Optogenetic stimulation of DANs

on Saturday, September 2nd, 2023 11:09 | by

With the focus on dopaminergic neurons, I conducted two experiments with two different driver lines to see how the naive gustatory behavior in Drosophila larvae is affected.

The first cross I tested was R58E02-Gal4 x UAS-ChR2-XXL, which gave me the following results:

As seen in the figures, there was no effect under blue light.

For the second experiment I crossed TH-Gal4 x UAS-ChR2-XXL with these results:

With this experiment, there was an effect on gustatory behavior as there is a clear significant difference between the experimental group and the control group.

Print Friendly, PDF & Email

Cloning via DNA Assembly

on Friday, September 1st, 2023 7:26 | by

DNA Assembly in a 1:2 ratio of vector to insert with gRNAs of rsh and rut (Q5 and template concentration: 640 pg/µl) and 100ng of pCFD6 BbsI AP (using QuickCIP). Heat-shock (hs) transformation into E. coli (DH5α competent) with 10 µl Assembly Reaction and 100 µl cells.

For Crtl, pCFD6 BbsI AP was wrongly used.

For Crtl, pCFD6 was wrongly used.

DNA Assembly in a 1:2 ratio of vector to insert with the gRNAs of rsh and rut (Q5 and template concentration: 640 pg/µl) and 100ng of pCFD6 BbsI AP (using FastAP and 2 extraction steps). Heat-shock (hs) transformation into E. coli (DH5α competent) with 10 µl Assembly Reaction and 100 µl cells.

For Crtl, pCFD6 BbsI AP was used in reaction.

Print Friendly, PDF & Email

Line verification SS56699

on Tuesday, August 29th, 2023 7:50 | by

After dissecting brains from the GAL4 driver line SS56699 with GFP staining and finding no fluorescence, I dissected them again and the immunohistochemical staining showed fluorescence. To check that the correct neurons were stained, I compared the images with the image in the paper by Hulse et al (2021; https://doi.org/10.7554/eLife.66039) . The three PPL1 dopaminergic dorsal fan-shaped body tangential neurons per hemisphere are stained, but there are some additional unidentified neurons (presumably PPM1 neruons) visible.

Hulse, B. K., Haberkern, H., Franconville, R., Turner-Evans, D., Takemura, S.-y., Wolff, T., Noorman, M., Dreher, M., Dan, C., Parekh, R., Hermundstad, A. M., Rubin, G. M., & Jayaraman, V. (2021). A connectome of the Drosophila central complex reveals network motifs suitable for flexible navigation and context-dependent action selection. eLife, 10, e66039. https://doi.org/10.7554/eLife.66039

Print Friendly, PDF & Email

Bachelor Blog / #4 is there something?

on Monday, August 7th, 2023 2:13 | by

The offspring of my first experimental fly cohort finally hatched! Below you find a few first pre-tests I ran last week :)

First, here are the results of a quick test to see if the offspring shows a preference for the parentally trained side after the first training period:

After that I played around with the laser a little bit to find the learning threshhold. I set the laser on 2,6V but the results I got look a bit weird:

-> untrained wtb flies

-> offspring of trained wtb flies

I´m optimistic however there is still a lot of work to do…

Print Friendly, PDF & Email

Preferences with higher salt concentration

on Monday, August 7th, 2023 12:46 | by

The following plots show the results after I increased the salt concentration from 1,5M to 2,5M. It shows that the difference between the control groups and experimental group are not significant anymore.

Print Friendly, PDF & Email

Success: rsh Stock has rsh1 Mutation

on Monday, August 7th, 2023 11:11 | by

Via gDNA analysis and PCR was the specific area of the rsh gene extracted and amplified where the nucleotide substitution: C to T (Folkers et al., 2006) should be for the rsh1 mutation. The amplicon was Sanger sequenced which proved the nucleotide substitution.

Print Friendly, PDF & Email

Bachelor Blog / #3 finally, data!

on Monday, July 24th, 2023 2:22 | by

Print Friendly, PDF & Email

Little by little

on Monday, July 24th, 2023 1:59 | by

Despite very warm weather, some flies did fly, even though the learning performance of the control flies was really poor. At least for now, it looks like all stocks are learning and that rut and rsh flies learn at least equally well as the Berlin flies. I’ve also managed to fix the positive preference problem:

Print Friendly, PDF & Email

Pure vs Sugar Preference results

on Monday, July 24th, 2023 9:24 | by

Print Friendly, PDF & Email

Creating gRNAs via PCR

on Friday, July 7th, 2023 6:38 | by

1% Agarose gel with 100bp marker and PCR1 rsh, PCR2 rsh and PCR3 rsh or PCR1 rut, PCR2 rut and PCR3 rut, respectively.
The template pCFD6 was used with a concentration of 640 pg/µl.
1% Agarose gel with 100bp marker and PCR1 rsh, PCR2 rsh and PCR3 rsh or PCR1 rut, PCR2 rut and PCR3 rut, respectively.
The template pCFD6 was used with a concentration of 64 pg/µl (1:10 dilution).
1% Agarose gel with 100bp marker and PCR1 rsh, PCR2 rsh and PCR3 rsh or PCR1 rut, PCR2 rut and PCR3 rut, respectively.
The template pCFD6 was used with a concentration of 64 pg/µl (1:10 dilution).
50µl of 5xQ5 High GC Enhancer was added to the PCR mix.
1% Agarose gel with 100bp marker and PCR1 rsh, PCR1 rut.
The template pCFD6 was used with a concentration of 128 pg/µl (1:5 dilution).
50µl of 5xQ5 High GC Enhancer was added to the PCR mix.
1% Agarose gel with 100bp marker and PCR1 rsh, PCR2 rsh and PCR3 rsh or PCR1 rut, PCR2 rut and PCR3 rut, respectively.
The template pCFD6 was used with a concentration of 640 pg/µl.
The Phusion DNA Polymerase was used instead of the Q5 High-Fidelity DNA Polymerase.
Print Friendly, PDF & Email