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A neural mechanism for deprivation state-specific expression of relevant memories in Drosophila

Nature Neuroscience volume 22, pages 2029–2039 (2019)Cite this article

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Abstract

Motivational states modulate how animals value sensory stimuli and engage in goal-directed behaviors. The motivational states of thirst and hunger are represented in the brain by shared and unique neuromodulatory systems. However, it is unclear how such systems interact to coordinate the expression of appropriate state-specific behavior. We show that the activity of two brain neurons expressing leucokinin neuropeptide is elevated in thirsty and hungry flies, and that leucokinin release is necessary for state-dependent expression of water- and sugar-seeking memories. Leucokinin inhibits two types of mushroom-body-innervating dopaminergic neurons (DANs) to promote thirst-specific water memory expression, whereas it activates other mushroom-body-innervating DANs to facilitate hunger-dependent sugar memory expression. Selection of hunger- or thirst-appropriate memory emerges from competition between leucokinin and other neuromodulatory hunger signals at the level of the DANs. Therefore, coordinated modulation of the dopaminergic system allows flies to prioritize the expression of the relevant state-dependent motivated behavior.

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Fig. 1: Thirst gates water memory expression.
Fig. 2: Leucokinin promotes thirst-dependent water memory expression.
Fig. 3: Leucokinin controls water memory expression by inhibiting two classes of dopaminergic neurons.
Fig. 4: Leucokinin regulates hunger-dependent sugar memory expression via other dopaminergic neurons.
Fig. 5: Dopaminergic neurons control state-appropriate memory expression by integrating leucokinin and other hunger signals.

Data availability

Data supporting the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank P. Cognigni (University of Oxford, UK) for experiments leading to those in Fig. 5. We thank G. Wright (University of Oxford, UK), J. Felsenberg (Friedrich Miescher Institute, Switzerland), E. Perisse (University of Montpellier, France), G. Das (National Centre for Cell Science, India), and V. Croset (University of Oxford, UK) for comments on the manuscript. We thank A. -S. Chiang (National Tsing Hua University, Taiwan), G. Rubin (Janelia Farm Research Campus, USA), Y. Aso (Janelia Farm Research Campus, USA) and FlyLight (Janelia Farm Research Campus, USA), the Bloomington Drosophila Stock Center, Vienna Drosophila RNAi center, Kyoto Stock Center, Harvard TRiP RNAi stock center, and Taiwan Fly Core for fly stocks. C.-L.W. is funded by the Ministry of Science and Technology, Taiwan (106-2311-B-182-004-MY3) and Chang Gung Memorial Hospital, Taiwan (CMRPD1G0341-3 and BMRPC75). S.W. is funded by a Wellcome Principal Research Fellowship (200846/Z/16/Z), an ERC Advanced Grant (789274), and the Bettencourt–Schueller Foundation. S.L. is funded by the Ministry of Science and Technology, Taiwan (105-2628-B-001-005-MY3 and 107-2311-B-001-042-MY3) and Academia Sinica, Taiwan.

Author information

Authors and Affiliations

  1. Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

    Bhagyashree Senapati & Suewei Lin

  2. Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

    Bhagyashree Senapati, Chang-Hui Tsao, Yi-An Juan & Suewei Lin

  3. Institute of Life Sciences, National Central University, Taoyuan, Taiwan

    Yi-An Juan

  4. Department of Biochemistry and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

    Tai-Hsiang Chiu & Chia-Lin Wu

  5. Department of Neurology, Chang Gung Memorial Hospital, Linkou, Taiwan

    Chia-Lin Wu

  6. Centre for Neural Circuits and Behaviour, The University of Oxford, Oxford, UK

    Scott Waddell

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  1. Bhagyashree Senapati
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Contributions

S.L., S.W., B.S., and C.-H.T. designed the study and analyzed the data. B.S. performed behavioral experiments. C.-H.T. performed imaging experiments. Y.-A.J. performed naïve water-seeking experiments. T.-H.C. and C.-L.W. assisted in the initial establishment of the water reward olfactory conditioning paradigm. S.L. directed the research. S.L. and S.W. wrote the paper.

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Correspondence to Suewei Lin.

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Peer review information Nature Neuroscience thanks Liqun Luo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Controls and additional experiments related to Fig. 2.

a, Knockdown of lk in all LK neurons (LK-GAL4) or in LHLK neurons (ap-GAL4) using a second RNAi line impairs 6 h water memory performance in thirsty flies (LK-GAL4: p<0.015; ap-GAL4: p<0.025; n = 8; one-way ANOVA with Tukey’s test). b, Permissive 23 ˚C control for experiments in Fig. 2a. No effect was observed (p>0.987, n = 8; one-way ANOVA with Tukey’s test). c, Permissive 23 ˚C control for experiments in Fig. 2c. No effect was observed (p>0.552, n = 8; one-way ANOVA with Tukey’s test). d, Expression of UAS-mCD8::GFP driven by LK-GAL4 (LK neurons, green) and LexAop-rCD2::RFP driven by MB247-LexA::P65 (mushroom bodies, magenta). Brain also stained with anti-Brp antibody (gray). Four brains were examined and show the same expression pattern. Scale bar 50 µm. e, Expression of UAS-mCD8::GFP driven by ap-GAL4 (green) and LexAop-rCD2::RFP driven by MB247-LexA::P65 (magenta). Four brains were examined and show the same expression pattern. Scale bar 50 µm. f, Knockdown of lk in LHLK neurons using ap-GAL4 impairs innate water-seeking behavior (p<0.023, n = 9-12; Kruskal-Wallis test with Dunn’s multiple comparisons test). g, RNAi knockdown of lk in LHLK neurons using ap-GAL4 does not affect resistance to desiccation (p>0.098, n = 8; two-way ANOVA with Tukey’s test). Mean ± SEM are shown. h, Permissive 23 °C control for experiments in Fig. 2h. No effect was observed (p>0.82, n = 8; one-way ANOVA with Tukey’s test). Temperature regimens shown above a-c and h. Box-plots: center line indicates median; box limits, upper and lower quartiles; whiskers, max to min range; dots, individual data points. See Supplementary Table 3 for statistics details.

Extended Data Fig. 2 Additional experiments related to Fig. 3.

a, RNAi knockdown of Lkr in PPL1-γ1pedc DANs (MB320C-splitGAL4) does not affect 6 h water memory performance (p>0.48, n = 7-8; one-way ANOVA with Tukey’s test). RNAi knockdown of Lkr in PPL1 (TH-GAL4 and MB060B-splitGAL4) or PAM (DDC-GAL4 and R58E02-GAL4) DANs impairs 6 h performance (p<0.02, n=7-14; one-way ANOVA with Tukey’s test). b, Knockdown of Lkr after eclosion in TH-GAL4- and DDC-GAL4-labeled DANs with tubp-GAL80ts and UAS-Lkr-RNAi impairs 6 h water memory performance in thirsty flies (TH-GAL4: p<0.032; DDC-GAL4: p<0.0001; n = 12; one-way ANOVA with Tukey’s test). c, Adult brain expression of UAS-mCD8::GFP driven by MB087C-splitGAL4 (PAM-β′2a, green) and LexAop-rCD2::RFP driven by MB247-LexA::p65 (mushroom bodies, magenta). d, Adult brain expression of UAS-mCD8::GFP driven by MB296B-splitGAL4 (PPL1-γ2α′1, green) and LexAop-rCD2::RFP driven by MB247-LexA::p65 (magenta). Both brains counterstained with anti-Brp antibody (gray). Four brains were examined for each splitGAL4 and their expression patterns are consistent. Scale bars 50 µm. e, Knockdown of Lkr in PAM-β′2a (MB087C-splitGAL4) and PPL1-γ2α′1 (MB296B-splitGAL4) DANs with a second RNAi line impairs 6 h water memory performance (p<0.03, n = 12; Kruskal-Wallis test with Dunn’s multiple comparisons test). Temperature regimens shown above b and e. Box-plots: center line indicates median; box limits, upper and lower quartiles; whiskers, max to min range; dots, individual data points. See Supplementary Table 3 for statistics details.

Extended Data Fig. 3 Controls and additional experiments related to Fig. 3.

a, Permissive 23 ˚C controls for experiments in Fig. 3b. No defect was observed (MB296B: p>0.38; MB087C: p>0.72; n = 8; one-way ANOVA with Tukey’s test). b, Activating PAM-β′2a (MB087C) and PPL1-γ2α′1 (MB296B) DANs with UAS-TrpA1 at 32 ˚C 10 min before and during training had no effect on 6 h water memory (MB087C: p>0.97; MB296B: p>0.9999; n = 8; one-way ANOVA with Tukey’s test). c, Activating PAM-β′2a (MB087C) and PPL1-γ2α′1 (MB296B) DANs with UAS-TrpA1 at 32 ˚C immediately after training until 30 min before testing did not affect 6 h water memory (MB087C: p>0.46; MB296B: p>0.9999; n = 8; Kruskal-Willis test with Dunn’s multiple comparisons test). d, Permissive 23 ˚C control for experiments in Fig. 3c. No enhancement was observed of 6 h water memory performance in water-satiated flies (p>0.9999, n = 8; Kruskal-Willis test with Dunn’s multiple comparisons test). e, Blocking PAM-β′2a (MB087C) 20 min before and during testing did not enhance 6 h water memory performance in thirsty flies (p>0.41, n = 8; one-way ANOVA with Tukey’s test). f, Blocking PPL1-γ2α′1 (MB296B) DANs 20 min before and during testing did not enhance 6 h water memory performance in water-satiated flies (p>0.73, n = 8; one-way ANOVA with Tukey’s test). g, Permissive 23 ˚C control for experiments in Fig. 3d. No effect was observed (p>0.97, n = 8; one-way ANOVA with Tukey’s test). Temperature regimens shown above a-g. Box-plots: center line indicates median; box limits, upper and lower quartiles; whiskers, max to min range; dots, individual data points. h, Incubating explant brains with 100 nM, but not 1 nM, LK decreases GCaMP6m signal in PPL1-γ2α′1 (MB296B) DANs (100 nM: p=0.0057, n = 12; 1 nM: p=0.8659, n = 14; two-tailed paired t-test). Pre: before incubating brains with peptides. Post: after incubating brains with peptides. i, Incubating explant brains with 100 nM, but not 1 nM, LK decreases GCaMP6m signal in PAM-β′2a (MB087C) DANs (100 nM: p=0.0225, n = 11; 1 nM: p=0.2423, n = 12; two-tailed paired t-test). Pre: before incubating brains with peptides. Post: after incubating brains with peptides. Representative images of GCaMP6m signal shown under the plots in h and i. See Supplementary Table 3 for statistics details.

Extended Data Fig. 4 Controls and additional experiments related to Fig. 4.

a, Knockdown of lk in all LK neurons (LK-GAL4) or only LHLK neurons (ap-GAL4) using a second RNAi line impairs 6 h sugar memory performance in hungry flies (LK-GAL4: p<0.0001; ap-GAL4: p<0.0025; n = 8; one-way ANOVA with Tukey’s test). b, RNAi knockdown of lk in all LK neurons (LK-GAL4) or only LHLK neurons (ap-GAL4) does not affect immediate sugar memory performance (LK-GAL4: p>0.98; ap-GAL4: p>0.81; n = 8; one-way ANOVA with Tukey’s test). c, Permissive 23 ˚C control for experiments in Fig. 4c. No effect was observed (LK-GAL4: p>0.68; ap-GAL4: p>0.66; n = 8; one-way ANOVA with Tukey’s test). d, RNAi knockdown of Lkr in PAM-β′2a (MB087C-splitGAL4), PPL1-γ2α′1 (MB296B-splitGAL4), or PPL1-γ1pedc (MB320C-splitGAL4) DANs does not affect 6 h sugar memory performance (MB087C: p>0.49, n = 8; MB296B: p>0.98; MB320C: p>0.7; n = 8; one-way ANOVA with Tukey’s test). e, RNAi knockdown of Lkr in DDC-GAL4- but not TH-GAL4-labeled DANs impairs 6 h sugar memory performance in hungry flies (DDC-GAL4: p<0.036, n = 8; TH-GAL4: p>0.97, n = 8; one-way ANOVA with Tukey’s test). f, Adult brain expression of UAS-mCD8::GFP driven by MB056B-splitGAL4 (PAM-β′2mp, green) and LexAop-rCD2::RFP driven by MB247-LexA::p65 (mushroom bodies, magenta). Counterstained with anti-Brp antibody (gray). Four brains were examined and show the same expression pattern. Scale bar 50 µm. g, Knockdown of Lkr in PAM-β′2mp DANs with MB056B-splitGAL4 and a second RNAi line impairs 6 h sugar memory (p<0.0002, n = 8; one-way ANOVA with Tukey’s test). h, Permissive 23 ˚C control for experiments in Fig. 4f. No effect was observed (p>0.079, n = 8; one-way ANOVA with Tukey’s test). i, Blocking PAM-β′2mp (MB056B) DANs with UAS-Shits1 at 32 ˚C 20 min before and during training does not affect 6 h sugar memory performance (p>0.437, n = 8; one-way ANOVA with Tukey’s test). j, Blocking PAM-β′2mp (MB056B) DANs with UAS-Shits1 at 32 ˚C immediately after training until 30 min before testing does not affect 6 h sugar memory performance (p>0.21, n = 8; one-way ANOVA with Tukey’s test). k, Permissive 23 ˚C control for experiments in Fig. 4g. No effect was observed (p>0.55, n = 8; one-way ANOVA with Tukey’s test). l, Adult brain expression of JFRC2-10XUAS-IVS-mCDGFP driven by R67C06-GAL4 (putative Lkr expressing neurons, green). Counterstained with anti-Brp antibody (gray). Confocal stack downloaded from JFRC FlyLight database38. Illustration below indicates position of R67C06 enhancer in fly genome. Scale bar 50 µm. Temperature regimens shown above a-c and g-k. Box-plots: center line indicates median; box limits, upper and lower quartiles; whiskers, max to min range; dots, individual data points. See Supplementary Table 3 for statistics details.

Extended Data Fig. 5 Controls and additional experiments related to Fig. 5.

a, Illustration of the anatomical and functional relationships between LHLK neurons and the three identified DANs that control water and sugar memory expression. b, Permissive 23 ˚C control for experiments in Fig. 5d. No effect was observed (dNPF-GAL4: p>0.63; R50H05-GAL4: p>0.48; n = 8; one-way ANOVA with Tukey’s test). Temperature regimens shown above the plot. Box-plots: center line indicates median; box limits, upper and lower quartiles; whiskers, max to min range; dots, individual data points. c, Top: incubating explant brains with 100 nM scrambled control peptide for dNPF does not change GCaMP6m signal in PAM-β′2a (MB087C>GCaMP6m) DANs (p=0.5186, n = 12; two-tailed Wilcoxon matched-pairs signed rank test). Bottom: incubating explant brains with solvent-only (control for serotonin (5-HT) experiments in Fig. 5h) does not change GCaMP6m signal in PPL1-γ2α′1 (MB296B>GCaMP6m) DANs (p=0.6645, n = 12; two-tailed paired t-Test). Pre: before incubating brains with peptides or control solvent. Post: after incubating brains with peptides or control solvent. Representative images of GCaMP6m signal shown to right of each plot. See Supplementary Table 3 for statistics details. d, RT-PCR of Lkr and Rpl32 transcripts extracted from third-instar larvae. Pan-neuronal da-GAL4 was used to drive UAS-Lkr-RNAi2. Intensity of the PCR bands was normalized to the internal control Rpl32. Quantification of intensities relative to the da-GAL4-control is shown. The experiment was repeated three times with consistent results.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Tables 1–3.

Supplementary video 1

Adult brain expression of LK-GAL4-driven mCD8::GFP (green), MB247-LexA-driven rCD2::RFP (magenta) and Brp (gray).

Supplementary video 2

Adult brain expression of ap-GAL4-driven mCD8::GFP (green) and MB247-LexA-driven rCD2::RFP (magenta).

Supplementary video 3

Adult brain expression of MB087C-splitGAL4-driven mCD8::GFP (green), MB247-LexA-driven rCD2::RFP (magenta) and Brp (gray).

Supplementary video 4

Adult brain expression of MB296B-splitGAL4-driven mCD8::GFP (green), MB247-LexA-driven rCD2::RFP (magenta) and Brp (gray).

Supplementary video 5

Adult brain expression of MB056B-splitGAL4-driven mCD8::GFP (green), MB247-LexA-driven rCD2::RFP (magenta) and Brp (gray).

Supplementary video 6

Registered expression of LK-GAL4-driven mCD8::GFP (LK neurons; green) and MB087C-splitGAL4-driven mCD8::GFP (PAM-β′2a DANs; magenta) in a standard brain.

Supplementary video 7

Registered expression of LK-GAL4-driven mCD8::GFP (LK neurons; green) and MB296B-splitGAL4-driven mCD8::GFP (PPL1-γ2α′1 DANs; magenta) in a standard brain.

Supplementary video 8

Registered expression of LK-GAL4-driven mCD8::GFP (LK neurons; green) and MB056B-splitGAL4-driven mCD8::GFP (PAM-β′2mp DANs; magenta) in a standard brain.

Supplementary video 9

Adult brain expression of Lkr Trojan-GAL4-driven mCD8::GFP (green).

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Senapati, B., Tsao, CH., Juan, YA. et al. A neural mechanism for deprivation state-specific expression of relevant memories in Drosophila. Nat Neurosci 22, 2029–2039 (2019). https://doi.org/10.1038/s41593-019-0515-z

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