Shock priming changes flies’ preference for novel odors.

(A-B) Electric shocks enhance the flies’ preference for a subsequent novel odor. In each panel, the training and test diagram is shown on the top, and the performance indices of mock-trained and primed flies are at the bottom. (A) Electric shocks paired with CS+ increased the preference for a novel odor when pure air was used as reference (n=10; paired t-test; ** P < 0.01). (B) Shocks alone also induced attraction to the novel odor (n=10; paired t-test; *** P < 0.001). (C) Enhanced preference for novel odors induced by shock experience lasts for minutes. Top: Schematic of priming with or without coincident odors. Bottom: Time course of the PI scores tested at different time points following training. ΔPI = primed PI – mock PI.

Both aversive and appetitive artificially substituted US induce priming effects.

(A-B) Optogenetic substitution of US results in different odor preference compared to naïve flies. (A) Top: schematic of experimental protocol. Bottom: PI scores of mock-trained (-LED) flies comparing with primed (+LED) ones. From left to right: optogenetic activation of UAS-CsChrimson expressed in empty split-GAL4 (+, control, n=12), PPL1-γ1pedc DANs (MB320C, aversive cue, n=12), and sugar sensory neurons (SS87269, appetitive cue, n=14). (B) Activation of aversive or appetitive neurons induces priming bidirectionally. ΔPI = PI+LED – PI-LED. (C-D) Flies change odor preference after optogenetic activation. (C) Top: experimental protocol. Bottom: PI scores of post-activation tests compared with pre-activation tests. From left to right: UAS-CsChrimson expressed in empty split-GAL4 (+, n=12), PPL1-γ1pedc DANs (MB320C, n=12), and sugar sensory neurons (SS87269, n=14). (D) Activation of aversive or appetitive neurons induces bidirectional priming. ΔPI = post PI – pre PI. (A and C: paired t-test within each genotype. B and D: one-way ANOVA. All ΔPI shown as mean ± SEM. The number of asterisks indicates the significance level: * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.)

Reward PAM-DANs function during aversive priming.

(A) Schematic of the in vivo calcium imaging setup (top) and the experimental protocol (bottom). (B) Sample image of GCaMP6s expressed by PAM-DAN driver 58E02 in γ-MB during odor presentation. (C) γ4-DANs, but not γ3-DANs or γ5-DANs, show increased odor response after shock priming. Top: calcium traces of odor-evoked responses in γ3, γ4, and γ5 compartments before and after electric shocks. Black and red dashed lines denote odor onset and offset, respectively. Data presented as mean (solid curve) ± SEM (shaded area). Bottom: comparison between pre-shock and post-shock odor responses (n=7; paired t-test; ** P < 0.01; ns, not significant). (D-E) Silencing reward DANs by shits disrupts aversive priming effect. (D) PI scores of mock-trained flies comparing with shock primed ones, with shits expressed in empty split-GAL4 (+), PAM-DANs (58E02), or in PAM07 and PAM08 (MB312B) at different temperatures (n = 13, 15, 9, 16, 10, 10, respectively; paired t-test). (E) Activation of shits in reward DANs at restrictive temperature disrupts priming (two-way ANOVA). ΔPI = primed PI – mock PI, shown as mean ± SEM (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant).

MBON21 shows reduced calcium activity during priming and conditioning.

(A) Confocal image of UAS-GFP expressed by MBON21 driver VT999036 in γ4γ5-MB in both hemibrains. (B) Shock priming results in reduced calcium in response to odors in MBON21. Left: schematic of the experimental protocol. Middle: calcium traces of odor response in MBON21 before and after electric shocks. Black and red dashed lines mark odor onset and offset, respectively. Data represented as mean (solid curve) ± SEM (shaded area). Right: comparison between pre-shock and post-shock odor response (n=8; paired t-test; *** P < 0.001). (C-D) MBON21 is plastic during spaced shock conditioning. (C) Top: experimental paradigm of 5x shock conditioning and test. Bottom: calcium traces of MBON21 in response to CS+ (orange) and CS-(green) during training cycle 1, 3, 5 and test. Calcium traces of training cycle 2 or 4 are not shown. Black and red dashed lines indicate odor onset and offset time. Data presented as mean (solid curve) ± SEM (shaded area). (D) Comparison between CS+ and CS-responses, shown as mean ± SEM (n=7,6,6,6,5,5, respectively; multiple paired t-test; *P < 0.05; ** P < 0.01; ns, not significant).

MBON21 is required for priming.

(A-B) Silencing MBON21 by TNT disrupts priming effect. (A) The experimental protocol, and the PI scores of mock-trained flies comparing with primed ones (n = 16 and 12; paired t-test). (B) Blocking MBON21 activity by TNT disrupts priming (unpaired t-test). (C-D) Silencing MBON21 by shits impairs priming effect. (C) PI scores of mock-trained flies comparing with primed ones (n = 11, 10, 8, 10, respectively; paired t-test). (D) Activation of shits in MBON21 at restrictive temperature disrupts priming (two-way ANOVA). (E-F) Activating MBON21 by TrpA1 disrupts priming effect. (E) PI scores of mock-trained flies comparing with primed ones (n = 10, 12, 11, 10, respectively; paired t-test). (F) Activation of TrpA1 in MBON21 at restrictive temperature disrupts priming (two-way ANOVA). All ΔPI = primed PI – mock PI, presented as mean ± SEM (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant).