Generation of a conditional knockout allele for Dop1R2.

A) Schematic representation of the receptor structure and interaction with the G-protein complex. The position of the HA-Tag is 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 two 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) 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) y, w, 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) antibody. Scale bar: 80 µm.

Single-cell Transcriptomic analysis of Dop1R2 and various G-proteins in the Mushroom Body neurons.

A-B) Co-expression of Dop1R2 and Gαo and Gαq respectively. Cells expressing Dop1R2 are depicted in red, while the G-proteins are depicted in blue. While cells expressing both Dop1R2 and one of the G-proteins is shown in magenta. C) Venn Diagram of cells expressing Dop1R2, Gαo and Gαq in pink, violent and green respectively. The size of the circles corresponds to the number of cells expressing proteins. D) umap clustering of Mushroom body neurons, γ-lobe neurons are shown in pink, α/β-lobe neurons are shown in green while α’/β’-lobe neurons are blue. E) DotPlot of Dopamine receptors and G-Proteins in the different MB lobes. The color and size of the dots represent the average expression and percentage of expression respectively.

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 Fig 2 S1 for sensory controls. Bar graphs represent the mean, and error bars represent the standard error of the mean. For each shown graph the N = 12. ns – not significant determined by a One-way ANOVA and Tukey HSD.

Sensory tests of the Dop1R2 conditional knock-out line.

A) Response of y w and Dop1R2cko to MCH. B) Response of y w and Dop1R2cko to Oct. A) Response of y w and Dop1R2cko to Shock. A) Response of y w and Dop1R2cko to Sugar. E) 0h aversive memory of y w and Dop1R2cko. F) 2h aversive memory of y w and Dop1R2cko. G) 0h reward memory of y w 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, K) 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: not significant) determined by One way ANOVA and 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 Fig 2 S1 for sensory controls. 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, ****p < 0.0001, ns: not significant) determined by One way ANOVA and Tukey HSD (Panels A-C, E-H) and Kruskal-Wallis with Dunn’s multiple comparisons test due to non-normal distribution (Panel D).

24h 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 aversive and appetitive 24h memory performance is impaired. Loss of Dop1R2 in the γ-lobe does not affect 24h memory. See Fig 2 S1 for sensory controls. Bar graphs represent the mean, and error bars represent the standard error of the mean. For each shown graph included in the reward training experiment the N = 12, while for the graphs included in the aversive training experiment the N = 14. Asterisks denote significant differences between groups (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, ns: not significant determined by Kruskal-Wallis with Dunn’s multiple comparisons test due to non-normal distribution (Panels A-C) and One way ANOVA and Tukey HSD (Panels D-H).

Memory retention upon single shock aversive training in Dop1R2 pan-neuronal knockout flies.

A) 6 hours memory performance of Dop1R2 pan-neuronal knockout flies using elav-Gal4. B) 24 hours memory performance of Dop1R2 pan-neuronal knockout flies using elav-Gal4. C) Memory retention over 24 hours in Dop1R2 pan-neuronal knockout flies. Lack of the receptor in the whole nervous system does not improve memory retention suggesting that Dop1R2 sparsely expressed outside of the MB might not be regulating any memory process. Single shock training is not sufficient to retain aversive memory for 24 hours in control flies either. For each graph shown the N = 14. Asterisks denote significant differences between groups (*p < 0.05, **p < 0.005, ***p < 0.001, ns: p>0.05) determined by unpaired t-test. The one-sample t-test showed no significant difference from zero for all the groups apart from the control line in the 6 hours memory experiment.

Gamma lobe development in the Dop1R2cko carrying flies.

A-C) FasII expression in a frontal view of whole brain immunostaining, highlighting the Mushroom body. The Dop1R2cko modification does not affect gamma lobe (white arrows) development. Images were taken at 63x magnification, Scale bar: 50 µm.

A temporally restricted knock-out of Dop1R2 in the mushroom Body rules out developmental defects.

A) and B) Aversive training of flies with whole Mushroom Body flip-out at 0 days post-eclosion, using the flies ;;Dop1R2cko and UAS-flp/+; tub-Gal80ts/+; Dop1R2cko as control and UAS-flp/+;tub-Gal80ts/+;Dop1R2cko; OK107-Gal4/+ the experimental, temporally restricted knockout flies. A) Short term memory tested 0 hours after training. B) Long term memory tested 24 hours after training. Loss of Dop1R2 in the whole mushroom body exclusively after eclosion had no effect on STM but significantly impaired memory retention at 24 hours. Bar graphs represent the mean, error bars represent the standard error of the mean. For the STM graph the N = 9, while for the LTM graph the N = 14. Asterisks denote significant differences between groups (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, ns: not significant) determined by One way ANOVA and Tukey HSD

Key Resource Table

Primers and gRNAs for generating Dop1R2 conditional knock-out lines.