Animals Eight-9-week-old male and female BALB/c mice were purchased from Koatech (Pyeong-Taek, Korea) and mated. On TP 12.5, preganant BALB/c mice were subcutaneously injected with a single dose of VPA (600mg/kg in saline) or vehicle saline (SAL). Mice were maintained under a 12 h light/dark schedule in specific pathogen-free facility at Seoul National University College of Medicine. All animal experiments were approved by the Animal Care Committee of Seoul National University, Seoul, Republic of Korea (Approval number: SNU-190426-10-1). Striatal tissues of PatDp+/- mice were generous gift from Dr. Jong-Cheol Rah (Korea Brain Research Institute).
RNA-Seq Ten-week-old male mice were anesthetized using Zoletil mix with Xylazine. The striatum was then isolated from ~1 mm thick coronal section, which was located 0.86-–0.14 mm anterior to the bregma as previously described [28]. Total RNAs were extracted by Qiazol reagent (Qiagen, Hilden, Germany). RNA library preparation, cluster generation, and sequencing were performed by TheragenEtex BiO Institute (Suwon, Korea). RNA quality was assessed by analysis of rRNA band integrity on an Agilent RNA 6000 Nano kit (Agilent Technologies, CA, USA). Ahead of cDNA library construction, the 2 μg of total RNA and magnetic beads with Oligo dT were used to enrich poly A mRNA from it. Then, the purified mRNAs were disrupted into short fragments, and the double-stranded cDNAs were immediately synthesized. The cDNAs were subjected to end-repair, poly A addition, and connected with sequencing adapters using the TruSeq RNA Library prep Kit (Illumina, CA, USA). The suitable fragments automatically purified by BluePippin 2% agarose gel cassette (Sage Science, MA, USA) were selected as templates for PCR amplification. The final library sizes and qualities were evaluated electrophoretically with an Agilent High Sensitivity DNA kit (Agilent Technologies, CA, USA) and the fragment was found to be between 350–450 bp. Subsequently, the library was sequenced using an Illumina HiSeq2500 sequencer (Illumina, CA, USA). Statistics for each gene in each of the differential expression analysis, including FDR corrected p-values are found in Table S1.
Bioinformatic analysis The gene ontology analyses of differentially expressed genes were performed using DAVID software (version 6.8). Differentially expressed genes were also analyzed for phenotypes using mouse genome informatics mammalian phenotype analysis in Enrichr (http://amp.pharm.mssm.edu/Enrichr/). p-value 0.05 was used as cutoff.
AQ treatment For an in vitro study, primary striatal neuron cultures were treated with 100 nM of AQ (Sigma-Aldrich, MO, USA) for 24 h. AQ was diluted in 0.9% saline and prepared before treatment.
For in vivo study, 6-week-old prenatally SAL or VPA-exposed mice were intraperitoneally injected with AQ (20 mg/kg), twice per day at 12 h intervals, for 2 weeks. The AQ dose (20 mg/kg) used in this study was referred from previous report regarding the activating effect of AQ on Nurr1 in rodents [29]. AQ was diluted in 0.9% saline and prepared before administration. Mice underwent behavioral testing 1 week after the final injection.
Stereotaxic injection of lentivirus Nurr1 shRNA lentiviruses and its control shRNA lentiviruses were purchased from Sirion Biotech (Martinsried, Germany). Mice received bilateral stereotaxic injections of virus (1.5 μl per side) into the striatum (coordinates: AP +0.3, ML ±1.9, DV -3.25 mm) at rates of 0.15 μl/min at each site (Kopf instruments, CA, USA). The needle was left in place for an additional 5 min and then was withdrawn gently.
Golgi staining After mice were transcardially perfused using heparin (100 U/ml) PBS solution, brains were dissected. Golgi staining was performed using the FD Rapid GolgiStain Kit (FD Neurotechnologies, MD, USA) according to manufacturer’s instructions to label neurons. The brains were sectioned in the coronal plane at 100 μm thickness on a cryostat. Images of neurons in the DMS or DLS were acquired using a LSM 510 confocal microscope (Zeiss, Oberkochen, Germany) with a Plan-Neofluar 100x/1.30 N.A. oil immersion objective and the bright-field setting. To assess spine density and phenotype, 8-–10 cells of each slice were randomly selected. 2-3 dendrites per neuron were analyzed. Stacks of 512 x 512 pixel 3-D images with an interval of 1 μm were then taken for each cell to include all visible dendritic branches in the Zen software. After 3D neuronal reconstruction, the secondary and tertiary dendrite spines were measured, wherein the distance to the soma varied from 20–80 µm.
Quantitative reverse transcription polymerase chain reaction (qPCR) Total RNA was extracted from the whole striatum using the Qiazol reagent (Qiagen, Hilden, Germany). RNA was converted to cDNA using AccuPower RocketScript RT PreMix (Bioneer, Daejeon, Korea). qPCR was performed using a CFX96 (Bio-Rad, CA, USA). Results are presented as △△Ct-values normalized to the 18S rRNA. Primers were designed using NCBI primer blast software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The specificity of the primer pairs was tested by PCR, and the PCR product were examined by agarose gel electrophoresis.
Western blot The striatum of mice was homogenized in ice-cold RIPA buffer (Elpis Biotech, Daejeon, Korea) with freshly added protease inhibitors (Roche, IN, USA), and phosphatase inhibitors (1 mM PMSF, 1 mM Na3VO4, 5 mM NaF). Protein was quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific, IL, USA). 20-50 μg of proteins were resolved on a 10% SDS-PAGE gel or tris-tricine gel and transferred to nitrocellulose or polyvinylidene fluoride membrane, followed by blocking with 5% skim milk. Antibodies used were Nurr1 (#PA5-13416, Thermo Fisher Scientific, IL, USA), Cbln1 (#ab-64184, Abcam, Cambridge, UK), D2 (#AB5084P, Millipore, CA, USA), D1 (#ab20066, Abcam, Cambridge, UK), Vesicular glutamate transporter 1 (VGLUT1, #48-2400, Invitrogen, CA, USA), Vesicular glutamate transporter 2 (VGLUT2, #135 403, Synaptic systems), Dopamine transporter (DAT, #MAB369, Millipore, CA, USA), Glutamate decarboxylase 67 (GAD67, #ab26116, Abcam, Cambridge, UK). Reletive intensity of blots was quantified using ImageJ software.
Behavioral assays
- Self-righting test was held on postnatal day 5-9 (P5-9) as described in previous literature [30]. Each mouse was placed in the supine position and gently held with all four limbs extended outwards at which time it was released. Time taken to right was recorded by the latency for all four paws touching the surface. A maximum score of 30 sec was recorded when the mouse failed to right in that period.
- Maternal scent preference test was conducted on P14 as described in previous research [31]. Each pup was moved from home cage to a fresh transparent polycarbonate cage (20 × 30 × 15 cm). The left third of the test cage was filled to a depth of 3 cm with litter from the mother’s cage, the center third contained clean litter, and the right third contained litter from the cage of a stranger dam. The placement of the test litters (mother and stranger) was alternated across subjects to control for any side preferences. Three 1 min trials, with inter-trial intervals of 10 sec, were administered for each pup. For the first trial, pups were placed in the center of the fresh litter facing the back wall of the test cage. For the second trial, pups were placed in the center of the fresh litter facing the section containing its mother’s cage litter. For the third trial, pups faced the section containing the litter of the stranger dam. Time spent in each section of the cage was recorded and averaged across the 3 trials. The pup was considered to be inside a section when all four paws were touching the litter within the specified region.
- Social interaction was assayed using the 3 chamber test. The apparatus was constructed of a Plexiglas box (60×45×22 cm) partitioned into 3 chambers with retractable doorways. Openings between the compartments allowed the animals access all three chambers. In the first phase, a mouse was placed in the center chamber and was allowed to freely explore with an age-matched male (familiar) for 10 min. In the second phase, the test mouse was gently guided to the center chamber, and the entrances were blocked. An age-matched stranger mouse then was placed in apposite chamber from the familiar mouse, and then the test mouse was similarly allowed to explore with the familiar and stranger mouse for an additional 10 min. The apparatus was cleaned with 70% ethanol between trials.
- Self grooming was performed at 9-10 weeks of age, and each mouse was placed individually into a clean transparent polycarbonate cage (20 × 30 × 15 cm) with a video camera placed 15 cm away from the cage. The duration of the test was 10 min after 10 min habituation. The time spent grooming was measured.
- Rotarod test was conducted with mice (9-10 weeks) following previously published research [17]. The test consisted of three trials per day over the course of 3 days. Rotarods were accelerated from 4–40 rpm in 300 s. Each trial ended when a mouse fell off, made one complete backward revolution while hanging on, or reached 300 sec.
- Open field test was conducted at 9-10 weeks of age. A square plastic box (100 cm × 100 cm × 40 cm) was used for this general locomotor activity test. The mice were put into the arena and its movements monitored with a video camera for 30 min. Tracking of mouse behavior was done using EthoVision XT (Noldus) tracking system. The open field was thoroughly cleaned with 70% alcohol between test animals.
Immunofluorescence and image analysis Mice were anesthetized using Zoletil/Xylazine and perfused using heparin (100 U/ml) phosphate buffered saline (PBS) solution. The brains from 9 to 10-week-old prenatally VPA- or saline-exposed mice were then removed and post-fixed in 4% paraformaldehyde (PFA) at 4 °C for 24 h before they were transferred to 30% sucrose-PBS 0.1 M, pH 7.3 solution at 4 °C. Afterwards, the brains were sectioned into 30 μm-thick coronal sections using a cryostat (Thermo Fisher Scientific, IL, USA), and three slices per mouse were used in all IF analyses (n=3–4 mice/staining).
The brain slices were incubated in 10 mM sodium citrate buffer (pH 6.0) for 10 min at 95°C for antigen retrieval, and blocked in PBS containing 2% BSA or 10% serum and 0.3% Triton X-100 for 1 h at room temperature (RT). Sections were then incubated in blocking buffer containing a primary antibodies diluted in blocking buffer at 4°C for overnight. Next, sections were incubated with the secondary antibodiesin PBS for 2-3 h at RT protected from light. Finally, sections were stained with Topro3 (diluted 1:1000; Thermo Fisher Scientific, IL, USA) or DAPI (1:1000, D1306, Thermo Fisher) in PBS. After final rinsing, sections were mounted and cover-slipped using mounting medium (#345789, Merck, Darmstadt, Germany). The images were acquired on an LSM510 confocal microscope (Zeiss, Oberkochen, Germany) using a Plan-Neofluar 40x/0.90 N.A. with a water immersion objective or on a Nikon A1 confocal microscope (Nikon, Melville, NY, USA) with a Plan fluor 20 × lens (0.75 numerical aperture). For quantification, 2–3 striatal regions were randomly selected for confocal imaging, wherein the intensity of each region was analyzed. The primary antibodies used were Nurr1 (#PA5-13416, Thermo Fisher Scientific, IL, USA), NeuN (#MAB377, Millipore, CA, USA), Iba-1 (#NB100-1028, Novusbio, CO, USA). Secondary antibodies used were goat anti-rabbit Alexa 555, goat anti-mouse Alexa 488, goat anti-rabbit Alexa 488, and donkey anti-goat Alexa 555 (Thermo Fisher Scientific, IL, USA).
Primary striatal neuron culture On E16-17, embryonic striatal tissues were dissected, dissociated with 0.25% trypsin, and plated onto plates or coverslips coated with poly-L-lysine. Neurons were grown in Neurobasal medium (Gibco, CA, USA) supplemented with B27 (Gibco, CA, USA), 2 mM GlutaMAX-I supplement (Gibco, CA, USA) and 100 μg/ml penicillin/streptomycin (Gibco, CA, USA) at 37 °C in a humidified environment of 95% O2/5% CO2.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay To determine cell viabilities, cells were treated with the MTT solution (0.5 mg/ml, Duchefa Biochemie, Haarlem, Netherlands) for 3 h at 37 °C. Formazan grains were solubilized with DMSO and the absorbance was measured at 570 nm using a microplate reader (Molecular Devices, CA, USA).
Statistical analysis The data are expressed as means ± SEM values and were analyzed with the SPSS 23 software (IBM, Chicago, IL, USA) using the Kruskal–Wallis test, one-way-ANOVA with LSD post-hoc analysis, two-way-ANOVA with LSD post-hoc analysis, or repeated measures (RM)-ANOVA with Bonferroni post-hoc analysis. The results were considered to be statistically significant if p < 0.05. n means a number of mice analyzed unless stated otherwise.