Supporting Information

Atucha et al. 10.1073/pnas.1710819114 SI Methods the floor. The starting compartment (31 cm) was made of opa- Subjects. Male Sprague–Dawley rats (280 to 320 g at time of que white plastic and was well-lit; the shock compartment surgery) from Charles River breeding laboratories were used. (60 cm) was made of two dark, electrifiable metal plates and was They were kept individually in a temperature-controlled (22 °C) not illuminated. The training context in which footshock was vivarium room and maintained on a 12-h:12-h light:dark regimen given (shock box) did not have any contextual modifications. The (0700 to 1900 hours lights on) with ad libitum access to food and safe training context (nonshock box) had four vertical white water. Training and testing were performed between 1000 and stripes (2 cm wide) taped in the dark compartment together with 1500 hours. All experimental procedures were in compliance tape placed on the floor, closing the gap between the two plates. with European Union Directive 2010/63/EU and approved by The novel box (used on the retention test only) had two white the Institutional Animal Care and Use Committee of Radboud circles (3.5-cm diameter) taped on each wall of the dark com- University, Nijmegen, The Netherlands. partment, and the gap between the plates was closed with tape (Fig. S1). All three inhibitory avoidance apparatuses were lo- Cannula Implantation. Rats were anesthetized with an s.c. injec- cated next to one another in a sound- and light-attenuated room. tion of ketamine (37.5 mg/kg; Alfasan) and dexmedetomidine For a detailed description of the task, see ref. 38. (0.25 mg/kg; Orion) and received the nonsteroidal analgesic For training, rats were initially placed in the starting compart- carprofen (4 mg/kg; Pfizer). They were positioned in a stereotaxic ment of the nonshock box and could freely explore the apparatus frame (Kopf Instruments), and two stainless steel guide cannulae for 20 s without footshock being delivered. Afterward, the rats were (23 gauge; Component Supply/SKU Solutions) were implanted removed from the apparatus and, after a delay of 2 min, placed in bilaterally with the cannula tips 2.0 mm above the basolateral the starting compartment of the second inhibitory avoidance ap- amygdala (BLA) [15 mm long; coordinates: anteroposterior (AP): paratus (shock box). After the rat stepped completely into the dark 2.8 mm posterior to Bregma; mediolateral (ML): ±5.0 mm lateral compartment, the sliding door was closed and a single inescapable to midline; dorsoventral (DV): 6.5 mm below skull surface] (59). footshock (0.60 mA; 1 s) was delivered. The rats were removed Some rats received additional bilateral guide cannulae implanted from the apparatus 20 s after termination of footshock and, after 1.5 mm above the dorsal hippocampus (11 mm long; coordi- drug treatment, returned to their home cages. On the retention nates: AP: −3.6 mm; ML: ±1.9 mm; DV: −2.6 mm). After sur- test, either 2 or 28 d after training, they were tested, in a ran- gery, the rats were administered atipamezole hydrochloride domized order and without delay, in the two training contexts (i.e., (0.25 mg/kg s.c.; Orion) to reverse anesthesia and 3 mL of sterile shock box and nonshock box) and in a novel box they had not visited saline to facilitate clearance of drugs and prevent dehydration. before. Previously, we have shown that the order of retention The rats were allowed to recover for a minimum of 7 d before testing in the different test environments does not affect retention commencement of training, and were handled three times for latencies (38). For all three boxes, the rats were placed in the 1 min each during this recovery period. starting compartment and their latency to enter the dark com- partment with all four paws (maximum latency of 600 s) was Drug and Infusion Procedure. Norepinephrine (NE; 1.0 μgin recorded. Longer latencies in the shock box compared with the 0.2 μL; Sigma-Aldrich) was dissolved in saline and infused into nonshock box were interpreted as indicating accurate memory of the BLA either immediately (0 h) or 3 h after the training. Drug the shock–context association. Different groups of trained rats dose and volume were based on previous findings from our were used for the 2- and 28-d retention tests. laboratory (9, 42). The GABAergic receptor agonist muscimol (3-hydroxy-5-aminomethyl-isoxazole; 0.5 μg in 0.5 μL; Sigma- Cannula Placement Verification. Rats were deeply anesthetized with Aldrich) was dissolved in saline and administered into the dor- sodium pentobarbital and perfused transcardially with 0.9% sa- sal hippocampus 20 min before retention testing (39). line, followed by 4% formaldehyde. The brains were removed and Bilateral infusions of drug or an equivalent volume of saline immersed in 4% formaldehyde. At least 24 h before sectioning, into the BLA were made by using 30-gauge injection needles brains were placed in a 25% sucrose solution for cryoprotection. connected to 10-μL Hamilton microsyringes by polyethylene Coronal sections of 50 μm were cut on a cryostat, mounted on (PE-20) tubing. The injection needles protruded 2.0 mm beyond gelatin-coated slides, stained with cresyl violet, and examined by the cannula tips, and a 0.2-μL injection volume per hemisphere light microscopy by an observer blind to drug treatment. Rats was infused over a period of 30 s by an automated syringe pump with needle tips located outside the boundaries of the BLA and/ (Stoelting). The injection needles were retained within the can- or hippocampus, or with extensive tissue damage at the target nulae for 20 s following drug infusion to maximize diffusion. areas, were excluded from final analyses. For hippocampal infusions, the infusion needles protruded For verification of cannula placement of flash-frozen brains, 1.5 mm beyond the guide cannulae, and a 0.5-μL injection volume 50-μm-thick coronal sections were cut, collected on gelatin- per hemisphere was infused over a period of 75 s. All drug so- coated slides, and let to dry. Sections were then fixed in 100% lutions were freshly prepared before each experiment. acetone for 30 min and stained with cresyl violet and processed as mentioned above. Inhibitory Avoidance Discrimination Task. Rats were subsequently trained in two contextually distinct inhibitory avoidance appa- Tissue Collection. Rats were administered an overdose of sodium ratuses within a single training session, but footshock was de- pentobarbital 30 min after training for Dnmt mRNA measure- livered only in the latter context. On the retention test, they were ments or immediately after retention testing for mRNA ex- tested in both training contexts as well as in a novel context (Fig. pression and DNA methylation measurements. Within 90 s after 1A). Each apparatus had the same geometry and consisted of a the pentobarbital injection, rats were decapitated, and the brains trough-shaped alley (91 cm long, 15 cm deep, 20 cm wide at the were rapidly removed and flash-frozen by submersion for 2 min top, and 6.4 cm wide at the bottom) divided into two compart- in a beaker filled with precooled isopentane on dry ice. Flash- ments, separated by a sliding door that opened by retracting into frozen brains were stored at −80 °C.

Atucha et al. www.pnas.org/cgi/content/short/1710819114 1of9 Brain tissue containing the hippocampus or ACC was cut into lease 3.3, package ragene20stprobeset.db 8.2.0). Probe set sum- 350-μm-thick coronal slices and further dissected using a 0.75-mm maries were further annotated by NetAffx biological annotation brain puncher (Stoelting). Bilateral punches from the dentate (Affymetrix). gyrus area of the hippocampus (AP: −2.64 to −3.86 mm) and Differential expression analysis was performed using linear ACC (AP: +3.00 to +1.92 mm) were collected from three con- models, with the limma R package (60), considering treatment secutives slices for a total of six punches. Punches were preserved (two levels: NE and saline), tissue (two levels: hippocampus and at −20 °C for at least 16 h and later in RNAlater-ICE (Ambion ACC), and time point (two levels: 2 and 28 d). From these models, Life Technologies). contrasts were built to assess temporal effects of posttraining NE treatment in different tissues. P values were corrected for mul- Tissue Homogenization and Nucleic Acid Extraction. Parallel DNA tiple testing (for all probe sets and 13 contrasts) using the and RNA isolation was performed using a chaotropic lysis pro- Benjamini and Hochberg (61) false discovery rate (FDR < 0.05). tocol. After removing RNAlater-ICE, tissue samples were flash- Additionally, a fold-change criteria ≥j1.8j was applied (median frozen in liquid nitrogen and disrupted using TissueLyser II jFCj observed for all significant contrasts). Transcripts’ FCs were (Qiagen) for 1 min at 28 Hz. Then, 420 μL of guanidinium calculated by summarizing all of the probe sets that fulfilled the thiocyanate disruption lysis buffer (4.5 M guanidinium thiocya- above criteria. nate, 2% N-lauroylsarcosine, 50 mM EDTA, pH 8, 25 mM Tris·HCl, pH 7.5, 0.1 M beta-mercaptoethanol, 0.2% antifoam Gene Set Enrichment Analysis. A memory gene set composed of A) was added. Disrupted tissue was solubilized by vortexing for 122 genes previously implicated in memory processes was created 1 min at maximum speed. The lysate was then split into two (35, 36, 40, 51, 62). For gene ranking based on their differential equal parts for RNA and DNA isolations. expression across the contrasts, we computed three different RNA isolation was performed by adding 600 μL TRI Reagent metrics from all associated probe sets: the mean difference in (Ambion Life Technologies) to 200 μL tissue lysate and pulse means, the mean of quotients in mean expression levels, and a vortexing. Then, 830 μL of absolute ethanol was added (final combined P value. Then, each region was assigned a metrics- ethanol concentration ∼51%) and the mixture was loaded on a specific rank, and a combined rank was computed as the maxi- Zymo-Spin IIC column. RNA was purified using a Direct-zol Kit mum value among the three metrics ranks. Next, GSEA was (Zymo Research) following the recommended procedure, in- performed (63). Briefly, the ranking of the genes was based on cluding DNase I treatment. The concentration and quality of the combined rank test statistics. Then, a weighted Kolmogorov– RNA were determined using a NanoDrop 2000 (Thermo Sci- Smirnov statistic was used as a basis for determining whether entific) and RNA 6000 Nano Kit on a Bioanalyzer 2100 in- genes from a prespecified memory gene set were overrepresented strument (Agilent). among the top or bottom of the list (Bioconductor release 3.3, DNA isolation was first performed with the QIAamp DNA package GO.db). Next, shuffling the phenotypes generated the Micro Kit (Qiagen). Two hundred microliters of Buffer AL background distribution of the gene set statistics and the gene set (Qiagen) and 200 μL absolute ethanol were added to 200 μL lysate analysis was repeated. Each randomly generated gene set for and mixed by pulse vortexing. DNA was purified on a QIAamp which its maximum deviation was higher than the original data MinElute column using the recommended protocol. An additional was counted and, after n iterations, the P value was computed purification was performed using a Genomic DNA Clean & (10,000 permutations). The procedure was repeated until all gene Concentrator Kit (Zymo Research) using the recommended sets were tested. For false discovery correction, we applied the protocol. The concentration and quality of the DNA were de- Benjamini–Hochberg procedure (61). termined using fluorometry (Qubit dsDNA BR Assay Kit; Invi- trogen) and gel electrophoresis. Quantitative Real-Time PCR. For measuring posttraining Dnmt ex- pression changes, total RNA from hippocampus punches was iso- Transcriptomic Analysis: Target Synthesis and GeneChip Hybridization. lated using the recommended TRIzol protocol (Life Technologies), Target synthesis was performed starting from 25 ng total RNA by including DNase treatment [1 μg RNA was treated with 2 U DNase using NuGEN kits: Ovation Pico WTA System V2 (3302-12), (Sigma-Aldrich), 1 h at 37 °C]. The quality of RNA samples was followed by an Encore Biotin Module (4200-12), using standard verified by agarose gel electrophoresis and UV spectrophotometric protocols. Synthesis reactions were carried out on a PCR machine analysis. cDNA was synthesized from 0.5 to 1 μgRNAbyusingthe (TProfessional TRIO; Biometra). Eighty-five microliters of mix- RevertAid First Strand cDNA Synthesis Kit standard protocol ture (25 ng/μL DNA) was loaded on an Affymetrix Rat Gene 2.0 (Fermentas). qPCR was performed with the GoTaq qPCR Kit ST Array (902124) and hybridized for 17 h (45 °C, 60 rpm) in a (Promega), including 2 μL of the reverse transcription reaction Hybridization Oven 640 (Affymetrix). (1:20 dilution), 1:50 dilution of CXR (carboxy-X-rhodamine refer- The arrays were washed and stained on a Fluidics Station 450 ence dye), and 200 nM each primer: for Dnmt3a, forward (FW)-5′- (Affymetrix) by using a Hybridization, Wash, and Stain Kit ATCGACGCCAAAGAAGTGTC-3′ and RV-5′-GCTATTCTG- (Affymetrix) under the FS450_0007 protocol. The GeneChips CCGTGTTCCAG-3′;forDnmt3b,FW-5′-AAAGTCGAAGACG- were processed with an Affymetrix GeneChip Scanner 3000 7G. CACAACC-3′ and reverse (RV)-5′-CTTACCGCAGGACAGA- DAT images and CEL files of the microarrays were generated CAGC-3′;forDnmt1,FW-5′-GGAGCAGATCGAGAAGGATG- using an Affymetrix GeneChip Command Console. 3′ and RV-5′-CTTGCACTTCCCACACTCAG-3′;andforCreb, FW-5′-TCAGCCGGGTACTACCATTC-3′ and RV-5′-TTCAGC- Transcriptomic Analysis: GeneChip Data Preprocessing. Preprocess- AGGCTGTGTAGGAA-3′. Samples were normalized to actin-β ing of the Gene ST arrays was done with the RMA workflow using transcript using the following primers: FW-5′-CCAACTGGGAC- the oligo R package (57). In short, background correction was GATATGGAG-3′ and RV-5′-AACACAGCCTGGATGGCTAC-3′. performed using the convolution of signal and noise distributions. Expression was quantified using the ABI PRISM 7900HT Sequence Log2-transformed values were obtained and further normalized Detection System (Applied Biosystems) and the following cycling using quantile normalization (57). Above-background expression conditions: 95 °C, 10 min; 40× (95 °C, 15 s; 60 °C, 1 min); 72 °C, 5 min. filtering was applied using the Mas5Calls algorithm: Probe sets Validation of expression changes of microarray significant called as absent (cutoff P ≥ 0.05) were excluded from all arrays genes from GSEA was performed starting from SPIA cDNA (Mas5Calls function from the affy R Package: https:/bioconductor. (Single Primer Isothermal Amplification; NuGEN). qPCR was org/packages/release/bioc/html/affy.html). For annotation purposes, performed using the SYBR FAST PCR Kit (KAPA Biosystems), we used the ragene20stprobeset.db 8.2.0 library (Bioconductor re- in a 12-μL final reaction volume, using 2 μL cDNA template, on

Atucha et al. www.pnas.org/cgi/content/short/1710819114 2of9 a RotorGene 6000A instrument (Corbett Research). Cycling 30-μL reactions containing the following: 1× PCR buffer II, 300 μM × conditions were as follows: 95 °C, 60 s; 32 (95°C,10s;10°C,15s; deoxynucleotide triphosphates, final 3.5 mM MgCl2,200μMeach 72 °C, 10 s); 72 °C, 5 min, followed by a melting curve analysis (61 to primer, and 20 ng BSC DNA. We used the following cycling con- 95 °C, rising by 0.7 °C/3 s). Threshold cycles were determined using ditions: 95 °C, 15 min; 50× (95°C,30s;55°C,30s;72°C,30s); RotorGene software version 6.1 (Corbett Research). Ywhaz and 72 °C, 10 min. PCR products were purified and sequenced using a Gapdh were selected as reference genes for normalization, with the PyroMark ID System (Biotage) following the manufacturer’ssug- following primer sets: Pkmζ,FW-5′-CTTAAAGGGACGGAA- gested protocol and sequencing primers: for Pkmζ,5′-GGG- GATG-3′ and RV-5′-TAGATGGACTCGGCTTTC-3′; Reln,FW- TTTTGGTTAGTTTTTATT-3′;andforReln,5′-ACATACAA- 5′-CTCCAGTTCAAGCTAAAC-3′ and RV-5′-CAGCATCATG- AAAAATAACTAACAAC-3′ (Microsynth). TGAATACT-3′; Gapdh,FW-5′-TCACCACCATGGAGAAGGC- Reln and Pkmζ gene promoter regions analyzed by bisulfite ′ ′ ′ Ywhaz 3 and RV-5 -GCTAAGCAGTTGGTGGTGCA-3 ;and , pyrosequencing are depicted in Fig. 3 G and H, respectively. The ′ ′ ′ FW-5 -TTGAGCAGAAGACGGAAGGT-3 and RV-5 -GAAG- Reln gene region analyzed (enlarged box) is located in the Reln ′ CATTGGGGATCAAGAA-3 (Microsynth). promoter in front of exon 1. The Pkmζ gene region analyzed Expression levels were normalized using a geometric mean level (enlarged box) is located in the alternative promoter in front of of expression, and fold differences were calculated using the delta- exon 1′ that carries the unique 5′-PKMζ mRNA sequence. Num- delta-Ct method (64), qBasePlus software (Biogazelle). Differen- bering is relative to the transcription start site +1 located in exon tial expression analysis was performed using linear models, limma 1′. The examined CpG sites are marked in bold. The sequencing R package (60), using the same contrasts as described above for the primer is marked in italics. Relative size and chromosomal coor- microarray data analysis. dinates are also depicted (Upper and Lower, respectively; RGSC To account for multiple testing, we applied Bonferroni 6.0/rn6). correction. Controlling for PCR temperature bias was done with a series of Pyrosequencing Analysis. DNA methylation was quantified by direct calibrator samples of known methylation levels. Briefly, unme- bisulfite pyrosequencing (58). Five hundred nanograms of high- thylated standards were prepared by using two rounds of linear purity, intact DNA was used for bisulfite conversion using an whole-genome amplification with an Ovation WGA System Kit EZ DNA Methylation-Gold Kit (Zymo Research) by following (NuGEN), starting from 10 ng of DNA, as recommended by the standard protocols. Bisulfite-converted (BSC) DNA quality and manufacturer. Methylated standards were made using the CpG concentration were determined using an RNA 6000 Pico Kit on a methyltransferase assay with M.SssI (New England Biolabs), Bioanalyzer 2100 instrument (Agilent Technologies) and Nano- starting from 2 μg of purified DNA, following the standard Drop 2000 (Thermo Scientific). BSC samples were normalized to protocol. Bisulfite conversion of standard samples was done as 10 ng/μL. described above. The following primer sets were used: for Pkmζ,FW-5′- All samples were analyzed in quadruplicate. Differential GATGTGATATTTTAAAGGTTGTTGAGTA-3′ and RV-5′ methylation analysis was performed using linear models, limma [biotin]-CACATCTCTACCTCCTCATATC-3′;andforReln,FW- R package (60), using the same contrasts as described above 5′-ATTTTGGTTTGGTGTTGAGTTTG-3′ and RV-5′[biotin]- for the microarray data analysis. DNA methylation levels were ATATCATACATAACCACTATCCCTAAAAT-3′ (Microsynth). averaged across CpGs in the examined promoter region. Promoter fragments were amplified using an AmpliTaq Gold Kit To account for multiple testing, we applied Bonferroni from Applied Biosystems (Life Technologies). PCR was done in correction.

Fig. S1. Inhibitory avoidance apparatus and contextual modifications. The shock box (purple) did not have any contextual modifications. Footshock was delivered in this apparatus only. The nonshock box (blue) and novel box (gray) had some distinct contextual modifications and served as nonshock safe training and/or test contexts. The nonshock box had four vertical white stripes taped on the wall of the dark compartment together with tape placed on the floor, closing the gap between the two plates along the entire length of the apparatus. The novel box had two white circles taped on each wall of the dark compartment, and the gap between the plates was closed with tape. The colored frames refer to the histograms in Figs. 1–3.

Atucha et al. www.pnas.org/cgi/content/short/1710819114 3of9 Fig. S2. Rats that had received muscimol infusions into the hippocampus (Fig. 2) showed recovered retention performance 24 h later. (A) Retention latencies (mean ± SEM in s) of rats tested 24 h after muscimol infusions on the 2-d retention test (n = 12 to 14 rats per group, two-way ANOVA followed by paired or ◆◆ ◆◆◆ unpaired t test; NE, F1,50 = 10.19, P = 0.004; context, F2,50 = 39.12, P < 0.0001; interaction, F2,50 = 10.01, P = 0.0002). **P < 0.01 vs. saline; P < 0.01, P < 0.001. (B) Retention latencies (mean ± SEM in s) of rats tested 24 h after muscimol infusions on the 28-d retention test (n = 11 to 12 rats per group, two-way

ANOVA followed by paired or unpaired t test; NE, F1,42 = 0.39, P = 0.54; context, F2,42 = 30.71, P < 0.0001; interaction, F2,42 = 10.01, P = 0.0002). *P < 0.05 vs. ◆◆ saline; P < 0.01.

Fig. S3. Posttraining NE and saline infusions into the BLA induce tissue-specific temporal changes in Reln and Pkmζ expression. (A and B) NE-specific temporal expression changes of Reln (A) and Pkmζ (B) in the ACC and hippocampus. (C and D) Saline-specific temporal expression changes of Reln (C) and Pkmζ (D)inthe ACC and hippocampus. Expression levels were based on averaged Affymetrix Rat Gene ST 2.0 data. Bars represent fold change in treatment- and tissue-specific expression at 2 and 28 d posttraining, relative to median array expression (log FC). Horizontal lines mark significant time-specific expression changes (28 vs. 2 d, t test). Red and green bars relate to Reln and Pkmζ, respectively. Pure and tinted color relate to the hippocampus and ACC, respectively. n = 3 pooled samples per group. Each pooled sample consisted of tissue punches from 6 to 10 independent animals. All bars are represented as mean ± SEM.

Atucha et al. www.pnas.org/cgi/content/short/1710819114 4of9 Fig. S4. Posttraining NE infusions into the BLA induce tissue-specific temporal changes in Pkmζ and Reln expression (qPCR validation). Temporal specificity of NE-induced expression changes in the hippocampus (A) and ACC (B). Bars represent tissue- and time point-specific fold expression changes of Pkmζ (tinted color) and Reln (pure color) in NE-treated rats relative to saline-treated rats (log FC). The asterisk below and above the bar represents changes significant upon correcting for multiple comparisons (t test, *PBonferroni < 0.05). Horizontal lines mark significant temporal changes (28 vs. 2 d, t test). Expression levels were based on constitutive, whole-gene expression. n = 3 pooled samples per group. Each pooled sample consisted of tissue punches from 6 to 10 independent animals. All bars are represented as mean ± SEM.

Fig. S5. Posttraining NE and saline infusions into the BLA induce tissue-specific temporal changes in Reln and Pkmζ DNA methylation. (A and B) NE-specific temporal changes of Reln (A) and Pkmζ (B) promoter DNA methylation in the ACC and hippocampus. (C and D) Saline-specific temporal changes of Reln (C)and Pkmζ (D) promoter DNA methylation in the ACC and hippocampus. Promoter DNA methylation levels were measured with bisulfite pyrosequencing. Analyzed promoter regions were chr4:9,346,306 to 9,346,547 and chr5:172,751,942 to 172,752,163 for Reln and Pkmζ, respectively (RGSC 6.0/rn6; adapted from refs. 35 and 41 for Reln and Pkmζ, respectively). DNA methylation levels were averaged across CpGs in the examined promoter region. Bars represent fold change in treatment- and tissue-specific promoter DNA methylation at 2 and 28 d posttraining, relative to median DNA methylation of the control, calibrator sample (log FC). Horizontal lines mark significant time-specific DNA methylation changes (28 vs. 2 d, t test). Red and green bars relate to Reln and Pkmζ, respectively. Pure and tinted color relate to the hippocampus and ACC, respectively. n = 3 pooled samples per group. Each pooled sample consisted of tissue punches from 6 to 10 independent animals. All bars are represented as mean ± SEM.

Atucha et al. www.pnas.org/cgi/content/short/1710819114 5of9 Fig. S6. Posttraining NE infusions into the BLA induce tissue-specific temporal changes in Dnmt3a expression. Temporal specificity of NE-induced Dnmt3a expression changes in the hippocampus (A) and ACC (B). Expression levels were based on averaged Affymetrix Rat Gene ST 2.0 data. Bars represent tissue- and time point-specific fold expression changes of Dnmt3a in NE-treated rats relative to saline-treated rats (log FC). The asterisk below the bar represents sig- nificant changes upon correcting for multiple comparisons (t test, *PBonferroni < 0.05). The horizontal line marks a significant temporal change (28 vs. 2 d, t test). Red and green bars relate to the hippocampus and ACC, respectively. n = 3 pooled samples per group. Each pooled sample consisted of tissue punches from 6 to 10 independent animals. All bars are represented as mean ± SEM.

Atucha et al. www.pnas.org/cgi/content/short/1710819114 6of9 Table S1. Memory-associated gene set External gene name Ensembl gene ID Chromosome Strand Start position End position P value

Abat ENSRNOG00000002636 10 −1 5895469 6002068 1.5E-03 Abcb6 ENSRNOG00000018697 9 −1 82143209 82151515 6.4E-03 Ache ENSRNOG00000050841 12 −1 24487852 24491390 NA Adora1 ENSRNOG00000003442 13 −1 56097928 56131500 8.1E-02 Adra1a ENSRNOG00000009522 15 −1 48203098 48295045 1.4E-02 Adra1d ENSRNOG00000021256 3 −1 130621199 130637207 2.1E-02 Atp1a1 ENSRNOG00000030019 2 −1 223440514 223469880 NA Atp2a2 ENSRNOG00000001285 12 1 41435157 41482924 3.0E-02 Atp2b2 ENSRNOG00000030269 4 −1 209001178 209104640 3.1E-03 Bcl2l11 ENSRNOG00000016551 3 −1 126571721 126601829 1.8E-02 Bdnf ENSRNOG00000047466 3 1 107390648 107421863 3.3E-01 C3 ENSRNOG00000046834 9 1 8728465 8754412 3.4E-02 C8b ENSRNOG00000007639 5 1 128159874 128196920 5.6E-02 CAMK2A ENSRNOG00000018712 18 1 55428528 55529045 NA Camk4 ENSRNOG00000020478 18 1 25464155 25680622 2.4E-02 Casp1 ENSRNOG00000007372 8 1 2627973 2636854 1.3E-01 Casp6 ENSRNOG00000009508 2 1 249539675 249551881 7.2E-02 Ccnl1 ENSRNOG00000011586 2 −1 177110681 177122888 7.7E-03 Ceacam1 ENSRNOG00000020578 1 −1 83589574 83605846 5.5E-03 Chrna3 ENSRNOG00000013829 8 −1 58175886 58189161 4.7E-02 Chrnb1 ENSRNOG00000014698 10 −1 56135720 56148237 2.8E-02 Chrnb4 ENSRNOG00000014427 8 −1 58192309 58211019 2.8E-02 Cit ENSRNOG00000001143 12 −1 48135859 48294184 5.6E-03 Cntn1 ENSRNOG00000004438 7 1 133085052 133270392 2.1E-02 Cntn2 ENSRNOG00000009033 13 −1 54360710 54389586 8.6E-02 Crh ENSRNOG00000012703 2 −1 124182919 124184783 2.0E-01 Ctnnd2 ENSRNOG00000010649 2 1 103069802 103662662 7.5E-03 Cttnbp2 ENSRNOG00000008440 4 −1 42516939 42680592 3.6E-02 Drd1 ENSRNOG00000023688 17 1 13212535 13214770 NA Drd4 ENSRNOG00000017927 1 1 221195844 221199031 9.6E-02 Dusp5 ENSRNOG00000014061 1 1 281658075 281671441 1.2E-03 Egr1 ENSRNOG00000019422 18 1 27369166 27372951 1.5E-01 Fgf18 ENSRNOG00000048389 10 −1 17932880 17948600 NA Gabbr1 ENSRNOG00000000774 20 −1 3996333 4025427 8.7E-02 Gabbr2 ENSRNOG00000008431 5 −1 66802348 67142203 6.4E-03 Gabra4 ENSRNOG00000002336 14 1 38966016 39040596 4.1E-01 Gabra5 ENSRNOG00000010803 1 −1 113844283 113955085 3.2E-02 Gabrb2 ENSRNOG00000003680 10 1 27818095 28026899 1.9E-02 Gabrg2 ENSRNOG00000003241 10 −1 26937103 27023929 5.1E-02 Gabrp ENSRNOG00000032417 10 −1 18369151 18391029 2.2E-02 Gfra2 ENSRNOG00000014010 15 1 56280942 56372307 7.6E-03 Gja1 ENSRNOG00000000805 20 1 39612108 39624547 9.9E-02 Grik1 ENSRNOG00000001575 11 −1 31426908 31828130 1.8E-02 Grik2 ENSRNOG00000000368 20 −1 55396403 56122333 3.8E-02 Grin1 ENSRNOG00000011726 3 −1 2489166 2515963 2.3E-02 Grin2a ENSRNOG00000033942 10 1 4527220 4936773 8.1E-02 Hmox1 ENSRNOG00000014117 19 1 25622556 25629372 3.8E-02 Homer1 ENSRNOG00000047014 2 1 42107429 42208021 NA Htr3a ENSRNOG00000006595 8 −1 51803897 51816207 8.2E-02 Htr6 ENSRNOG00000049761 5 −1 161242626 161257707 9.6E-02 Htt ENSRNOG00000011073 14 −1 81796508 81942093 5.7E-03 Icam1 ENSRNOG00000020679 8 1 22092135 22103927 1.1E-02 Ifng ENSRNOG00000007468 7 1 61333602 61337640 1.0E-01 Igfbp2 ENSRNOG00000016957 9 1 79888614 79915188 2.0E-01 Il15 ENSRNOG00000003439 19 −1 34532034 34598725 1.3E-01 Il1b ENSRNOG00000004649 3 1 128133909 128140349 4.6E-02 Il2ra ENSRNOG00000047647 17 −1 72202947 72250588 3.0E-01 Ins1 ENSRNOG00000012052 1 1 280213615 280214178 3.3E-01 Insr ENSRNOG00000029986 12 −1 3852577 3989125 1.8E-02 Itpr1 ENSRNOG00000007104 4 1 204714826 205047900 2.2E-02 Jak1 ENSRNOG00000011157 5 −1 123865518 123966233 3.0E-02 Junb ENSRNOG00000042838 19 −1 37069075 37070109 3.0E-01 Kcna5 ENSRNOG00000019719 4 −1 226075051 226076859 8.6E-01

Atucha et al. www.pnas.org/cgi/content/short/1710819114 7of9 Table S1. Cont. External gene name Ensembl gene ID Chromosome Strand Start position End position P value

Kcna6 ENSRNOG00000047736 4 1 232324385 232357644 2.4E-02 Kcnb1 ENSRNOG00000046949 3 −1 170010756 170094516 1.8E-01 Kcnd2 ENSRNOG00000029610 4 1 48100008 48607643 1.9E-02 Kcnh5 ENSRNOG00000009542 6 −1 107294666 107578241 4.2E-03 Kcnj11 ENSRNOG00000021128 1 −1 103188484 103189656 2.5E-01 Kcnj16 ENSRNOG00000004713 10 1 99084253 99086021 7.8E-02 Kcnj4 ENSRNOG00000013869 7 −1 120710788 120738030 1.9E-02 Kcnn2 ENSRNOG00000016675 18 1 38985930 39129190 1.9E-01 Lsamp ENSRNOG00000031852 11 −1 62956510 63592532 1.0E-01 Mag ENSRNOG00000021023 1 −1 90500839 90516262 1.3E-01 Map1b ENSRNOG00000017428 2 −1 48837124 48930483 1.6E-03 Map2 ENSRNOG00000011841 9 −1 73841103 73922677 4.9E-02 Mapk14 ENSRNOG00000000513 20 1 7968539 8028708 3.8E-03 Mapk9 ENSRNOG00000002823 10 1 35114480 35145312 2.7E-02 Mprip ENSRNOG00000003226 10 1 45775202 45890063 5.4E-03 Ncan ENSRNOG00000048036 16 1 20900031 20925162 3.0E-02 Ncs1 ENSRNOG00000008761 3 −1 15914623 15960622 2.8E-02 Nes ENSRNOG00000018681 2 1 206747073 206755585 1.9E-02 Nos2 ENSRNOG00000049980 10 −1 65423626 65455464 NA Nr1h2 ENSRNOG00000019812 1 −1 101619967 101625287 1.2E-01 Nr3c1 ENSRNOG00000014096 18 −1 31408742 31430004 8.3E-02 Oprl1 ENSRNOG00000016768 3 1 180934268 180940194 9.6E-04 Pak1 ENSRNOG00000029784 1 1 168974734 169089649 2.2E-02 Pik3r1 ENSRNOG00000018903 2 −1 50895264 50965217 4.0E-03 Pld1 ENSRNOG00000028156 2 1 133400609 133554180 1.0E-01 Plec ENSRNOG00000023781 7 −1 117215919 117275610 1.2E-02 Pkmζ ENSRNOG00000015480 5 −1 176118465 176228206 1.7E-04 Ppp1cc ENSRNOG00000001269 12 −1 41744539 41761986 1.1E-01 Ppp3ca ENSRNOG00000009882 2 1 260462929 260735288 5.1E-02 Rab1b ENSRNOG00000050510 1 −1 227413058 227413663 NA Rb1 ENSRNOG00000016029 15 −1 58804732 58942062 9.8E-02 Reln ENSRNOG00000021441 4 1 9349481 9775752 9.9E-05 Scn9a ENSRNOG00000006639 3 −1 59207262 59286961 3.6E-03 Slc1a1 ENSRNOG00000014816 1 1 254195390 254275293 4.4E-02 Slc1a3 ENSRNOG00000016163 2 −1 79348445 79422645 5.4E-02 SLC24A2 ENSRNOG00000008169 5 −1 109317349 109564102 NA Slc32a1 ENSRNOG00000015393 3 −1 160500060 160504727 5.6E-02 Slc6a4 ENSRNOG00000003476 10 −1 62851326 62872481 6.4E-02 Snap25 ENSRNOG00000006037 3 1 136083976 136269421 5.5E-02 Sncg ENSRNOG00000010437 16 −1 9049951 9054488 6.1E-02 Sod3 ENSRNOG00000003869 14 −1 61071313 61077045 1.8E-01 Stat4 ENSRNOG00000050942 9 −1 54051157 54167491 4.2E-02 Stx12 ENSRNOG00000011804 5 −1 154712006 154740248 1.3E-02 Stx2 ENSRNOG00000000936 12 1 33261860 33285512 6.2E-02 Stx5 ENSRNOG00000018847 1 1 231876955 231893829 4.6E-02 Stx8 ENSRNOG00000003849 10 1 54258230 54498841 3.7E-02 Svop ENSRNOG00000000693 12 1 50074360 50132727 5.4E-02 Synj1 ENSRNOG00000002051 11 −1 34717258 34791435 2.0E-03 Syt2 ENSRNOG00000004756 13 1 56621946 56629657 NA Syt4 ENSRNOG00000017333 18 −1 23893019 23902428 1.3E-02 Syt7 ENSRNOG00000026432 1 1 233382666 233440845 8.1E-03 Syt8 ENSRNOG00000020245 1 1 222500771 222503063 9.3E-02 Syt9 ENSRNOG00000019613 1 1 178591098 178768239 6.1E-02 Tacr1 ENSRNOG00000005853 4 1 177782405 178095041 2.6E-02 Tgfbr3 ENSRNOG00000002093 14 1 3509601 3683814 6.9E-02 Thra ENSRNOG00000009066 10 1 86465253 86480926 8.4E-03 Tph1 ENSRNOG00000011672 1 −1 103746878 103774100 3.8E-02 Tubb5 ENSRNOG00000000821 20 1 5519551 5523476 2.8E-02 Xcl1 ENSRNOG00000002964 13 −1 88079753 88083196 3.0E-01

For the gene set enrichment analysis, we created a memory-associated gene set composed of genes previously implicated in memory processes (adapted from ref. 40; expanded from refs. 35, 36, 51, and 62). The final gene set consisted of 122 memory genes. Columns show MGS gene symbols, Ensembl identifiers, chromosome, strand, start and end positions, and global-test nominal P values. NA, not available.

Atucha et al. www.pnas.org/cgi/content/short/1710819114 8of9 Table S2. Time- and tissue-specific differential expression of the memory-associated gene set Log FC CI.L CI.R AveExpr tPAdjPB setName

2.266 0.662 3.869 19.414 2.842 0.007 0.02 −2.202 MemoryGeneSet

Posttraining NE administration into the BLA induced differential expres- sion of the memory-associated gene set specifically in the hippocampus and to remote memory (global test: 28 vs. 2 d; controlled for saline effects and

changes in the ACC). Columns show differential expression log2 fold change (Log FC), upper- and lower-bound confidence intervals (CI.L and CI.R, respec- tively), average expression (AveExpr; standardized to the median array ex- pression), t value (t), P value (P), adjusted P value (AdjP; SI Methods), B statistic (B), and gene set name (setName).

Atucha et al. www.pnas.org/cgi/content/short/1710819114 9of9