a recovery time of 1h instead of 5h. A short pilot for control groups showed that Tdc2virgins crossed to w1118males have a flight defect.
Arnim Jenett (Janelia Farm Research Campus), Kazunori Shinomiya, Kei Ito (both Tokyo University), and other anatomists made a great site with a 3D-viewer of adult Drosophila brains available. You have the chance to scroll threw a whole mount stack while ticking different brain areas. Those brain areas are listed next to the stack. Different areas are coloured differently, so that you can look at the location of several areas in the same brain. On the main page you can find simply explained tutorials about the usage of the site. It is correlated to the anatomical search engine of the Janelia farm GAL4 collection.
Because it was very helpful to me to learn all the synonyms of relevant areas and because I think it is very helpful to learn more about the structure of the Drosophila brain in general I wanted to advertise the site here.
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.
The loss of tßh in adult flies leads to decreased walking speed and an increase in stripe fixation. Rescuing the gene by a heat shock construct (flies from Henrike Scholz, Cologne) increases walking speed back to wild type level but cannot change stripe fixation.
It is possible that the phenotype in stripe fixation is not exclusively tßh-dependent but more due to other problems the tßh-mutants have, e.g. developmental defects. That would ask for another heat shock timing.
– 1st cross
– 2nd cross