Reagents and Antibodies

TSA and a Glutamate Assay Kit were utilized.

The following antibodies were used: rabbit anti-EAA2 (GLT-1) (Abcam) (ab205248; 1:3000 for blotting), rabbit anti-EAAT1 (GLAST) (Abcam) (ab181036; 1:5000 for blotting), rabbit anti-GAPDH Abcam) (ab9485; 1:10000 for Western blotting), rabbit anti-H3 (Abcam) (ab1791; 1:1000 for Western blotting; 1:200 for ChIP); rabbit anti-H3k18ac (active motif) (39130; 1:1000 for Western blotting); mouse anti-H3k27ac (active motif) (39085; 1:1000 for Western blotting); anti-H4 (Abcam; ab17036; 1:1000 for Western blotting; 1:200 for ChIP); rabbit anti-H4k8ac (active motif) (61104; 1:1000 for Western blotting); and rabbit anti-H4k12ac (active motif) (39928; 1:1000 for Western blotting).

Animals and Usage of ITSA

Eight- to twelve-week-old male C56BL/6 mice were utilized for the study. This study was approved by the Ethics Committee of the Affiliated Hospital of Guangxi University of Science and Technology. ITSA was dissolved in DMSO and administered by intraperitoneal injection at a dose of 5 mg/kg.

Cannula Implantation

Adult male mice were anesthetized with isoflurane (maintenance: 2–3%), and their heads were fixed in a stereotaxic device (RWD Life Science, Inc.). After an incision was made in the scalp, a small hole was drilled into the skull, and a guide cannula (IO: 0.48 mm; RWD Life Science. Inc.) was implanted into the right amygdala (coordinates: anteroposterior, − 1.22 mm; dorsoventral, − 4.5 mm; mediolateral, 3 mm (relative to bregma)) and cemented onto the skull with dental cement. The mice were then returned to their home cages for at least 1 week.

Seizure Induction and Behavioural Monitoring

To establish a kainic acid (KA)-induced acute seizure model, mice in which guide cannulas had been implanted were gently restrained, and an infusion cannula (IO: 0.3 mm; RWD Life Science. Inc.) was inserted into the amygdala through the guide cannula. A total of 0.15 µl of KA (3 mg/ml) was infused into the amygdala at a rate of 2 nl/s under the control of a microinjector (Nanoject III, Drummond Scientific). The cannula was kept in the right amygdala for two minutes after completion of the infusion, after which it was withdrawn slowly to minimize reflux along the injection tract. Seizures were classified on the basis of the criteria described by Racine [16] and scored every 5 min by a blinded investigator as follows: stage 0, no seizure; stage 1, arrest and rigid posture; stage 2, head nodding; stage 3, sporadic full-body shaking and spasms; stage 4, chronic full-body spasms; stage 5, jumping, shrieking, and falling over; and stage 6, violent convulsions or death.

Diazepam (8 mg/kg) was intraperitoneally injected into mice with KA-induced spontaneous recurrent seizures (SRSs) 1 h after status epilepticus (SE) induction. Then, TSA was administered intraperitoneally. Three weeks later, the mice were video-monitored from 8 am to 8 pm each day for 2 weeks. SRSs were defined as seizures with a score ≥ 5 and were counted by an investigator blinded to the groups by reviewing video files.

Electrophysiological Recording

Adult male mice were anaesthetized with isoflurane. The brains were quickly removed and chilled in ice-cold modified artificial cerebrospinal fluid (ACSF) containing the following (in mM): 120 choline-Cl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 25 NaHCO3, and 10 glucose. Coronal hippocampal slices (300 μm) were sectioned in ice-cold modified ACSF using a VT-1000 S vibratome (Leica, Germany) and transferred to a storage chamber containing regular ACSF (containing (in mM) 126 NaCl, 3 KCl, 1 MgSO4, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose) at 32 °C for 30 min and at room temperature (24 ± 1 °C) for an additional 1 h before recording. All the solutions were saturated with 95% O2/5% CO2 (vol/vol).

Slices were placed in the recording chamber and superfused (2 ml/min) with ACSF. Whole-cell patch-clamp recordings of CA1 pyramidal neurons were visualized with infrared optics using an upright microscope equipped with an infrared-sensitive CCD camera (DAGE-MTI, IR-1000E). Pipettes were pulled by a micropipette puller (P-97, Sutter instrument) with a resistance of 3–5 MΩ. Recordings were made with a MultiClamp 700B amplifier and 1440 A digitizer (Molecular Devices).

For spontaneous excitatory postsynaptic current (sEPSC) recording, pyramidal neurons were held at − 70 mV in the presence of 20 µM RS-95,531 using a pipette solution containing the following (in mM): 125 Cs-methanesul fonate, 5 CsCl, 10 HEPES, 0.2 EGTA, 1 MgCl2, 4 Mg-ATP, 0.3 Na-GTP, 10 phosphocreatine and 5 QX314 (pH 7.40, 285 mOsm). Evoked excitatory postsynaptic currents (eEPSCs) were recorded by stimulating the Schafer collateral (SC)-CA1 pathway with gradually increasing stimulation intensities by using a concentric bipolar electrode (FHC) ~ 200 μm away from the recording pipette.

To record spontaneous inhibitory postsynaptic currents (sIPSCs), pyramidal neurons were held at − 70 mV in the presence of 20 µM CNQX and 100 µM AP-5 using a pipette solution containing (in mM) 140 CsCl, 10 HEPES, 0.2 EGTA, 1 MgCl2, 4 Mg-ATP, 0.3 Na-GTP, 10 phosphocreatine and 5 QX314 (pH 7.40, 285 mOsm). To measure evoked inhibitory postsynaptic currents (eIPSCs), a stimulation electrode was positioned in the Schafer collateral (SC)-CA1 pathway ~ 100 μm away from the recording pipette.

In all the experiments, the series resistance was maintained below 20 MΩ and not compensated. Cells were rejected if the membrane potential was more than.

−60 mV or if the series resistance fluctuated by more than 20% of the initial value. The data were filtered at 1 kHz and sampled at 10 kHz.

Western Blotting

Brain tissue homogenates were prepared in RIPA buffer containing (in mM) 50 Tris–HCl, pH 7.4; 150 NaCl; 2 EDTA; 1 PMSF; 50 sodium fluoride; 1 sodium vanadate, 1 DTT with 1% sodium deoxycholate, 1% SDS and 1% protease inhibitor cocktails. The samples were resolved via SDS‒PAGE and transferred to nitrocellulose membranes, which were subsequently incubated in TBS buffer containing 0.1% Tween-20 and 5% milk for 1 h at room temperature before being incubated with primary antibodies (overnight at 4 °C). After washing, the membranes were incubated with HRP-conjugated secondary antibodies (Absin ImmunoResearch) in the same TBS for 1 h at room temperature. The immunoreactive bands were visualized using enhanced chemiluminescence (Pierce) and captured using a Genesys imaging system (Gene Company Limited, UK). The densities of the protein bands of interest were normalized to those of the loading control band.

Timm staining

We performed Timm staining using an FD Rapid TimmStain™ Kit (Biosensis) following the manufacturer’s instructions. Images of the dorsal hippocampus were randomly taken with a microscope (CTR6, Leica). The staining intensity was quantified via ImageJ. The extent of mossy fibre sprouting was quantified using the Timm index, which represents the ratio of the total area of Timm-positive granules to the total dentate gyrus length.

Measurement of Changes in Glutamate Levels in the Interstitial Space in the Hippocampus of KA-treated Mice Infused with TSA into the Hippocampus via Microdialysis

A probe was stereotaxically inserted into the hippocampus of each mouse (AP: 2.2 mm, ML: 1.6 mm, DV: 1.6 mm). After continuous perfusion with ACSF, the interstitial fluid was collected and subsequently lyophilized. A total of 20 µL of reconstituted tissue fluid was prepared and derivatized with 2,4-dinitrofluorobenzene at 60 °C for 30 min. Then, 50 mM dihydrogen phosphate was added to the microdialysis sample to stop the reaction. A UV detector was then used to analyse the glutamate concentration in the microdialysis samples at an absorbance of 360 nm. On the basis of a standard curve, the glutamate concentration was calculated using LC Solution software.

Analysis of Changes in Glutamic Acid Levels in the Hippocampus of KA-treated Mice Administered TSA via Metabolomics Analysis of the Hippocampus

Mice were euthanized for metabolomics analysis. Hippocampal samples were accurately weighed and transferred to an Eppendorf tube. After 500 µL of extraction solvent (precooled at -20 °C, acetonitrile/methanol/water, 2:2:1) was added, the samples were vortexed for 30 s, homogenized at 45 Hz for 4 min via a TissueLyser LT instrument, and sonicated in an ice–water bath for 5 min. The homogenization‒sonication cycle was repeated 3 times, followed by incubation at -20 °C for 1 h and centrifugation at 12,000 rpm and 4 °C for 15 min. A 100 µL aliquot of the clear supernatant was transferred to an autosampler vial for HPLC‒MS analysis.