Reagents and antibodies
Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. DL-AP5 (0105), CNQX (0190) were purchased from Tocris Bioscience. Following antibodies were used: Mouse anti-Nrg1 (Santa Cruz Biotechnology) (sc-393006; 1:1000 for blotting); Mouse anti-ErbB4 (Santa Cruz Biotechnology) (sc-8050; 1:1000 for blotting); Rabbit anti-P-ErbB4 (Cell Signaling Technology) (Tyr1284; 1:1000 for blotting); Mouse anti-GAPDH (Santa Cruz Biotechnology) (sc-32233; 1:1000 for blotting); Rabbit anti-PSD95 (Cell Signaling Technology) (#3450; 1:1000 for blotting); Mouse anti-Gephyrin (Santa Cruz Biotechnology) (sc-25311; 1:1000 for blotting); Rabbit anti-Synaptotagmin-1 (Cell Signaling Technology) (#14,558; 1:1000 for blotting); Rabbit anti-GABARa1 (Millipore) (3,108,661; 1:1000 for blotting); Rabbit anti-GluN2B(NMDAR2B) (Cell Signaling Technology) (#14,544; 1:1000 for blotting); Rabbit anti-GluN2A (Cell Signaling Technology) (#4205; 1:1000 for blotting); Rabbit anti- GluR1 (Cell Signaling Technology) (#13,185; 1:1000 for blotting).
Eight-to twelve-week-old male mice were used for experiments. The detailed information about mouse strains has been described previously: ErbB4f/f ; PV::Cre . Genotyping primer sequences were as follows: ErbB4f/f, 5′-AAA TCA TCC TCT TGT GTG CTT TTG TAC-3′ and 5′-CTC GGT ACT GCT GTT CCA GGT CAG A-3′; PV::Cre, 5′-AAA TGC TTC TGT CCG TTT GC-3′ and 5′-CAG AGC AGG CAT GGT GAC TA-3′ and 5′-AGT ACC AAG CAG GCA GGA GA-3′ and 5′-ATG TTT AGC TGG CCC AAA TG-3′. In all experiments, significant efforts are made to minimize the total number of animals used while maintaining statistically valid group numbers. Animal housing conditions were maintained at a temperature of 22 ± 1 °C, at > 30% humidity and a standard 12 h light/dark cycle. All animal experimental protocols were approved by the Animal Ethics Committee of Guangzhou Medical University.
Diets and feeding
The contents of control diet (CD) and ketogenic diet (KD) (Research Diets) are described previously . Briefly, CD (D10070802) (per-calorie macronutrient) contained: 10% protein, 80% carbohydrates, and 10% fat; KD (D10070801) was constituted by: 10% protein and 90% fat. Sources of fat are Soybean oil and cocoa butter. Micronutrient content, fiber, and preservatives are matched according to a per-calorie basis. During experiments, CD or KD was placed in the food well of the cage-top wire lid (stick-like texture). Diets were changed every week.
Blood ketones and glucose
Blood ketone levels were measured using the blood glucose and ketone monitoring system (FreeStyle Optium Neo, Abbott) according to the manufacturer’s instructions. Briefly, after sterilization with 70% ethanol, the tail tips of mice under tests were cut using a clean scissor, and a drop of blood was collected. Using the test strip (Abbott), levels of β-hydroxybutyrate or glucose were determined.
Adult male mice were maintained anesthetized with isoflurane (2–3%) and head-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 inside 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. Mice were then returned to their homecages for at least 1 week.
Seizure induction and behavioral monitoring
For kainic acid (KA)-induced acute seizure model, mice with guide cannulas were gently restrained and an infusion cannula (IO: 0.3 mm; RWD Life Science.Inc) was inserted into the amygdala through the guide cannula. 0.15 µl of KA (3 mg/ml) was infused into the amygdala at the flow rate of 2 nl/s controlled by microinjector (NanojectIII, Drummond Scientific). The cannula was kept in the right amygdala for two additional min after completion of infusion and withdrew slowly to minimize reflux along the injection tract. Behavioral seizures were classified based on the criteria described by Racine  and scored every 5 min by a blinded investigator: stage 0, no seizure; stage 1, arrest and rigid posture; stage 2, head nodding; stage 3, sporadic full-body shaking, spasms; stage 4, chronic full-body spasms; stage 5, jumping, shrieking, falling over; stage 6, violent convulsions or death.
For KA-induced spontaneous recurrent seizure (SRS) model, diazepam (8 mg kg−1) was i.p. injected 1 h after status epilepticus (SE) induction. The food for mice was switched from standard chow diet to CD and KD, respectively. Three weeks later, mice were video-monitored from 8 am to 8 pm each day for 2 weeks. SRS was defined with score ≥ 4 and counted by review of video files by a blinded investigator.
We performed Timm staining using FD Rapid TimmStain™ Kit (Biosensis) and followed manufacturer’s instruction. Images were randomly taken from dorsal hippocampus with microscope (CTR6, Leica). Staining intensities were quantified using image J. The extent of mossy fiber sprouting was quantified by Timm index, which denote the ratio between total area of Timm granules and total dentate gyrus length .
Adult male mice were anesthetized with isoflurane. Brains were quickly removed and chilled in ice-cold modified artificial cerebrospinal fluid (ACSF) containing (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-1000S vibratome (Leica, Germany) and transferred to a storage chamber containing regular ACSF (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 additional 1 h before recording. All solutions were saturated with 95%O2/5%CO2 (vol/vol).
Slices were placed in the recording chamber superfused (2 ml/min) with ACSF. Whole-cell patch-clamp recording from CA1 pyramidal neurons was 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 MultiClamp 700B amplifier and 1440A digitizer (Molecular Device).
For spontaneous excitatory postsynaptic current (sEPSC) recording, pyramidal neurons were held at −70 mV in the presence of 20 μM RS-95531, with the pipette solution containing (in mM): 125 Cs-methanesulfonate, 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 Schaffer Collaterals (SC)-CA1 pathway with gradually increasing stimulation intensities by using a concentric bipolar electrode (FHC), ~ 200 µm away from recording pipette.
To record spontaneous inhibitory postsynaptic current (sIPSC), pyramidal neurons were held at −70 mV in the presence of 20 μM CNQX and 100 μM AP-5, with the 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), stimulation electrode was positioned on the Schaffer Collaterals (SC)-CA1 pathway, ~ 100 µm away from recording pipette.
In all experiments, series resistance was maintained below 20 MΩ and not compensated. Cells would be rejected if membrane potentials were positive more than −60 mV; or if series resistance fluctuated more than 20% of initial values. Data were filtered at 1 kHz and sampled at 10 kHz.
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 inhibitors cocktails. Samples were resolved on SDS/PAGE and transferred to nitrocellulose membranes, which were incubated in TBS buffer containing 0.1% Tween-20 and 5% milk for 1 h at room temperature before incubating with primary antibodies (overnight at 4 °C). After wash, the membranes were incubated with HRP-conjugated secondary antibodies (Absin ImmunoResearch) in the same TBS buffer for 1 h at room temperature. Immunoreactive complex bands were visualized using enhanced chemiluminescence (Pierce) and captured using the Genesys imaging system (Gene Company Limited, UK). Band densities of interested proteins were normalized with loading control.
RNA sequencing and transcriptome analysis
Total RNA was extracted from hippocampi using Trizol (Life Technologies, Carlsbad, CA, USA). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments of preferentially 250–300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
The library preparations were sequenced on an Illumina Hiseq 4000 and 150 bp paired-end reads were generated. Image analysis and base calling were performed with Illumina CASAVA pipeline, and finally 150 bp paired-end reads were generated. Feature Counts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis was performed using the DESeq2 R package (1.10.1). Genes with an P-value < 0.05 found by DESeq2 were assigned as differentially expressed. Note that the raw sequencing data have been deposited in the Gene Expression Omnibus (GEO) database (GSE156239).
qRT-PCR was performed as described previously . Briefly, total RNA was isolated by using TRIzol reagent (15596–026, Invitrogen). RNA (1 µg) was reverse-transcribed with oligo dT-primers using Maxima reverse transcriptase (EP 0742, Fermentas) followed by q-PCR with SYBR Green detection (K 0222, Fermentas). Samples were assayed in triplicates, with each plate having loading standards in duplicate. RNA levels of ErbB4 and Nrg1 were normalized to those of GAPDH. Primer sequences were: ErbB4, 5′-CAT GGC CTT CCA ACA TGA CTC TGG-3′ and 5′-GGC AGT GAT TTT CTG TGG GTC CC-3′; Nrg1, 5′-ATG TGC AAA GTG ATC AGC AAG -3′ and 5′-TGA GGA CAC ATA GGG TCT TT -3′; GAPDH, 5′-GGT TGT CTC CTG CGA CTT CA-3′ and 5′-CCA CCA CCC TGT TGC TGT AG -3′.
Anesthetized mice were transcardially perfused with PBS followed by 4% PFA. Removed brains were fixed in 4% PFA at 4 °C for 8 h. After dehydration by 30% sucrose, brain blocks were frozen and cut into 30-μm-thick sections on a cryostat (CM1950, Leica). Sections were permeabilized with 0.3% Triton X-100 and 5% BSA in PBS and incubated with primary antibodies at 4 °C overnight. After washing with PBS for 3 times, samples were incubated with Alexa Fluor-conjugated secondary antibodies (1:800, Jackson ImmunoResearch) for 1 h at room temperature. Samples were mounted with Vectashield mounting medium (Vector lab) and images were captured using Leica immunofluorescent microscope (CTR6, Leica).
Experimental design and statistical analysis
Adult male mice (≥ 2 months) were used in the present study. Animal or replicate numbers for each experiment and results of the statistical analyses, including degrees of freedom and exact p-values were mentioned in the figure legends. Statistical analyses were performed using GraphPad Prism version 8.0.1 (GraphPad Software). Sample size choice was made based on previous studies [36, 37]. Student’s t test was used to compare data from two groups. Repeated two-way ANOVA was for KA-induced seizure development studies. Regular two-way ANOVA was used for electrophysiological studies that analyze more than two parameters. All tests were two-sided. All data represent mean ± SEM, unless otherwise stated. P < 0.05 was considered to be statistically significant.