Necdin deficient mice were maintained on the C57BL/6 J background and the paternal mutation was transmitted by crossing Ndn males with C57BL/6 J WT females just as previously described [19]. Thy1-GFP mice was obtained from Jackson Laboratory (#0221069). All animals were housed under controlled conditions: temperature of 20 ± 2 °C, relative humidity of 50-60%, and a 12:12-h light-dark (LD) cycle (lights on at 7:00 AM and off at 7:00 PM). Food and water were provided ad libitum. All animal studies were approved by the Institutional Animal Care and Use Committee of Central South University, China.
All behavioral tests were carried out on male mice at 6-10 weeks of age. Prior to testing, the mice were transferred to the behavioral room and given a minimum of one hour to acclimate.
The three-chamber social interaction apparatus comprises three interconnected compartments (20 × 40 cm). The side chambers contain an inverted pencil cup (11 cm height ×10.5 cm bottom diameter) to house the social or nonsocial stimulus.
A fresh paper ball was placed beneath each cup, and the test mouse was placed in the central chamber to explore freely for 10 min.
A novel conspecific (stranger 1) was placed under one cup, and the test mouse was returned to the apparatus to explore for another 10 min.
A second novel mouse (stranger 2) was introduced under the previously empty cup, and the test mouse was allowed another 10 min to explore.
The test mouse was gently removed between each phase and returned to its home cage for a 5-min rest. The apparatus and cups were cleaned with 40% ethanol to eliminate olfactory cues. Exploration time at each cup was recorded, and the preference index was calculated as described in reference [20].
On Day 1 (habituation), mice were allowed to explore an empty arena (40 × 40 × 40 cm) for 10 min. On Day 2 (familiarization), two identical objects were placed in the center of the arena, and the mouse was allowed to explore for 10 min. On Day 3 (test), one familiar object and one novel object (with a different shape) were placed in the same arena. Each trial was followed by cleaning the arena with 40% ethanol. Exploration time for each object was recorded, and the preference index was calculated as in reference [20].
The open field test (OFT) was performed in a white plexiglass arena (72 × 72 × 36 cm) subdivided into a central zone (24 × 24 cm) and a surrounding periphery. Mice were allowed to explore freely for 10 min. Total distance traveled, movement speed, time, and distance in the central zone, and the number of central zone entries were quantified using Anilab software (Anilab, China).
The maze consisted of two open arms (30 × 5 cm) and two enclosed arms (30 × 5 × 15 cm) connected by a central platform (5 × 5 cm), elevated 50 cm above the floor. Mice were placed on the central platform and allowed to explore for 10 min. Time spent, distance traveled in open arms, and the number of open arm entries (defined as all four paws entering) were recorded using Anilab software.
The apparatus consisted of a light compartment (30 × 20 × 25 cm, 300 lux) and a dark compartment (15 × 20 × 25 cm), connected by an opening (10 × 5 cm). Mice were placed in the dark compartment and allowed to explore for 10 min. The number of entries, time spent, and distance traveled in the light compartment were recorded.
Transverse brain slices (300-350 µm) were prepared from 8-12-week-old male mice as described previously [21]. The mice underwent deep anesthesia with pentobarbital sodium (50 mg/kg) and were decapitated. Brains were quickly removed and placed in ice-cold sucrose-based artificial cerebrospinal fluid (ACSF) containing: 2.5 mM KCl, 2 mM MgSO, 2 mM CaCl, 26 mM NaHCO, 1.25 mM NaHPO·HO, 10 mM glucose, and 213 mM sucrose. Slices were then transferred to an incubation chamber containing standard ACSF composed of: 126 mM NaCl, 2.5 mM KCl, 2 mM MgSO, 2 mM CaCl, 26 mM NaHCO, 1.25 mM NaHPO, 25 mM glucose (pH 7.2-7.3). Slices were incubated at 34-35 °C for 45-60 min and subsequently maintained at room temperature until use. During recordings, slices were perfused with oxygenated ACSF at a flow rate of 2 ml/min at 34 °C.
Patch pipettes (resistance 4-7 MΩ) were filled with internal solution containing: 140 mM K-gluconate, 3 mM KCl, 2 mM MgCl₂, 0.2 mM EGTA, 10 mM HEPES, and 2 mM Na₂ATP (pH 7.2-7.25, 285-295 mOsm). Layer V pyramidal neurons, identified by their morphology and prominent apical dendrites, were visually selected under IR-DIC optics (BX-51WI, Olympus). Cell selection was confirmed based on anatomical location, cellular shape, and electrophysiological properties assessed using pClamp's online membrane test. Cells with series resistance <20 MΩ were included. Voltage- and current-clamp recordings were performed using a MultiClamp 700B amplifier (Molecular Devices). Signals were digitized with a Digidata 1550B system and acquired using pClamp software. Voltage-clamp signals were filtered at 10 kHz; current-clamp signals at 2.2 kHz; all were sampled at 50 kHz.
For evoked responses, a bipolar tungsten electrode was placed 150-300 µm away from the recorded neuron within the same layer. Stimulation was delivered using a stimulus isolator (Model 2100, A-M Systems) with pulse intensities ranging from 40 to 150 µA and durations of 0.1 ms.
mEPSCs were recorded at a holding potential of -70 mV in the presence of 1 μM tetrodotoxin (TTX; Beijing Jiangchen) and 50 μM picrotoxin (Cayman, #20771). mIPSCs were recorded under the same conditions with the addition of 1.5 mM kynurenic acid (KynA; Sigma, #K3375) instead of picrotoxin. Pipettes were filled with an internal solution containing: 120 mM CsCl, 10 mM HEPES, 1 mM EGTA, 4 mM MgATP, 0.3 mM Na₃GTP, 2 mM QX-314, 10 mM TEA-Cl, and 10 mM Na₂-phosphocreatine (pH adjusted to 7.3 with CsOH, ~285 mOsm). After viral overexpression of Syngap1 in the medial prefrontal cortex, recordings were performed specifically from GFP-positive pyramidal neurons in Layer V.
To assess presynaptic release probability, paired EPSCs were evoked using stimuli set to twice the threshold intensity at a holding potential of -70 mV. Ten consecutive traces were recorded with an inter-stimulus interval of 50 ms. The paired-pulse ratio was calculated as the amplitude of the second EPSC divided by that of the first.
Total RNA was extracted from medial prefrontal cortex (mPFC) tissue and subjected to RNA sequencing using Illumina platform technology. Read counts for all genes across different samples were used for differential expression analysis with the DESeq2 package. Genes were considered differentially expressed if they met the following criteria: absolute log₂(fold change) > 0.3612 and -log₁₀(p-value) > 1.301. GO term enrichment analysis was performed using the DAVID Bioinformatics Resources, applying the Benjamini-Hochberg correction for multiple testing with a false discovery rate (FDR) cutoff of 0.05. Enriched GO terms were visualized using graphical representations.
Total RNA was extracted from mPFC tissue using TRIzol® reagent (Life Technologies, USA), and cDNA synthesis was performed with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, K1622). Quantitative PCR (qPCR) was carried out using Fast SYBR™ Green Master Mix (Thermo Fisher, 4385612) on a C1000 Touch Thermal Cycler. Relative mRNA expression levels were calculated from cycle threshold (Ct) values and converted to fold change using the comparative Ct (ΔΔCt) method.
mPFC tissues from wild-type and knockout mice, as well as transfected Neuro2a (N2a) cells, were homogenized using a Dounce homogenizer in lysis buffer containing 150 mM NaCl, 1% NP-40, 2 mM EDTA, 50 mM Tris-HCl (pH 8.0), and protease inhibitor cocktail. Lysates were incubated overnight at 4 °C with an anti-Necdin antibody, tag antibodies, or a normal IgG control. Immunocomplexes were captured with Protein A/G agarose beads (Sigma), followed by centrifugation and extensive washing with a buffer consisting of 50 mM Tris-HCl (pH 7.4), 0.1% Triton X-100, 500 mM NaCl, 5 mM EDTA, and protease inhibitors. After washing, the beads were resuspended in 2× SDS loading buffer and heated. Proteins were separated by SDS-PAGE and analyzed by immunoblotting with appropriate primary antibodies.
After immunoprecipitation with a Necdin-specific antibody, protein samples were dissolved in DB protein buffer (8 M urea, 100 mM TEAB, pH 8.5) and digested with trypsin. The resulting peptides were centrifuged, desalted using C18 columns, and lyophilized. The dried peptide mixture was reconstituted in 10 µL of 0.1% formic acid, centrifuged at 14,000 × g for 20 min at 4 °C, and 1 µg of supernatant was subjected to LC-MS analysis. Chromatographic separation was performed using an EASY-nLC 1200 nano-UHPLC system equipped with a homemade pre-column (4.5 cm × 75 μm, 3 μm) and analytical column (15 cm × 150 μm, 1.9 μm). Mass spectrometry was conducted on a Q Exactive HF-X system with Nanospray Flex™ (ESI), using a spray voltage of 2.1 kV and transfer tube temperature of 320 °C. Data were acquired in data-dependent acquisition (DDA) mode with a full MS scan (m/z 350-1500) at a resolution of 60,000 (at m/z 200). The top 40 precursor ions were fragmented by higher-energy collisional dissociation (HCD) at a collision energy of 27%. MS/MS scans were acquired at 15,000 resolution, with dynamic exclusion set at 20 s. Raw data files (raw) were collected for further analysis.
N2a cells (ATCC) were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO₂. To promote differentiation, cells were pre-incubated in serum-free DMEM for 24 h prior to transfection. For transfection, a medium consisting of DMEM and Opti-MEM (Gibco) mixed at a 1:1 ratio and supplemented with 5% FBS was used. Plasmid or siRNA transfection was carried out using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol.
N2a cells were co-transfected with Flag-tagged Necdin (or siNecdin), Myc-tagged SynGAP, and HA-tagged ubiquitin constructs. MG132 (10 µM) was added for 8 h prior to lysis. Cells were lysed in denaturing buffer I (2% SDS, 10 mM Tris-HCl pH 7.5, 150 mM NaCl) by heating at 100 °C for 10 min. Lysates were then diluted tenfold with buffer II (1% Triton X-100, 10 mM Tris-HCl pH 7.5, 2 mM EDTA, 150 mM NaCl) and subjected to immunoprecipitation followed by Western blotting.
Cells or tissue samples were lysed in 2× SDS buffer (2% SDS, 63 mM Tris-HCl, 10% glycerol) and incubated for 15 min. Lysates were cleared by centrifugation at 13,000 rpm for 10 min. Equal amounts of protein were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked and incubated overnight at 4 °C with primary antibodies, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using the Pierce™ ECL Western Blotting Substrate (Thermo Scientific, 32106).
Primary cortical neurons were isolated from individual E16.5 wild-type or Necdin knockout mouse embryos and cultured on poly-D-lysine-coated coverslips as previously described [22]. Briefly, cerebral cortices were dissected and placed in cold HBSS, followed by digestion with 0.025% trypsin at 37 °C for 20 min with gentle agitation every 5 min. After enzymatic digestion, cells were dissociated using flame-polished glass Pasteur pipettes, filtered through a 40 μm nylon cell strainer, and counted. Neurons were plated at a density of 4 × 10⁵ cells per well in 24-well plates and maintained in Neurobasal medium supplemented with 2% B27 and 2 mM GlutaMAX at 37 °C in a 5% CO₂ incubator.
Recombinant adenoviruses encoding pCMV/Syngap1-P2A-EGFP (Ad-Syngap1; 8.0 × 10 pfu/mL) or pCMV/EGFP (Ad-EGFP; 5.6 × 10¹⁰ pfu/mL) were obtained from Changsha Bochu Biotech Co., Ltd. For stereotactic injections, wild-type or Ndn mice were anesthetized with isoflurane and positioned in a stereotaxic frame. After scalp incision and skull exposure under aseptic conditions, a small burr hole was drilled at the coordinates targeting the mPFC (1.98 mm anterior to Bregma, ±0.4 mm lateral). A total volume of 0.4 µL of either Ad-GFP or Ad-Syngap1 was slowly injected at a depth of 1.65 mm over 30 min. Mice were sutured post-injection and kept on a warming pad until recovery. Behavioral tests were performed 1-2 weeks after virus administration.
Mice were deeply anesthetized and transcardially perfused with PBS, followed by 4% paraformaldehyde (PFA). Brains were post-fixed in 4% PFA overnight, rinsed in PBS, and cryoprotected in 30% sucrose at 4 °C. Coronal sections ( ~ 30 μm) were cut using a cryostat. For BrdU staining, the brain sections were incubated with 1 M HCl for 15 min on ice and with 2 M HCl for 30 min at 37 °C. Sections were permeabilized in 0.5% Triton X-100 for 30 min, blocked in 5% BSA with 0.1% Triton X-100 for 1 h, and incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. Following PBS washes, sections were incubated with fluorophore-conjugated secondary antibodies for 1 h at room temperature. Primary antibodies included anti-GFP (Aves; 1:400), anti-Necdin (Abcam; 1:500), anti-c-Fos (Synaptic Systems; 1:500), anti- BrdU (Abcam, 1:500), anti-Ctip2 (Oasis, 1:200), anti-Tbr1(Oasis, 1:200), anti-Satb2 (Abcam, 1:300). Images were acquired with a ZEISS LSM880 confocal microscope using a Plan-Apochromat 63×/NA 1.4 oil immersion objective. Quantification of c-Fos⁺ neurons was performed using ImageJ (NIH).
Coronal brain sections ( ~ 100 μm) were collected from Thy1-GFP wild-type and Ndn mice and subjected to immunofluorescence staining using a GFP antibody to enhance the signal. For Syngap1 overexpression experiments, EGFP-positive neurons were whole-cell patched, and biocytin (0.2%) was included in the internal pipette solution. After fixation, slices were stained with Alexa Fluor 555-conjugated streptavidin (Invitrogen). Z-stack images (0.5 μm step size) were acquired using a 63× oil objective on the ZEISS LSM880 confocal microscope. Spine density was quantified using Imaris 9.01 (Bitplane).
Cultured neurons were transfected with an EGFP-expressing plasmid using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. After 48 h, cells were fixed with 4% paraformaldehyde overnight, permeabilized with 0.3% Triton X-100 in PBS for 15 min, and blocked in 10% Normal Goat Serum (NGS) for 1 h. Neurons were incubated overnight at 4 °C with a primary antibody against GFP (Abcam; 1:500), followed by fluorophore-conjugated secondary antibodies for 1 h at room temperature. Dendritic arbors were traced using the Simple Neurite Tracer plugin in ImageJ. Dendritic complexity was quantified by Sholl analysis with a step radius of 10 μm.
All data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 8.0. Normality was assessed using the Kolmogorov-Smirnov test. Comparisons between two groups were performed using Student's t-test. For comparisons involving more than two groups, one-way ANOVA followed by Bonferroni post hoc tests was used. When two independent variables were involved, two-way ANOVA with Bonferroni correction was applied.