It remains unclear how memory engrams are altered by experience, such as new learning, to cause forgetting. Here, we report that short-term aversive memory in Drosophila is encoded by and retrieved from the mushroom body output neuron MBOn-γ2α′1. Pairing an odor with aversive electric shock creates a robust depression in the calcium response of MBOn-γ2α′1 and increases avoidance to the paired odor. Electric shock after learning, which activates the cognate dopamine neuron DAn-γ2α′1, restores the response properties of MBOn-γ2α′1 and causes behavioral forgetting. Conditioning with a second odor restores the responses of MBOn-γ2α′1 to a previously learned odor while depressing responses to the newly learned odor, showing that learning and forgetting can occur simultaneously. Moreover, optogenetic activation of DAn-γ2α′1 is sufficient for the bidirectional modulation of MBOn-γ2α′1 response properties. Thus, a single DAn can drive both learning and forgetting by bidirectionally modulating a cellular memory trace.

Here, we use in vivo functional imaging of MBOn physiology before and after learning and forgetting paradigms using aversive US stimuli, optogenetic control of DAns, and memory-retrieval assays to determine whether a single DAn can both form and disrupt memory traces. We report that coincidence of odor and electric shock creates an immediate cellular memory trace in MBOn-γ2α′1, and the retrieval of aversive memories immediately after learning require this MBOn. This memory trace manifests as a fully depressed MBOn-γ2α′1 response specifically to the paired odor, likely due to changes in odor-specific MBn:MBOn-γ2α′1 synapses. In addition, subsequent activation of DAn-γ2α′1 using electric shock or optogenetics restores the normal odor-response properties of MBOn-γ2α′1. This represents the disruption of the odor-specific memory trace. Parallel conditioning experiments indicate that this cellular memory trace and its disruption are relevant to learning and forgetting.

After aversive learning, the initially robust memory is forgotten rather rapidly across ∼24 hr. We previously discovered that activity within a small set of PPL1 DAns, including DAn-γ2α′1, is critical not only for the formation of aversive memory but also for the forgetting of these memories (). These findings suggest that these DAns, activated during learning, promote the formation of cellular memory traces in their corresponding MBn:MBOn compartments, with their subsequent activity disrupting these memory traces to cause forgetting. Other studies have shown that artificial DAn activation can alter odor-specific plasticity in some MBOns (), presumably through the modulation of MBn:MBOn synapses. However, these studies did not synthesize the formation and disruption of cellular memory traces in MBOns with acquisition and forgetting of associative memory. Even in cases in which a cellular memory trace forms in parallel with learning, behavioral forgetting may function by disrupting the memory trace or by overriding an existing memory trace through changes in other parts of the circuit. In addition, it remains unknown what happens to a memory trace formed with one odor when a second odor is learned.

Changes in the response properties of an MBOn involved in memory retrieval that are created by aversive learning represent cellular memory traces that are likely components of the aversive memory engram (). The full aversive memory engram is undoubtedly spread across many MBOns, and the full list of MBOns involved in aversive memory is presently lacking. DAns belonging to the PPL1 cluster that are activated by punishing electric shock and are critical to aversive memory formation correspond to MBOns that drive approach, suggesting that these compartments are likely sites for aversive memory storage. While prior work has implicated some of these approach MBOns in aversive memory, curiously one, the MBOn-γ2α′1, was recently shown to play a role in appetitive memory but not aversive memory (). Thus, despite the fact that its cognate DAn belongs to the PPL1 cluster and is strongly activated by electric shock, the role of MBOn-γ2α′1 in aversive memory remains in question.

Fruit flies form strong negative olfactory associations when an odor (conditioned stimulus, CS) is paired with a punishing electric shock (unconditioned stimulus, US), and these memories critically depend on the mushroom bodies (MBs) for their formation, storage, and retrieval (). Within the MB, odors activate sparse and unique sets of MB neurons (MBns) from an array of ∼2000 in each hemisphere that in turn provide synaptic input to the dendrites of multiple MB output neurons (MBOns) via discrete axonal compartments along the length of the axon (). The majority of the MBOn network appears divided into two mutually antagonistic classes that drive either approach or avoidance behavior (). Each MBn:MBOn compartment is innervated by specific cognate dopamine neurons (DAns) (), many of which respond to either rewarding or punishing stimuli and are necessary and sufficient, as a US, for memory formation (). Therefore, the MB is thought to store associative memory engrams as sparse and DAn-mediated modifications in the connectivity of MBns to the MBOn network, and MBOns are the primary MB effectors of behavior during memory retrieval (). Consistent with this model, several MBOns have been implicated in the retrieval of aversive or appetitive memories, and the response properties of some MBOns are altered to odors paired with rewarding or punishing US, or artificial DAn activation ().

Animals have evolved associative memory systems to rapidly and robustly adapt their behavior in response to environmental cues that accompany and thus potentially predict impactful outcomes. However, a lifetime in a complex world would inevitably produce a large number of erroneous associations or an ever-increasing load of correct associations that could saturate the memory system. For optimal cognitive flexibility, memory circuits must balance the formation of strong and enduring memories with the removal or updating of memories required by a changing environment (). Disruptions in forgetting processes would lead to the loss of critical memories or the persistence of harmful ones, creating pathological conditions that could include dementia, drug addiction, and post-traumatic stress disorder. Thus, it is critical to understand the biological mechanisms for memory formation, memory updating, and memory removal.

Electric shock, our US above, activates multiple DAns and likely many other types of neurons in the fly brain (). While DAn-γ2α′1 is robustly activated by this type of US ( Figure 2 D) and is the only DAn with axonal terminals covering MBOn-γ2α′1 dendrites, we tested whether optogenetic activation of the DAn-γ2α′1 itself, in lieu of electric shock, would be sufficient to modulate MBOn-γ2α′1 connectivity. We used the red light-activated cation channel, Chrimson, fused to tdTomato (ChrT;) for this purpose. When we expressed ChrT along with GCaMPspecifically in DAn-γ2α′1 and pulsed a red-orange light-emitting diode (LED) (617 nm) onto the heads of flies through a fiber optic cable ( Figures 5 A and 5B ), we found robust and immediate Catransients in the DAn synaptic terminals, while flies lacking ChrT expression in DAn-γ2α′1 showed no LED-induced activation ( Figures 5 C and 5D). These data indicate that short LED pulses can activate DAn-γ2α′1 with temporal precision through the head cuticle. Next, we expressed GCaMPin the MBOn-γ2α′1 while simultaneously expressing ChrT specifically in the DAn-γ2α′1 ( Figures 5 E and 5F). To minimize unwanted activation of ChrT from Caimaging, we scanned a small section of the MBOn-γ2α′1 axon tracts outside the ChrT-enriched DAn-γ2α′1 terminals with infrared light (two-photon imaging) and sampled odors at only three time points. Mimicking the short odor-US pairing protocol used in Figure 4 , we sampled the odor responses of MBOn-γ2α′1 before (pre), after a learning period (post 1), and finally after a forgetting period (post 2; Figure 5 G). Similar to our results above, obtained with electric shock stimuli, pairing LED pulses with odor (MCH) led to a complete depression of CSresponses for MBOn-γ2α′1, with no effect on the CSresponses ( Figures 5 H and 5I, paired > decay or paired > unpaired). After this initial depression, LED pulses presented unpaired with odor led to the restoration of the CSresponses of MBOn-γ2α′1, but had no effect on the CSodor responses. There was no significant modulation of MBOn-γ2α′1 responses when no LED was presented ( Figures 5 H and 5I), and flies lacking ChT expression in DAn-γ2α′1 exhibited no significant depression or response restoration to the CSodor ( Figures S5 A and S5B). These results indicate that output from DAn-γ2α′1 directly modulates MBOn-γ2α′1 odor responses in a bidirectional manner, presumably through MBn:MBOn synaptic connectivity, with the directionality depending on its association or lack of an association with MBn activation.

(H) Time course of axonal GCaMP 6f responses during a 5-s odor exposure (gray-shaded region) to MCH or OCT at the pre (gray lines), post 1 (light-colored lines), and post 2 (dark-colored lines) time points shown in (G) for flies expressing ChrT in DAn-γ2α′1 and GCaMP 6f in MBOn-γ2α′1.

(G) Stimulus protocols used for experiments shown in (H) and (I). MCH (5 s) and OCT (5 s) odor stimuli were presented with a 45-s ITI. The red bars represent 3-s light stimuli (LED). The MBOn-γ2α′1 axon was scanned before (pre) and after (post 1) a learning period (learn) during which odor and light stimuli were paired or light was not presented (no LED), and after (post 2) a forgetting period (forget) during which either three light pulses were presented unpaired with MCH (22 s prior) (paired > unpaired) or light was not presented (no LED and paired > decay). Timing of odor trials is the same as the timeline in Figure 4 C.

(F) Example images of GCaMP 6f (left) expression in MBOn-γ2α′1 using R25D02-lexA and Chrimson:tdTomato (right, ChrT) expression in DAn-γ2α′1 using MB296B-gal4. The yellow box indicates the two-photon scanning region of the MBOn-γ2α′1 axon to prevent unwanted activation of ChrT expressed in the DAn-γ2α′1 terminals (area outlined in white).

(D) Mean response of DAn-γ2α′1 during LED light exposure from data shown in (C). Two-way repeated-measures ANOVA with Bonferroni post hoc tests. ∗ p < 0.01, ∗∗ p < 0.0001; n = 8.

(C) Time course of the mean (±SEM) for axonal GCaMP 6f responses in DAn-γ2α′1 during a 3-s train (2 Hz) of 1 or 5 ms LED pulses presented to flies with (ChrT) or without ChrT expression (n = 8).

(B) GCaMP 6f (left) and Chrimson::tdTomato (ChrT, right) expression in DAn-γ2α′1 with the region of interest outlined and analyzed in (C) and (D).

The CS-specific depression remained somewhat stable in the absence of additional shock stimuli after this learning period for at least 15 min ( Figures 4 B [paired→—-], 4 C, and S4 B). We note that repeated odor exposures beyond trial 11 caused a rapid decline in responsiveness to OCT ( Figures 4 B [paired→—- and paired→unpaired] and 4 C) or if no shocks are ever given to the animals (data not shown). This resembles the “repetition suppression” phenomenon that was recently reported for the MBOn-α′3 (). Therefore, we have focused our analysis on trials 1–11. Similar to what was observed with weak 45 V, 12× learning ( Figures 3 B, 3C, S3 C, and S3D), shock exposure unpaired with the CSsignificantly restored the response of MBOn-γ2α′1 to odor, with no measurable change to the CS(trials 9–15, paired→unpaired, Figures 4 B, 4C, and S4 C). When the shock pulses were paired with the CS(OCT) and unpaired with the CS, we observed the expected depression to the CS(trials 8–10, paired→Rev, Figures 4 B, 4C, and S4 D), but this protocol also produced a simultaneous restoration of the CS(MCH) responses, similar to the unpaired protocol (trials 10–15, paired→Rev, Figures 4 B, 4C, and S4 D). These data indicate that three pairings of a strong shock with an odor creates an immediate depression in the responsiveness of MBOn-γ2α′1 that is vulnerable and easily disrupted by three subsequent and unpaired electric shocks. Furthermore, these results suggest that new learning can cause forgetting through the disruption of previously formed memory traces.

To extend our results and further test the flexibility of this compartment to update MBn:MBOn connectivity to changing CS/US associations, we mimicked the stimulus protocols used in long-term depression/long-term potentiation (LTD/LTP) experiments in which we repeatedly measured MBOn-γ2α′1 responses to two odors before, during, and after both a learning period and a forgetting period ( Figure 4 A). During the learning period, all of the groups received three pairings of a single US shock with short odor exposure (MCH, the CS). During forgetting, the groups were separated into one with no subsequent shock stimuli (no shock), one with three shock exposures unpaired with the CS(unpaired ×3), and one with three shock exposures unpaired with the CSbut paired with the CS(OCT) of the learning period (reversal ×3; Figure 4 A). Given our results as detailed above, we anticipated that single shock pairing with brief odor presentation would create weak associations, such that a depressed CSresponse of MBOn-γ2α′1 to odor would be more easily disrupted by unpaired DAn-γ2α′1 activation. We found that as few as three of these weak pairings were required to completely depress the response of MBOn-γ2α′1 to the odor CS Figure 4 C). This protocol also allowed us to measure MBOn-γ2α′1 responses during the association (i.e., acquisition) process itself. We found that the depression appeared immediately during the first CS/US pairing (trial 3) and that this depression grew stronger with each pairing, while leaving responses to the CSodor unaffected ( Figures 4 and S4 A). These data suggest that memory is both stored in and retrieved from MBn:MBOn-γ2α′1 synapses immediately (within seconds) after aversive learning.

Blue arrows indicate depression or recovery of responses. Blue dashed lines show the initial response level. Two-way repeated-measures ANOVA with Bonferroni post hoc tests. ∗ p < 0.05 between both unpaired and Rev groups relative to the control decay; n = 8.

(C) The mean dendritic Ca 2+ responses of MBOn-γ2α′1 (MB077B-gal4 > GCaMP 6f ) during odor trials 1–15 for MCH (top) and OCT (bottom) depicted using the color scheme shown in (B).

(B) Conditioning protocols used to collect data in (C). P, paired; UP, unpaired; Rev, reversal. The gray-shaded areas of the protocols highlight the learning trials (trials 3–6) and the forgetting trials (trials 7–11).

(A) Simplified diagram of conditioning protocols used to collect data for (C) and Figure S4 , consisting of three short odor-shock pairings (learning) with a 45-s intertrial interval (ITI) between CSand CSfollowed by several forgetting protocols (forgetting). The red bar represents a single 3-s, 90-V shock.

We conclude from these results that pairing odor with a weak shock stimulus depresses subsequent MBOn-γ2α′1 responses to the learned odor and that this depression is disrupted by DAn activation through strong shock stimulation. Moreover, the odor/USpairing produces conditioned behavioral responses to the odor, and these responses are reversed by DAn activation through strong shock stimulation. Because the effects of strong shock were specific to the CSodor and not control odors ( Figures 3 B, 3C, S3 C, and S3D), we propose that depressed synapses are preferentially sensitive to DAn output, while other synapses in the MBn:MBOn synaptic network are less affected. Our data also indicate that strong memories, like those produced after standard conditioning with 90 V, are more resistant to DAn-mediated forgetting, while weaker memories and their memory traces are more vulnerable. This feature would allow the γ2α′1 compartment to store strong odor memories with little interference from forgetting processes, while weaker memories are removed.

To demonstrate the behavioral relevance of these physiological results, parallel memory experiments were conducted mimicking the above protocol ( Figure 3 D). Pairing of odor with a weak US (45 V, 12×) produced substantial aversive memory, and this performance was lost when a strong shock stimulus (150 V, 12×) was presented after learning ( Figure 3 E). To confirm that memory formation and its subsequent removal by the strong shock was a result of changes in behavioral response specifically to the paired odor, odor avoidance was measured in a parallel group of flies. We found that pairing an odor A (MCH) with an aversive US causes a robust increase in avoidance only to the paired odor and that this increased avoidance is completely reversed with the subsequent strong shock stimulus. Avoidance to two other odors, OCT and ethyl lactate (EL), were not altered by this MCH pairing and subsequent strong shock ( Figure 3 F), paralleling the memory trace changes seen in MBOn-γ2α′1.

Studies in our lab and other labs have provided clear evidence that strong activation of DAns after learning causes behavioral forgetting (). We therefore asked whether activating DAn-γ2α′1 with strong electric shock stimuli after formation of the MBOn-γ2α′1 depression could disrupt the cellular memory trace. We exposed flies to the CS(MCH) in association with either a weak (45 V, 12×) or strong US (90 V, 12×) without CSexposure to form the MBOn-γ2α′1 memory trace ( Figure 3 A). Then, we attempted to disrupt this trace with a subsequent and strong shock stimulus (150 V, 12×). We found that training with both weak and strong US stimuli produced a strong depression to the CSinput, with no effect on the response to a control odor ( Figures 3 B, 3C, S3 A, and S3B). Furthermore, the strong post-learning 150-V shock stimulus significantly restored responses to the CSproduced by conditioning with a 45-V US. However, conditioning with a 90-V US produced an MBOn-γ2α′1 memory trace that was refractory to the 150-V post-learning stimulus ( Figures 3 C, S3 C, and S3D). We conclude from this that the strong post-learning shock is sufficient to disrupt the memory trace produced by weak but not strong training.

(F) Avoidance index (AI) of w 1118 flies measuring the preference between the paired odor A (MCH), or one of two novel odors, OCT and ethyl lactate (EL), and fresh air after the conditioning protocols illustrated in (D).

(E) Performance index (PI) measuring preference of w 1118 flies between the paired odor A (e.g., MCH) and a second odor (e.g., OCT) after the conditioning protocols illustrated in (D). A positive PI indicates aversion of the flies to odor A. One-way ANOVA with Bonferroni post hoc tests. ∗∗ p < 0.0001; n = 8–12.

(D) Behavioral conditioning protocols used to collect data in (E) and (F). Twelve electric shock pulses (45 V) were paired with an odor A (e.g., MCH) and then flies were tested for memory performance (E) or odor avoidance (F) after no intervening stimulus (paired A) or after electric shock stimulation (150 V, 12×) (paired A→150 V stim). Timeline (bottom) indicates time points relative to the start of CS/US association, including the start of 150 V shock and the start of behavioral testing.

(C) Mean dendritic response during the three odor exposures relative to time of learning for data from (A) (light-colored lines, no 150 V; dark-colored lines, 150 V stim). Two-way repeated-measures ANOVA with Bonferroni post hoc tests (comparing across 150 V stim or no 150 V conditions, black asterisk, ∗ p < 0.05; or comparing across time points, colored asterisk matched to condition, ∗ p < 0.05, ∗∗ p < 0.001; ns, not significant; n = 10–11).

(B) Time course of dendritic Ca 2+ responses in MBOn-γ2α′1 for 45 V pairing experiments upon odor exposure (gray-shaded regions) to MCH or OCT before (pre, gray lines), after odor/45 V shock pairing (post 1, light-colored lines), and after either 150 V shock or no shock (post 2, dark-colored lines). The blue arrow indicates the recovered response due to the 150 V stimulation.

(A) Conditioning protocols used to collect data shown in (B) and (C) and Figures S3 A–S3D. GCaMPresponses in MBOn-γ2α′1 (MB077B-gal4 > GCaMP) to odor (pre, post 1, and post 2) were monitored before odor/shock pairing, after the pairing (45 V, 12× or 90 V, 12×), and after either strong 150 V, 12× electric shock (150 V stim) or no strong shock (no 150 V), respectively. Timeline (bottom) indicates time points relative to the start of CS/US association, including the start of pre, post 1, and post 2 responses, and the start of 150 V shock.

These data support the model that odor-activated MBn:MBOn synapses become depressed when odor-driven MBn activation is paired with US-driven activation of DAn-γ2α′1, which is similar to observations using artificial DAn activation (). Given that optogenetic stimulation of MBOn-γ2α′1 drives approach behavior (), this large depression in the CSinput compared to the CSwould shift the MBOn network away from approach and toward avoidance during memory retrieval.

We performed additional conditioning protocols to test the hypothesis that the presence of both odor and electric shock in temporal alignment was necessary and sufficient for the MBOn-γ2α′1 depression ( Figure S2 B). We found that odor (CS) temporally aligned with shock created a similar depression with or without the CS, and that shock only or time by itself (no stimuli) did not produce the depression ( Figures S2 B and S2C). We did note an unexpected increase in the response of MBOn-γ2α′1 to odors if they were unpaired with shock or when no stimuli were given ( Figures S2 A, S2Bii–S2Bv, S2C, 2 I, and 2J). It may be that MBn:MBOn-γ2α′1 connectivity increases across time as a nonassociative effect. Because this trend is opposite the depression observed with CS/shock presentation, our conclusions on the effects of CSare not altered. In addition, we found that minimal US pairing with odor (1 shock, 5-s odor) caused a weak but significant depression ( Figures S2 Biii and S2C), highlighting a correlation between the strength of the conditioning stimuli and MBOn-γ2α′1 plasticity. Finally, standard conditioning leads to MBOn-γ2α′1 depression that decays to nonsignificant levels by 1 hr ( Figures S2 D and S2E), displaying kinetics similar to DA-mediated forgetting ().

To investigate the physiological responses of MBOn-γ2α′1 during learning and forgetting, we developed an in vivo functional imaging approach allowing simultaneous exposure of flies to odor and electric shock (the US) via the same shock grid used in standard behavioral assays ( Figure 2 A). We chose to use electric shock in our initial experiments because it represents a true aversive stimulus to the fly, rather than using artificial methods to activate DAns (). DAn-γ2α′1 responded to the standard 90 V, 12× shock protocol used in the field with robust Catransients in its synaptic terminals as detected by GCaMPexpression ( Figures 2 B–2D), validating our setup and confirming prior studies (). Next, we expressed GCaMPin MBOn-γ2α′1 ( Figures 2 E and 2F) and measured its response to MCH and OCT odors before (pre) and after (post) the standard associative learning paradigm, pairing an odor (CS) with 12 electric shock pulses (US) followed by an unpaired odor (CS) in the absence of shock pulses (paired group, P, Figure 2 G). This associative conditioning schedule produces robust aversive memory, whereas an unpaired schedule (unpaired group, UP), which eliminates the contiguity between the CSand US, produces no conditioned behavior. We found that MBOn-γ2α′1 (both dendrites and axon tracts) in naive flies responds robustly with a Caincrease followed by a decline during odor exposure ( Figures 2 H and 2I), with a rebound in Caactivity at the termination of odor exposure. We found that responses to CSodor exposure were completely depressed after associative conditioning, while the CSresponses remained unaffected, regardless of the odor used as CS Figures 2 H–2J). This associative plasticity was specific to Casignaling during odor exposure, with little effect on Casignaling occurring after odor cessation; this indicates that conditioning alters input to MBOn-γ2α′1 specifically during odor exposure. In addition, this depression extended to the axon tracts of MBOn-γ2α′1 ( Figures 2 H and S2 A), supporting the proposition that action potential propagation and MBOn-γ2α′1 output are suppressed during exposure to the learned odor, thus contributing to reduced approach behavior.

(J) Mean dendritic response during odor exposure before (pre) and after (post) conditioning protocols from data shown in (I).

(I) Time course of the dendritic Ca 2+ responses of MBOn-γ2α′1 during a 5-s exposure (gray-shaded regions) to either MCH (top) or OCT (bottom) before (light-colored line) and after (dark-colored line) paired or unpaired protocols. Traces show the average response (±SEM) across all flies tested. The blue arrows indicate the depressed response due to paired conditioning.

(H) Pseudocolored peak responses of MBOn-γ2α′1 axons and dendrites to the CS + (MCH) and CS − (OCT) before and after a paired protocol.

(G) Paired and unpaired conditioning protocols used to collect data shown in panels (H)–(J). Odor A and B are defined as the first and second odors given during the training, respectively. GCaMP 6f responses were monitored before conditioning (pre) to establish basal responses to odor stimulation. The CS + was presented paired or unpaired with electric shock pulses. A CS − odor was presented as a control. Changes in response properties were measured during odor stimulation after conditioning (post). Timeline (bottom) indicates time points relative to the start of CS/US association, including the start of pre and post responses and the end of shock in the unpaired protocol.

(F) Mean time series projection of baseline GCaMP 6f driven by MB077B-gal4 in the MBOn-γ2α′1 dendrites and its axon tracts (tdTomato was also expressed but not used in the analysis). The area labeled “Axon Tract” resides outside the MB neuropil and probably contains axon terminals in addition to the axon tracts, explaining its breadth.

(E) Schematic of circuits and tools used to collect data shown in (F)–(J). Odors (CS) activate sparse MBns that stimulate MBOn-γ2α′1 (blue) via synaptic connections (circles). Electric shock (US) activates DAn-γ2α′1 (red) with DA release modulating MBn:MBOn-γ2α′1 functional connectivity.

(D) Time course of GCaMP 6f responses in DAn-γ2α′1 terminals with electric shock exposure (90 V, 12×) used in (G) showing the mean and SEM of n = 8 animals.

(C) GCaMP 6f specifically expressed in DAn-γ2α′1 axon terminals using MB296B-gal4 (red-dotted outline). Mean time series projection of GCaMP 6f fluorescence from a 30-s time window before electric shock.

(B) Schematic of circuits and tools used to collect data in (C) and (D).

(A) Photograph of the in vivo functional imaging setup showing a fly standing on an electric shock grid prepared to receive odor and/or electric shock stimuli. The top of the head capsule is obscured but positioned just below the microscope objective.

A recent study failed to find a role for MBOn-γ2α′1 in aversive memory when tested at 2 hr after learning (). To probe this neuron’s role in short-term aversive memory, we blocked MBOn-γ2α′1 synaptic output via expression of temperature-sensitive shibire (shi), either during acquisition and retrieval 1.5 min later ( Figure 1 E) or specifically during retrieval 5 min after acquisition ( Figure 1 F). We found that blocking MBOn-γ2α′1 output in either case caused a significant disruption of short-term memory performance. Because blocking only during retrieval was sufficient to disrupt short-term memory, these behavioral data strongly support the conclusion that short-term memory is encoded in and retrieved by MBOn-γ2α′1 and is a potential site of a short-term cellular memory trace.

MBOn-γ2α′1 consists of two unipolar neurons in each hemisphere, with dendritic projections inside and presynaptic terminals outside the MB neuropil. The dendrites of these neurons innervate the MB neuropil at the junction between the vertical and horizontal lobes ( Figures 1 A–1C and S1 A;). This architecture suggests that the MBOn-γ2α′1 dendrites likely integrate synaptic input across the dense array of MBn axons.have shown functional connectivity between MBns and MBOn-γ2α′1. To test that the functional connectivity is due to a direct synaptic interaction, we used the synaptic GRASP (GFP reconstructed across synaptic partners,) technique, coupled with confocal ( Figure S1 B) and structured illumination microscopy (SIM; Figures 1 D and S1 C). We found that the MBn axons do form presynaptic connections with MBOn-γ2α′1 that broadly cover the γ2 and α′1 compartments.

Previously, we demonstrated that the PPL1 DAns, including those in the γ2α′1 circuit (also known as MV1), play a dual role in both the learning and forgetting of aversive olfactory memory (). Therefore, we hypothesized that a part of the aversive memory engram is likely stored in the MBn:MBOn-γ2α′1 compartment ( Figure 1 A) as a cellular memory trace, and that subsequent activation of DAn-γ2α′1 may alter and disrupt this memory trace.

Time in protocols is with respect to the beginning of odor-shock association. Bar plots represent the mean with error bars equal to + SEM in this figure and others. Two-way ANOVA followed by Bonferroni post hoc tests. ∗ p < 0.01, ∗∗ p < 0.0001; n = 7–8. ns, not significant; UAS, upstream activation sequence.

(E and F) MBOn-γ2α′1 output was blocked either during both acquisition, A, and retrieval, R, of short-term aversive memory (E), or specifically during the retrieval of short-term aversive memory (F).

(D) Reconstituted GFP across the MBn to MBOn-γ2α′1 synapses visualized using GRASP immunostaining (left) overlaid on the collection of MBn presynaptic terminals detected by syb::spGFP 1–10 immunostaining (right).

(B) Simplified circuit diagram of (A), with odor input being conveyed by MBns, and approach behavior biased by the output of MBOn-γ2α′1.

(A) Schematic diagram of the γ2α′1 compartment (also referred to as the junction) of the MB (gray shading) showing relevant neurons and pathways. The red objects represent DAn innervation, conveying information about electric shock to this neuropil compartment. The DAn axon terminals overlap the region of innervation by the dendrites of the MBOn-γ2α′1 neuron (red region outlined in blue). Circles represent MBn to MBOn synapses. Odors A, B, and C activate axon fibers from individual MBns (colored lines).

Discussion

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Rubin G.M. Dopaminergic neurons write and update memories with cell-type-specific rules. Recent studies in the mouse have indicated that forgetting of long-term fear memories after protein synthesis inhibition () or during Alzheimer disease () occurs through the disruption of retrieval and not erasure of the engram. In this study, we focused on short-term aversive memory, and our data suggest that the cellular effects (i.e., a depressed odor-specific input) underlying memory encoding and its retrieval can be disrupted by DAn-mediated mechanisms after learning. These disruptions of the cellular memory trace, in turn, likely cause failure of memory retrieval, and thus forgetting. We cannot exclude the possibility that after this disruption some hidden cellular memory traces remain that may allow for the reconstitution of this memory trace at a later time, perhaps by retraining, similar to “savings” memory, originally proposed by. While this short-term memory trace is not long-lasting, other compartments have been implicated as loci for the storage of stable long-term memory (e.g.,). Future efforts to examine how DAns alter memory traces in these compartments and the permanence of these effects should prove interesting.