Animals and ethical permissions

C57BL/6 female mice were obtained from Charles River Laboratories (Sulzfeld, Germany). Pups were delivered with their respective dams and were separated at weaning (PND21). Mice were kept under standard temperature, humidity, and daylight conditions (12-h light:dark cycle) and were provided with food and water ad libitum. All experiments were conducted in accordance with the national and European laws for the use of animals in research (EU Directive 2010/63/EU) and were approved by the local ethical committee (Ethics Committee on Animal Research, Stockholm North). The ethical identification numbers were: N9–12, N248–13 and N163–15 for the in vivo study, and N284–11 and N190114 for the in vitro study.

Irradiation procedure

Mice were anaesthetized using isoflurane at 4% for induction followed by 1.5–2% throughout the procedure. Mice were placed on a custom-made Styrofoam frame in prone position (head to gantry), and the frame placed inside an X-ray system (Precision X-RAD 320, North Branford, CT, USA) setup in-house for in vivo targeted radiotherapy research with an energy of 320KV, 12.5 mA and a dose rate of 0.75 Gy/min. The whole brain was irradiated with a radiation field of 2 × 2 cm. A single dose of 4 Gy was delivered to each animal on postnatal day (PND) 21. The source-to-skin distance was approximately 50 cm. The sham-irradiated mice were anesthetized but not irradiated.

Lithium in vivo administration

Female littermates (4–6 animals in each cage) were randomly assigned to lithium chow (2.4 g/kg Li 2 Co 3 , 0.24%, TD.05357 Lithium Carbonate Diet 2018, Harlan laboratories, Netherlands) or control chow diet (T.2918.CS, Harlan laboratories, Venray, The Netherlands). This regimen was determined in our previous study [81] and was sufficient to yield a lithium serum concentration of 0.7–0.9 mM in mice, which is equivalent to the commonly used 0.6–1.2 mM therapeutic range in humans. The lithium chow was maintained for 4 weeks, from PND 49 to PND 77 (Fig. 1a in vivo study design).

Immunohistochemistry

Animals were injected intraperitoneally with 50 mg/kg BrdU (5-Bromo-2′-Deoxyuridine) (B5002, Sigma-Aldrich, St. Louis, MO, USA) for 5 days starting from PND 72. Animals were sacrificed for analysis at three different time points, each separated 2 weeks apart: PND 77, PND 91, and PND 105.

Immunohistochemistry and quantification were performed as previously described [81]. The following primary antibodies were used: rat anti-BrdU (1:500, AbD Serotec, Kidlington, UK) and goat anti-DCX (1:100, Santa Cruz Biotechnology Inc., CA, USA). The following secondary antibodies were used: biotinylated donkey anti-goat IgG (H + L) (Molecular Probes, Paisley, UK) and biotinylated donkey anti-rat IgG (H + L) (Jackson ImmunoResearch Europe, Suffolk, UK). For immunofluorescence, the following primary antibodies were used: rat monoclonal anti-BrdU (1:500, AbD Serotec, Kidlington, UK), goat anti-DCX (1:200, Santa Cruz Biotechnology Inc., Dallas, TX, USA), mouse monoclonal anti-NeuN (neuronal nuclei) (1:200, Merck Millipore, Billerica, EMD Millipore Corporation, Temecula, CA, USA) and rabbit polyclonal anti-S100β calcium binding protein Abcam, Cambridge, UK). The following secondary antibodies were used: Alexa Fluor® 555 donkey anti-mouse IgG (H + L), Alexa Fluor® 488 donkey anti-rat IgG (H + L) (Invitrogen, Life technologies, Carlsbad, CA, USA), Alexa Fluor® 555 donkey anti-goat IgG (H + L) (Biotium, Hayward, CA, USA), and Alexa Fluor® 633 donkey anti-rabbit IgG (H + L) (Biotium, Hayward, CA, USA). Sections were mounted in ProLong® Gold Antifade Reagent with DAPI (#8961, Cell Signaling Technology, Danvers, MA, USA). For DCX+ cell analysis and density measurements, a fluorescent microscope was used and the total number of cells and contour areas were estimated using unbiased counting software (Stereo Investigator, MicroBrightField Inc.; Colchester, VT, USA). The other fluorescent analyses were conducted using a confocal microscope as previously reported (van Praag, Kempermann, and Gage 1999) (Axio Observer-Z1 with ZEN lite software, Carl Zeiss AG, Oberkochen, Germany). Only cells with an entire, clearly visible cell body were counted.

Dendrite reconstruction and morphometric analyses

Imaging of dendritic arbors on PND 77 and PND 91 was performed using a confocal microscope by acquiring images with 1 µm intervals using a 20X objective lens (X20/0.8 Plan-Apochromat lens, Carl Zeiss) (1024 × 1024 pixels). The entire dendrites and cell bodies of each neuron in the captured images were traced manually using Neurolucida® software (MBF Bioscience, Williston, VT, USA). Three-dimensional analysis of the reconstructed neurons was performed and the total dendritic length, dendritic complexity and the cell body area were measured using Neurolucida Explorer® software (MBF Bioscience). A branch order was assigned to each dendrite and then the dendritic complexity was calculated as follows; dendritic complexity = [Sum of the terminal orders + Number of terminals] × [Total dendritic length/Number of primary dendrites] (Pillai et al. 2012). This analysis was performed after DCX staining of three animals (1:12 series) randomly and blindly chosen in each group; 15–20 cells per animal were randomly chosen by an investigator blinded to the treatment of the animals. To measure the extent of dendritic arborization away from the soma at different distances, Sholl analysis (Sholl 1953) was conducted by counting the number of dendritic intersections for a series of concentric spheres at 10-µm intervals. The center of a concentric sphere was placed at the centroid of the soma. Immature neurons which satisfied the following criteria were selected for analysis; (i) fully labeled DCX+ cells at a postmitotic stage [63], (ii) neurons were relatively isolated from neighboring DCX-positive neurons to avoid interfering with analysis, (iii) the soma located in the SGZ or within the inner one-third of granular cell layer of the upper blade of the dentate gyrus, to compare the neurons which are at the same stage of immaturity. Truncated cells were excluded from the analysis. To assure impartiality morphometric analysis was performed blindly by the same investigator.

Protein quantification

Hippocampal tissue was processed for protein quantification as previously described [82]. The Wes capillary electrophoresis system (Protein Simple-Bio Techne, San Jose, CA) was used for all protein quantitation. Sample aliquots were thawed and diluted to 0.2 μg/μl for all targets using 0.1× Sample Buffer and 5× Master Mix (1:1 mix of 400 mM DTT and 10× Sample Buffer) according to manufacturer’s instructions. Samples were denatured at 95 °C for 5 min. Rabbit α-GAD65 (PA5-22260, Thermo Fisher Scientific, USA) and Rabbit α-Tppp (ab92305, Abcam, USA) were used at a concentration of 1:200 and 1:2000, respectively. Rabbit α-Vinculin was used as housekeeping control at a concentration of 1:200,000. An α-rabbit secondary antibody was provided in the kit and was used according to manufacturer’s instructions. For protein quantification the total chemiluminescent peak area was normalized to the respective reference capillary of the housekeeping control. The normalized peak area under the curve was used for protein quantification.

Embryonic telencephalic NSPC culture and LiCl exposure procedures

Primary cultures of NSPCs were established as previously described [83, 84]. Cells were obtained from embryonic telencephalon (n = 10–12/cell preparation) dissected in HBSS (Life Technologies, Carlsbad, CA, USA) from timed pregnant Sprague Dawley rats (Harlan Laboratories, Harlan, The Netherlands), (Ethical Permit: N284/11 and N190114) at E15.5 (the day of copulatory plug defined as E0). The tissue was mechanically disrupted, and meninges and larger cell clumps were allowed to sediment for 10 min. The cells were plated at a density of 40,000/cm2 on dishes precoated with poly-l-ornithine and fibronectin (both from Sigma-Aldrich, St. Louis, MO, USA; Stockholm, Sweden). Cells were kept in enriched N-2 medium with 10 ng/ml of basic fibroblast growth factor (R&D systems, Minneapolis, MN, USA) added every 24 h and medium changed every alternate day to keep the cells in an undifferentiated and proliferative state. Cells were passaged every 5 days by detaching through scraping in HBSS. Thereafter, the cells were gently mixed in N-2 medium and plated at 1:4 density. To investigate LiCl (Sigma-Aldrich, St. Louis, USA) effects, we exposed passage 3 (P3) NSPCs from 12 h before irradiation to LiCl (3 mM), as previously described [41]. A photon 60Co irradiation source was used to expose the NSPCs at a set distance of 80 cm and an absorbed dose of 2.5 Gy. P3 cells were harvested 24 h after irradiation for gene expression analysis (Fig. 3a, in vitro study design).

RNA, cDNA, and RT-qPCR

For real-time qPCR, total RNA from culture NSPCs was extracted using RNeasy Mini Kit (Qiagen) and stored at −80 °C until further use. Integrity and concentration of extracted RNA were measured using Qubit (Thermo Fisher Scientific). cDNA was synthesized from extracted RNA using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher) according to the manufacturer’s protocols. Quantitative real-time PCR was performed with Platinum SYBR Green qPCR Supermix-UDG (Thermo Fisher Scientific) together with site-specific primers. Expression levels were normalized to housekeeping gene, TATA-box binding protein levels.

RNA sequencing

Illumina TruSeq Stranded mRNA sample preparation kit with 96 dual indexes (Illumina, CA, USA) was used to prepare RNA libraries for sequencing, respectively four biological samples per condition (Sham, ShamLi, Irr, IrrLi), for a total of 16 samples. The protocols were automated using an Agilent NGS workstation (Agilent, CA, USA) using purification steps as described previously [85, 86]. Quality control was checked with 2100 Bioanalyzer (Agilent) with all 16 samples having RIN values of 8 or above. Libraries were sequenced on HiSeq 2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) with a 2 × 126 setup using HiSeq SBS Kit v4 chemistry to an average depth 32.3 M reads (30.4–34.5). The data were briefly processed as following: FastQC/0.11.5 quality check was performed on raw sequencing reads, Star/2.5.1b was used to align the reads to the reference genome Rattus norvegicus genome Rnor_6.0 and QualiMap/2.2 was used to evaluate the quality of this alignment. Reads overlapping fragments in the exon regions were counted with featureCounts (subread/1.5.1) using default parameters, i.e., fragments overlapping with more than one feature and multi-mapping reads were not counted.

Differential expression analyses were performed under R/3.3.3 using EdgeR/3.16.5 package. Low count reads were filtered by keeping reads with at least 1 read per million in at least two samples. Counts were normalized for the RNA composition by finding a set of scaling factors for the library sizes that minimize the log-fold changes between the samples for most genes, using a trimmed mean of M values (TMM) between each pair of samples. Design matrix was defined based on the experimental design, genes-wise glms models were fitted and likelihood ratio tests were run for the selected group comparisons. RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-7238.

MeDIP-qPCR

MeDIP-qPCR was performed using MagMeDIP kit (Diagenode) according to the manufacturer’s instructions. Briefly, rat NSPCs cells were lysed and the DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich), purified using Purelink Genomic DNA kits (Invitrogen, now Thermo Fisher), fragmented using Bioruptur (Diagenode), and immunoprecipitated with the antibody anti-5′-methylcytosine (Diagenode), following MagMeDIP kit settings. DNA concentration was measured using Qubit dsDNA HS Assay Kit (Thermo Fisher). Immunoprecipitated DNA was quantified using RT-qPCR, as described above, and the temperature profile used was: 95 °C for 7 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min, followed by 1 min 95 °C. Tppp, Gad2 promoter primers (Qiagen) and Methylated DNA and unmethylated DNA control primers (Diagenode) were used as internal controls (Supplementary Fig. 3). The efficiency of methyl DNA immunoprecipitation was expressed as a relative to the percentage of the input DNA using the following equation:

$$ \% \left( {meDNA \,-\, IP \div Total\;input} \right) \\ =\, 2 \wedge \left[ {\left( {Ct\left( {10\% input} \right) \,-\, 3.32} \right) \,-\, Ct\left( {meDNA \,-\, IP} \right)} \right] \times 100\%$$

Behavioral assessment

Three different cohorts (n = 15–16/cohort) of mice were used for the behavioral study. From each litter employed, animals were randomly assigned to all four experimental groups. Animals were group-housed (n = 3–4 mice/cage) and in each cage animals from different experimental groups were included. All animals were naïve to the tasks. Testing took place between 10.00 and 16.00 under low illumination (100–150 Lux) to reduce stress but strong enough to provide proper visibility of environmental cues. On the days of testing, animals were brought in their home cages to the testing room and allowed to rest and habituate for at least 1 h before the beginning of the experiment. The estrous phase was determined, based on vaginal smears, on the last day of testing to avoid additional stress to the animals. We verified that all groups of animals showed all stages of the estrous cycle without any apparent differences in their relative frequencies (data not shown).

Morris water maze

The MWM was performed as previously described with slight modifications [87,88,89,90]. Mice were trained in the reference and reversal memory versions of the MWM to locate a hidden escape platform (10 cm × 10 cm) in a circular pool (120 cm diameter). The pool was placed in a room with stable temperature and humidity. The platform was submerged 1 cm under the water surface and its position was fixed relative to extra-maze 3D visual cues (O’Hara & Co. ltd, Tokyo, Japan). Water was made opaque with non-toxic white dye (Opacifier for MWM, cat. number OP301, Viewpoint, Civrieux, France) and kept at a temperature of 22 ± 1 °C. On the first day, the cued version of the MWM took place, during which each mouse was allowed four trials to locate the platform guided by a credit card size plastic “flag”. The reference version of the MWM followed for 6 consecutive days. The platform was moved 90° counterclockwise from the position used during the cued test (Fig. 5a). Each session (day) consisted of four trials, separated by 15-min intervals, each trial having a maximum duration of 60 s. In each trial the starting position was different, in a pseudorandom order, and each day included entry points in all four quadrants of the pool. Each trial ended when either the animal found the platform or after 60 s, in which case the animal was guided to the platform by the experimenter. In either case, it remained on the platform for 20 s and was then returned to its home cage. On day 8, a reversal version of the MWM followed for 3 consecutive days. The platform was moved 180°, to the quadrant opposite to the one used during reference memory training, and the animals entered the water maze again from four different entry points per day in a pseudorandom order. The behavior of the animals was recorded and analyzed using the Noldus Ethovision software (Ethovision XT 14.0, Noldus Information Technologies, Wageningen, Netherlands). For each trial, for each mouse, the latency (time in sec to climb onto the platform) and the distance swam (total distance in cm to climb on the platform) were determined and an average was calculated for each day. For the first trial of the reversal learning, we calculated the % of time spent in the quadrant where the platform was during the learning phase, and the number of crossings over the “previous position” of the platform. In order to exclude the possibility that any learning or memory deficits observed in the MWM were due to deficits in motor activity, swim speed (cm/sec) was calculated.

Statistical analysis

Statistical differences in immunohistochemistry, protein quantitation, and gene expression analysis were calculated using a two-way ANOVA analysis followed by a Bonferroni post hoc test for multiple comparisons correction using GraphPad Prism® (La Jolla, CA, USA). For methylation analysis, Kruskal–Wallis test for multiple comparisons, followed by Dunn´s multiple comparisons test was performed. For dendritic analysis, linear mixed models with a random intercept for each animal were used to account for within-individual dependencies, using R version 3.3.3 (The R Foundation for Statistical Computing, Vienna, Austria). As data were not normally distributed, natural logarithmic transformation was used to satisfy the requirement for normality. To ensure normal distributions, variables were plotted as Q–Q plots using SPSS (IBM SPSS Statistics, version 21.0, Chicago, IL, USA). For Sholl analysis, two-way ANOVA followed by post hoc Bonferroni test was performed using GraphPad Prism (version 7.03, GraphPad Software, La Jolla, CA, USA). All quantifications were done in a blinded fashion. The analysis of behavior was performed using SPSS v. 24 software (SPSS Inc., Chicago, IL, USA) by an investigator blinded to the group identity of the animals. Any group effects on the latency and distance to reach the platform, as well as on the velocity during the learning and reversal learning in the MWM, were assessed using the generalized estimated equations model. The animal ID was used as a subject variable, the day as a within-subject variable, the group, day and group × day as predictor factors, and the group (litter) as build nested predictor factor. Any group effects on latency and distance moved during cued learning as well as on the % time spent in the quadrant where the platform was during learning, and on the number of crossings over the old position of the platform during the first trial of the reversal learning, were assessed using the generalized linear model, with the group as predictor factor and the group (litter) as a build nested predictor factor. In the case of statistically significant effects, Bonferroni post hoc tests were used to determine specific group differences. In all analyses, the data represent the mean ± SEM. The level of significance was set at p < 0.05.