Volume 22, Issue 9, Pages 2346-2358 (February 2018) Two Parallel Pathways Assign Opposing Odor Valences during Drosophila Memory Formation  Daisuke Yamazaki, Makoto Hiroi, Takashi Abe, Kazumichi Shimizu, Maki Minami-Ohtsubo, Yuko Maeyama, Junjiro Horiuchi, Tetsuya Tabata  Cell Reports  Volume 22, Issue 9, Pages 2346-2358 (February 2018) DOI: 10.1016/j.celrep.2018.02.012 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 22, 2346-2358DOI: (10.1016/j.celrep.2018.02.012) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 γCRE-p Neurons Play Pivotal Roles in 2-hr Aversive Memory (A) Schematic showing MB lobes and γCRE-p and γCRE-n neurons (left). The expression patterns of γCRE-p (middle) and γCRE-n neurons (right) are shown in green. MBs were co-labeled using MB247-DsRed, shown in magenta. In these and subsequent confocal images, images were compressed and contrasts were linearly (green channel) or non-linearly (magenta channel) enhanced for visibility. See also Movies S1 and S2. Background non-mushroom body cells are labeled with asterisks in the γCRE-n panels. These cells do not synapse onto the MBs (see Movie S2) and are not labeled with an independent MB607 driver (see Figure S4). (B–D) Inhibition of γCRE-p neurons during memory acquisition (B), consolidation (C), or retrieval (D) causes 2-hr aversive memory defects (∗∗∗∗p < 0.0001, ∗p < 0.05, n = 6–14 for all data). (E–G) Inhibition of γCRE-n neurons does not affect acquisition (E) or consolidation (F) of 2-hr aversive memory, but does affect retrieval (G). Total γ inhibition resulted in the same effect. (G, ∗∗∗∗p < 0.0001, n = 8–14 for all data). (H–J) Thermal activation of γCRE-n neurons using dTrpA1 impaired acquisition (H), consolidation (I), and retrieval (J) of 2-hr aversive memory (∗∗∗∗p < 0.0001, n = 8–12 for all data), whereas activation of γCRE-p neurons did not affect any phases. Data are expressed as the means ± SEM. Cell Reports 2018 22, 2346-2358DOI: (10.1016/j.celrep.2018.02.012) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Opposing Functions of γCRE-p and γCRE-n Neurons during Appetitive Memory (A–C) Inhibiting activity of γCRE-n neurons disrupted acquisition (A), consolidation (B), and retrieval (C) of 2-hr appetitive memory (pale orange bar, ∗∗∗∗p < 0.0001). Activation of γCRE-p neurons had a similar effect (deep blue, ∗∗∗∗p < 0.0001). Activation of γCRE-n neurons (brown) or inhibition of γCRE-p neurons (pale blue) had no effect on the 2-hr appetitive memory. n = 8–12 for all data. Data are expressed as the means ± SEM. (D) Schematic summary of the effects of γCRE-n and γCRE-p manipulation on the acquisition and consolidation of 2-hr memory. (1) γ neurons are subdivided into γCRE-n and γCRE-p types. (2) Blocking activity of γCRE-p neurons disrupts aversive memory, while (3) blocking activity of γCRE-n neurons disrupts appetitive memory. (4) Blocking activity of both types of neurons has no effects. (5) Activation of γCRE-n neurons disrupts aversive memory, while (6) activation of γCRE-p neurons disrupts appetitive memory. (7) These results suggest that γCRE-p neurons promote aversive memory by disinhibiting γCRE-n neurons, and γCRE-n neurons promote appetitive memory by disinhibiting γCRE-p neurons. Cell Reports 2018 22, 2346-2358DOI: (10.1016/j.celrep.2018.02.012) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Opposing Activities of γCRE-p and γCRE-n Neurons in Valence Assignment (A) Inhibition of γCRE-n activity during odor exposure produces aversive associations in both fed and starved flies (pale orange bars, ∗p < 0.05, ∗∗∗∗p < 0.0001). (B) Strong inhibition of γCRE-p activity during odor exposure induces appetitive associations in starved flies (shaded purple bar, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (C) The same effect as in (B) can be seen by activation of γCRE-n neurons in starved flies (shaded brown bar, ∗∗∗∗p < 0.0001). (D) Conversely, activation of γCRE-p neurons during odor exposure induces aversive associations, similar to the result seen in (A) (blue bar, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). (E) Inhibition of all γ neurons induces aversive associations in fed flies (pale green bar, ∗∗∗∗p < 0.0001) and appetitive associations in starved flies (shaded pale green bar, ∗∗p < 0.01). n = 6–13 for all data. Behavioral experiments inhibiting (A) and activating (B) γCRE-n were performed at the same time so genetic control (−/− and γCRE-n -GAL4/+) data are the same in (A) and (C). Likewise, genetic control data are the same in (B) and (D). Data are expressed as the means ± SEM. Cell Reports 2018 22, 2346-2358DOI: (10.1016/j.celrep.2018.02.012) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Functional Ca2+ Imaging Reveals Mutual Inhibition between γCRE-n and γCRE-p Neurons (A and B) Dorsal views of γCRE-p (A) and γCRE-n (B) neurons labeled using CsChrimson::tdTomato. Only the γ3–γ5 region is shown, and the pixel values of the background field (outside of the GCaMP6f signals) were set to zero. (C) Ca2+ responses during light activation of γ lobes as a function of tdTomato expression. CsChrimson::tdTomato was expressed in γCRE-n neurons, and Ca2+ responses were measured in the presence of TTX. Pixels in the γ3–γ5 region were divided into 10 populations (each 10th percentile) based on tdTomato signal intensities. The magnitudes of the Ca2+ responses upon 588 nm light stimulation increased monotonically as the pixel intensity of the tdTomato signal increased. Ca2+ responses were normalized to the peak intensity in each fly. The lowest 15% of tdTomato signals were defined as γCRE-p neurons. (D) Relationship between tdTomato fluorescence and peak δF/F0 GCaMP6f responses in γCRE-p > CsChrimson::tdTomato and γCRE-n > CsChrimson::tdTomato γ3–γ5 regions. (E and F) Distribution of pixels with the highest 15% and lowest 15% tdTomato fluorescence in γCRE-p > UAS-CsChrimson::tdTomato, MB247-LexA > LexAop-GCaMP6f (E) and γCRE-n > UAS-CsChrimson::tdTomato, MB247-LexA > LexAop-GCaMP6f flies (F). (G) Activation of γCRE-p neurons inhibits Ca2+ signals in γCRE-n pixels. The horizontal red bar shows the light stimulation period (20 s), and the vertical scale bar represents 0.1 δF/F0. n = 16 for the experimental group and n = 19 for the control group. (H) γCRE-n activation decreases Ca2+ signals in γCRE-p pixels. After an initial increase, there is a reduction in GCaMP6f fluorescence in γCRE-n > UAS-CsChrimson::tdTomato, MB247-LexA > LexAop-GCaMP6f flies. n = 20 for the experimental group and n = 20 for the control group. Vertical black bar, 0.1 δF/F0. (I and J) TTX treatment prevents inhibition between γCRE-n and γCRE-p. Traces in (I) and (J) were obtained by subtracting control traces in (G) and (H) from experimental traces. Treatment with TTX prevents inhibitory responses. n = 7 for CRE-p > CsChrimson::tdTomato flies and n = 19 for control flies in the presence of TTX. n = 14 for CRE-n > CsChrimson::tdTomato group and n = 20 for the control group. The horizontal red bar shows the light stimulation period (20 s). Vertical black bar, 0.05 δF/F0. Cell Reports 2018 22, 2346-2358DOI: (10.1016/j.celrep.2018.02.012) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 MBON-γ5β′2α/β′2mp Is Required for All Phases of Aversive Memory, and MBON-γ2α′1 Is Required for All Phases of Appetitive Memory (A and B) GFP expression patterns driven from MB077B (A, MBON-γ2α′1) and MB210B (B, MBON-γ5β′2a/β′2mp). The MB structure is shown in magenta from MB247-DsRed. Scale bars, 10 μm. (C–E) Inhibiting output from MBON-γ2α′1 during acquisition (C), consolidation (D), or retrieval (E) using shits resulted in impairment of 2-hr appetitive memories (pale purple bar, ∗∗∗∗p < 0.0001), but not 2-hr aversive memory. Inhibiting output from MBON-γ5β′2α/β′2mp caused deficits in 2-hr aversive memory (pale green, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001), but not 2-hr appetitive memory. n = 8–10. Data are expressed as the means ± SEM. Cell Reports 2018 22, 2346-2358DOI: (10.1016/j.celrep.2018.02.012) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 MBON-γ5β′2a/β′2mp Functions Downstream of γCRE-p Neurons and MBON-γ2α′1 Functions Downstream of γCRE-n Neurons (A–C) Top: blocking output from MBON-γ2α′1 (MB077B) restored the aversive memory defects in acquisition (A), consolidation (B), and retrieval (C) caused by the inhibition of γCRE-p neurons (blue versus pale purple bar), while blocking γCRE-n neurons failed to restore the aversive memory defects in acquisition (A), consolidation (B), and retrieval (C) caused by the inhibition of MBON-γ5β′2a/β′2mp (MB210B) neurons (yellow versus pale green bar). n = 8–10, ∗∗∗∗p < 0.0001. Bottom: blocking output from MBON-γ5β′2a/β′2mp (MB210B) restored the appetitive memory defects in acquisition (A), consolidation (B), and retrieval (C) caused by the inhibition of γCRE-n neurons, while blocking γCRE-p neurons did not restore the appetitive memory defects in acquisition (A), consolidation (B), and retrieval (C) caused by the inhibition of MBON-γ2α′1 (MB077B) neurons. n = 8–10, ∗∗∗∗p < 0.0001. Data are expressed as the means ± SEM. (D) Schematic summary of epistatic analyses. (E) Model of γCRE-p and γCRE-n circuit function. γCRE-p and γCRE-n neurons mutually inhibit each other, through a mechanism that may be mediated by recurrent loops mediated by APL neurons or MBONs. γCRE-p neurons acting through MBON-γ5β′2a/β′2mp promote aversive memory, while γCRE-n neurons acting through MBON-γ2α′1 promote appetitive memory. This circuit is a part of a multi-layered network (gray color) including other KCs, MBONs and DANs. It remains unknown how valence-coding DANs converge on this circuit. Cell Reports 2018 22, 2346-2358DOI: (10.1016/j.celrep.2018.02.012) Copyright © 2018 The Author(s) Terms and Conditions