Supplementary information 1. Supplementary material and methods 2. Supplementary references 3. Table S1: Primers used for quantitative PCR 4. Table S2: Body weight and novelty suppressed feeding test measurements 5. Figure S1: Correlations between novelty suppressed feeding test and expression of myelination-related genes 6. Figure S2: Correlations between time spent in the corner of the open field and expression of myelination-related genes 7. Figure S3: Correlations between time spent in the center of the open field and expression of myelination-related genes 8. Figure S4: Potential pathway for fluoxetine affecting myelination-related genes Supplementary materials and methods RNA extraction and Double-Stranded cDNA Synthesis Animals of group 2 were sacrificed at PND 128, brains were removed and immediately placed on dry ice and stored at -80°C. Hippocampal tissue was dissected in 8 consecutive slices of 200 µm using a 2 mm punch needle. Tissue from 2 rats was pooled for total RNA isolation with QIAzol (RNeasy lipid tissue kit; QIAGEN, Venlo, The Netherlands) according to the manufacturer's recommendations. Total RNA was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit; QIAGEN), followed by DNaseI treatment (QIAGEN) and fragmentation by hydrolysis (5× fragmentation buffer: 200mM Tris acetate, pH8.2, 500mM potassium acetate and 150mM magnesium acetate) at 94°C for 90 s. Fragmented mRNA was purified (RNeasy MinElute Kit; QIAGEN) and used for cDNA synthesis with 5 μg random hexamers by Superscript III Reverse Transcriptase (Invitrogen Life technologies, Bleiswijk, The Netherlands). Double stranded cDNA synthesis was performed in second strand buffer (Invitrogen) according to the manufacturer's recommendations and purified using the Minelute Reaction Cleanup Kit (QIAGEN) according to the manufacturer's protocol. Sequencing DNA samples were prepared for RNA-seq by end repair of 20 ng DNA as measured by Qubit dsDNA HS (Invitrogen). Adaptors were ligated to DNA fragments, followed by size selection (~300 bp) and 14 cycles of PCR amplification. Quality control of DNA libraries prepared for sequencing was made by qPCR and by running the products on a Bioanalyzer (Bio-Rad, Veenendaal, The Netherlands). Cluster generation and sequencing (36 bp, single read) was performed with the Illumina Genome Analyzer IIx (GAIIx) platform according to standard Illumina protocols. Samples were sequenced to a depth of approximately 15 million uniquely mapped tags per sample. Sequences were aligned to the rat rn4 reference genome with the Illumina Analysis Pipeline allowing one mismatch. Only the tags aligning to one position on the genome were considered for further analysis. The output data were converted to Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing. Data analysis RNA-seq data were analyzed using Genomatix software (www.genomatix.de). The number of sequence reads for each transcript was quantified and additionally a standardized normalized expression (NE) value per transcript was calculated (based on the number of reads located in the exons of the transcript and normalized to the length of the transcript and the density of the data set). NE values of vehicle and fluoxetine samples (group 2) were used to calculate fold change values for each transcript. Genes were identified as differentially expressed if they showed a DESeq 1 P-value < 0.05 and fold change (FC) > 1.5 (among two biological replicates for both fluoxetine and vehicle treatment). The Database for Annotation, Visualization and Integrated Discovery (DAVID, http://david.abcc .ncifcrf.gov/) was used for gene ontology (GO) analysis. Quantitative Reverse Transcription PCR RNA-seq validation was performed by RT-qPCR analysis of selected genes using GoTaq® qPCR Master Mix (Promega Benelux b.v. Leiden, The Netherlands). Primers were designed using Primer3 online software (http://frodo.wi.mit.edu). See table S1 for primer sequences. Complementary DNA (cDNA) was synthesized using 1 μg of total RNA in a reverse transcription reaction using iScript cDNA Synthesis Kit according to manufacturer’s protocol (Bio-Rad). qPCR reactions were performed in a 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA, U.S.A) using the SYBR Green fluorescence quantification system (GoTaq® qPCR Master Mix, Promega). Thermal cycling was initiated with incubation at 95C for 10 min followed by 40 cycles of 95C for 30 sec and 60C for 1 min. To normalize the cDNA content of the samples, we used the comparative threshold cycle (CT) method 2, which consists of the normalization of the number of target gene copies versus two endogenous reference genes Ywhaz and Hprt1. Table S1: Primers used for quantitative PCR Gene symbol Ref-seq number Forward primer Reverse primer Olfm1 NM_053573.1 AGACCTCAGGCTCAAGGTTC CACCATGGACTTGTACTCACG Grina NM_153308.4 AGGCTCTTCTGCGTCTTCC AACTCTTTTCATGGGACATGG Syn2 NM_001034020.1 ATGCGGATGGAACCTACG GGATGAGCACGAAGTCTGG Adcy1 NM_001107239.1 CTGTGTGGAGATGGGACTTG CACACGCATGTTCAGGTCTAC Src NM_031977.1 GGAATCAGAGCGGCTACTTC TTTCACATTTAGGCCCTTGG Tspan2 NM_022589.1 CAGCTCATTGGAATTGTTGG AGTTCCGTATTGCACAGCAG Prkcd NM_133307.1 GCCTTTGTCCTGAATGTGG CCTTCCTCACCCATCTCATC Cldn11 NM_053457.2 CGCATACAGGAAACCAGATG CTGGGGTGCTCCTTATTCTG Cpne4 NM_001109003.1 TCATCCTCAAGATGCAATCC CCACCGTAAACAGCTTTGAG Nts NM_001102381.1 CTGCTTGTCAGAAGGCTGAG GATCTGCCTCCAGGACTCTC Sult5a1 NM_001201369.1 CAGAGTCACCCATCTTGGAC ACCAGAGTCAGGGCAAGTTC Cntf NM_013166.1 CTTGCCACTGGTACACCATC TCGTTCAGACCTGACTGCTC Egr2 NM_053633.1 TGCCCATGTAAGTGAAGGTC TGATCAGATGAACGGAGTGG Igf1 NM_001082479.1 AAAGTCAGCTCGTTCCATCC TCTTGTTTCCTGCACTTCCTC Plp1 (ex2-3) NM_030990.2 TCTCCAAAAACTACCAGGACTATG GGCCCCATAAAGGAAGAAG Plp1 (ex3-5) NM_030990.2 TTTGGGAAAATGGCTAGGAC TGCAGATGGACAGAAGGTTG Olig2 NM_001100557.1 TCACAGGAGGAACCGTGTC TGCTGGAGGAAGATGACTTG Sox10 NM_019193.2 TCTTTGGGGTGGTTGGAG GCTGCTATCCAGGCTCACTAC Mag NM_017190.4 AGACAATGGCAATCAGGATG TTTGTACCTCCAGGAACCTCTAC Tf NM_001013110.1 GGAAAGTGCAGGCTTCTAGG CAGAGATGACACCAAGTGTTTG Mog NM_022668.2 GAGGTTCTCGGATGAAGGAG CAGGGTTGATCCAGTAGAAGG Cnp NM_012809.2 GGCAGAAGAATATGCCCAAC TCACAAAGAGGGCAGAGATG Hprt1 NM_012583.2 GCAGACTTTGCTTTCCTTGG CGAGAGGTCCTTTTCACCAG Ywhaz NM_013011.3 TTGAGCAGAAGACGGAAGGT GAAGCATTGGGGATCAAGAA Table S2: Body weight and novelty suppressed feeding test measurements 76 75 74 73 72 71 70 69 68 67 66 65 64 63 62 61 60 59 58 57 56 55 54 Fluo xetine Fluoxetine Fluoxetine Fluoxetine Fluoxetine Fluoxetine Fluoxetine Fluoxetine Fluoxetine Fluoxetine Fluoxetine Fluoxetine Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle Vehicle 294 267 117 160 166 203 207 165 169 176 81 269 234 96 246 442 184 235 258 277 407 289 292 284 294 283 305 293 303 300 305 300 290 300 293 290 294 295 292 300 300 297 301 296 288 287 294 283 290 279 302 285 300 290 300 299 289 301 301 290 300 299 294 295 300 297 301 294 287 BW (g) 2 288 296 280 289 282 299 293 302 298 302 298 294 305 304 296 302 301 297 299 304 300 303 299 288 BW (g) 3 290 300 285 293 283 307 290 305 295 300 300 300 310 309 297 311 303 302 307 311 303 305 302 293 BW (g) 4 291 300 288 291 287 313 294 307 299 299 299 298 316 312 306 316 304 304 313 315 306 309 307 300 BW (g) 5 297 300 290 293 285 307 294 308 295 304 299 299 319 312 306 315 307 306 313 313 307 314 305 300 BW (g) 6 296 306 292 295 290 316 300 312 301 302 305 305 321 319 313 322 317 314 321 323 315 319 317 308 BW (g) 7 294 307 293 292 287 314 302 317 298 305 302 305 327 320 311 325 316 314 320 318 311 322 314 305 BW (g) 8 291 303 292 294 284 310 299 310 300 299 304 304 324 319 313 328 320 314 322 324 315 320 317 310 BW (g) 9 296 309 295 295 288 317 302 316 300 303 300 307 330 325 317 324 318 320 323 324 314 317 317 308 297 307 297 295 285 316 306 322 308 302 307 305 333 325 322 328 324 321 328 328 318 330 320 316 299 312 295 298 290 307 309 323 305 305 308 308 337 330 321 331 328 325 327 335 323 333 320 314 290 312 298 300 290 324 310 323 303 304 310 315 342 335 324 336 329 333 331 334 329 332 322 322 302 317 302 300 289 321 307 329 307 306 312 315 341 337 327 340 332 329 332 342 327 339 330 323 306 320 305 305 290 323 317 328 308 306 315 315 345 341 331 341 334 332 341 346 328 340 333 325 301 314 307 307 290 323 321 331 306 307 322 319 345 341 335 343 341 335 337 345 336 345 334 329 308 320 307 307 294 328 320 332 317 311 319 320 348 345 336 346 342 341 346 351 342 347 342 335 306 323 312 309 295 329 326 339 313 313 327 320 348 348 339 343 342 343 346 355 338 351 338 337 307 322 314 310 294 330 327 336 312 311 324 319 351 353 337 351 346 346 349 351 348 353 342 334 307 324 314 311 297 332 328 337 314 314 317 326 355 353 342 346 344 349 352 356 342 357 345 344 310 331 314 313 300 322 325 342 315 315 333 322 352 352 348 354 352 348 353 359 348 355 345 338 BW BW BW BW BW BW BW BW BW BW BW BW (g) 10 (g) 11 (g) 12 (g) 13 (g) 14 (g) 15 (g) 16 (g) 17 (g) 18 (g) 19 (g) 20 (g) 21 NSF BW Rat nr Treatm ent Latency (s) (g) 1 77 Fig. S1: Correlations between latency to start eating in the novelty suppressed feeding test (NSFT) and expression of myelination-related genes in adult fluoxetine-exposed rats. Spearman correlations between latency to start eating (s) and expression of myelination-related genes (2ΔCT). A significant negative correlation was found for latency to start eating and expression of Cldn11 (p<0.05) and a trend for a negative correlation was found for latency to start eating and expression of Tspan2 (p<0.1). Fig. S2: Correlations between time spent in the corner (OFC) and expression of myelinationrelated genes in neonatally fluoxetine-exposed rats. Spearman correlations between OFC (s) and expression of myelination-related genes (2ΔCT). A significant negative correlation was found for OFC and expression of Cldn11, Cnp, Plp1_3_5 and Plp1_2_3 (p<0.05) and a trend for a negative correlation was found for Mag (p<0.1). Fig. S3: Correlations between time spent in the center (OFCe) and expression of myelinationrelated genes in neonatally fluoxetine-exposed rats. Spearman correlations between OFCe (s) and expression of myelination-related genes (2ΔCT). A trend for a positive correlation was found for OFCe and expression of Cldn11, Plp1_3_5, Plp1_2_3 and Mog (p<0.1). Fig S4: Potential pathway for fluoxetine affecting myelination-related genes. It has been shown that fluoxetine can stimulate the 5-HT2B receptor on astrocytes, which results in activation of its downstream signaling cascades3. The 5-HT2B receptor is Gq/11 protein coupled and stimulates the diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) pathway (IP3-DAG). DAG is the physiological activator of protein kinase C (PKC), which in turn can activate the mitogen-activated protein kinase (originally called ERK, extracellular signal-regulated kinase) pathway (MAPK-ERK1/2)3. A third signal transduction pathway activated by fluoxetine-induced stimulation of the 5-HT2B receptor is the PI3K-AKT pathway3../../Users/Yvet/Downloads/fnbeh-09-00025.pdf. Activation of these signal transduction pathways results in transcription factor activation and transcription of neurotrophic factors. MAPK-ERK1/2 activation by fluoxetine results in transcription of glial-derived nerve factor (GDNF)3, 4../../Users/Yvet/Downloads/fnbeh-09-00025.pdf. In addition, transcription of brain derived neurotrophic factor transcription is induced, but not through the MAPK-ERK1/2 pathway4. Possibly also transcription and release of ciliary neurotrophic factor (CNTF) is triggered by fluoxetine. The released CNTF can bind to the CNTF receptor triggering intracellular signaling through three major signal transduction pathways: JAK- STAT, MAPK-ERK1/2 and PI3K-AKT5. These signal transduction pathways all mediate different responses. Studies have shown that CNTF-activated astrocytes release an astrocyte specific factor (>30 kD), which promotes proliferation and survival of oligodendrocyte precursor cells6. Furthermore, studies have shown that CNTF can induce maturation of oligodendrocytes7, 8. An increase in the number of oligodendrocyte precursors and mature oligodendrocytes in our hippocampus tissue might explain the enhanced expression of myelinationrelated genes. It has been shown that the expression of Nkx6.2 in oligodendrocytes is strongly induced after CNTF treatment (grey dotted line)9 and that Plp1 is required for transport and expression of SIRT2 in myelin (grey dotted line)10. Supplementary references 1. Anders S, Huber W. Differential expression analysis for sequence count data. Genome biology 2010; 11(10): R106. 2. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25(4): 402408. 3. Hertz L, Rothman DL, Li B, Peng L. Chronic SSRI stimulation of astrocytic 5-HT2B receptors change multiple gene expressions/editings and metabolism of glutamate, glucose and glycogen: a potential paradigm shift. Frontiers in behavioral neuroscience 2015; 9: 25. 4. Mercier G, Lennon AM, Renouf B, Dessouroux A, Ramauge M, Courtin F, et al. MAP kinase activation by fluoxetine and its relation to gene expression in cultured rat astrocytes. J Mol Neurosci 2004; 24(2): 207-216. 5. Askvig JM, Watt JA. The MAPK and PI3K pathways mediate CNTF-induced neuronal survival and process outgrowth in hypothalamic organotypic cultures. Journal of cell communication and signaling 2015. 6. Albrecht PJ, Enterline JC, Cromer J, Levison SW. CNTF-activated astrocytes release a soluble trophic activity for oligodendrocyte progenitors. Neurochemical research 2007; 32(2): 263-271. 7. Mayer M, Bhakoo K, Noble M. Ciliary neurotrophic factor and leukemia inhibitory factor promote the generation, maturation and survival of oligodendrocytes in vitro. Development 1994; 120(1): 143-153. 8. Stankoff B, Aigrot MS, Noel F, Wattilliaux A, Zalc B, Lubetzki C. Ciliary neurotrophic factor (CNTF) enhances myelin formation: a novel role for CNTF and CNTF-related molecules. J Neurosci 2002; 22(21): 9221-9227. 9. Rivera FJ, Kandasamy M, Couillard-Despres S, Caioni M, Sanchez R, Huber C, et al. Oligodendrogenesis of adult neural progenitors: differential effects of ciliary neurotrophic factor and mesenchymal stem cell derived factors. J Neurochem 2008; 107(3): 832-843. 10. Werner HB, Kuhlmann K, Shen S, Uecker M, Schardt A, Dimova K, et al. Proteolipid protein is required for transport of sirtuin 2 into CNS myelin. J Neurosci 2007; 27(29): 7717-7730.