Establishment of ASD Model Mice

All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Southeast University (Ethics code: 20210311002). Eight-week-old male and female C57BL/6J mice, weighing ~20–26 g, were obtained from Sino-British SIPPR/BK Lab Animal Ltd (Shanghai, China). Male mice were used for experiments involving neural activity activation, while female mice were used to breed VPA-induced ASD mouse models.

Pregnant female mice received a single intraperitoneal (i.p.) injection of 600 mg/kg sodium valproate (1069-66-5, Sigma-Aldrich) post-conception to induce autism-like behavior in the offspring. Each pregnant female produced six to nine pups; the day of birth was recorded as day 0. Pups were weaned and group-housed (3–5 mice/cage) by biological sex into cages on postnatal day 26 (PND26). Behavioral tests were conducted between 14:00 and 18:00, following a 120-min acclimation period in the testing room to minimize environmental stress. For detailed protocols and analyses, refer to Note S1 in the supplementary information. All animal care and experimental procedures strictly adhered to institutional ethical guidelines and were approved by the Institutional Animal Care and Use Committee of Southeast University (Ethics code: 20210311002).

Cell Cultures

Neuro-2a cells (mouse neuroblastoma cells, N2a, zl-056516) were sourced from Wuhan Pricella Biotechnology Co. and cultured in Dulbecco’s modified Eagle’s medium (DMEM, 11965118, Gibco), supplemented with 10% fetal bovine serum (A5670701, Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (15140122, Gibco). Upon reaching 80–90% confluency, SPIONs were added and co-incubated for 24 h.

For pyramidal hypothalamic neuronal culture, embryos were collected in a sterile environment, and brains were dissected to obtain hypothalamic tissue, which was placed in ice-cold dissection solution. The tissue was digested with 0.125% trypsin (25200072, Gibco) at 37°C for 10 min, then neutralized with three times the volume of DMEM containing 10% fetal bovine serum. The tissue was triturated into a single-cell suspension, centrifuged, and resuspended in Neurobasal medium with 2% B-27 supplement (17504044, Gibco). Cells were plated at a density of 1×105 per mL and incubated under 5% CO2 at 37 °C, with medium changes every other day.

Cytotoxicity Assessment of Superparamagnetic Nanoparticles

SPIONs were prepared at concentrations of 1, 2, 4, 8, and 16 mg/mL in sterile PBS. N2a cells were seeded in 96-well plates (density: 5×103 cells/well) and allowed to adhere for 24 h. Then, SPION solutions (1 μL per well) were added to the culture medium and co-incubated with cells for 24 h at 37°C under 5% CO₂. After incubation, 10 μL CCK-8 reagent (Beyotime, China) was added to each well according to the manufacturer’s protocol. Plates were incubated at 37°C for 30 min, and absorbance was measured at 450 nm using a microplate reader. Cell viability (%) was calculated as:

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Construction of Precise Magneto-stimulation System (pMSS) Suitable for Mouse Brain

The pMSS system utilized γ-Fe2O3 SPIONs and a pair of disk-like magnets. The γ-Fe2O3 nanoparticles (brand name, Ferumoxytol) are approved by the FDA as an intravenous iron supplement. A rotating magnetic field was generated using an NdFeB (neodymium, iron, and boron) static magnet and a motor from Innuvo (China). For brain stimulation, mice were exposed to a 50 mT magnetic field, measured with a Gauss meter (HT20; Hengtong, China).

Parameters for rTMS Modulation of N2a Neuronal Activity

The rTMS procedure was carried out using a CCY-I magnetic stimulator (Yiruide Medical Equipment Co., Ltd, Wuhan, China) equipped with a specially designed circular induction coil (model Y064). The coil exhibited concentric dimensions of 18 mm (inner diameter) and 57 mm (outer diameter), featuring a parallel-wound solenoidal configuration with multilayer winding architecture. Technical specifications included: axial height = 20.4 mm, conductor cross-sectional area = 18 mm2, and 30 total turns arranged in 6 vertically stacked layers (5 turns/layer) [24]. Stimulation parameters were configured as follows: The coil assembly was positioned 12 mm above the plane of the N2a cell culture substrate. High-frequency repetitive TMS was delivered at 10 Hz with intensity calibrated to 50% of the device's maximum output (2.60 T measured at a coil surface contact point). Each stimulation protocol comprised 50 burst trains, with individual sequences containing 10 pulses followed by 9 s inter-train intervals, yielding 500 total pulses per experimental session.

Calcium Imaging

Ca2+ ion endocytosis under pMSS was monitored using Fluo-4 AM (S1060, Shanghai Biyuntian Biotechnology Co.), a fluorescent probe for Ca2+ ions. Fluo-4 AM, which emits green fluorescence upon hydrolysis by intracellular esterases, was used to stain N2a cells. N2a cells were incubated with Fluo-4 staining solution at 37°C for 30 min in the dark, and Ca2+ flow was dynamically recorded under a fluorescence microscope (Eclipse Ti2, Nikon, Fluo-4 AM excitation/emission = 490/525 nm).

Ca2+ images were processed using MATLAB (MathWorks). The Ca2+ trace of each active neuron was extracted by defining a circular region of interest (ROI) with a 15 mm diameter, centered on the detected cell position. The average fluorescence of the surrounding circular region was used as the baseline background fluorescence (F<ring>). The Ca2+ signal of each mouse (Fsig) was calculated by subtracting the baseline background fluorescence (F<ring>) from the fluorescence of the ROI (F<ROI>): Fsig = F<ROI> − F<ring>. Each Ca2+ trace ΔF/F0 was calculated as Fsig divided by the baseline fluorescence over the neuron ROI (F0). F0 was estimated by subtracting the original Ca2+ signal F<ROI> from the video background luminance curve (Fb), and Fb was formed by considering the minimum value of the fluorescence of each frame of the video. Given that the signal may contain multiple Ca2+ transients, F0 was determined using the 15% quantile instead of the mean, i.e., F0 = F<ROI> − Fb.

Virus Injection and Fiber/GRIN Lens Implantation

Under anesthesia, mice were positioned on a stereotaxic apparatus (71000-S, RWD Life Science). The virus was injected using a modified syringe at a rate of 0.1 μL/min, with the needle remaining in place for 5 min post-injection. A total of 0.5 μL of rAAV-hSyn-GCaMP6f (BC-0079, Braincase) or AAV9-hSyn-OT1.0 (PT-2741, BrainVTA) was bilaterally injected into the target area, with coordinates (mm from bregma): PVN: −0.82 anteroposterior (AP); ±0.25 mediolateral (ML); −4.85 dorsoventral (DV). Two weeks following the surgery, SPIONs were delivered into the PVN at the same coordinates. An optical fiber (400-µm, FOC-W-1.25-400, Inper) or GRIN lens (1-mm, GT-LFRL-100-025-50-NC, GRINTECH GmbH) was inserted into the injection site and secured using adhesive dental cement (S380, C&B Metabond).

Fiber Photometry and MiniScope Recordings

Three weeks after GRIN lens implantation, the miniscope was attached to the mouse's head and adjusted for clear focus across the field of view. The miniscope body was detached and reattached to the base for each imaging experiment using a side-locking screw. During experiments, Ca2+ activity at the single neuron level in the PVN was recorded as the mice underwent pMSS.

In Vivo Microdialysis

Mice were anesthetized with pentobarbital and isoflurane, then surgically implanted with a guide cannula (AG-X, Eicom, Japan) into the PVN. After surgery, mice were allowed to recover for five days. On the sixth day, microdialysis was applied using a probe (FZ-X-Y, Eicom, Japan) at 1.5 μL/min. Artificial cerebrospinal fluid of the following composition was used (in mmol/L): 147 NaCl, 2.7 KCl, 1.2 CaCl2, 0.85 MgCl2, pH 7.4. Samples were collected and stored at −80 °C until testing for oxytocin secretion using an ELISA kit (E-EL-0029, Elabscience Biotechnology, China).

Oxytocin Administration

Oxytocin (HY-17571A, Tocris Bioscience) was administered either intravenously (20 mg/kg) [25] or by local infusion into the PVN. The mice in this experiment weighed ~25.5 g, so ~510 μg of oxytocin was injected into each mouse via the tail vein. Since the mass of the PVN of C57BL/6J mice couldn't be measured and no relevant literature was available, the mass of the hypothalamus was taken as the reference for in situ oxytocin injection. Given that the PVN mass of adult C57BL/6J mice is ~10 mg, ~0.2 μg of oxytocin was injected into the PVN of each mouse.

Administration of Oxytocin Receptor Antagonists

Oxytocin receptor antagonists (MCE, L-372662) were delivered to the medial prefrontal cortex (mPFC; coordinates (in mm): AP +2.0, ML ±0.3, DV −2.0) and lateral ventricle (LV; coordinates: AP −0.6, ML ±1.0, DV −2.0) of ASD mice via stereotaxic surgery. The injection protocol followed previously established parameters [26], with a concentration of 2 μg/μL and a total volume of 1 μL per site.

Histology

Following the injection of magnetic nanoparticles into the PVN (AP −0.82, ML ±0.25, and DV −4.5) using a modified needle, mice were allowed to recover for 1 week before sacrifice. The mice were anesthetized (i.p.) and perfused with 50 mL of 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) via intracardiac puncture. Brains were then post-fixed in 4% PFA at 4°C for 24 h, then dehydrated in 30% sucrose. Sections were cut at 5 μm and stained with hematoxylin and eosin (H&E; C0105S, Beyotime) and a Prussian Blue Iron Stain Kit (G1420, Solarbio).

For immunofluorescence, 50 μm-thick sections were mounted on slides and permeabilized with 0.5% Triton X-100 in Tris-buffered saline (TBS). Then they were incubated with primary and secondary antibodies as required, and the nuclei were stained with 6-diamidino-2-phenylindole (DAPI). Images were captured using an FV1200 confocal microscope (Olympus). Antibody details are listed in Table S1 in the Supplementary Information. In addition, the percentage of c-Fos positive cells was calculated using the following formula: percentage of c-Fos positive cells = (number of positive cells/ total DAPI-stained nuclei) ×100%.

For Golgi-Cox staining, the FD Rapid GolgiStain™ Kit (PK401, FD NeuroTechnologies) was used. Brain tissues were soaked in an AB mixture (A + B, 1:1 ratio, 10 mL/mouse) for 21 days, followed by soaking in C solution for 72 h. Slices were cut at 150 μm at −80°C and mounted on gelatin-coated slides. Tissue dehydration was achieved through a gradient of ethanol concentrations (50%, 75%, 95%, and 100%). Transparency was attained using xylene, and slides were sealed with neutral resin. Dendritic spine images were captured with a 63× objective lens (Zeiss Axio Observer 7, Germany). Dendritic spine density in PVN neurons was quantified using ImageJ software; ~8 neurons were analyzed from 3 mice in a blinded manner.

RNA-Sequencing

After 7 days of continuous pMSS, the PVN of the mouse brain was collected for RNA sample preparation. Three mice from each experimental group (pMSS-OFF/pMSS-ON) were used. RNA sequencing was carried out by Lianchuan Biotech (Hangzhou, China). In brief, RNA was reverse-transcribed into cDNA, fragmented, and specific adapters were attached to form a sequencing library. This library was sequenced using a high-throughput sequencer, generating extensive sequence data. Quality-filtered data underwent bioinformatics analysis, including functional annotation, gene enrichment, and transcription factor analysis. Differentially-expressed genes (P < 0.05, Student's t test) were identified and classified by expression profiles. Functional enrichment analysis was applied using Gene Ontology (GO) (http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (https://www.kegg.jp/kegg/pathway.html).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from mouse brain using the TRIzol reagent (15596026CN, Invitrogen) in accordance with the manufacturer's instructions. Subsequently, cDNA synthesis was carried out using the HiScript® III RT SuperMix (R222-01, Vazyme) for qPCR (+gDNA wiper) reverse transcription kit (Vazyme) with 1 mg of total RNA. A qRT-PCR was performed using the ChamQTM SYBR® qPCR Master Mix kit (Q311-02, Vazyme) on a Step OnePlus thermal cycler (4376600, Applied Biosystems). The expression levels of the target genes were normalized to glyceraldehyde 3-phosphate dehydrogenase as the reference gene. The primer sequences for the target genes can be found in Table S1. Each experiment was repeated six times to ensure the reliability and reproducibility.

Western Blot

For protein analysis, a 5% concentrated gel and a 10% separation gel were prepared, and each gel lane was loaded with 40 μg of total protein. After electrophoretic separation, proteins were transferred onto a polyvinylidene fluoride membrane. The membrane was rinsed with Tris Buffered Saline with Tween (TBST) for 5 min and blocked with TBST containing 5% bovine serum albumin (BSA) for 1 h. The corresponding primary antibody diluted 1:1000 in TBST solution with 5% BSA was added and incubated overnight at 4°C. The membrane was then rinsed three times for 10 min each with TBST. Thereafter, the corresponding labeled secondary antibody diluted 1:1000 in PBS was added and incubated for 2 h at room temperature. The membrane was subsequently rinsed three times with TBST for 10 min each. Finally, densitometric analysis was applied using TotalLab software, and the experiment was repeated three times to ensure the reliability and reproducibility of the results. Information regarding the antibodies used can be found in Table S1 in the Supplementary Information.

Quantification and Statistical Analysis

Data were analyzed using one-way or two-way analysis of variance (ANOVA), followed by post hoc tests for multiple comparisons or t tests. Results are presented as the mean ± standard error of the mean. A statistically significant difference was set at P < 0.05, with asterisks denoting significance levels (*P < 0.05, **P < 0.01, *** P < 0.001, ### P < 0.001).