Re-exposure to reward re-evaluates related memories

Abstract

To adapt behavior in changing environments, animals must continuously re-evaluate previously learned associations. This flexibility of memory systems has been identified as a promising strategy to target maladaptive memories. Here, we show that re-exposure to an unconditioned stimulus (US) alone, a sugar reward, can re-evaluate appetitive memories in Drosophila melanogaster. Using olfactory conditioning, we demonstrate that unpaired US exposure after memory formation reduces conditioned responses to multiple odor-reward associations. This reduction is specific to the re-exposure of the trained US and does not result from an altered motivational state or generalized behavioral suppression. Importantly, this US-induced memory devaluation engages mechanisms distinct from dopamine-driven modulation of memory accessibility, indicating a separate process of memory re-evaluation. Moreover, we find that sugar re-exposure diminishes both short- and long-term memory phases and can act on consolidated memories, suggesting broad temporal applicability. Notably, this devaluation does not change the reward-memory trace in specific mushroom body output neurons, implying that the underlying memory trace remains intact despite behavioral suppression. Our findings reveal a mechanism by which reward re-experience pervasively devalues associated memories, offering a potential approach to target multiple memories without requiring re-exposure to individual cues. This work provides insight into how experience can broadly reshape memory networks and may inform future approaches for persistent memory modification.

Keywords: Drosophila melanogaster; dopaminergic neurons; extinction; memory; mushroom body; reconsolidation; retrieval; reward; unconditioned stimulus; update.

Figure 1

Reward re-exposure diminishes the expression of olfactory reward memory (A) Re-exposing flies to the sucrose reward (US) for 5 min at 3 h after training leads to a reduction of 6-h memory performance (n ≥ 10). (B) Re-exposing flies to the sucrose at 3 or 24 h after training leads to diminished memory retrieval in a 27-h test (n ≥ 10). (C) Exposing flies to sucrose 3 h before training does not affect 6-h memory (n ≥ 25). (D) In a test against a novel odor, the approach to the CS+ is reduced after sucrose re-exposure (n ≥ 12). (E) Although re-exposing flies to sucrose for 5 min at 3 h after training leads to memory devaluation, feeding flies regular fly food instead does not affect 6-h memory performance (n ≥ 11). (F) Re-exposing flies to sweet but non-caloric sugar arabinose for 5 min 3 h after training does not reduce memory performance at 6 h (n ≥ 12). (G and H) Sucrose re-exposure after two sets of training using different odor sets leads to the devaluation of both memories (n ≥ 10). In all figures, data represent the mean ± SEM. Individual n indicated by circles. Asterisks denote significant differences (p < 0.05, t test or ANOVA). See also Figure S1.

Figure 2

Activity of reward DANs bidirectionally influences sugar-memory retrieval (A) Activating PAM-DANs 3 h after sucrose training reduces 6-h memory retrieval (n ≥ 12). (B) Depiction of the different time protocols. (C) Artificially activating PAM-DANs (R58E02-GAL4) 15 min after training reduces 1-h memory performance. Likewise, activating PAM-DANs for 30 min at 15 min or 24 h after sucrose training diminishes 27-h memory (n ≥ 9). (D) Activating STM-DANs (R48B04-GAL4) 15 min after training leads to memory reduction in a 1-h memory test (data from two independent experiments; Figure S2D), whereas activating STM-DANs 15 min or 24 h after sucrose training leaves 27-h memory unaltered (n ≥ 9). (E) Activation of LTM-DANs labeled by R15A04-GAL4 15 min after training diminishes 1-h memory retrieval. Likewise, activating LTM-DANs 15 min or 24 h after sucrose training lowers 27-h memory (n ≥ 10). (F) When LTM- or STM-DANs are activated 3 h after sucrose training, 6-h memory retrieval stays intact (n ≥ 16). (G) When blocking R58E02-GAL4-labeled neurons (using shi^ts) during sucrose re-exposure, sucrose-mediated memory devaluation still occurs (n ≥ 12). Only blocking PAM-DANs for 30 min at 3 h after training enhances 6-h memory expression (n ≥ 12). (H) When output from PAM-DANs is restricted while artificially reactivating neurons at 3 h for 30 min, DAN activity-mediated memory reduction is impaired (n ≥ 6). (I) Blocking NOS activity between training and sucrose re-exposure does not impact sucrose-mediated memory devaluation (n ≥ 8). In all figures, data represent the mean ± SEM. Individual n indicated by circles. Asterisks denote significant differences (p < 0.05, t test or ANOVA). See also Figure S2.

Figure 3

Memory trace is intact after US-re-exposure (A) Scheme of experimental procedure. (B) Re-exposure to sucrose 1 h after learning leads to diminished memory in a 2-h test (n ≥ 18). (C) Illustration of the setup for head-fixed flies under two-photon microscope (top). Depiction of MBON-β′2mp and MBON-γ5β′2a neurons (bottom). (D) Calcium responses in MBON-β′2mp and MBON-γ5β′2a at 2 h after training show differences between the CS− and the CS+ in trained flies but not in flies receiving mock treatment. These differences in the responses are still present when flies are re-exposed to the sucrose reward 1 h after training. Normalized peak responses to the paired and unpaired odors are shown. Peak responses are normalized to the group average of the mock group for that odor and to the response to the air puff (STAR Methods) (n ≥ 9) Shown is the mean ± SEM. Individual n indicated by circles. Asterisks denote significant differences (p < 0.05, t test). See also Figure S3.

Figure 4

Sucrose-mediated memory devaluation is context dependent (A) Sucrose memory is diminished when sucrose re-exposure is performed in housing vials. Conversely, when sucrose is presented in the T-maze context, the memory stays intact. (B) When flies are trained in housing vials, re-exposure to sucrose in the same vials leads to memory devaluation (n ≥ 10). (C) Artificial reactivation of PAM-DANs in the T-maze does lead to diminished memory independent of the context (n ≥ 12). (D) Exposing flies to a novel odor during sucrose re-exposure in housing vials leaves the memory intact (n ≥ 18). In all figures, data represent the mean ± SEM. Individual n indicated by circles. Asterisks denote significant differences (p < 0.05, t test or ANOVA).