Multiple neurons encode CrebB dependent appetitive long-term memory in the mushroom body circuit
- University of Fribourg, Switzerland
Figures
Figure 1 with 1 supplement
Generation of a conditional knockout allele for CrebB.
(A) Schematic representation of the CrebB gene locus with nine different transcript isoforms. The location of the two used CRISPR sites are highlighted in green. (B) The CrebB conditional knockout …
Figure 1—figure supplement 1
Expression pattern of CrebB::GFP.
A whole-mount Drosophila adult brain stained for GFP (green), CrebB (red) and Discs large (blue). Scale bar: 100 μm.
Figure 2 with 1 supplement
CrebB is dispensable for STM and MTM, but required for LTM.
CrebB::GFP was removed from neurons and antibody stainings were conducted along with appetitive olfactory conditioning experiments. (A, B) Brains were stained with anti-GFP antibodies (green), …
Figure 2—figure supplement 1
LTM is not impaired in non-induced CrebB knockout flies.
Non-induced control flies for the pan-neuronal CrebB knockout experiment, that were kept at 18°C, were tested for 0 h (A; N ≥ 8), 3 h (B; N ≥ 8) and 24 h memory (C; N ≥ 8). No significant memory …
Figure 3 with 3 supplements
CrebB is required in MB α/β and MB α’/β’ neurons for LTM formation.
(A, B) Brains were stained using anti-GFP (green), anti-Discs large (Dlg, blue) and anti-Eyeless (Ey, red) antibodies, which labels Kenyon cells. (A–A’’) CrebB::GFP is expressed in Kenyon cells of Cr…
Figure 3—figure supplement 1
STM is not affected by CrebB knockout in Kenyon cells.
(A) Induced knockout of CrebB in MB neurons did not impair STM. Memory scores of the tested genotypes did not differ from each other (Welch two sample t-test). N = 8. (B) Non-induced CrebB knockout …
Figure 3—figure supplement 2
Non-induced controls display normal 24 h memory.
Flies that were used for CrebB knockout experiments in Kenyon cells were tested for LTM under non-induction conditions. 24 h memory performance of non-induced experimental flies was similar to the …
Figure 3—figure supplement 3
Successful removal of CrebB::GFP from MB γ neurons.
Confocal sections of adult brains showing the Kenyon cell (KC) region stained for GFP (green), the KC marker Eyeless (Ey, red) and Dlg to outline the neuropile (blue). (A, A’, A’’) In control flies, …
Figure 4 with 2 supplements
The CrebB gene is required for LTM in MBON α3.
(A) Deleting CrebB in PAM neurons using the driver GMR58E02-Gal4 did not affect LTM formation. Performance indices did not differ between the groups. N ≥ 9. (B) CrebB knockout in MBON α3 with G0239-G…
Figure 4—figure supplement 1
Effective CrebB::GFP knockout in PAM neurons.
Brains were stained with anti-GFP (green) and anti-TH antibodies (red). Central brain regions, where TH-positive dopaminergic PAM neurons are located, are shown. Control flies displayed GFP-positive …
Figure 4—figure supplement 2
Non-induced controls for the CrebB knockout experiment in PAM neurons and MBON α3 show normal LTM.
Memory performance measured 24 h after conditioning was assessed in flies with non-induced FLP expression. Control flies were not significantly different from experimental flies for the PAM neurons …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | CrebBcKO | this paper | See Materials and Methods section: Creation of CrebBcKO | |
| Genetic reagent (D. melanogaster) | c739-Gal4 | Hiromu Tanimoto (Tohoku University) | RRID:BDSC_7362 | |
| Genetic reagent (D. melanogaster) | OK107-Gal4 | Kyoto stock center | RRID:DGGR_106098 | |
| Genetic reagent (D. melanogaster) | nSyb-Gal4 | Bloomington stock center | RRID:BDSC_51635 | |
| Genetic reagent (D. melanogaster) | c305a-Gal4 | Bloomington stock center | RRID:BDSC_30829 | |
| Genetic reagent (D. melanogaster) | 5-HTR1B-Gal4 | Bloomington stock center | RRID:BDSC_27636 | |
| Genetic reagent (D. melanogaster) | GMR58E02-Gal4 | Bloomington stock center | RRID:BDSC_41347 | |
| Genetic reagent (D. melanogaster) | G0239-Gal4 | Bloomington stock center | RRID:BDSC_12639 | |
| Genetic reagent (D. melanogaster) | tubGal80ts | Bloomington stock center | RRID:BDSC_7019 | |
| Genetic reagent (D. melanogaster) | nos-Cas9 | Bloomington stock center | RRID:BDSC_54591 | |
| Genetic reagent (D. melanogaster) | UAS-FLP (Chr 2) | Bloomington stock center | RRID:BDSC_55806 | |
| Genetic reagent (D. melanogaster) | UAS-FLP (Chr 3) | Bloomington stock center | RRID:BDSC_55804 | |
| Antibody | guinea pig anti-CrebB | this paper | polyclonal anti-CrebB antibody raised against the CrebB full-length protein sequence of isoform F; 1:400 | |
| Antibody | rabbit anti-Eyeless | Uwe Waldorf (Saarland University) | 1:400 | |
| Antibody | chicken anti-GFP | Abcam | Cat. #: ab13970 RRID:AB_300798 | 1:1000 |
| Antibody | rabbit anti-GFP | Invitrogen | Cat. #: A-6455 RRID:AB_221570 | 1:1000 |
| Antibody | rabbit anti-Tyrosine hydroxylase | Merck | Cat. #: AB152 RRID:AB_390204 | 1:100 |
| Antibody | mouse anti-Repo | Developmental Studies Hybridoma Bank | Cat. #: 8D12 RRID:AB_528448 | 1:20 |
| Antibody | mouse anti- Discs large | Developmental Studies Hybridoma Bank | Cat. #: 4F3 RRID:AB_528203 | 1:50 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.39196.014
