For the human study, the institutional review board of the Second Affiliated Hospital School of Medicine Zhejiang University approved this study (license ID: 2020-235). A written patient consent was obtained from all included patients with SEEG implantation. All animal care regimens and experiments were approved by the Animal Care and Use Committee of Zhejiang Chinese Medical University, and were in complete compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
FCD cases were retrospectively recruited in the epilepsy center, the Second Affiliated Hospital of School of Medicine, Zhejiang University from January 2014 to June 2022. The inclusion criteria were as follows: all patients (1) underwent presurgical SEEG recordings; (2) underwent epilepsy surgery with pathologically diagnosed FCD I/II, according to the 2011 International League Against Epilepsy FCD classification.40 At the time of surgery, all patients had a comprehensive evaluation including a detailed clinical history, neurological examination, routine MRI, surface EEG and fluorodeoxyglucose-positron emission tomography. A decision to proceed to SEEG was made at the individual level when noninvasive data was discordant and typically when the presumed epileptogenic zone could not be confidently limited to a single lobe. The surgical plans were made with a multidisciplinary team. Patients were followed up for seizure relapses after surgery. Freedom from seizures corresponded to Engel classification I at the 1-year follow-up.41 Acute postoperative seizures during the first week were not taken into consideration.
SEEG recording and analysis
We further selected patients who underwent SEEG exploration to quantify the seizure network in FCD subtypes. Intracerebral multiple contact electrodes (8–16 contacts, 2 mm in length, separated by 1.5 mm, and 0.8 mm in diameter, HKHS, Beijing, China) were placed according to the Talairach stereotactic method.42 The anatomical targeting of electrodes was established according to information from the noninvasive study and clinical hypotheses about the localization of the EZ. A postoperative CT scan or MRI was used to verify the position of electrodes. SEEG signal was recorded by a 256-channel long-term monitoring system (Nihon-Kohden EEG-1200C, Tokyo, Japan) with sampling rate at 2000Hz, and a SEEG bipolar montage was created and applied to all channels. Channels showing artifacts were excluded from analysis. Two electroclinical seizures with both video and SEEG data were analyzed for each patient. For patients with >2 captured seizures, all seizures were reviewed, of which 2 representative seizures were analyzed, so as to keep the same proportion of each distinct electroclinical seizure type. Seizure onset was manually defined as the first unequivocal SEEG signal change from the background in the setting of a sustained rhythmic discharge,43 and the first 15 s of SEEG onset were used for further analysis. Seizure onset without a fast discharge was excluded from subsequent analysis. We computed EI for all selected seizures using AnyWave Software (available at http://meg.univ-amu.fr/wiki/AnyWave). It is a normalized value that ranges from 0 (no epileptogenicity) to 1 (peak epileptogenicity). Electrodes with an EIå 0.3 is considered to have a high epileptogenicity.11 The number of brain regions/lobes with a high epileptogenicity is used to denote the extension of the seizure network.
Timed pregnant (embryonic day 17–18, E17–18) female Sprague-Dawley rats of 8-9 weeks old were anesthetized with isoflurane (1.5 ml/min of oxygen and 3.5% isoflurane). We established the prenatal freeze lesion model as adapted from Takase et al.20 In brief, uterine horns were exposed. A liquid-nitrogen-cooled bronze probe was applied longitudinally on 2 points on the scalp of a rat embryo, from outside of the uterus wall. The uterus was then returned to the abdominal cavity. The freeze-lesioned pups were born at around E22 and weaned at postnatal day 21 (P21). Pups of time-pregnant rats not exposed to freeze lesioning were used as age-matched controls. All behavioral experiments were performed at around P50.44 No statistical method was used to predetermine sample size; sample sizes were estimated based on our previous studies for similar types of behavioral, biochemical, and electrophysiological analyses.24,34 No method of randomization was used.
Electrode implantation, seizure induction and EEG recordings
Bipolar electrodes (each 0.2 mm in diameter; A.M. Systems, USA) were implanted into the right hippocampus (AP −5.28 mm, L −5.0 mm, V −6.0 mm) and one screw was placed over the motor cortex (AP: + 2.5 mm; ML: −2.5 mm) to record hippocampal and cortical EEGs respectively. Another screw was placed over the cerebellum to serve as the ground reference. All the above coordinates were measured from bregma according to the Paxinos and Franklin’s Rat Brain Atlas.45 Rats were allowed for recovery for 1 week after surgery.
To test the seizure severity, single-dose PTZ was injected intraperitoneally for seizure induction (60 mg/kg PTZ was used unless otherwise specified). Continuous EEG recording of each rat started 10 min before the PTZ injection and lasts for an additional 30 min using PowerLab system (AD Instruments, Australia).34 Ictal events were defined electrographically as a spike in frequency (≥2 Hz), high amplitude (>3 x baseline), rhythmic epileptiform, activity with a minimum duration of 10 s.46 Seizure severity was scored according to a modified Racine’s scale:47 1, mouth and facial movement; 2, head nodding; 3, forelimb clonus. 4, rearing with forelimb clonus. 5, rearing and falling with forelimb clonus. 6, fully tonic-clonic generalized seizure or death. Stages 1–3 were focal seizures and stages 4–6 were considered GS. A trained observer who was unaware of the experimental groupings scored the seizure severity. All data were collected and analyzed in a double-blind manner.
The stereotactic viral delivery was strictly performed according to our previous studies.48 Briefly, anesthetized rats were mounted in a stereotaxic apparatus (Stoelting, USA). Virus was injected into the DG (AP: −3.0 mm, ML: −1.5 mm, V: −4.2 mm) with a 1-μL microliter syringes (Gaoge, China) controlled by an injection pump (Micro 4, World Precision, USA) at 100 nl/min. AAV-SOM-Cre and AAV-PV-Cre were purchased from BrainVTA Co., Ltd (Wuhan, China). All other viruses were purchased from OBiO Technolog Corp.,Ltd. (Shanghai, China).
For fluorometric monitoring the calcium activity of DG SOM + or PV + interneurons during seizures, a viral cocktail (1:1, 1 μl) of AAV-SOM-Cre (serotype: AAV2/9, viral titers: 2.50 × 1012 vg/mL) or AAV-PV-Cre (serotype: AAV2/9, viral titers: 3.38 × 1012 vg/mL) and AAV-EF1a-DIO-GCaMP6s (serotype: AAV2/9, viral titers: 2.05 × 1012 vg/mL) was stereotactically injected.25
To optogenetically inhibit SOM + or PV + interneurons in the DG, a viral cocktail of (1:1, 1 μl) of AAV-SOM-Cre or AAV-PV-Cre and AAV-CAG-FLEX-ArchT-GFP (serotype: AAV2/8, viral titers: 1.3 × 1013 vg/mL) was stereotactically injected.
To optogenetically activate SOM + or PV + interneurons in the DG, a viral cocktail of (1:1, 1 μl) of AAV-SOM-Cre or AAV-PV-Cre and AAV-EF1a-DIO-hChR2(H134R)-EYFP (serotype: AAV2/8, viral titers: 1.58 × 1013 vg/mL) was stereotactically injected.
Stereotactic fiber/cannula implantation surgery on rats was described in detail in our previous study.48 For fluorometric monitoring, an optical fiber (300 μm O.D., 0.37 mm FOC-C-1.25-200-0.37-6.0, Inper, China) was implanted into the DG. For in vivo optogenetic manipulation, the guide cannulas (RWD, China), used to aid in optical fiber (diameter, 200 μm; Thorlabs, USA) insertion in optogenetic studies, were implanted in the DG. Electrode location and viral expression were histologically verified in all animals, and only rats with correct locations were taken into analysis. Co-labeling with anti-SOM or anti-PV antibodies was further performed in randomly selected subjects to verify the sensitivity and specificity of the viruses.
Fiber photometry was performed as our previous study.15 Briefly, the fiber photometry system (Nanjing-Thinkertech, China) used a 488-nm diode laser (Coherent, USA), reflected by a dichroic mirror (Thorlabs, USA) and coupled into the optical fiber using a x10 objective lens (Olympus, USA) and fiber launch (Thorlabs, USA). The laser intensity at the interface between the fiber tip and the animal ranged from 0.01–0.03 mW to minimize bleaching. The GCaMP fluorescence was bandpass filtered (Thorlabs, USA) and collected by a photomultiplier tube (Hamamatsu, Japan). An amplifier (Hamamatsu, Japan) was used to convert the photomultiplier tube current output to voltage signals, which was further filtered through a low-pass filter (100 Hz cut-off). Photometry data were exported to MATLAB Mat files for further analysis. We segmented data based on individual trial of seizures and derived the values of fluorescence change (ΔF/F) by calculating (F − F0)/F0, which were presented with heatmaps or average plots. Only rats with GCaMP expression within the target region (DG) were taken into analysis (Supplementary Fig. S6).
Photostimulation was applied according to the previous report.15 Blue (473 nm) or yellow (589 nm) laser light was delivered through a 200 μm diameter optic fiber connected to the laser (BL473T3-050 or YL589T3-050, Shanghai Laser & Optics Century, China). The optic fiber was flat cut, and the laser power was adjusted to about 5 mW. The blue light (473 nm, 20 Hz, 10 ms/pulse) or DC yellow-light (589 nm) stimulation was delivered immediately after PTZ injection.
Histology and quantification
The immunohistochemistry was performed strictly according to our previous studies.48 Briefly, rats that had undergone behavioral analysis were deeply anesthetized with pentobarbital (50 mg/kg, i.p.). We removed the brains and obtained coronal 20-μm sections with a sliding freezing microtome (Leica, Japan). Free-floating samples were used for Fig. 3f. Slide-mounted sections were used for the rest of figures. We processed sections for immunofluorescence for PV (1:400, Swant PV27), SOM (1:400, Santa cruz sc-13099), NeuN (1:400, Millipore MABN140), CaMKII (1:300, Invitrogen 13-7300) by incubating the sections with primary antibodies, and then incubated with a Alexafluor 488 conjugated secondary fluorescent antibody (1:400, Molecular Probes, USA) at room temperature. Fluoroshield Mounting Medium with DAPI (Sigma-Aldrich, USA) was used as a nuclear stain. We assessed the immunofluorescence with Leica SP8 laser confocal microscope.
Image analyses and quantification were performed using ImageJ (version 1.52a) software. For assessing the cell numbers, two coronal sections showing dorsal (Bregma −2.4 mm to −2.8 mm) and ventral hippocampus (Bregma −3.2 mm to −4.0 mm) were chosen. The data were averaged per rat. The number of rats used in each experiment was indicated in figures.
Immunofluorescence staining for neuronal apoptosis
To better assess neuronal apoptosis, immunofluorescent double staining of TUNEL and SOM was conducted to determine the colocalization of apoptotic cells and SOM + neurons. In brief, frozen sections were immunostained with SOM (1:400, Santa cruz sc-13099) at 4 °C overnight and subsequently subjected to TUNEL staining using an In Situ Cell Death Detection kit (Roche, South San Francisco, CA, USA) according to the manufacturer’s suggested protocol. Finally, the sections were covered with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Positive cells were calculated per square millimeter from four random microscopic fields of each section (two sections per animal) under a fluorescence microscope (Olympus). The results were presented as the apoptotic ratio of the total neurons (SOM-TUNEL double stained cells/SOM-stained cells).
Fluorescence in situ hybridization (FISH) by RNAscope
Rats were perfused with saline and 4% paraformaldehyde in PBS (pH 7.4). The harvested brains were fixed in 4% paraformaldehyde for another day before consecutive dehydration in 10, 20, and 30% sucrose. Fresh frozen rat brain slices with 14-μm thickness were subjected for FISH. RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, USA) was used for duplex hybridization by combing the CaMKII probe (ACDBio, 445231) with a VGAT probe (ACDBio, 319191). For further double labeling that combines FISH and immunofluorescence, the slices were then incubated with antibodies against SOM (1:400, Santa cruz sc-13099). We obtained images using Leica SP8 laser confocal microscope.
In vitro electrophysiology
In vitro electrophysiology was strictly performed as previously reported.24,49 To obtain acute hippocampal slices, rats were anesthetized and decapitated, the brains were then quickly removed and submerged in oxygen-saturated artificial cerebrospinal fluid (ACSF) containing in mM: 120 NaCl, 11 Dextrose, 2.5 KCl, 1.28 MgSO4, 3.3 CaCl2, 1 NaH2PO4, and 14.3 NaHCO3. Coronal slices (300 μm) were cut using a vibratome (Leica, Germany) and incubated at 25°C for 1 h. The slices were transferred into a recording chamber at 25 °C for patch clamp recording. The glass patch pipettes (4–8 MΩ resistance) were pulled by a two-stage puller (PC-10, Narishige). For action potential recordings, the intracellular solution contained (in mM): 35K-gluconate, 110KCl, 104-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2MgCl2, and 2Na2ATP, 10ethylene glycol tetra-acetic acid (pH 7.4). For AP properties, step depolarized currents were injected through the pipettes in 5pA increments from 0pA to 100pA. We analyzed the first spike induced by the minimum depolarizing current (Rheobase) for threshold, amplitude, and the half-width. AP amplitude was defined as the voltage from the AP threshold to the AP peak, and the AP half-width was calculated as the duration at half-maximal amplitude.
For gramicidin-perforated patch-clamp recording, pipettes were back-filled with the same intracellular solution containing gramicidin (50 μg/ml) prepared freshly prior to recording. GABAA receptor-mediated postsynaptic currents were isolated with AMPA receptor antagonist CNQX (20 μM) and NMDA receptor antagonist D-AP5 (50 μM). Whole-cell gramicidin perforated patch-clamp recordings were performed from visually and electrophysiologically identified granule cells in the DG. GABAA receptor-mediated IPSCs were evoked through a concentric bipolar electrode placed 50–100 μm lateral to the recorded neuron (stimulation rate 0.1 Hz at 100 μA, and 100 μs duration). To identify the EGABA, the holding potential was systematically varied from −80 to 30 mV in 10-mV steps at the same time as the stimulation. The membrane potential was verified after breaking the perforated patch following the recording.
To record evoked synaptic currents, a low divalent ion ACSF (in mM: 125 NaCl, 3.5 KCl, 1.25 NaH2PO4, 0.5 MgCl2, 26 NaHCO3, 25 Dextrose, and 1 CaCl2) and cesium-based internal fluid (in mM: 100 CsCH3SO3, 20 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Tris-GTP, 7 Tris2-Phosphocreatine, and 3 QX-314) were used. To examine whether the granule cells in the DG received excitatory synaptic input from SOM + interneurons, we injected a viral cocktail of (1:1, 1 μl) of AAV-SOM-Cre and AAV-EF1a-DIO-hChR2(H134R)-EYFP into the DG. Three weeks after viral injection, we performed whole-cell patch clamp recording. EPSCs were recorded at a holding potential of −60 mV and IPSCs were recorded at +10 mV.24,49 eEPSCs and eIPSCs were recorded from visually identified granule cells upon blue light stimulation (473 nm, 10 ms pulse width). Only granule cells with eIPSCs were considered having a direct connection with the stimulated SOM + interneurons, and were thereby included in the final analysis. To confirm whether the light-evoked currents were monosynaptic, TTX (1 μM) and 4-AP (100 μM) were applied. AMPA receptor antagonist CNQX (20 μM) and NMDA receptor antagonist D-AP5 (50 μM) were added to block excitatory synaptic transmission. All patch-clamp recordings were performed using an EPC10 patch-clamp amplifier (HEKA Instruments) with a low-pass filter at 3 kHz and a sample rate of 10 kHz.
Statistical comparisons were performed using Graphpad Prism (version 9.0) with appropriate methods as indicated in the figure legends. Number of experimental replicates (n) is indicated in figures and refers to the number of experimental subjects used and independently treated in each experimental condition. For all experiments, at least two times experiment was repeated independently with similar results. Data were presented as number and percentages for categorical variables, and continuous data were expressed as mean ± s.e.m, unless otherwise specified. A two-tailed P value < 0.05 was considered statistically significant.