Re-evaluation of learned information in Drosophila - PubMed (original) (raw)

Extended Data Figure 1 related to Figure 1, 2, 3 and 4

Extended Data Figure 1 related to Figure 1, 2, 3 and 4. Extinction and reconsolidation of reward memory requires distinct subsets of dopaminergic neurons that are directed by recurrent and hierarchical connections within the mushroom body output network.

a, Aversively reinforcing DANs in the paired posterior lateral 1 (PPL1) cluster innervate discrete regions of the vertical MB lobe whereas individual rewarding DANs in the protocerebral anterior medial (PAM) cluster innervate unique zones on the horizontal lobe. b, Each zone innervated by a particular DAN houses the dendritic field of a corresponding MBON. Aversive DANs overlap with the dendrites of MBONs directing behavioral approach whereas rewarding DANs overlay the dendrites of MBONs driving avoidance. c, The presynaptic fields of many MBONs overlap with the dendrites of DANs that innervate the same MB zones suggesting the presence of local recurrent feedback loops. d, The weight of behavioral drive to approach and avoidance directing MBONs is balanced in naïve flies (orange and blue circles of equal size). e, Sugar conditioning engages rewarding DANs that innervate the tips of the horizontal MB lobes and that drive depression of synaptic connections between odor-activated MB Kenyon cells and MBONs. f, Following reward conditioning the CS+ drive to avoidance directing MBONs is reduced (smaller orange circle) thereby favoring activation of odor-driven behavioral approach pathways. g, The reward learning induced skew in the MBON network is expressed when flies re-encounter the CS+ odor. Preferential CS+ drive of approach directing MBONs in turn activates aversively reinforcing PPL1 DANs which feed back to encode a competing aversive odor memory. h, We propose that, after this extinction process, reduced CS+ drive to approach directing MBONs (smaller blue circle) equals, and so neutralizes, the previously coded approach memory (smaller orange circle). i, Example of fly behavior. Naïve flies approach odors equally as a result of equal drive to avoidance and approach MBON pathways. Reward conditioned flies exhibit odor preference as a result of reduced CS+ drive to avoidance MBONs. Extinction restores the balance by reducing the CS+ drive to the approach MBONs. j, Sugar conditioning establishes enhanced CS- odor-drive to γ2α′1 MBONs. k, During memory reactivation the CS- odor drives the γ2α′1 MBON which activates the MV1 DAN that feeds back and releases dopamine within the same MB compartment. This activity is required at the time of odor re-exposure to induce memory reconsolidation. l, CS- memory reactivation of the γ2α′1 MBON also activates rewarding DANs that innervate the horizontal MB lobe tips, and that were earlier required for the formation of the original reward memory. The output of these rewarding DANs is required for a restricted period of time after odor exposure to reconsolidate memory.

Extended Data Figure 2 related to Figure 1

Extended Data Figure 2 related to Figure 1. Extinction of reward memory requires negatively reinforcing dopaminergic neurons.

a, 6 h reward memories for other odors (IAA and EB) can also be extinguished with two CS+ odor exposures 3 h after training (n≥4). b, One or three odor exposures 3 h after training, varying the memory reactivation regimen of Fig. 1, abolish odor preference behaviour of trained flies measured 3 h later (n≥6). c, Memory extinguished with two odor exposures at 3 h remains low 24 h after training (n=8). Spontaneous recovery of the initial reward memory is not obvious with our current training and extinction protocols . d, Two odor exposures, matching the memory reactivation regimen of Fig. 1, do not change the odor preference behaviour of naïve flies measured 3 or 21 h later (n=7). e, Blocking a reinforcement signal from rewarding R58E02-GAL4 DANs during retraining does not induce memory extinction (n≥9). f, Permissive temperature control for Fig. 1c. No differences in CS+ directed extinction or approach behavior following CS- exposure are apparent when the experiment in Fig. 1c is performed at permissive 23°C throughout (n≥7). g, Exposing flies to novel odors, IAA or EB, while MB504B-GAL4 PPL1 aversive DANs are blocked does not significantly impact 6 h memory performance (n≥7). h, Blocking aversive PPL1 DANs during odor pre-exposure in naïve flies does not attach a value to the pre-exposed odor (n≥9).

Extended Data Figure 3 related to Figure 2

Extended Data Figure 3 related to Figure 2. Reconsolidation of reward memory is triggered by CS- exposure and requires MB-MV1 dopaminergic neurons.

a, Reward memories formed with other odors (IAA and EB) can also be rendered sensitive to cold-shock by reactivating them with CS- exposure 3 h after training (n=9). b, Reward memories can also be made labile by reactivation 21 h after training (n=10). c, Extinction of reward memory is insensitive to blocking small groups (<3 neurons per hemisphere) or individual classes of aversive PPL1 DANs during CS+ driven memory reactivation. Blocking c, MB-MP1 (n≥10); d, MB-V1 (b, n≥9) or e, PPL1-α3 and PPL1-α′3 (n≥6) during CS- reactivation leaves 6 h memory performance unaltered. f, Manipulating the MB-MV1 DANs with the alternative driver R73F07-GAL4 during reactivation confirms a specific role in CS- driven memory reconsolidation as seen with MB296B-GAL4 in Fig. 2c. Blocking R73F07-GAL4 neurons during CS+ reactivation does not affect reward memory extinction (n≥14). g, Blocking MB-MV1 DANs (MB296B-GAL4) 1.5 h after CS- exposure does not impair reconsolidation (n≥12). h, Permissive temperature control for Fig. 2c and Supplementary Fig. 3f. CS- reactivation at permissive temperature does not change 6 h approach memory performance (n≥8). i, MB-MV1 neurons are not required to form a 3 h sugar-rewarded memory (n≥8).

Extended Data Figure 4 related to Figure 3

Extended Data Figure 4 related to Figure 3. Reward memory extinction requires V2 MBONs that drive negatively reinforcing dopaminergic neurons.

a, Blocking the GABAergic MVP2 MBONs (MB112C-GAL4) during CS- or CS+ triggered memory reactivation does not significantly impact 6 h conditioned approach behavior or CS+ driven extinction (n≥8). b, Permissive temperature control for Fig. 3a. Presenting the CS+ exposure at 23°C does not change the extinction of reward memory in V2 MBON MB052B GAL4; uas-_shi_ts1 flies (n≥8). c, Light-triggered activation (red bar) of R65B09-LexA V2 MBONs or d, R24H08-LexA V2 MBONs evokes calcium responses in PPL1 DANs. For c and d * denotes significant difference (p<0.05) between pre- and post activation responses. e, Sugar-reward training does not alter CS+ or CS- odor-evoked calcium responses in V2 MBONs (n≥11). Responses to CS-, CS+ and novel odor were measured in a section through the α2 region of the vertical MB lobe (example traces, lower left panel). Calcium transients during CS- and CS+ re-exposure were normalized to responses recorded in the same preparation to novel odor (IAA). f, Sugar-reward training does not alter CS+ or CS- odor-evoked calcium responses in MB-MV1 or MB-MP1 DANs (n≥7). Responses to CS-, CS+ and novel odor were measured in a section through the α2 or γ1 region of the MB (example traces, lower left panel). Calcium transients during CS- and CS+ re-exposure were normalized to responses recorded in the same preparation to novel odor (IAA). N.B. Order of CS+ and CS- odor presentation is reversed for MB-MV1 and MB-MP1 experiments.

Extended Data Figure 5 related to Figure 4

Extended Data Figure 5 related to Figure 4. The γ2α′1 MBONs orchestrate CS- triggered reconsolidation.

a, Blocking the cholinergic MBON-γ2α′1 (MB077C-GAL4) after CS- exposure does not impair memory reconsolidation (n≥10). b, Permissive temperature control for Fig. 4b. No defect in 3 h memory performance is apparent when the entire experiment is conducted at permissive 23°C (n≥11).

Extended Data Figure 6 related to Figures 1-4

Extended Data Figure 6 related to Figures 1-4. The expression patterns of all GAL4 and LexA lines used in this study.

Panels a-k show GFP expression driven by the relevant GAL4 (green), LexA driven RFP expression in MB Kenyon cells (red) and general neuropil stained with an antibody to the Bruchpilot presynaptic marker (blue). a, R58E02-GAL4 broadly labels rewarding DANs in the PAM cluster including PAM-α1, PAM-β1(MVP1), PAM-β1ped, PAM-β2, PAM-β'1ap, PAM-β'1m, PAM-β'2a PAM-β'2m, PAM-β'2p, PAM-γ3, PAM-γ4<γ1γ2, PAM-γ4, PAM-γ5. **b,** TH-GAL4 broadly labels DANs throughout the brain including all six MB innervating PPL1-DANs, PPL1- γ1pedc (MB-MP1), PPL1- γ2α'1, PPL1-α'2α2 (MB-V1), PPL1-α3, PPL1-α'3. **c,** MB504B-GAL4 labels PPL1- γ1pedc (MB-MP1), PPL1- γ2α'1, PPL1-α'2α2 (MB-V1), PPL1-α3. **d,** MB296B-GAL4 and **e,** R73F07-GAL4 label PPL1- γ2α'1 neurons. **f,** c061-GAL4:MBGAL80 labels PPL1-γ1pedc (MB-MP1). **g,** MB058B-GAL4 labels PPL1-α'2α2 (MB-V1). **h,** MB308B-GAL4 includes PPL1-α'3 and weak expression in PPL1-α3. **i,** MB122C labels MBON-γ1pedc>α/β (MB-MVP2). j, MB052B-GAL4 labels MBON-α'1, MBON-α2sc (MB-V2α), MBON-α2p3p, MBON-α'3ap (MB-V2α'3) and MBON-α'3m (MB-V2α'3). k, MB077C-GAL4 labels MBON- γ2α'1. Panels l-p show GFP expression driven by the relevant LexA (green) and general neuropil stained with an antibody to the Bruchpilot presynaptic marker (blue). l, R65B09-LexA labels MBON-α'1, MBON-α2sc (MB-V2α), MBON-α2p3p, MBON-α'2 (MB-V4), MBON-α'3ap (MB-V2α'3) and MBON-α'3m (MB-V2α'3). m, R71D08-LexA includes MBON-α2sc (MB-V2α), MBON-α'3ap (MB-V2α'3) and MBON-α'3m (MB-V2α'3). n, R24H08-LexA includes MBON-α'1, MBON-α'3ap (MB-V2α'3) and MBON-α'3m (MB-V2α'3). o, R58E02-LexA includes PAM-α1, PAM-β1(MVP1), PAM-β1ped, PAM-β2, PAM-β'1ap, PAM-β'1m, PAM-β'2a, PAM-β'2m, PAM-β'2p, PAM-γ3, PAM-γ4<γ1γ2, PAM-γ4, PAM-γ5. p, R25D01-LexA includes MBON-γ2α'1.

Figure 1

Figure 1. Extinction of reward memory requires negatively reinforcing dopaminergic neurons.

a, Only CS+ evoked memory reactivation at 3 h leads to extinction of appetitive memory (n≥8). b, Blocking rewarding DANs in the protocerebral anterior medial (PAM) cluster during 3 h CS- or CS+ re-exposure did not alter extinction or 6 h learned approach (n≥15). c, Blocking aversive DANs in the paired posterior lateral 1 (PPL1) cluster during CS+ reactivation significantly impairs extinction, while block during CS- reactivation leads to loss of memory (n≥10). d, Blocking PPL1 DANs without reactivation does not alter 6 h performance (n≥10). Unless otherwise noted, in all figures data represent the mean ± standard error of the mean (s.e.m.). Asterisks (*) denote significant difference (p<0.05, ANOVA) between groups of same genotype treated differently, hash (#) denotes significant difference (p<0.05, ANOVA) between different genotypes treated identically. A break in the x-axis indicates independent experiments.

Figure 2

Figure 2. Reconsolidation of reward memory is triggered by CS- exposure and requires MV1/PPL1-γ2α′1 dopaminergic neurons.

a, Re-exposing trained flies to CS- odor renders reward memory sensitive to cold-shock anesthesia (n≥7) b, Memory remains sensitive 30 min after CS- reactivation but returns to a cold-shock resistant state by 90 min (n=10). c, Blocking MB-MV1 DANs during CS- reactivation abolishes 6 h learned approach but blocking during CS+ reactivation leaves extinction intact (n≥12). d, MB-MV1 block after CS- reactivation does not significantly impair 6 h performance (n≥23). e, MB-MV1 output is dispensable during 3 h memory retrieval (n≥14). f, Blocking MB-MV1 DANs during CS- reactivation abolishes 6 h approach towards the CS+ (n≥11).

Figure 3

Figure 3. Reward memory extinction requires V2 MBONs that drive aversively reinforcing dopaminergic neurons.

a, Blocking V2 cluster MBONs significantly impairs CS+ driven extinction but spares CS- induced reconsolidation (n≥9). b, V2 MBONs are not essential to express 3 h memory performance (n≥9). c-e, Light-triggered activation (red bar) of lexAop-CsChrimson expressing R71D08-LexA V2 MBONs reproducibly evoked calcium responses in 2 of 3 MB504B-GAL4/ uas-GCaMP6f PPL1 DAN cell bodies per animal (Region of interest, ROI, indicated on schematic as dashed box). Data points from cells recorded in same individual fly shown in same color. One neuron was activated (cell 1), one inhibited (cell 3) and responses in the other (cell 2) did not reach significance. Time points (arrows) quantified before and after LED. * denotes significant difference (p<0.05, t-test) between pre- and post activation responses.

Figure 4

Figure 4. The γ2α′1 MBONs orchestrate CS- triggered reconsolidation.

a, Blocking γ2α′1 MBONs during CS- reactivation significantly impairs reconsolidation of 6 h memory but spares CS+ driven extinction (n≥12). b, γ2α′1 MBON output is required for 3 h memory expression (n≥15). c, Light-triggered activation (red bar) of R25D01-LexA or MB077C-GAL4 γ2α′1 MBONs evokes calcium responses in aversive MB296B-GAL4 MB-MV1 DANs and in d, rewarding R58E02-LexA DANs. Arrows, time points quantified before and after LED. For c and d * denotes significant difference (p<0.05, t-test) between pre- and post activation responses. e, Sugar-reward training specifically enhances CS- odor-evoked calcium responses in γ2α′1 MBONs (n≥13). Responses to CS-, CS+, novel odor were measured in projections outside the MB (example traces, lower left panel). Calcium transients during CS- and CS+ re-exposure were normalized to responses recorded in the same preparation to novel odor (IAA). Solid lines, mean response; shaded area, s.e.m., color background, odor presentation. Groups trained with MCH or OCT are shown separately. f, Blocking rewarding DANs immediately, but not 1.5 h, after CS- re-exposure abolishes reconsolidation of 6 h memory (n≥9).