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. 2013 Mar 27;33(13):5821-33.
doi: 10.1523/JNEUROSCI.4004-12.2013.

MicroRNA-276a functions in ellipsoid body and mushroom body neurons for naive and conditioned olfactory avoidance in Drosophila

Wanhe Li  1 , Michael Cressy, Hongtao Qin, Tudor Fulga, David Van Vactor, Josh Dubnau
Affiliations

MicroRNA-276a functions in ellipsoid body and mushroom body neurons for naive and conditioned olfactory avoidance in Drosophila

Wanhe Li et al. J Neurosci. .

Abstract

MicroRNA (miRNA)-mediated gene regulation plays a key role in brain development and function. But there are few cases in which the roles of individual miRNAs have been elucidated in behaving animals. We report a miR-276a::DopR regulatory module in Drosophila that functions in distinct circuits for naive odor responses and conditioned odor memory. Drosophila olfactory aversive memory involves convergence of the odors (conditioned stimulus) and the electric shock (unconditioned stimulus) in mushroom body (MB) neurons. Dopamine receptor DopR mediates the unconditioned stimulus inputs onto MB. Distinct dopaminergic neurons also innervate ellipsoid body (EB), where DopR function modulates arousal to external stimuli. We demonstrate that miR-276a is required in MB neurons for memory formation and in EB for naive responses to odors. Both roles of miR-276a are mediated by tuning DopR expression. The dual role of this miR-276a::DopR genetic module in these two neural circuits highlights the importance of miRNA-mediated gene regulation within distinct circuits underlying both naive behavioral responses and memory.

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Figures

Figure 1.
Figure 1.
Genetic and behavioral characterization of miR-276a gene locus. A–C, The dme-miR-276a gene falls into a large intergenic region (A). The p{lacW} element that causes the miR-276aRosa mutant phenotype is inserted 1.2 kb upstream of the dme-mir-276a precursor region. The structure of each mutant allele or transgene is illustrated, including hypomorphic allele miR-276aRosa, null allele miR-276aD8, and precise excision alleles miR-276aA6 and miR-276aD2.2. The structure and relative sizes of rescue constructs, including three BAC rescue clones and a 4.7 kb genomic fragment, also are shown (A). The dme-miR-276 gene family produces two miRNA precursors, which give rise to three miRNAs (miRBase: http://www.mirbase.org): dme-miR-276a, dme-miR-276b, and dme-miR-276* (B, C). D, The relative expression level of miR-276a in the heads of WT, miR-276aRosa/Rosa, miR-276aD8/D8, and miR-276aD8/Rosa animals, measured by QPCR. *p < 0.05; N = ∼3–8. E, miR-276aD8 and miR-276aRosa alleles failed to complement each other for LTM measured 24 h after 10 spaced training sessions. *p < 0.05; N = 16. F, miR-276aD8 and miR-276aRosa alleles failed to complement each other for olfactory avoidance test with MCH (1.0 × 10−3 v/v). *p < 0.05; N = ∼4–6. G, Shock avoidance (60 V) appeared normal in WT, miR-276aRosa/+, miR-276aD8/+, and miR-276aD8/Rosa animals. n.s., not significant; N = 4. H, Two precise excision alleles, miR-276aA6 and miR-276aD2.2, can reverse the olfactory avoidance defect seen in miR-276aRosa/D8 mutant animals. *p < 0.05; N = 6. I, Olfactory avoidance tests with MCH, OCT, and BA at various concentrations for WT, miR-276aRosa/+, miR-276aD8/+, and miR-276aD8/Rosa animals. N = ∼6–10.
Figure 2.
Figure 2.
Transgene rescue of miR-276a mutants. A, A copy of ∼75 kb BAC rescue clone CH321-46B15 fully rescues the olfactory avoidance behavior defect in miR-276aD8/Rosa mutant animals. *p < 0.05; N = ∼12–16. B, The two ∼20 kb BAC rescue clones, CH322-151H13 and CH322-133G18, failed to rescue the olfactory avoidance defect in miR-276aD8/Rosa mutant animals. *p < 0.05; N = 4. C, Expression levels of miR-276 are restored with the ∼75 kb BAC CH321-46B15 but not with the ∼20 kb BAC CH322-133G18. *p < 0.05; n.s., not significant; N = 4–8.
Figure 3.
Figure 3.
Postdevelopment miR-276a function in EB neurons for naive olfactory responses. A–D, The inducible miRNA “sponge” system in Drosophila is illustrated (Loya et al., 2009) (A). Quantification of early embryo hatching rate from crossing UAS::miR-276a-4.7Kb transgene to elav with or without UAS::EGFP::miR-276aSPONGE indicated that the miR-276a “sponge” can efficiently “soak up” excessive miR-276a (B). “Sponge” as a sensor to report endogenous brain expression of miR-276a. EGFP expression in a representative brain from the progeny of crossing elav to UAS::EGFP::SCRAMBLED (C) and EGFP expression in a representative brain from the progeny of crossing elav to UAS::EGFP::miR-276aSPONGE (D). E, F, Neuronal expression of UAS::EGFP::miR-276aSPONGE with elav GAL4 causes a similar behavior defect, as observed in miR-276a mutant animal, and expression of control UAS::EGFP::SCRAMBLED transgenes does not affect olfactory avoidance (E; *p < 0.05; N = 8). Animals heterozygous for each transgene exhibit normal olfactory avoidance behavior (F; n.s., not significant; N = 6). G, Postdevelopment expression of miR-276a is sufficient to restore normal naive olfactory avoidance. Temperature-shift induction scheme is illustrated (left; for detail, see Results and Materials and Methods). Olfactory avoidance was partially rescued when miR-276a function was turned on postdevelopmentally (right; *p < 0.05; n.s., not significant; N = ∼4–12). H–J, To map the circuitry where miR-276a is required for naive olfactory avoidance, UAS::EGFP::miR-276aSPONGE transgenes were expressed with a collection of GAL4 lines (Or83b, GH146, GH298, OK107, c747, c232, and c547), each of which label defined cell types that make up the Drosophila olfaction or olfactory memory circuit (H). Expression of UAS::EGFP::miR276aSPONGE transgenes with c547 GAL4, which labels the R2/4m neurons in EB, phenocopies the naive olfactory avoidance defect observed in miR-276a mutant animals (I; *p < 0.05; N = 8). In contrast, other cell types did not affect this olfactory behavior (I; n.s., not significant; N = 4). c547 expression pattern is demonstrated in J.
Figure 4.
Figure 4.
miR-276a regulates DopR expression level. A, miR-276a target prediction was obtained from four published methods: Pictar, TargetScan, ElMMo, and miRanda (Pictar: http://pictar.mdc-berlin.de/; TargetScanFly6.0: http://www.targetscan.org/fly/; ElMMo: http://www.mirz.unibas.ch/ElMMo2/; and miRanda: http://www.microrna.org/microrna/home.do) (Enright et al., 2003; Grün et al., 2005; Gaidatzis et al., 2007; Ruby et al., 2007; Betel et al., 2010). We selected several targets with known neuronal or olfactory functions, including zfh2, dpr, DopR, Pino, and Nf1 (Nakamura et al., 2002; Rollmann et al., 2005; Kim et al., 2007b; Andretic et al., 2008; Lebestky et al., 2009; Kong et al., 2010; Qin et al., 2012). B–G, To test whether the predicted target gene expression is regulated by miR-276a, we used a heat-inducible hs-GAL4 line in combination with a UAS::miR-276a-4.7Kb transgene to overexpress miR-276a. Because high level of neuronal expression through development is lethal (Fig. 3B), we used a mild-temperature regime (B; see Results and Materials and Methods) to provide a less severe heat induction during development. We then tested the effects on target gene transcript levels by shifting animals to lower temperature postdevelopment. In the temperature-shifted groups (29 to 18°C) we see significantly induced levels of target transcript levels for Zfh2 and DopR, compared with groups maintained at 29°C (C and D; *p < 0.05; N = ∼2–3). Transcript levels of Zfh2 and DopR were not affected by the temperature shift in control hs-GAL4/+ animals (C and D; n.s., not significant; N = ∼2–3). With dpr, we observed induction in temperature-shifted groups for both hs-GAL4/UAS::miR-276a-4.7Kb and hs-GAL4/+ animals, indicating temperature shift, but not miR-276a, can alter dpr expression levels (E; *p < 0.05; N = ∼2–3). For Pino and Nf1, no change in transcript levels was observed for any genotype or treatment (F and G; n.s., not significant; N = ∼2–3). H–J, Another temperature-shift regimen was also used to test whether miR-276a regulates Zfh2 and DopR gene expression (H). Forty-minute heat-shock induction of miR-276a expression at 37°C in adult hs-GAL4/UAS::miR-276a-4.7Kb animals caused a significant downregulation of transcript levels of Zfh2 and DopR, which are predicted to be targets of miR-276a (I and J; *p < 0.05; n.s., not significant; N = 3).
Figure 5.
Figure 5.
DopR is a downstream target of miR-276a for naive olfactory responses. A, miR-276a seed sequence recognizes a highly conserved 6 nt target site in the 3′UTR of the DopR transcript. The predicted miR-276a-binding site (shaded boxes) is conserved within the 3′UTR sequences of DopR transcripts from 12 different Drosophila species (TargetScanFly6.0). B, Removing a copy of DopR gene by introducing a strong hypomorphic allele of DopR (the DopRdumb2 allele) was sufficient to fully suppress the naive olfactory avoidance defect in miR-276aD8/Rosa mutant animals. *p < 0.05; n.s., not significant; N = ∼8–16.
Figure 6.
Figure 6.
miR-276a regulates DopR in MB for LTM. A, B, DopR expresses in MBs and EB of the fly brain. C, D, Expression of UAS::EGFP::miR276aSPONGE transgenes with MB GAL4 line OK107 results in elevated DopR expression in MB (D compared with C). Immunohistochemistry for control (C) and experimental (D) brains were performed in parallel and images were taken at identical confocal settings. E–J, Expression of UAS::EGFP::miR276aSPONGE transgenes with MB GAL4 line OK107 causes impaired LTM performance compared with WT and heterozygous controls (E; *p < 0.05; N = 8). In contrast, memory measured 24 h after 10 massed training sessions (F) or short-term memory measured 3 min after a single training session (G) each appeared normal (F; n.s., not significant; N = 7; G, n.s., not significant; N = 8). As with the OK107 GAL4 line, expression of UAS::EGFP::miR276aSPONGE with c747, a second MB-expressing GAL4 line resulted in significantly impaired LTM performance. The LTM defect can be fully suppressed in the presence of one copy of DopRdumb1 strong mutant allele (H; *p < 0.05; n.s., not significant; N = 14). Olfactory acuity and shock reactivity appeared normal when UAS::EGFP::miR276aSPONGE transgenes were expressed with OK107 (I; n.s. not significant; N = 4–8). OK107 and c747 expression patterns are shown in J and K. L, A PiggyBac (PBac) insertion within DopR produces the DopRdumb2 allele, which is defective in forming LTM (*p < 0.05; N = 8). This PBac insertion contains a UAS-enhancer element that can drive DopR expression when Gal4 is present. Expression of DopR in OK107-labeled MB neurons via the UAS element contained within this PBac is sufficient to restore both short-term memory and LTM (Qin et al., 2012). To investigate the effects of higher levels of overexpression of DopR in OK107-labeled MB neurons, we tested the effects of combined expression from an additional UAS::DopR transgene and the PBac insertion line. The addition of the UAS::DopR transgene results in LTM defect. *p < 0.05; N = 8. M, Postdevelopment induction of miR-276a function was accomplished with a GAL80ts transgene. Animals were grown at 29°C to allow expression of the UAS::EGFP::miR276aSPONGE under control of the c747 GAL4 line. After development, animals were either maintained at 29°C or shifted to 18°C for 3 d to allow miR-276a function to return. Subsequent 10× spaced training sessions for LTM experiment were conducted at 25°C. Trained animals were kept at 18°C before testing for 24 h memory at 25°C (left; for details, see Results and Materials and Methods). The LTM defect resulting from expression of UAS::EGFP::miR276aSPONGE can be fully restored in the temperature-shift group in which miR-276a function was turned back on postdevelopmentally. LTM performance was normal in control GAL80ts/+; OK107/+ animals (right; *p < 0.05; n.s., not significant; N = 8).
Figure 7.
Figure 7.
A model in which miR-276a tunes DopR levels in EB and MB for naive olfactory arousal response and LTM. Distinct dopaminergic fibers innervate EB and MB respectively. In EB, DA modulates arousal thresholds via activation of DopR. In MB γ neurons, DA release conveys the unconditioned stimulus reinforcement, which is mediated by DopR. Too much or too little DopR expression (red dots) within EB or MB is sufficient to disrupt the normal responses to external stimuli and the formation of LTM. DopR levels normally are tuned by miR-276a to provide an optimal balance. An intriguing hypothesis is that long-lasting plasticity in DopR levels within EB may affect retrieval of LTM by altering arousal to the CS+ odor, such as MCH.
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