1. Long-Term Memory Engram Cells Are Established by c-Fos/CREB Transcriptional Cycling 
Tomoyuki Miyashita, Emi Kikuchi, Junjiro Horiuchi, Minoru Saitoe 
Cell Reports 
Volume 25, Issue 10, Pages e3 (December 2018)
DOI: /j.celrep
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2. Cell Reports 2018 25, 2716-2728.e3DOI: (10.1016/j.celrep.2018.11.022)
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3. Figure 1
Increases in c-Fos and CREB during Spaced Training Are Mutually Dependent
(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).
(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.
(D) c-fos expression during spaced and massed training. n = 4–8.
(E) Increases in c-fos during spaced training are inhibited by expressing a CREB repressor isoform (hs-dCREB2b). n = 6.
(F) dCREB2 activator expression during spaced and massed training. n = 4–8.
(G) FBZ prevents spaced training-dependent increases in dCREB2 activator expression. n = 6.
(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.
Error bars indicate SEM and ∗∗∗p ≤ 0.001, ∗∗p ≤ 0.01, ∗p ≤ 0.05, and n.s., not significant for all figures.
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4. Figure 2
Increased Expression of c-fos and dCREB2 during Spaced Training Requires ERK Activation
(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.
(C) Increased expression of c-fos during spaced training is prevented by MEK inhibitors. n = 4.
(D) Increased dCREB2 activator expression after the eighth training trial is prevented by MEK inhibitors. n = 4.
(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.
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5. Figure 3
ERK Is Activated during Rest Intervals and Inhibited during Training
(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.
(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.
(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
(D) Quantification of the results shown in (C). n = 5.
(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.
(F) Overexpressing MAPK phosphatase in α/β KCs inhibits LTM formation. elav > DMKP3 expresses DMKP3 pan neuronally, while 30Y > DMKP3 expresses DMKP3 throughout the MBs. n = 5.
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6. Figure 4
Sustained pERK Activity Bypasses the Requirement for Rest Intervals during LTM Formation
(A and B) CaNB2/+ and PP1/+ mutants have sustained pERK activity during spaced training.
(A) Schematic diagram and example western blots measuring pERK and ERK amounts at indicated time points.
(B) Quantification of results described in (A). n = 4.
(C) Memory 24 hr after 10× massed training is increased in CaNB2/+ and PP1/+ mutants compared to wild-type controls. n = 9–10.
(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.
(F) c-fos expression increases after 10× massed training in CaNB2/+ and PP1/+ mutants. This increase is prevented by MEK inhibitor feeding. n = 4.
(G) Improved memory in CaNB2/+ and PP1/+ mutants is sensitive to the protein synthesis inhibitor CXM. n = 9–10.
(H) Improved memory in CaNB2/+ and PP1/+ mutants is suppressed by U0126 feeding before 10× massed training. n = 4.
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7. Figure 5
Coexpression of c-fos and CREB in pERK-Positive KCs after Spaced Training
(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. Gal80ts 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.
(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/tubp-GAL80ts flies. Scale bars represent 20 μm.
(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.
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8. Figure 6
LTM Retrieval Requires Reactivation of c-Fos/CREB Cycling Cells
(A) Schematic of the experimental protocol. Training was performed as described in Figure 5A. 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.
(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.
(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.
(D) Schematic diagram of the experimental protocol. shits was expressed in kayak-positive neurons during the latter half of spaced training. This protocol permitted expression of shits, 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).
(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.
(F) T-maze apparatus in which flies are exposed to blue and green light and choose between the two.
(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.
(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.
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9. Figure 7
Prolonged pERK Activation Activates c-Fos/CREB Cycling to Induce LTM
(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.
(B) kayak expression increases at both early and late time points during 48× massed training in PP1/+ and CaNB2/+ mutants. n = 4–12.
(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.
(D) LTM formed after 48× massed training in CaNB2 mutants is suppressed by expressing dominant-negative c-fos (FBZ). n = 10.
(E) pERK distribution and expression of kayak and dCREB2 in KCs 7 min after 48× massed training in CaNB2EP774/UAS-mCD8-GFP; kayak-GAL4/tubp-GAL80ts and control UAS-mCD8-GFP; kayak-GAL4/tubp-GAL80ts flies. Scale bars represent 20 μm.
(F) 48× massed training increases the percentage of pERK-positive KCs expressing both kayak and high amounts of dCREB2 in CaNB2/+ flies.
(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 6H, replacing spaced training with 48× massed training of CaNB2EP774/UAS-ChR2-mCherry;kayak-GAL4/tubp-GAL80ts and UAS-ChR2-mCherry/+;kayak-GAL4/tubp-GAL80ts flies. n = 7.
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