Training-dependent increases in c-fos have been used to identify engram cells encoding long-term memories (LTMs). However, the interaction between transcription factors required for LTM, including CREB and c-Fos, and activating kinases such as phosphorylated ERK (pERK) in the establishment of memory engrams has been unclear. Formation of LTM of an aversive olfactory association in flies requires repeated training trials with rest intervals between trainings. Here, we find that prolonged rest interval-dependent increases in pERK induce transcriptional cycling between c-Fos and CREB in a subset of KCs in the mushroom bodies, where olfactory associations are made and stored. Preexisting CREB is required for initial c-fos induction, while c-Fos is required later to increase CREB expression. Blocking or activating c-fos-positive engram neurons inhibits memory recall or induces memory-associated behaviors. Our results suggest that c-Fos/CREB cycling defines LTM engram cells required for LTM.

In this study, we found that the relationship among ERK, CREB, and c-Fos in LTM formation is more complex than previously proposed. Repeated and prolonged activation of ERK produces transcriptional cycling between c-fos and CREB. CREB is phosphorylated by phosphorylated ERK (pERK) to induce c-fos expression, while c-Fos is phosphorylated by ERK to form the transcription factor AP-1, which promotes expression of CREB (). The first c-fos induction step is required to form LTM, while the second CREB induction is required to prolong LTM to last at least 7 days in flies. Thus, our data indicate that memory engram cells in flies are established by cell-autonomous ERK-dependent c-Fos/CREB cycling.

LTM increases as a function of the number of training sessions and rest interval length, reaching a maximum in flies at ten training sessions and 10-min rest intervals (). A previous study has shown that the appropriate length of rest intervals is determined by the activity of protein tyrosine phosphatase 2 (SHP2), which is encoded by the corkscrew (csw) gene in Drosophila and is associated with increased activity of ERK (extracellular signal-regulated kinase), a member of mitogen-activated protein kinase (MAPK) family (). Phosphorylation of CREB by ERK is critical for CREB-dependent gene expression (), suggesting that ERK may activate CREB to induce c-fos.

Although c-fos activity has been useful in identifying engrams, it has been less clear how learned experiences activate c-fos to induce LTM. Drosophila can form LTM of an aversive olfactory association by repeatedly exposing them simultaneously to an odor and electrical shocks. LTM formation requires repetitive training sessions with rest intervals between each session, a training paradigm known as spaced training (). These rest intervals are critical for c-fos-dependent engram formation, because repetitive training without rest intervals, known as massed training, produces a different type of long-lasting memory, anesthesia-resistant memory (ARM), which is formed even in the presence of transcriptional and translational inhibitors ().

Long-term memories (LTMs) are thought to be stored in the brain in specific neural networks that have been modified during learning or consolidation to alter their outputs relative to the naive state (reviewed by). These networks are known as memory engrams (). Because formation and consolidation of LTMs requires de novo transcription and translation (), researchers have identified potential LTM engrams by identifying cells in which memory-associated transcription factors are activated (). In particular, the immediate early gene product, c-Fos, is rapidly expressed during LTM formation (), and c-fos reporters and c-fos promoter-regulated activity modulators have been used to identify and perturb engrams (). The cyclic AMP (cAMP)-response element binding protein (CREB) and other transcription factors have also been used to study engrams (). Although it has been unclear whether CREB expression is highly altered upon learning, various studies have demonstrated that neurons with high CREB activity are preferentially recruited into LTM engrams ().

Our results suggest that 48 massed trainings in CaNB2/+ flies produce LTM by inducing c-Fos/CREB cycling. Consistent with this idea, expression of dominant-negative FBZ during training in CaNB2/UAS-FBZ;elav-GS/+ flies suppressed 24-hr memory ( Figure 7 D). Furthermore, we found significant colocalization of training-dependent kayak and dCREB expression in pERK-positive KCs in CaNB2/+ flies (CaNB2/UAS-mCD8-GFP; kayak-GAL4/tub-GAL80) after 48× massed training ( Figures 7 E and 7F). Optogenetic reactivation of these kayak-positive neurons using CaNB2/UAS-ChR2-mCherry;kayak-GAL4/tubp-GAL80flies induced avoidance behavior in these flies ( Figure 7 G). These results again support a model in which engram cells encoding LTMs are determined by c-Fos/CREB cycling.

As shown in Figures 4 C and 4F, in CaNB2/+ and PP1/+ flies, 10× massed training is sufficient to produce protein synthesis-dependent LTM that lasts at least 24 hr. However, we noticed some differences between LTM produced in wild-type flies after spaced training and LTM produced in CaNB2/+ and PP1/+ flies after massed training. In particular, LTM induced by spaced training lasts at least seven days, while LTM after massed training in mutant flies does not ( Figure S6 ). Because 10× massed training takes 35 min to complete and spaced training takes more than 3 hr, the total time of ERK activation is significantly different in wild-type spaced trained flies and CaNB2/+ and PP1/+ massed trained flies. Our data indicate that 35 min is sufficient for c-Fos induction but insufficient for subsequent dCREB2 induction ( Figures 1 D and 1F). Thus, 10× massed training may only be able to start, but not complete, c-Fos/CREB cycling in CaNB2/+ and PP1/+ flies, leading to a shorter-lasting form of LTM. To examine whether differences in training time may explain differences in LTM perseverance, we next performed 48× massed training trials in CaNB2/+ and PP1/+ flies, which takes the same amount of time as 10 spaced trainings ( Figure 7 A). 7-day memory scores after 48 massed trainings of CaNB2/+ flies were equivalent to 7-day memory scores after 10 spaced trainings of wild-type flies ( Figure S6 ), indicating that these extra training sessions were sufficient to induce normal LTM. We observed increased c-fos expression in both PP1/+ and CaNB2/+ flies after the 9th massed training trial (equivalent in time to the 2nd training trial of spaced training) ( Figure 7 B) but no increase in dCREB at this time ( Figure 7 C). After the 40th massed training trial, equivalent in time to the 8th trial of spaced training, both c-fos and dCREB expression were increased in these flies ( Figures 7 B and 7C). These results suggest that PP1/+ and CaNB2/+ mutations are sufficient to bypass the requirements for rests during training but that pERK still needs to be activated for a sufficient amount of time to allow c-Fos/CREB cycling to occur.

(G) Optogenetic activation of engram cells produced during 48× massed training of an aversive association in CaNB2/+ flies induces avoidance behavior. The experimental protocol was modified from Figure 6 H, replacing spaced training with 48× massed training of CaNB2/UAS-ChR2-mCherry;kayak-GAL4/tubp-GAL80and UAS-ChR2-mCherry/+;kayak-GAL4/tubp-GAL80flies. n = 7.

(E) pERK distribution and expression of kayak and dCREB2 in KCs 7 min after 48× massed training in CaNB2 EP774 /UAS-mCD8-GFP; kayak-GAL4/tub p -GAL80 ts and control UAS-mCD8-GFP; kayak-GAL4/tub p -GAL80 ts flies. Scale bars represent 20 μm.

(C) dCREB2 activator expression increases at late time points, but not early time points, during 48× massed training in PP1/+ and CaNB2/+ mutants. n = 4–12.

(A) Diagram indicating that 48× massed training takes the same amount of time as 10× spaced training. a and b refer to time points used for sample harvesting.

If memory is encoded in c-Fos/CREB cycling neurons, artificial activation of these neurons after spaced training may induce memory-associated behaviors in the absence of odor exposure. To examine this possibility, we generated transgenic UAS-ChR2/tub-GAL80;kayak-GAL4 flies, which express photo-sensitive channelrhodopsine2 (ChR2) under kayak promoter control at the GAL80restrictive temperature (32°C). We subjected these flies to spaced training at 32°C so that ChR2 is expressed in c-Fos/CREB cycling cells. ChR2 is activated by blue light in the presence of all-trans retinal (ATR), and naive transgenic flies strongly prefer blue light to green light when allowed to choose between the two in a T maze ( Figures 6 F and 6G). However, 24 hr after spaced training, ATR-fed flies avoided blue light, while flies not fed ATR maintained their preference of blue light ( Figure 6 H). This suggests that spaced aversive training induces c-Fos/CREB cycling in specific neurons and artificial activation of these neurons induces recall of this aversive association, leading to avoidance behavior. In control experiments, GAL80-dependent inhibition of kayak was relieved at a different time from when flies were spaced trained ( Figure S5 F, top panel). In this situation, flies maintained their preference for blue light, indicating that avoidance behavior requires ChR2 activation in training-dependent engram cells ( Figure S5 F).

To support the conclusion that c-Fos/CREB cycling neurons are required for memory recall, we inactivated synaptic outputs from kayak-positive neurons during recall using a temperature-sensitive mutant of shibire (shi). shiis a variant of dynamin that dominantly disrupts synaptic vesicle recycling at the restrictive temperature (32°C) (). We expressed shiin kayak-positive neurons using tubp-GAL80;kayak-GAL4 > UAS-shiflies and raised these flies at 18°C to prevent background shiexpression. As earlier, we shifted the temperature to 32°C after the fifth spaced training trial to allow kayak promoter-dependent expression of shiand then tested these flies for memory recall 24 hr later at both restrictive (32°C) and permissive (18°C) temperatures ( Figure 6 D). Similar to TNT, blocking synaptic output from kayak-positive neurons during retrieval using shiimpaired LTM scores ( Figure 6 E). LTM scores were improved when synaptic output from kayak-positive neurons was restored at the permissive temperature ( Figure 6 E).

To determine whether reactivation of kayak-positive neurons is required for LTM retrieval, we next generated transgenic UAS-TNT/UAS-mCD8-GFP; kayak-GAL4/tub-GAL80flies to express GFP and tetanus toxin (TNT) in kayak-positive neurons at permissive temperatures (32°C). TNT blocks synaptic transmission by cleaving neuronal synaptobrevin (), and we searched for conditions in which TNT and GFP are transcribed in c-Fos/CREB cycling neurons during spaced training but are not yet translated. Translation of GFP and TNT after training should label kayak-positive neurons and prevent synaptic outputs from these neurons during recall. We raised UAS-TNT/UAS-mCD8-GFP; kayak-GAL4/tub-GAL80flies at 18°C to inhibit background expression and then shifted to 32°C from the end of the fifth training trial until the end of training to induce TNT and GFP in kayak-positive neurons ( Figure S5 B). This induction protocol did not produce GFP signals immediately after spaced training (data not shown) but instead did so 24 hr after spaced training ( Figure S5 C). If production of TNT mirrors that of GFP, TNT should not be produced during spaced training but should inhibit synaptic outputs 24 hr later during memory recall. Consistent with this idea, we found that our induction protocol did not affect memory retrieval immediately (5 min) after spaced training ( Figure S5 D), while it significantly suppressed LTM retrieval measured 24 hr after spaced training ( Figure S5 E). Thus, reactivation of c-Fos/CREB cycling neurons is required for recall of LTM.

To determine whether cells in which c-Fos/CREB cycling occurs display characteristics of memory engram cells, we next examined whether these cells are reactivated during LTM retrieval. We again used transgenic UAS-mCD8-GFP; kayak-GAL4/tub-GAL80flies to identify neurons that had high c-fos expression during spaced training ( Figure 6 A), and examined whether these neurons are reactivated during exposure to the shock-paired odor (CS+). When we monitored neural activity during memory retrieval, 24 hr after training, by exposing flies to odors and assaying for pERK, we found that more than 80% of kayak-positive KCs were reactivated upon exposure to the CS+ ( Figures 6 B and 6C). This percentage was significantly lower when flies were exposed to the unpaired odor (CS−) or to an unrelated odor. Thus, neurons that undergo c-Fos/CREB cycling during spaced training are preferentially reactivated upon memory recall.

(H) ChR2 was expressed in kayak-positive neurons during spaced training of an aversive association. Later reactivation of these neurons with blue light in the presence of ATR induced avoidance behavior. n = 20.

(G) Naive flies expressing ChR2 under kayak promoter control prefer blue light in the presence or absence of all-trans retinal (ATR). Positive avoidance scores indicate avoidance of blue light, while negative avoidance scores indicate preference of blue light. n = 7.

(F) T-maze apparatus in which flies are exposed to blue and green light and choose between the two.

(E) Memory 24 hr after spaced training is significantly impaired when synaptic output from kayak-positive cells is suppressed (Test1). Memory improves when output from these cells is restored (Test2). n = 8.

(D) Schematic diagram of the experimental protocol. shi ts was expressed in kayak-positive neurons during the latter half of spaced training. This protocol permitted expression of shi ts , but not inhibition of activity, during training. Testing was performed twice in succession, once at the restrictive temperature (32°C) and later at the permissive temperature (18°C).

(C) Quantification of the results shown in (B). OCT-BEN refers to flies trained to octanol and exposed to the unrelated odor, benzaldehyde 24 hr later. n = 5–9.

(B) Memory recall preferentially reactivates cells that expressed kayak during spaced training. OCT-OCT: flies trained to associate octanol with aversive shocks and exposed to octanol 24 hr later. OCT-MCH: flies trained to octanol and exposed to MCH. Filled white arrowheads indicate kayak-expressing (GFP positive) KCs in which pERK is reactivated upon odor exposure. Non-filled arrowheads indicate kayak-expressing KCs in which pERK is not reactivated upon odor exposure. Scale bars represent 20 μm.

(A) Schematic of the experimental protocol. Training was performed as described in Figure 5 A. 24 hr after training, flies were exposed to odors for 1 min and harvested and fixed 8 min later. GFP signals were used to identify cells that were activated during training, and pERK signals were used to identify cells activated upon odor exposure 24 hr later.

If pERK-dependent c-Fos/CREB cycling is required for LTM formation, pERK-positive cells expressing high levels of dCREB2 and kayak may function in encoding LTM engrams. Thus, we next monitored GFP expressed from a kayak-GAL4 driver during spaced training in transgenic UAS-mCD8-GFP; kayak-GAL4/tub-GAL80flies ( Figure 5 A). We did not find significant pERK or GFP (kayak) expression in KCs in naive flies ( Figure 5 B), a result consistent with previous studies showing that ERK phosphorylation and kayak expression reflect neural activity (). dCREB2 amounts were more variable in KCs from naive animals, with some KCs displaying high amounts. When we examined expression 8 min after the 10th spaced training session, we found significant increases in pERK signals, kayak expression, and dCREB2 amounts in the MBs ( Figure 5 B). Among pERK-positive KCs, we found a significant overlap in KCs expressing c-Fos and high amounts of dCREB2, suggesting that c-Fos/CREB cycling occurs cell autonomously in potential memory engram-encoding cells ( Figure 5 C). In contrast, 8 min after massed training, kayak expression was unaltered compared to naive animals, and overlapping kayak and dCREB2 signals were not observed ( Figures 5 B and 5C). When we examined kayak expression (kayak>GFP) throughout the brain after spaced training, we found kayak signals predominantly in KCs, suggesting that engram cells are found primarily in this area ( Figure S5 A).

(C) An increase in pERK-positive KCs expressing both kayak and high amounts of dCREB2 after spaced training. After massed training, 1.3% ± 0.4% of pERK-positive KCs express kayak, 6.5% ± 0.5% express high amounts of dCREB2, and there is no overlap between these two populations. After spaced training, a new population of KCs (18.9% ± 1.2% of pERK-positive KCs) expressing both kayak and high amounts of dCREB2 is observed.

(B) pERK distribution and expression of c-fos (kayak) and dCREB2 in KCs. pERK and dCREB2 were monitored using monoclonal antibodies, and kayak expression was monitored using monoclonal antibodies for GFP in UAS-mCD8-GFP; kayak-GAL4/tub p -GAL80 ts flies. Scale bars represent 20 μm.

(A) Schematic diagram for identifying neurons with increased kayak expression after training. mCD8-GFP is expressed under Gal4 control, and Gal4 is expressed from the kayak promoter. Gal80 ts is expressed from a tubulin promoter and suppresses Gal4 activity at permissive temperature (18°C). Training is performed at 32°C, so GFP expression should reflect kayak expression. The black arrowhead indicates the time point when flies were fixed and dissected.

c-Fos and pERK, which is a better marker for neuronal activation and central sensitization after noxious stimulation and tissue injury?.

How does training suppress ERK phosphorylation? Activity-dependent ERK phosphorylation is suppressed by calcineurin (CaN) and protein phosphatase (PP1) () ( Figure S7 B). Thus, we next examined ERK phosphorylation during training in heterozygous CaN (CanB2/+) () and PP1 (Pp1-87B/+) () loss-of-function mutants and found that phosphorylation was maintained during training trials in these flies ( Figures 4 A and 4B ). CanB2/+ and Pp1-87B/+ flies show normal learning after single-cycle training ( Figure S4 A) and normal LTM after spaced training ( Figure S4 B) but increased memory 24 hr after massed training ( Figure 4 C). Consistent with the idea that this increased memory consists of LTM, massed training increased ERK phosphorylation ( Figures 4 D and 4E) and kayak expression ( Figure 4 F) in CanB2/+ and Pp1-87B/+ flies. Furthermore, this increased memory was sensitive to both the protein synthesis inhibitor, cycloheximide (CXM), and the MEK inhibitor, U0216 ( Figures 4 G and 4H). These results suggest that the function of rest intervals during spaced training is to increase pERK activity and that artificially increasing pERK activity bypasses the need for rests.

(D and E) Massed training increases pERK in CaNB2/+ and PP1/+ mutants. Samples were taken from naive animals and after the 5th and 10th training trials in indicated genotypes. (D) shows an example western blot, while (E) shows the average of 4 independent experiments. n = 4.

Different types of MB Kenyon cells (KCs) project axons to distinct α/β, α′/β′, and γ lobes. Previous studies have shown that KCs projecting to the α/β lobes are critical for LTM formation (). Consistent with these studies, pERK-positive signals after spaced training localized mainly to KCs projecting to the α/β lobes, not to the α′/β′ and γ lobes ( Figures 3 C and 3D). Furthermore, suppressing ERK activity by expressing Drosophila MAPK phosphatase (DMKP) () ( Figure 3 E) in α/β KCs, but not in the other KCs, disrupted LTM formation ( Figure 3 F).

To determine whether training actively suppresses ERK activity, we next examined pERK amounts during spaced training with 5-min rest intervals, in which a subsequent training trial starts before pERK reaches peak amounts. pERK amounts decreased upon starting a new training trial ( Figure 3 A). Consistent with a prior study (), spaced training with 5-min rest intervals produced lower memory scores than spaced training with normal 15-min rest intervals ( Figure 3 B). Altogether, these results suggest that training trials not only suppress ERK activity but also trigger subsequent ERK activation.

(E) Heat shock (hs)-dependent expression of Drosophila MAPK phosphatase (DMKP) prevents pERK increases during rest intervals. Indicated flies were heat-shocked at 37°C for 30 min 3 hr before spaced training.

(C) pERK is found in α/β KCs during spaced training. Flies were harvested and fixed 8 min after the 10th spaced training trial. C305a>GFP flies express GFP in α′/β′ KCs, C739>GFP flies express GFP in α/β KCs, and 72B08>GFP flies express GFP in γ KCs. pERK signals were seen in α/β KCs, but not α′/β′ or γ KCs. Scale bar represents 20 μm

(B) 24-hr memory after massed and spaced training with indicated RIs. 5-min RIs produce reduced LTM, which can be inhibited by cycloheximide (CXM) feeding. n = 7–10.

(A) Left panel, top: diagram showing time points at which flies were harvested during training. Left panel, bottom: western blots showing ERK and pERK amounts. Right panel: quantification of pERK amounts during training with 5- and 15-min rest intervals (RIs). n = 4.

previously suggested that ERK activity increases specifically during the rest intervals between trainings and decreases during actual training. Thus, ERK activity increases during spaced training, but not during massed training, which lacks rest intervals. Consistent with their results, we found that pERK amounts started to increase after training, reached a peak 8 min into the rest interval, and then decreased back to basal amounts during the subsequent training session ( Figure S3 A). We further found that dumb1 and dumb2 mutations, which disrupt Drosophila D1 dopamine receptors, and rutabaga (rut) mutations, which disrupt a Ca/calmodulin regulated adenylyl cyclase (AC), prevent rest interval-dependent increases in pERK ( Figures S3 B and S3C). Furthermore, training-dependent increases in dCREB2 were inhibited in rut mutants ( Figure S3 D), suggesting that dopamine signaling may activate the rut-AC, which then stimulates rest interval-dependent ERK activation and subsequent c-Fos/CREB cycling.

Activity of both c-FOS and CREB is regulated by phosphorylation by ERK (). ERK activity increases upon phosphorylation by MEK (MAPK kinase) (). In agreement with a previous study by, we found a significant increase in pERK during spaced training, but not massed training ( Figure S2 ). To determine whether this increase in pERK is required for increased expression of c-fos and dCREB2 during spaced training, we used two MEK inhibitors, PD98059 and U0126 (). Feeding flies either inhibitor before spaced training abolished training-dependent increases in ERK phosphorylation ( Figures 2 A and 2B ) and c-fos and dCREB2 expression ( Figures 2 C and 2D). Furthermore, while neither MEK inhibitor affected initial learning ( Figure 2 E), short-term memory (STM) ( Figure 2 F), or ARM after massed training ( Figure 2 G), both significantly suppressed LTM after spaced training ( Figure 2 H). These results suggest that ERK activity during spaced training is primarily required for c-Fos/CREB cycling and LTM formation.

(E–H) Effect of MEK inhibitors on 3-min memory (E) and 1-hr memory after single-cycle training (F), 24-hr memory after 10× massed training (G), and 24-hr memory after 10× spaced training (H). n = 5 for all data.

(A and B) ERK phosphorylation increases during spaced training. This increase is prevented by MEK inhibitor feeding. Flies were harvested during the rest interval after the eighth training trial. (A) shows a typical western blot, and (B) shows the means ± SEMs of 3 independent experiments.

To test whether increased dCREB2 expression during spaced training requires c-Fos, we next induced expression of FBZ in neural cells (elav-GS>UAS-FBZ). While FBZ expression did not affect baseline expression of dCREB2, it significantly suppressed the increase in dCREB2 expression during spaced training ( Figure 1 G). Altogether, our results indicate that c-Fos/CREB cycling occurs during spaced training, dCREB2 activity induces an increase in kayak expression early during spaced training, and the subsequent increase in c-Fos activity induces a later increase in dCREB2 expression. If this type of mutually dependent expression occurs, kayak expression early during spaced training should depend on activation of baseline, preexisting dCREB2, while expression late during spaced training may depend on dCREB2 produced from earlier c-Fos activity. If this is the case, FBZ should not inhibit increased kayak expression at early time points during spaced training but should inhibit expression at later time points. Consistent with this idea, the initial increase in kayak expression after the second training session occurred normally in elav-GS>UAS-FBZ flies, but this increase was not maintained into the eighth training session ( Figure 1 H).

c-Fos and another leucine zipper protein, Jun, form the transcription factor AP-1. AP-1 positively regulates synaptic strength and numbers and has been reported to regulate CREB expression (). Thus, we next measured expression of the dCREB2 activator (dCREB2a and dCREB2d) and repressor (dCREB2b) isoforms during spaced training. Unlike kayak expression, expression of dCREB2 activators did not increase significantly after the second training session. Instead, expression increased more gradually and became significant after the fifth training session ( Figure 1 F), before rapidly decaying back to baseline within an hour after training ( Figure S1 C). dCREB2 repressor expression also increased during spaced training but was delayed compared to activator expression ( Figure S1 D), resulting in an increased activator-to-repressor ratio during the latter half of training ( Figure S1 E). In contrast to spaced training, massed training did not significantly affect expression of any dCREB2 isoform.

To determine whether increased kayak expression requires CREB, we expressed a repressor isoform of Drosophila CREB (dCREB2b) and examined kayak expression. Heat shock induction of dCREB2b before training significantly reduced increases in kayak ( Figure 1 E), suggesting that training activates CREB, which then induces kayak.

In mammals, LTM formation requires activity of c-Fos (), a CREB-regulated immediate early transcription factor (). To determine whether c-Fos is also required for LTM in Drosophila, we conditionally expressed a dominant-negative version (FBZ) (), either pan-neuronally (elav-GS>UAS-FBZ) or specifically in mushroom body (MB) neurons (MB-GS>UAS-FBZ), and measured memory after spaced and massed training ( Figure 1 A). Memory 1 day after spaced training was significantly inhibited, while memory after massed training was unaffected ( Figures 1 B and 1C), indicating that c-Fos is required for LTM, but not ARM. In Drosophila, c-Fos is encoded by the kayak gene, and we showed that conditional knockdown of kayak expression in the MBs (MB-GS > kayak RNAi) inhibited 24-hr memory after spaced training, but not massed training ( Figure S1 A). We next examined kayak expression during spaced training and observed a significant increase in expression after the second spaced training trial that was maintained throughout training ( Figure 1 D). In contrast, kayak expression did not change during massed training. Expression of a second immediate early gene, arc, whose expression is reported to be CREB independent (), did not increase during spaced training ( Figure S1 B) but instead increased after the end of training (data not shown).

(H) FBZ does not affect increases in kayak expression early during spaced training but prevents this increase from being maintained at later time points of training. n = 6–7.

(B and C) 24-hr memory is inhibited by acute expression of a dominant-negative form of c-Fos (FBZ) before spaced training. FBZ was expressed in all neurons (B) or in KCs (C) upon RU486 (RU+) feeding 12 hr before training. 24-hr memory was measured by allowing flies to choose between the shock-paired odor and the non-paired odor and calculating a performance index (PI). n = 5–6.

(A) Schematic diagram of spaced training and massed training protocols. Blue and red boxes represent single training trials. Spaced training consists of ten training trials with 15-min rest intervals between trials, while massed training consists of ten training trials without rest intervals. Expression of c-fos and CREB activators was measured at time points indicated by arrowheads for (D) and (F) and at time points a and b for (E), (G), and (H).

Contribution of Egr1/zif268 to activity-dependent Arc/Arg3.1 transcription in the dentate gyrus and area CA1 of the hippocampus.

Discussion

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Ostroff L.E.

LeDoux J.E. Molecular mechanisms of fear learning and memory. Why does ERK activity increase during rest intervals, but not during training? ERK is phosphorylated by MEK, which is activated by Raf. Amino acid homology with mammalian B-Raf () suggests that Drosophila Raf (DRaf) is activated by cAMP-dependent protein kinase (PKA) and deactivated by CaN. Our current results indicate that ERK activation requires D1-type dopamine receptors and rut-AC, while a previous study demonstrates that ERK activation also requires Cainflux through glutamate NMDA receptors (). Thus, training-dependent increases in glutamate and dopamine signaling may activate rut-AC, which produces cAMP and activates PKA. PKA activates the MAPK pathway, resulting in ERK phosphorylation. At the same time, training-dependent increases in Ca/CaM activate CaN and PP1 to deactivate MEK signaling (). The phosphatase pathway may predominate during training, inhibiting ERK phosphorylation. However, phosphatase activity may deactivate faster at the end of training compared to the Rut/PKA activity, resulting in increased ERK activation during the rest interval after training ( Figure S7 B).

Zhang et al., 2008 Zhang X.

Li Q.

Wang L.

Liu Z.J.

Zhong Y. Active protection: Learning-activated Raf/MAPK activity protects labile memory from Rac1-independent forgetting. In this study, we examined the role of ERK phosphorylation and activation in LTM and did not observe significant effects of ERK inhibition in short forms of memory ( Figure 2 F). However,previously reported that ERK suppresses forgetting of 1-hr memory, suggesting that ERK may have separate functions in regulating STM and LTM.

Ryan et al., 2015 Ryan T.J.

Roy D.S.

Pignatelli M.

Arons A.

Tonegawa S. Engram cells retain memory under retrograde amnesia. Roy et al., 2016 Roy D.S.

Arons A.

Mitchell T.I.

Pignatelli M.

Ryan T.J.

Tonegawa S. Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease. Roy et al., 2017 Roy D.S.

Kitamura T.

Okuyama T.

Obata Y.

Yoshiki A.

Tonegawa S. Distinct neural circuits for the formation and retrieval of episodic memories. c-Fos/CREB cycling distinguishes engram cells from non-engram cells, and we suggest that this cycling functions to establish and maintain engrams. However, studies in mammals () indicate that transcription and translation after fear conditioning is required for establishing effective memory retrieval pathways instead of memory storage. Thus, c-Fos/CREB cycling may be required for establishment and maintenance of engrams or for retrieval of information from engrams.