I am starting to look the data generated by Isabelle to generate the final graphs and the definitive analysis of it. This is a sample of a possible plot.

To test if diazepam may alter also photopreference in the T-maze, I first tried to replicate a previous experiment published recently in “Ancient Anxiety Pathways Influence Drosophila Defense Behaviors“, to ensure the method of delivering diazepam to the flies was effective.

The published methodology was only slightly modified. I used a round glas petri dish (diameter 100 mm, height 15 mm) above a luminous plate instead of a square plastic chamber (10 x 10 x 1.6 mm) which was lit from the sides. Also, the flies fed on filter paper soaked with a 5mM diazepam solution (in 10% ethanol, 5% sucrose, 5% yeast and food dye), instead on the CAFE capillaries. After starvation over night and 6h diazepam treatment, WTB flies were tested at 25°C.

(D = Diazepam; nD = no Diazepam)

In the paper, it was shown that the speed of flies which got the diazepam treatment gradually decreases. In this data here there can also be seen that the median speed is slightly lower than in the control group, suggesting that the treatment seems to be working.

The following heatmaps also show a tendency that diazepam treated flies may be spending more time in the center of the dish than the control flies.

However, these effects are not nearly of the size as those in the published paper, so I’ll have to try and play around some more with the size of the container to see if this is the reason why my effects are so tiny.

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)

Figure 1 shows the results of the tbh-subgroups screen, tested at RT and 24 h later at 32°C.

All genotypes with n=8, expect tbh(39942) x Shi^TS (n=4).

Fig 1: tbh subgroups screen

Figure 1 shows the newest results of the screen of the tbh-subgroups.

Fig. 1: tbh-subgroups tested at RT and 32°C

In addition I dissected brains of these flies.

Fig 2: 39940 x GFP (nucl)

Fig 3: 41306 x GFP (nucl)

Fig 4: 48333 x GFP (nucl)

Fig 5: 45904 x GFP (nucl)

Fig 6: 46972 x GFP (nucl)

Fig 7: 47755 x GFP (nucl)

As a next step I’ll compare my “stainings” to the ones from flybase.

As I said in my last post, I started doing the Benzer again but instead of giving them 15 seconds for every single choice , they had 1 minute.

In Fig. 1 you see the cumulative proportions for the tubes 3-0. All of them have an N of 2.

Fig. 1 cumulative proportions

In the following graph you see the final results of the trials done to investigate the genetic background of different wildtype lines.

Figure 1: different genotypes tested at different temperatures. (n = 8)