This is an update of the T-maze experiments for flies with OA and DA neurons manipulated.

1. TH-G4s x shi^TS.  Except for 2 groups the N is at least 5.

2. TbH-lexAs x shi^TS.  Last year I tried to complete this experiment and several problems appear. Now I am starting to do it again and it seams that it works. N=4.

3. I am starting to test the same TbH lines crossed to TrpA1. So far, not much to say.

After finishing the first round of the screen to find out the DA substrate of the photopreference, I decided to change the genetic background of the GAL4s and UAS. Now all of them are in WTB background.

Here are the first results. It is only an n of 1.

Update:

As a control for my T-Maze experiments I decided to test if treated flies were able to fly at the same conditions they were tested in the T-Maze (32°C).

As in the T-Maze experiments, I put the flies 15 min at 32°C before testing them at the same temperature. The, I measure the duration of the first spontaeous flight (Blue). If within 5min the fly doesn’t start to flight, I gently remove the steroform ball from the legs (Red), simulating a takeoff.

Details from 0 s to 200 s.

As a control for my T-Maze experiments I decided to test if treated flies were able to fly at the same conditions they were tested in the T-Maze (32°C).

As in the T-Maze experiments, I put the flies 15 min at 32°C before testing them at the same temperature.

The dashed line at 11 min indicates when a T-Maze experiment would have ended.

Almost all flies tested flew more than the time required to perform a T-Maze experiment.

In previous experiments, I found one TβH(lexA)>shi^TS  combination that recapitulated TDC2>shi^TS T-Maze results (https://lab.brembs.net/2015/09/looking-for-the-da-oa-neurons-involved-in-phototactic-flexibility/). Here I present the expression pattern of those two TβH-lexA drivers used.

TβH⁵⁴⁹⁵⁴-LexA (Brain)

TβH⁵⁴⁹⁵⁴-LexA (VNC)

TβH⁵⁴⁰⁷⁵-LexA (Brain & VNC)

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.

In order to find which dopaminergic and octopaminergic neurons are related to light preference and the switch on it seen after clipping the wings, I decided to use a tool called CaLexA.

Here we can see my first attempt to use it. We can see the CNS from TH>CaLexA flies with and without wings.

I did not see any special signal in the brains, but I still have to play around a little bit more.

With Wings

Without Wings

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.
3rd

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.

Results:

–          1^st cross

–          2^nd cross

Flies:

–          1^st cross (with heterozygous controls):

                  males                         females                                          females

w1118,tßh/y ;; HStßh  x  w+,tßh//FM7   ->    w1118,tßh//w+,tßh ;; HStßh//+
w1118,tßh/y                    x  w+,tßh//FM7   ->    w1118,tßh//w+,tßh
w1118,tßh/y ;; HStßh  x  w+                        ->   w1118,tßh//w+ ;; HStßh//+
w1118,tßh/y                    x  w+                        ->    w1118,tßh//w+

– 2^st cross (only mutants):

w1118,tßh/y ;; HStßh  x  w+,tßh//FM7    ->    w1118,tßh//w+,tßh ;; HStßh//+
w1118,tßh/y                    x  w+,tßh//FM7    ->    w1118,tßh//w+,tßh

Heat shock timing: