Research ArticleNEUROSCIENCE

A sperm peptide enhances long-term memory in female Drosophila

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Science Advances  20 Nov 2019:
Vol. 5, no. 11, eaax3432
DOI: 10.1126/sciadv.aax3432
  • Fig. 1 Aversive LTM is impaired in virgin females.

    (A) Left: LTM performance at 24 hours after 5× spaced training cycles of 3-day-old wild-type (wt) virgin females is significantly decreased in comparison to mated females of the same age (t test, t43 = 2.8, P = 0.006; n = 19 to 25). Right: Scheme to illustrate the time points of fly selection, mating, and the memory test for virgin and mated female groups. (B) Females mated to SP0 mutant males fail to increase their LTM performance. Memory scores at 24 hours after 5× spaced training are similar to those of virgin females and significantly different from females mated to wt males [one-way analysis of variance (ANOVA), F2,34 = 6.57, P = 0.004; n = 11 to 13]. Fly selection, mating, and the memory test were all performed as in (A). (C) Left: Scheme to illustrate the time point of fly selection, SP injection, and memory testing for virgin and mated female groups. Right: Injection of virgin females with synthetic SP rescues the LTM defect of virgins injected with Ringer’s solution (mock group). The memory performance of SP-injected virgins is indistinguishable from Ringer’s-injected females mated to wt males (one-way ANOVA, F2,30 = 5.5, P = 0.009; n = 11). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ns, not significant. Asterisks indicate the results from a two-tailed unpaired t test or the least significance level in a Newman-Keuls post hoc comparison of indicated groups.

  • Fig. 2 SPR in the SPN is involved in aversive LTM formation.

    (A) Left: Scheme of the sensory pathway of the postmating switch. Right: SPR knockdown in the SPSN driven by VT003280-Gal4 using UAS-SPRRNAi1 and UAS-SPRRNAi2 has no effect on LTM performances (one-way ANOVA, F4,37 = 0.12, P = 0.91; n = 8). (B) Left: Scheme to illustrate SPN anatomy in the brain. The inset illustrates the control of LTM consolidation: After LTM training, Dnc PDE default activity is inhibited, PKA levels rise, and serotonin [5-hydroxytryptamine (5HT)] signaling from the SPN allows downstream consolidation processes. Right: Immunolabeling of VT057280-Gal4 (SPR-Gal4) flies driving UAS-mCD8::GFP shows expression in the SPN (white arrows), as revealed by anti-GFP staining (green). Scale bar, 50 μm. (C) The cell body of the SPN visualized with anti-GFP staining (green) of VT026326-Gal4>UAS-mCD8::GFP flies colocalizes with a marker for SPR (Anti-SPR; magenta). Simultaneous knockdown of SPR using UAS-SPRRNAi1 driven by VT026326-Gal4 reduces SPR signals in the SPN cell body. The images represent a single 1-μm z-stack. Scale bars, 10 μm. Quantification of the normalized intensity of anti-SPR staining reveals a significant decrease in the SPN after SPR knockdown using VT026326-Gal4>UAS-SPRRNAi1 as compared to controls (t test, t10 = 2.4, P = 0.03; n = 6). (D) SPR knockdown in the SPN of adult flies with 3 days of induction using UAS-SPRRNAi1 driven by either tub-G80ts;GH298-Gal4 or tub-G80ts;VT026326-Gal4 impairs LTM performances (one-way ANOVA, SPR-RNAi1: F4,73 = 5.27, P = 0.0009; n = 13 to 18). (E) SPR knockdown in the SPN using UAS-SPRRNAi1 driven by SPNsplit-Gal4 impairs LTM performances (one-way ANOVA: F2,51 = 4.28, P = 0.023; n = 18). Data are presented as means ± SEM. *P < 0.05. Asterisks indicate the results from a two-tailed unpaired t test or the least significance level in a Newman-Keuls post hoc comparison of indicated groups.

  • Fig. 3 SPR in the SPN inhibits Dnc PDE.

    (A) Schematic illustration of the Dnc PDE activity readout using PKA imaging. Left: In the event of high Dnc PDE activity, intracellular PKA activity level is low. After 3-isobutyl-1-methylxanthine (IBMX) injection to inhibit PDE, PKA levels are no longer restricted by Dnc PDE and consequently rise. In contrast, if Dnc PDE activity is inhibited in the SPN after spaced training, injection of IBMX does not lead to any further PDE inhibition and PKA levels show little change. (B) Dnc PDE is not inhibited in virgin females or females mated to SP0 males after 5× spaced training. In vivo PKA imaging was conducted on flies expressing UAS-AKAR2 in the SPN using VT057280-Gal4 (SPR-Gal4). Time traces of PKA activity are shown, including the point at which IBMX is applied on the brain (dashed lines) to inhibit PDE. After 5× spaced training, mated females displayed a weak increase in PKA activity in response to IBMX, because Dnc PDE is inhibited by spaced training. In both virgin females and females mated to SP0 mutant males, IBMX evoked PKA activity in the SPN after 5× spaced training, which was significantly higher than in wt mated controls (one-way ANOVA: F2,26 = 6.8, P = 0.004; n = 9 to 11). (C) SPR is involved in Dnc PDE inhibition after LTM training. PKA imaging reveals that Dnc PDE activity is high in naïve flies and inhibited after 5× spaced training. Under SPR knockdown conditions using UAS-SPRRNAi1 driven by VT057280-Gal4, IBMX evoked an increase in PKA activity after 5× spaced training in mated females that was significantly greater in comparison to the trained wt control and indistinguishable from naïve flies (one-way ANOVA: F2,21 = 17.1, P < 0.0001; n = 7 to 9). Data are presented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.0001. Asterisks indicate the results from a two-tailed unpaired t test or the least significance level in a Newman-Keuls post hoc comparison of indicated groups.

  • Fig. 4 MIP signaling from the SPN is involved in aversive LTM formation.

    (A) Immunolabeling of MIP-Gal4 flies driving UAS-mCD8::GFP shows expression in the SPN (white arrows), as revealed by anti-GFP staining (green). Scale bar, 50 μm. (B) MIP knockdown in the SPN of adult flies with 3 days of induction using UAS-MIPRNAi1 driven by either tub-G80ts; GH298-Gal4 or tub-G80ts; VT026326-Gal4 impairs LTM performances (one-way ANOVA: F4,75 = 6.15, P = 0.0002; n = 13 to 19). (C) MIP knockdown in the SPN using UAS-MIPRNAi1 driven by SPNsplit-Gal4 also impairs LTM performances (one-way ANOVA: F2,33 = 7.59, P = 0.002; n = 12). Data are presented as means ± SEM. *P < 0.05; **P < 0.01. Asterisks indicate the results from a two-tailed unpaired t test or the least significance level in a Newman-Keuls post hoc comparison of indicated groups.

  • Fig. 5 Dnc PDE knockdown in the SPN of adult flies rescues the LTM performance of virgin females.

    (A) LTM performances of virgin females at 24 hours after 5× spaced training cycles with conditional knockdown using UAS-DncRNAi in the SPN and driven by either tub-G80ts; GH298-Gal4 or tub-G80ts; VT026326-Gal4 are significantly higher than the performances of the respective genetic control virgin females and are indistinguishable from mated females (one-way ANOVA: F4,50 = 6.31, P = 0.003; n = 10 to 12). Note that the memory performances of virgin females and genetic controls are relatively low, which is probably due to the fact that the high temperatures used to induce RNAi also compromise virgin behavior. (B) LTM performances of virgin females at 24 hours after 5× spaced training cycles with knockdown using UAS-DncRNAi driven specifically in the SPN by SPNsplit-Gal4 are significantly higher than the performances of the respective genetic control virgin females and are indistinguishable from mated females (one-way ANOVA: F2,35 = 6.69, P = 0.004; n = 12 to 13). (C) Schematic illustration of the molecular modulation in the SPN by SP transferred to the female during mating. Thereby, the SPN is switched into an activable state and can be triggered by LTM training. Data are presented as means ± SEM. *P < 0.05; **P < 0.01. Asterisks indicate the least significance level in a Newman-Keuls post hoc comparison of indicated groups.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaax3432/DC1

    Fig. S1. The memory defect of virgin females is specific to LTM.

    Fig. S2. Controls for SPR knockdown in SPN.

    Fig. S3. Dnc PDE is inhibited by mating.

    Fig. S4. Controls for MIP knockdown in SPN.

    Table S1. Sensory acuity of virgin females.

    Table S2. Sensory acuity of flies after SPR knockdown in SPN.

    Table S3. Sensory acuity of flies after SPR knockdown with SPNsplit-Gal4.

    Table S4. Sensory acuity of flies after MIP knockdown in SPN.

    Table S5. Sensory acuity of flies after MIP knockdown with SPNsplit-Gal4.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. The memory defect of virgin females is specific to LTM.
    • Fig. S2. Controls for SPR knockdown in SPN.
    • Fig. S3. Dnc PDE is inhibited by mating.
    • Fig. S4. Controls for MIP knockdown in SPN.
    • Table S1. Sensory acuity of virgin females.
    • Table S2. Sensory acuity of flies after SPR knockdown in SPN.
    • Table S3. Sensory acuity of flies after SPR knockdown with SPNsplit-Gal4.
    • Table S4. Sensory acuity of flies after MIP knockdown in SPN.
    • Table S5. Sensory acuity of flies after MIP knockdown with SPNsplit-Gal4.

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