Methods that were required to generate Supplementary Data are provided in the Supplementary Material.

Animals

For the in vivo experiments, young (4 months) and old (20 months) male transgenic DCX-DsRed2 Fisher 344 rats were used. They express the fluorescent reporter DsRed2 under control of the neuronal precursor cell-specific promoter of the doublecortin (DCX) gene and were originally generated at the transgenic mouse facility of the University of Heidelberg, Germany, and previously described64. For all other studies on rat tissue, young (4 months) and old (20 months) male wild-type Fisher 344 rats (Charles River, Germany) were used. All rats were bred and housed in the animal facility of the PMU Salzburg under standard conditions of a 12 h light/dark cycle with food and water ad libitum. All animal care and use was in accordance with the European Community Council Directive (86/609/EEC) and approved by the Federal State Land Salzburg, Austria (20901-TVG/40/6–2010). The numbers of rats used to result in statistically significant differences were calculated using G*Power 3.1.7 software. Standard power analyses with α=0.05 and a power of 0.8 were performed to calculate power and sample sizes. For neurosphere culture experiments, FoxO1/3/4fl mice65 (P56, male, N=6 animals) were obtained from Ronald A. DePinho (University of Texas MD Anderson Cancer Center, Houston, US) and GPR17−/− GFP mice66 (2 months old, male, N=10 animals) were provided by Douglas Fields (NICHD, Bethesda, USA). Mouse tissue was collected in conformity with the Austrian Federal Law for experiments on living animals (TVG2012§2).

Montelukast treatment and BrdU injections

Montelukast sodium powder (Sigma), dissolved in ethanol for maximum solubility and then further diluted (1:9 ratio) with a 0.9% saline (NaCl) solution, was administered daily per oral gavage (p.o.) at a dose of 10 mg kg−1 of body weight for 42 days to young adult (4 months) and old (20 months) rats (N=7–10 rats/group). Age-matched control rats received oral gavage of the vehicle solution (10% ethanol in 0.9% NaCl). For the analysis of cell survival and cell differentiation, all rats received intra-peritoneal injections of BrdU (Sigma) at 50 mg kg−1 of body weight dissolved in 0.9% saline solution on days 14, 15, 16 and 17. Weight and general appearance of the rats was recorded daily during the course of the experiment. Criteria for early termination of treatment were obvious signs of pain, apathy, and loss of weight more than 15%. During the course of this experiment, one vehicle-treated rat (20 months old) was killed because of a weight loss of more than 15% for unknown reasons.

Behavioural tests

28 days after starting the montelukast treatment, several standardised behavioural tests were carried out. All parameters analysed were recorded via the use of the video tracking software Ethovision 9.0 (Noldus Information Technology).

In the open field test (day 29 after the first montelukast administration), spontaneous changes in general locomotor activity were detected. The open field was a black circular plastic plate with a diameter of 1 m and a 5 cm high wall, set in the middle of the testing room. Before a new trial was begun, the open field was cleaned with 70% ethanol. Each rat was placed into the centre of the plate and was allowed to explore the apparatus for 5 min. The total distance moved was recorded.

The elevated plus maze (performed on day 30 after the first montelukast administration) was used to test anxiety of the rats. The wooden maze had four arms of the same dimensions (50 × 10 cm) and was situated 50 cm above the floor. Two opposite arms were enclosed by 20 cm high opaque walls (closed arms). To reduce olfactory influences, the maze was cleaned with 70% ethanol after every trial. The rats were allowed to perform trials with 10 min of free exploration starting in the central intersection facing an open arm. The camera software recorded the time spent in the closed arms, in the open arms, and in the centre zone, and the overall distance moved.

The forced swim test was performed on day 33 to analyse depressive-like behaviour. Rats were forced to swim for 10 min in a square plastic tank (40 cm side length) filled to a depth of 30 cm with tap water (20±1 °C). During the forced swimming session, the behaviour was recorded by a video system and scored by a trained observer, quantifying absolute time measurements. The behaviour of the animals was assigned to one of the three following behavioural categories: (1) ‘struggling’, defined as movements during which the forelimbs broke the surface of water; (2) ‘swimming’, defined as movement of the animal induced by movements of the fore and hind limbs without breaking the water surface; and (3) ‘immobility time’, defined as the behaviour during which the animal used limb movement just to keep its equilibrium without any movement of the trunk. After the 10 min swimming session, animals were gently dried using a towel and returned to their home cage.

The Morris water maze test (days 35–41) was used to assess the ability of spatial learning and memory. The apparatus consisted of a circular swimming pool built of black plastic (170 cm diameter, 30 cm height), filled with 21 °C±1 °C tempered water. The tank was virtually divided into four equal quadrants, with a submerged hidden 10 × 10 cm fibreglass platform placed 3 cm below the water surface in the middle of the target quadrant. The position of the platform was kept unaltered throughout the learning sessions. In the testing room, several big black cue symbols were put on each wall for spatial orientation. The water maze task was carried out twice a day for 5 consecutive days. One day before starting the learning experiment (day 0), each rat was put into the water and was allowed to locate the submerged platform for 60 s. If the animal failed to find the platform within the 60 s, it was guided onto the platform and allowed to remain there for 10 s. For the learning tasks on days 1–5, each rat was put into the water at one of four starting positions, the sequence of which being selected randomly. In each trial, a ceiling time of 60 s to find the platform was defined. The escape latency time to locate the hidden platform and the distance moved during the trial were recorded with the camera software as indices of spatial learning. On day 6, after the learning phase of the experiment, a probe trial was performed. Here, the platform was removed and each animal was allowed to explore the pool for 60 s. From the probe trial, several parameters were recorded as indices of memory: time spent in platform quadrant, number of crossings of the former platform location, number of crossings of ‘zone P’ (a defined circular area with 20 cm diameter enclosing the former platform location) and time spent in zone P.

A learning index was calculated for every rat based on the individual learning curve obtained in the water maze spatial learning paradigm. For this, the value of the area under the curve between days 0 and 5 was calculated and subtracted from 300, which represents the value for the worst possible performance. Thus, high values indicate successful learning, while low learning indices illustrate poor spatial learning.

For the object location memory test, a test of hippocampus-dependent memory functions, a different cohort of old (20 months) male Fisher 344 rats was used. Animals received montelukast and vehicle administration as well as BrdU injections as described above (N=5 per group), and on days 35–39 of montelukast treatment, the object location memory test was performed. Prior to training, rats habituated to the experimental apparatus (a 70 × 70 × 40 cm open top plastic box) for 5 min day−1 in the absence of objects for 3 days. During the training period, rats were placed into the box with two identical objects (beverage cans; 13.5 × 5.2 cm) and allowed to explore for 15 min. During the long-term retention test, that is, 24 h following the training period, one of the object locations was shifted and the rats were allowed to explore the experimental apparatus for 10 min. Exploration was scored when a rat’s head was oriented toward the object within a distance of 2 cm or when the nose was touching the object. The relative exploration time (t) of the newly positioned object was recorded and expressed as a novelty index (NI=t novel /(t novel +t familiar ) × 100%).

Perfusion and tissue processing

On day 42, all rats were deeply anaesthetized using a ketamine (20.38 mg ml−1), xylazine (5.38 mg ml−1) and acepromazine (0.29 mg ml−1) mixture. Transcardial perfusion was performed with 0.9% NaCl solution, followed by a 4% paraformaldehyde, 0.1 M sodium phosphate solution (pH 7.4). The brains were dissected and post-fixed in the paraformaldehyde solution overnight at 4 °C. Tissues were then cryoprotected in a 30% sucrose solution. Brains were cut into 40 μm saggital sections using a sliding microtome on dry ice. Sections were stored at −20 °C in cryoprotectant solution (ethylene glycol, glycerol, 0.1 M phosphate buffer pH 7.4, 1:1:2 by volume).

Immunohistochemistry

Free-floating sections were treated with 0.6% H 2 O 2 in tris-buffered saline (TBS: 0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5) for 30 min, followed by three washes with TBS. For immunological detection of PCNA and BrdU, sections were incubated in 0.3 M NaCl/30 mM citrate buffer (pH7.0)/ 50% formamide at 65 °C for 2 h, rinsed in 0.3 M NaCl/30 mM citrate buffer (pH 7.0), incubated in 2 N HCl at 37 °C for 30 min, rinsed in 0.1 M borate buffer (pH 8.5) for 10 min and again rinsed in TBS.

All sections were blocked with a solution composed of TBS, 0.1% Triton X-100, 1% bovine serum albumin (BSA), and 0.2% teleostean gelatin (Sigma) for 1 h. This buffer was also used during the incubation with primary antibodies, which were applied overnight at 4 °C. For chromogenic immunodetection, the sections were washed extensively and further incubated for 1 h with a biotin-conjugated species-specific secondary antibody. Sections were then incubated for 1 h in a peroxidase-avidin complex solution (Vectastain Elite ABC kit; Vector Laboratories). The peroxidase activity of immune complexes was revealed with a solution of TBS containing 0.25 mg ml−1 3,3 diaminobenzidine (Vector Laboratories), 0.01% H 2 O 2 , and 0.04% NiCl 2 . The following antibodies and final dilutions were used. Primary antibodies: rat anti-BrdU (1:500, BU1/75, Serotec), rabbit anti-CD68 (1:300, ab125212, Abcam), rabbit anti-CysLTR1 (1:100, SP4109P, Acris), rabbit anti-Doublecortin (1:300, 4604, Cell Signaling), goat anti-Doublecortin (1:200, sc-8066, Santa Cruz), mouse anti-5-LOX (1:100, 610695, BD Transduction Laboratories), guinea pig anti-GFAP (1:500, GP52, Progen), rabbit anti-GPR17 (1:400, 10136, Cayman), rabbit anti-Iba1 (1:300, 019–19741, Wako), goat anti-Iba1 (1:250, ab107159, Abcam), mouse anti-NeuN (1:500, A60, Millipore), goat anti-Olig2 (1:300, AF2418, R&D Systems), goat anti-PCNA (1:250, C-20, Santa Cruz), goat anti-Sox2 (1:500, Y-17, Santa Cruz). Secondary antibodies: donkey anti-goat, -mouse, -rabbit Alexa 488, donkey anti-guinea pig Alexa 568, donkey anti-goat, -guinea pig, -mouse, -rabbit Alexa 647 (all 1:1000, Invitrogen), donkey anti-rat Cy5, donkey anti-mouse, -goat, -rabbit, -rat biotinylated (all 1:500, Jackson Immuno Research). Nuclear counterstaining was performed with 4', 6'-diamidino-2- phenylindole dihydrochloride hydrate at 0.25 μg μl−1 (DAPI; Sigma). Secondary antibody controls, in which the first antibody was omitted, were performed for all experiments to ensure specificity of the antibody staining. Chromogenic immunodetection was photodocumented using a Zeiss Axioplan microscope (Zeiss,) equipped with the Zeiss AxioVision imaging system. Epifluorescence observation was performed on a confocal scanning laser microscope (LSM 710, Zeiss) with LSM software (ZEN 2011).

Quantitative analysis of immunohistological data

For quantification of different cell types in the DG, analyses were performed blinded on coded slides. Every tenth section (400 μm interval) of one hemisphere was selected from each animal and processed for immunohistochemistry. To analyse cell proliferation and survival, PCNA- or BrdU-labelled cells within the DG were counted on a Zeiss Axioplan microscope. The reference volume of the DG was determined by tracing the DG area on each analysed section. To estimate the numbers of Sox2+, DCX+, BrdU+/NeuN+, BrdU+/GFAP+, Iba1+, Iba1+/PCNA+ and Iba1+/CD68+ cells in the DG, four randomly selected visual fields per animal were photographed using a confocal scanning laser microscope (LSM 710, Zeiss) with LSM software (ZEN 2011). From these representative pictures, all positively labelled cells were counted. The corresponding tissue area was measured and multiplied by 40 μm to obtain the tissue volume. To assess cell densities, the total number of cells counted was divided by the sample volume and represented as cells per mm3. Because of the low level of neurogenesis in old animals, in case the total number of BrdU-positive cells remained below 50, every BrdU-labelled cell detected was examined. For assessment of possible alterations in the activation state of microglia within the hippocampus, the soma sizes of the Iba1+cells within the DG were determined using the ‘Analyze Particles’ function of ImageJ 1.45 s (ImageJ website: http://imagej.nih.gov/ij/). For each animal at least 40 microglial cells were assessed. The ‘Analyze Particles’ function of ImageJ was also used to analyse the mean size of CD68+ particles in four randomly selected visual fields (maximum projection of the z-stack across the whole section). For each animal, at least 120 particles were measured.

Human post-mortem brain tissue

Human hippocampal tissue sections from autopsy samples of young adult (<35 years, N=5) and elderly (>60 years, N=5) humans with a post-mortem interval <24 h were used. Human post-mortem tissue was obtained from the collection of the Department of Neuropathology of the University Hospital Erlangen (Germany). Written informed consent was obtained from the patients’ next of kin. The use of these specimen for scientific purposes was in accordance with institutional ethical guidelines and was approved by the ethics committee of the University of Erlangen (Germany). All samples used were obtained from individuals without any neurological or psychiatric diagnoses. After tissue extraction, the brain samples were fixed overnight in 10% formalin and subsequently processed into liquid paraffin. All tissue samples were cut at 5 μm on a rotation microtome and stored at 4 °C.

Formalin-fixed paraffin-embedded immunohistochemistry

Formalin-fixed paraffin-embedded human tissue sections were deparaffinized by incubation in xylene, rehydrated by a graded series of ethanol and rinsed in distilled water for 5 min. For antigen retrieval, the slides were steamed in 0.01 M sodium citrate buffer, pH 6.0 at 100 °C for 20 min, followed by three washes in TBS. Endogenous peroxidases were quenched with 0.3% hydrogen peroxide for 20 min. Sections were blocked with a solution composed of TBS, 0.1% Triton X-100, 1% BSA and 0.2% teleostean gelatin (Sigma) for 20 min, and incubated overnight at 4 °C with a rabbit anti-5-LOX antibody (1:50; 610695, BD Pharmingen) diluted in the blocking solution. After incubation, the sections were washed in TBS, incubated with a biotinylated rabbit anti-mouse antibody (1:500, Jackson Immuno Research) for 1 h, washed in TBS, and incubated for 1 h in a peroxidase-avidin complex solution (Vectastain Elite ABC kit; Vector Laboratories). The peroxidase activity of immune complexes was revealed with a solution of TBS containing 0.25 mg ml−1 3,3 diaminobenzidine (Vector Laboratories), 0.01% H 2 O 2 , and 0.04% NiCl 2 . Sections were mounted with Neo-Mount (Merck).

RNA extraction and quantification of 5-LOX mRNA

To detect 5-LOX mRNA levels in different brain regions of young and old rats, total RNA was extracted from rat hippocampus, subventricular zone and cortex from 4-month- and 20-month-old Fisher 344 wild-type rats. Animals were decapitated and the tissues of interest were dissected. Brain samples were homogenized in 1 ml of Trizol (TRI Reagent; Sigma). For phase separation, 200 μl of 1-bromo-3-chloropropane were added, vortexed and centrifuged (15 min at 12,000 g). After transferring the aqueous phase into a new tube and adding 350 μl of ethanol, RNA extraction was performed using the QIAGEN RNEasy Mini Kit (Qiagen) and cDNA was synthesized using Promega reverse transcription Kit (Promega).

Quantitative 5-LOX gene expression analysis was performed by the TaqMan gene expression assay kit (Catalogue number 4369514, Applied Biosystems) and a specific validated 5-LOX gene expression assay (Catalogue number Rn00689111_m1, Applied Biosystems). GAPDH was used as endogenous control gene for rat 5-LOX quantification. The following temperature profile was used: activation of polymerase 95 °C, 10 min; 40 cycles of denaturing 95 °C, 15 s, and annealing/extension 60 °C, 60 s. Data were obtained with a Rotor-Gene 6000R Corbett Research (geneXpress) and analysed by delta delta Ct method67.

BV-2 cell culture experiments

Cells from the murine microglial cell line BV-2 (ref. 68) were originally obtained from Banca Biologica e Cell Factory, IRCCS Azienda Ospedaliera Universitaria San Martino, Genua, Italy, and tested for mycoplasma contamination (Myco Alert, Lonza) before further use. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (Life Technologies) supplemented with 10% fetal bovine serum and antibiotics (penicillin 100 U ml−1, streptomycin 100 U ml−1, HVD Life Sciences) under standard culture conditions (95% relative humidity with 5% CO 2 at 37 °C). For RNA expression studies, subconfluent BV-2 cells were treated for 24 h either with 100 nM LTD4 (Sanova Pharma), 15 μM montelukast (Sigma), or with a combination of LTD4 and montelukast, in which the cells were pretreated with 15 μM montelukast (Sigma) for 15 min before 100 nM LTD4 were added. The controls received only the vehicle solution (ethanol) for 24 h.

Quantitative gene expression analyses were performed using TaqMan PCR with reverse transcription technology. RNA was isolated with the RNeasy Mini Plus Kit (Qiagen) and transcribed to cDNA using the Reverse Transcription System (Promega) according to the manufacturer’s instructions. Technical duplicates containing 10 ng of reverse transcribed RNA were amplified with the GoTAQ Probe qPCR Master Mix (Promega) using a two-step cycling protocol (95 °C for 15 s, 60 °C for 60 s; 40 cycles, Bio-Rad CFX 96 Cycler). The following gene expression assays were employed: Arg-1 (Mm00475988_m1, Applied Biosystems), CCL2 (Mm.PT.56a.42151692, Integrated DNA Technologies), NOS-2 (Mm00440485_m1, Applied Biosystems), TGFb1 (Mm.PT.56a.11254750, Integrated DNA Technologies), TNF (Mm00443258_m1, Applied Biosystems) as well as the following validated housekeepers: HPRT1 (Mm00446968_m1, Applied Biosystems), PSMD4 (Mm.PT.56.13046188, Integrated DNA Technologies), SDHA (Mm01352366_m1, Applied Biosystems). Quantification analyses were performed with qBase Plus (Biogazelle) using geNorm algorithms for multi-reference gene normalization followed by normalization to control conditions.

FoxO1/3/4fl/fl mouse neurospheres

A mouse line with floxed alleles for FoxO1, FoxO3 and FoxO4 in FVB/N background was used (FoxO1/3/4fl/fl mice) for the preparation of neural stem cells. Six male FoxO1/3/4fl/fl mice (P56) were killed via cervical dislocation and brains were kept in ice-cold PBS. The subependymal zone was isolated under binocular microscope and kept further in ice-cold PBS. Neural stem cells were isolated and cultured as described before69. Briefly, cells were resuspended in Neurobasal (NB) medium (Gibco BRL) supplemented with B27 (Gibco BRL), 2 mM L-glutamine (PAN), 100 U/ml penicillin/0.1 mg l−1 streptomycin (PAN), hereafter referred to as NB/B27. For maintenance and expansion of the cultures, the NB/B27 was further supplemented with 2 μg/ ml heparin (Sigma), 20 ng/ml FGF-2 (R&D Systems) and 20 ng/ml EGF (R&D Systems) (NB-A). Cultures were maintained in T-25 culture flasks at 37 °C in a humidified incubator with 5% CO 2 . Passages 2–10 were used througout this study. For passaging these cells, the culture medium containing floating neurospheres was collected in a 15- ml Falcon tube and centrifuged at 120 × g for 5 min. The pellet was resuspended in 200 μl of 1% Accutase (PAA) and triturated approximately 10 times using a pipette. Dissociated cells were centrifuged at 120 × g for 5 min, resuspended and reseeded.

For retroviral transduction of neural stem cells, mouse neurospheres from FoxO1/3/4fl/fl mice were passaged and dissociated into single cells as described above. Cells were counted and resuspended in 2 ml culture medium. Virus particles of a GFP/Cre-Recombinase-expressing retrovirus or an only GFP-expressing retrovirus corresponding to the number of counted cells were added and the cells were incubated at 37 °C for 30 min until the virus particles were inactive. The transduction was controlled after 4–7 days via fluorescence microscopy. The cells were FACS sorted to obtain homogenous populations. Knockout of the cells transduced with the GFP/Cre-Recombinase-expressing retrovirus was controlled via genotyping. In the following, cells from FoxO1/3/4fl/fl mice that have been successfully transduced with the GFP/Cre-Recombinase-expressing retrovirus and that show a FoxO1/3/4 knockout, are termed ‘FoxO1/3/4−/−’ or ‘FoxO1/3/4 KO’.

Overexpression of FoxO1 and rescue of FoxO1/3/4 KO

Both control cells, which were transduced with a GFP-expressing retrovirus, and FoxO1/3/4 KO cells, which had been transduced with a GFP/Cre-Recombinase-expressing retrovirus, were transduced additionally with a lentivirus coding for a dominant active form of FoxO1 (Lenti-FoxO1-ADA) as described above. This lentivirus was constructed by cloning the FoxO1-ADA of the pCMV-FoxO1-ADA70 into the expression cassette of the pLL3.7 lentivirus (Addgene).

For quantitative gene expression analyses, total RNA was isolated from control and FoxO1/3/4 KO neurospheres (with and without FoxO1 rescue) by using the RNeasy Mini-Kit (Quiagen) according to the manufacturer’s protocol. Possible genomic DNA contamination was eliminated by on-column DNAse treatment using the RNAse-free DNAse-Set according to the manufacturer’s protocol. cDNA was transcribed using the Fermentas RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Quantitative PCR with reverse transcription was performed using the StepOnePlusTM Real-Time PCR System. Brilliant II Fast SYBR Green quantitative PCR (qPCR) Master Mix was used for PCR reactions according to the manufacturer’s protocol. qPCR primers were designed using the software’s Primer3 ( http://primer3.sourceforge.net) and NetPrimer ( http://www.premierbiosoft.com). Amplicon sizes ranged from 100–250 bp. Suitability of qPCR primer was analysed by evaluation of melting curves and by determination of the efficiency via a standard curve. For quantitative expression analysis, the delta delta CT method67 was applied to determine the relative quantity of target sequence using a reference sample (control) and an endogenous control target sequence. RPL27 was used as endogenous control target. Primers for qPCR were as follows: RPL27: forward, CCTGGATAAACTGTGGACATTGG; reverse, TGTAGTAGCCTGATCGAACAACA; GPR17: forward, CGACTCACTGGCTTCCTCTT; reverse, GCCAGGTGAGCATAGAGAGG, CysLTR1: Forward, CCTCTCCGTGTGGTCTATTATGT; reverse: ACCGGAAAAAGCTCATGGCT.

GPR17−/− neurospheres

Neurospheres were obtained from 2-month-old male GPR17−/− GFP mice (N=10), in which the entire GPR17 coding exon is replaced by an h2bGFP/neo cassette via homologous recombination66. Age-matched C57BL/6 wild-type mice (N=10) were used as controls. Neurospheres were isolated and cultured as described for FoxO1/3/4fl/fl mouse neurospheres above. Passage numbers 2–8 were used throughout this study.

Single-cell cultures—single-cell neurosphere assay

To culture a neurosphere out of a single neural stem cell, neurospheres were diluted as 1 cell per 25 μl neurosphere media with 20 ng ml−1 growth factors (EGF and FGF-2) with or without montelukast (10 μM, Sigma), and seeded into the wells of a 60-well Nunc MicroWell MiniTray (Thermo Scientific) plate. After seeding the cells, the wells were examined under a binocular microscope/fluorescent microscope and those wells that contained one or two cells were marked. After 7 days, the marked miniwells were examined again under a binocular microscope/fluorescent microscope and the number of formed neurospheres was counted.

Neurosphere bulk assay

A total of 5 × 104 cells were seeded in a 25-cm2 culture flask in 5 ml medium under proliferative conditions (with 20 ng ml−1 EGF and FGF-2). Montelukast was added to a final molarity of 10 μM on days 0, 2, 4 and 6 (control cells received medium only). After 7 days, neurospheres were harvested and dissociated into single cells. Total numbers of cells were counted and normalized to untreated control cells.

Analysis of cell proliferation—MTS-assay

After passaging, cells were seeded in 96-well culture plates at a concentration of 5 × 104 cells/ml in a volume of 100 μl NB/B27 medium under proliferative conditions (with 20 ng ml−1 EGF and FGF). The plates were then maintained at 37 °C in a humidified incubator (Heraeus) with 5% CO 2 for 7 days. At days 0, 2, 4 and 6, montelukast was added to a final molarity of 10 μM to the wells (control cells received medium only). At day 7, proliferation was assessed using an MTS assay kit (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega) according to the manufacturer’s instructions. After 4 h of incubation, optical density was measured at 490 nm using a plate reader (Emax Precision Microplate Reader, Molecular Devices).

Adult rat neurosphere cultures

2–4 month-old female Fischer 344 rats (N=6) (Charles River) were decapitated and hippocampi were aseptically removed and dissociated. Neurosphere cultures were obtained as for FoxO1/3/4fl/fl mouse neurospheres described above. Cultures from passage numbers 2–8 were used throughout this study.

Immunocytochemistry

For immunocytochemical analysis of BV-2 cells and mouse neurospheres from FoxO1/3/4fl/fl mice, fixed cells were washed in TBS (0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5), then blocked with a solution composed of TBS, 1% BSA and 0.2% Teleostean gelatin (Sigma) (fish gelatin buffer, FSGB). The same solution was used during the incubations with antibodies. Primary antibodies were applied overnight at 4 °C. Fluorochrome-conjugated species-specific secondary antibodies were used for immunodetection. The following antibodies and final dilutions were used. Primary antibodies: rabbit anti-CysLTR1 (1:500, SP4109P, Acris), chicken anti-GFP (1:1,000; GFP1020, Aves Labs) and rabbit anti-GPR17 (1:400, 10136, Cayman).Secondary antibodies: donkey anti-rabbit Alexa 568, donkey anti-chicken Alexa 647 (all 1:1,000, Invitrogen). Nuclear counterstaining was performed with 4', 6'-diamidino-2- phenylindole dihydrochloride hydrate at 0.25 μg μl−1 (DAPI; Sigma). Specimens were mounted on microscope slides using a Prolong Antifade kit (Molecular Probes). Epifluorescence observation was performed on a confocal scanning laser microscope (LSM 710, Zeiss,) with LSM software (ZEN 2011).

Statistical analysis

Histological and behavioural experiments were randomized and performed blinded. Groups were unblinded at the end of each experiment before statistical analysis. Statistical analyses were performed using the GraphPad Prism 5.0 software (GraphPad Software) and IBM SPSS Statistics 20 software (IBM Corporation). Data were tested for normality using the Kolmogorov–Smirnov or the Shapiro–Wilk test, and equality of variance was confirmed using the F-test. Means between two groups were compared by the two-tailed unpaired Student’s t-test or, in case of non-gaussian distribution, by using the two-tailed Mann–Whitney U-test. Data from multiple groups were analysed by one-way ANOVA, and two-way ANOVA, followed by the appropriate post hoc tests (as indicated in the figure legends) when necessary. Learning index correlation analyses were performed with the ‘Pearson Product Moment Correlation test’.