Figures and data
Generation of a conditional knockout allele for Dop1R2.
A) Schematic representation of the receptor structure and interaction with the G-Protein. The FRT sites and HA-Tag are indicated B) Schematic representation of the conditional knock-out system. The endogenous Dop1R2 was replaced using CRISPR-Cas9 mediated homology-directed repair (HDR) from a donor plasmid. The plasmid contained the common coding exons of Dop1R2 with an HA-Tag in the C-Terminus and two FRT sites flanking this sequence. In the resulting Dop1R2cko allele the inserted HA-Tag and Dop1R2 sequence can be removed by flippase (FLP) recombinase in cells of interest. C) Schematic representation of the Dop1R2 gene locus with 3 different transcript isoforms. The location of the two used CRISPR sites are highlighted in red. The positions of the transmembrane domains in the isoforms and in the donor plasmid, are indicated.D-F) Dop1R2::HA expression in a frontal brain confocal section of D yw, E Dop1R2cko or F) UAS-flp;; dop1R2cko, OK107-Gal4 flies aged one week. The HA-Tag was visualized using an anti-HA-Tag antibody (green). Brain structures were labeled with anti-N-cadherin (nCad, magenta) antibodies. Scale bar: 18 µm.
Short-term memory of flies with knock-out of Dop1R2 in the mushroom Body.
A-D) Aversive training, E-H) reward training. A) and E) Whole MB flip-out using OK107-Gal4 and parental controls. B) and F) γ-lobe flip-out using 5HTR1B-Gal4 and parental controls. C) and G) α/β-lobe flip-out using c739-Gal4 and parental controls. D) and H) α’/β’-lobe flip-out using c305a-Gal4 and parental controls. No performance impairment was observed in any of the tested conditions. See S2 for sensory controls and S1 Table for the data. Bar graphs represent the mean, and error bars represent the standard error of the mean. For each shown graph the N = 12. Asterisks denote significant differences between groups (*p < 0.05, **p < 0.005, ***p < 0.001, ns: p>0.05) determined by ANOVA Tukey HSD.
2h memory of flies with knock-out of Dop1R2 in the mushroom Body.
A-D) Aversive training, E-H) reward training. A) and E) Whole MB flip-out using OK107-Gal4 and parental controls. B) and F) γ-lobe flip-out using 5HTR1B-Gal4 and parental controls. C) and G) α/β-lobe flip-out using c739-Gal4 and parental controls. D) and H) α’/β’-lobe flip-out using c305a-Gal4 and parental controls. For whole MB flip-out, α/β-lobes and α’/β’-lobes both aversive and appetitive 2h memory performance is impaired. Loss of Dop1R2 in the γ-lobe does not affect 2h memory. See S2 for sensory controls and S1 Table for the data. Bar graphs represent the mean, and error bars represent the standard error of the mean. For each shown graph the N = 12. Asterisks denote significant differences between groups (*p < 0.05, **p < 0.005, ***p < 0.001, ns: p>0.05) determined by ANOVA Tukey HSD.
24h reward memory of flies with knock-out of Dop1R2 in the mushroom Body.
A) Whole MB flip-out using OK107-Gal4 and parental controls. B) γ-lobe flip-out using 5HTR1B-Gal4 and parental controls. C) α/β-lobe flip-out using c739-Gal4 and parental controls. D) α’/β’-lobe flip-out using c305a-Gal4 and parental controls. For whole MB flip-out, α/β-lobes and α’/β’-lobes appetitive 24h memory performance is impaired. Loss of Dop1R2 in the γ-lobe does not affect 24h memory. See S2 for sensory controls and S1 Table for the data. Bar graphs represent the mean, and error bars represent the standard error of the mean. For each shown graph the N = 12. Asterisks denote significant differences between groups (*p < 0.05, **p < 0.005, ***p < 0.001, ns: p>0.05) determined by ANOVA Tukey HSD.
Sensory tests of the dop1R2 conditional knock-out line.
A) Response of yw and Dop1R2cko to MCH B) Response of y,w and Dop1R2cko to Oct A) Response of yw and Dop1R2cko to Shock A) Response of y,w and Dop1R2cko to Sugar. E) 0h aversive memory of yw and Dop1R2cko F) 0h aversive memory of y,w and Dop1R2cko G) 0h aversive memory of yw and Dop1R2cko. In all tested conditions the Dop1R2cko shows no significant difference to the control line. H-K) Sensory responses of flies with Dop1R2 knock-out in the whole MB alongside the parental control lines. H) Response to MCH, I) Response to Oct, J) Response to Shock, H) Response to Sugar. Loss of Dop1R2 in the whole MB does not affect sensory responses. See S1 Table for the data. Bar graphs represent the mean, and error bars represent the standard error of the mean. For each shown graph the N = 12. Asterisks denote significant differences between groups (*p < 0.05, **p < 0.005, ***p < 0.001, ns: p>0.05).
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