Been working on 17d for the past month.

The experiments in the flight simulator. Self-learning performance indices in a two-minute test with the heat switched off after 4 and 8 minutes of training, indicated impairment of 17d-TNT flies.

The flies also showed clear impairments in their flight performance. To quantify this I assessed both possible alterations in their motor coordination (using climbing assay) as well as flight performance. The climbing assay relies on walking rather than flying. Both experiments show reduced ability of motor coordination and flight performance.

To confirm the specificity of the 17d-Gal4 fly I used the trans-tango flies.

Neuron, 96 (2017) 783-799. doi:10.1016/j.neuron.2017.10.011

The trans-tango is notorious for having a low expression in adult flies, which was also observed by me. The image is taking without any GFP-antibody.

These are all the graphs I could fetch from other articles referring to TH-D’ expression.

from Galili et al. 2014:

from the table in B, the expression of TH-D’-G4 is different from TH-F3-G4 in PPL1 regions projecting to alpha and alpha’ as well to dorsal Fan Shaped Body (dFB) and DP. I would not focus on the alpha projections into the Mushroom bodies (MB), because the other G4s targeting the alpha lobes did not yield any effect.

In PPM3 only is different the projection to the ellipsoid body (EB). In addition from graph A, PPM1 and PAL regions are stained by TH-D’ but might not be targeted by the other drivers shown.

from Liu et al. 2012:

We see that other drivers that were also tested in our screen (like TH-D4 and TH-G1) also stain the PPL1->dFB, PPL1->DP and PPM3->EB. Since these two drivers did not have a phenotyp,e we might not attribute the effect of TH-D’ because of these projections.

From Pathak et al. 2015:

We have a different pattern where they do not describe expression in regions like PPM2, PPM1 or PAL. They point out the expression in PPM3 and PPL1 but we already discarded these regions as the ones involved in reinforcement in the graphs above in this post. They also observed expression in PPL2, which might be a region also stained by TH-G4, TH-D1 and TH-C’, but we do not know how they overlap. A few TH+ neurons in the ventral ganglia are also targeted by TH-D’.

From White et al. 2011?:

We see more general dopaminergic anatomical properties, like the number of neurons in each dopaminergic cluster. In the second graph one can see where the PPL1, PPM3, PPM1/2 and PAL project to.

from Xie et al. 2018:

I would say that the only two interesting columns are 1&2 and 2&3 which finds common regions for TH-C vs TH-D and TH-D vs TH-F, respectively. The only conclusion I would take from the first is that a few PPM2 regions are discarded as interesting, and from the second that the whole PPL1 does not seem to have differential expression in TH-D’.

Confocal image MAX stack of one of the brains at 20x

and at 40x

In this link we have a video of a 3D stainning pattern          zoomed_CC

In addition I add here teh boxplots from the final results of the Ping Pong ball setup with these experiments

The first figure shows each of the central complex ring neurons types (Martín Pena et al., 2014). The c105-G4 targets the R1 neurons and the c232-G4 targets the R2 and the R4d neurons

This is the c105-G4 stainning from Martín Pena et al., 2014

232-G4 stainning from Kahsai et al., 2012

Axel stainning from c232-G4 alone

Axel stainning from c105-G4 alone

Axel stainning from both drivers together. I would say it really contains both driver lines.

This are both driver lines together as well from Axel. To me it seems that only c105 is present

This are my stainnings at the fluorescence microscope (no confocal). This is to show that in all of the 10-12 brains I have looked at, they all had the c232 pattern present

In addition, they had many more neurons outside from the central complex which I believe belong to the c105-G4 line. This is my only proof to show that c105 is also present, since the R1 neurons seem to be hidden when R2 neurons are stained.

I was also looking to the youtube video you have online, Björn. To me it seems I can only see the R1 ring neuron from the c105

Here I used the CaLexA tool with the elav driver, and reared the flies on constant darkness or under L-D cycles.

L-D Cycle

D-D

I recently combined the two TbH-lexA  lines (54954 & 54075) with CD8GFP, and the TDC2-GAL4 line with CD8RFP, in order to compare their expression patterns. Here I present some of the dissections. The confocal is not working properly, but it is relatively good to draw some conclusions.

TDC2>GFP and anti-TβH (Scholz’s Lab)

TDC2>GFP (Gerber’s Lab)

anti-TDC2 (Goodwin’s Lab)

After refining my DA screening, I end up having three interesting GAL4s which lead to changes in photopreference after expressing Shibire and rising the temperature. What I am trying to do now is to understand if they label the same neuronal population or not.

In order to study the role of OA and DA in photopreference I am constantly looking for new drivers that label subpopulations of these groups. I recently found a TbH driver from the QF system, and wanted to know how representative of the TDC2-G4 neurons was. I have established a QUAS-mCherry;TbH-QFs line (Magenta) and crossed it with TDC2-G4;UAS-GFP (Green). In both cases, what is shown is the endogenous expression.

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)