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Learn more: PMC Disclaimer | PMC Copyright Notice Epilepsia . 2025 Sep 20;67(4):2007–2021. doi: 10.1111/epi.18626 Search in PMC Search in PubMed View in NLM Catalog Add to search Synaptic ultrastructural alterations in human focal cortical dysplasia: Insights from volume electron microscopy Gyu Hyun Kim Gyu Hyun Kim 1 Neural Circuits Research Group, Korea Brain Research Institute, Daegu, South Korea 2 Department of Neuroscience, Korea University College of Medicine, Seoul, South Korea Find articles by Gyu Hyun Kim 1, 2 , Na‐Young Seo Na‐Young Seo 1 Neural Circuits Research Group, Korea Brain Research Institute, Daegu, South Korea Find articles by Na‐Young Seo 1 , Seung‐Ki Kim Seung‐Ki Kim 3 Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, Seoul, South Korea Find articles by Seung‐Ki Kim 3 , Jae‐Kyung Won Jae‐Kyung Won 4 Department of Pathology, Seoul National University Hospital and College of Medicine, Seoul, South Korea Find articles by Jae‐Kyung Won 4 , Yang Hoon Huh Yang Hoon Huh 5 Center for Electron Microscopy Research, Korea Basic Science Institute, Cheongju, South Korea Find articles by Yang Hoon Huh 5, ✉ , Ji Yeoun Lee Ji Yeoun Lee 3 Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, Seoul, South Korea 6 Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea Find articles by Ji Yeoun Lee 3, 6, ✉ , Kea Joo Lee Kea Joo Lee 1 Neural Circuits Research Group, Korea Brain Research Institute, Daegu, South Korea Find articles by Kea Joo Lee 1, ✉ Author information Article notes Copyright and License information 1 Neural Circuits Research Group, Korea Brain Research Institute, Daegu, South Korea 2 Department of Neuroscience, Korea University College of Medicine, Seoul, South Korea 3 Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, Seoul, South Korea 4 Department of Pathology, Seoul National University Hospital and College of Medicine, Seoul, South Korea 5 Center for Electron Microscopy Research, Korea Basic Science Institute, Cheongju, South Korea 6 Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea * Correspondence , Kea Joo Lee, Neural Circuits Research Group, Korea Brain Research Institute, Daegu, South Korea. Email: [email protected] , Yang Hoon Huh, Center for Electron Microscopy Research, Korea Basic Science Institute, Cheongju, South Korea. Email: [email protected] , Ji Yeoun Lee, Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, Seoul, South Korea. Email: [email protected] ✉ Corresponding author. Revised 2025 Aug 18; Received 2025 Mar 10; Accepted 2025 Aug 19; Issue date 2026 Apr. © 2025 The Author(s). Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. PMC Copyright notice PMCID: PMC13075618 PMID: 40974555 Abstract Objective Focal cortical dysplasia (FCD) is a developmental malformation of the cerebral cortex and a leading cause of drug‐resistant epilepsy in children and young adults. Disruption of the excitation–inhibition (E–I) balance is a hallmark of neuronal hyperexcitability in FCD, yet the underlying synaptic ultrastructural changes remain poorly understood. This study aimed to investigate synaptic architecture and associated organelle alterations in epileptogenic cortex affected by FCD. Methods Using volume electron microscopy, we performed a detailed morphological assessment of synaptic density, size, and organelle distribution within synapses in the temporal cortical layer III of a patient with FCD. Comparative analyses were conducted between dysplastic and nondysplastic cortical regions. Results The dysplastic cortex exhibited a lower density of excitatory synapses but contained unusually large excitatory synapses with an increased number of synaptic vesicles. Inhibitory synapses were positioned farther from the nearest excitatory synapses along distal dendrites, potentially reducing the effectiveness of shunting inhibition in the dysplastic area. Presynaptic boutons in the dysplastic region showed increased mitochondrial density and abnormal mitochondrial morphology, whereas the proportion of postsynaptic protrusions containing a spine apparatus was reduced. These changes suggest potential deficits in intracellular calcium handling, metabolic homeostasis, and synaptic plasticity in the epileptogenic cortex. Additionally, maladaptive myelination was a prominent feature in the dysplastic region. Significance This study identifies distinct synaptic and subcellular structural abnormalities in FCD that may contribute to E–I imbalance and neuronal hyperexcitability. These findings provide novel ultrastructural insights into the pathophysiology of FCD and may inform future therapeutic strategies targeting synaptic and metabolic dysfunction. Keywords: cortex, electron microscopy, epilepsy, hyperexcitability, mitochondria, synapse Key points. Volume electron microscopy revealed ultrastructural synaptic and organelle changes in the cortex of a patient with FCD. The dysplastic region showed fewer excitatory synapses on distal dendrites, with some abnormally enlarged and vesicle‐rich. Inhibitory synapses were positioned farther from excitatory synapses, potentially reducing inhibitory control and increasing excitability in the epileptic region. Presynaptic boutons displayed altered mitochondrial density and morphology, and fewer postsynaptic spines contained a spine apparatus, suggesting disrupted calcium regulation and synaptic plasticity. 1. INTRODUCTION Focal cortical dysplasia (FCD) is a neurodevelopmental disorder of the neocortex characterized by disruptions in neuronal migration and differentiation. 1 It is recognized as the leading cause of drug‐resistant epilepsy in both pediatric and adult populations. 2 , 3 Due to the poor response of these seizures to antiepileptic medications, surgical removal of the affected cortical region remains the most effective treatment. The International League Against Epilepsy (ILAE) classifies FCD into three primary subtypes based on a updated multilayered diagnostic scheme integrating imaging data and genetic information with histopathologic features 1 : type I, involving abnormal vertical and/or horizontal cortical layering without significant cytological abnormalities; type II, marked by cortical disorganization along with dysmorphic neurons and/or balloon cells; and type III, which occurs alongside other neuropathological conditions such as hippocampal sclerosis, tumors, or vascular malformations. Epilepsy is characterized by excessive synchronized neuronal discharges leading to hyperexcitability. 4 A key mechanism implicated in epileptogenesis is the disruption of the balance between excitatory and inhibitory synaptic activity. 5 Although previous electrophysiological and histological studies have provided valuable insights into epilepsy mechanisms in FCD, 6 , 7 , 8 , 9 , 10 , 11 the exact cellular alterations responsible for hyperexcitability remain unclear, partly due to limited data on the intricate synaptic architecture of the human cortex. Immunocytochemical studies using light microscopy have reported a reduced number of basket and chandelier cells—key inhibitory interneurons that target the soma and axon initial segments of pyramidal neurons—in both animal models and human epileptic cortex. 4 , 11 , 12 , 13 , 14 , 15 Consistent with these findings, electrophysiological analysis has demonstrated a reduction in inhibitory postsynaptic currents in pyramidal neurons within dysplastic cortical regions, 16 suggesting an overall shift toward increased excitation. However, there exist some conflicting reports regarding glutamate receptor expression; some analyses indicate upregulation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid and N‐methyl‐D‐aspartate receptor subunits (GluA2/3, GluN1, GluN2A/B) in the dysplastic cortex, 11 , 17 , 18 whereas others describe a reduction in GluA2/3 immunoreactivity in epileptic neocortex. 19 Similarly, electron microscopic (EM) studies have yielded contradictory results, with one reporting an increase in excitatory (asymmetric) synapses in the epileptogenic cortex, 20 whereas others have found a decrease in excitatory synapses or dendritic spines—the primary postsynaptic sites of glutamatergic synapses. 21 , 22 These inconsistencies suggest that the mechanisms underlying hyperexcitability in FCD involve complex, heterogeneous changes in excitatory synaptic organization. Given these uncertainties, a precise three‐dimensional (3D) ultrastructural analysis of excitatory synapses is essential for elucidating the pathogenesis of epilepsy linked to FCD. In this study, we employed volume electron microscopy to examine local synaptic alterations in human temporal cortex tissue resected from a patient with FCD type Ia. This subtype, characterized by cortical dyslamination without significant cytological abnormalities, may serve as an ideal model for investigating intrinsic synaptic mechanisms of hyperexcitability, independent of additional confounding pathologies. We focused on cortical layer III, where pyramidal neurons play a key role in corticocortical propagation of neuronal activity 23 and have been previously shown to undergo seizure‐associated neuronal remodeling. 4 , 24 , 25 We hypothesize that synaptic density and morphology in the dysplastic cortex would be altered in a manner favoring excessive excitation. To test this hypothesis, we conducted within‐patient comparisons between the epileptic focus and an adjacent histologically normal region. Using serial block‐face scanning electron microscopy (SB‐SEM) and high‐voltage electron microscopy (HVEM), we analyzed synapse types, density, morphology, and intrasynaptic organelle distribution in the dysplastic region, aiming to identify structural changes that may contribute to neuronal hyperexcitability in FCD. 2. MATERIALS AND METHODS 2.1. Tissue acquisition and preparation for microscopy Surgery was performed after obtaining informed consent, in accordance with the Declaration of Helsinki. All procedures and the use of human tissue were approved in advance by the institutional review board (Ethics Committee, Seoul National University Medical Center, IRB No. H‐0507‐509‐153). Brain tissue samples were collected from a 15‐year‐old male patient diagnosed with FCD and experiencing intractable epilepsy. Seizure onset was at 12 years of age, approximately 3 years prior to surgical intervention. Seizure localization was determined based on concordant findings from preoperative magnetic resonance imaging (MRI), scalp video‐electroencephalography (EEG), and invasive intracranial monitoring. Preoperative MRI showed blurring of gray–white matter discrimination and reduced subcortical white matter signal in the anteromedial aspect of the inferior temporal lobe, without hippocampal asymmetry (Figure S1 ). The seizure focus was mapped through video‐EEG monitoring, and left temporofrontal lobe epilepsy was suspected. Further evaluation using invasive intracranial monitoring—via subdural grid and a depth electrode targeting the hippocampus—was conducted over 96 h. Ictal onset was suspected to be from the uncus, parahippocampal gyrus, and posterolateral temporal cortex. No epileptic discharges were detected from the depth electrode in the hippocampus. The patient underwent a standard left anterior temporal lobectomy, including resection of the uncus and temporal roof, while sparing the hippocampus. In addition, a posteriorly extended lateral cortisectomy of the temporal lobe was performed. Postoperatively, the patient remained seizure‐free for >2 years, and as of 7 years after surgery, experienced only rare, nondisabling seizures. The long‐term seizure outcome was hence classified as Engel class IC. Tissue from the uncus was used as the epileptogenic region, and the adjacent control cortex was taken from the anterior temporal lobe within the same resected specimen. This control region was located >2 cm away from the uncus in the gross surgical specimen, was histologically verified as nondysplastic, and showed no evidence of ictal discharges during intracranial monitoring. Histological evaluation was performed on biopsy specimens using hematoxylin and eosin (H&E) staining and immunohistochemistry with an anti‐NeuN antibody, enabling neuropathological classification of FCD type. Cell density in cortical layer III was estimated from H&E‐stained sections using a systematic random sampling with an unbiased counting frame. Cell bodies within the frame or intersecting the inclusion lines (but not exclusion lines) were counted. Random fields within layer III, corresponding to the regions used for electron microscopy, were analyzed using Fiji/ImageJ (National Institutes of Health). For volume electron microscopy, tissue samples were immediately immersion‐fixed to preserve ultrastructural integrity. Samples were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in .15 mol·L −1 cacodylate buffer (pH 7.4) and sectioned into 150‐μm slices with a vibratome (Leica VT 1000S). The slices were further dissected into small blocks containing cortical layer III. These blocks were washed in .15 mol·L −1 cacodylate buffer, postfixed in 2% osmium tetroxide/1.5% potassium ferrocyanide for 1 h, incubated in 1% thiocarbohydrazide (Sigma‐Aldrich, Cat #223220) for 20 min, and treated with 2% osmium tetroxide for 30 min. Samples were subsequently incubated in 1% uranyl acetate at 4°C overnight and in lead aspartate at 60°C for 30 min. Following serial ethanol dehydration, the specimens were infiltrated with acetone and embedded in 7% (wt/vol) conductive Epon 812 resin (EMS, Cat #14120) mixed with Ketjen black powder to enhance conductivity, as described previously. 26 Specimens were mounted on metal stubs and polymerized at 60°C for 48 h. To assess cortical layering, semithin 100‐nm sections were stained with toluidine blue and examined under light microscopy. 2.2. SB‐SEM imaging and analysis SB‐SEM imaging was conducted using a Merlin 3View SEM system (Carl Zeiss Microscopy). Serial images were acquired with a 30‐μm aperture, high vacuum, a voltage of 1.5 kV, an image size of 5000 × 5000 pixels, a dwell time of 3.5 μs, and an x–y resolution of 9 nm at a nominal section thickness of 50 nm. Stacks of 200 serial images of cortical layer III were obtained from the control and epileptogenic region, covering a volume of 20 250 μm 3 per each stack. In the dysplastic region, where cortical layering was less distinct, images were obtained from 550 to 900 μm beneath the pial surface, corresponding to cortical layer III. 21 Image stacks were processed using ImageJ and Fiji plugins ( http://fiji.sc/wiki/index.php/Fiji ), with TrakEM2 used for image alignment. Spiny dendritic segments, averaging 15.94 ± 3.56 μm in length, were randomly selected. A total of 20 dendritic branches (10 per condition) were manually reconstructed using open‐source reconstruction software ( https://synapseweb.clm.utexas.edu/ ) by annotators blinded to experimental conditions. Quantification of spine density, presence or absence of presynaptic boutons, and surface area of postsynaptic density (PSD) was performed. Synapses were classified as asymmetric or symmetric based on PSD thickness. 27 Additional analyses included spine volume, length, spine head and neck diameters, and classification of extra‐large synapses (top 20th percentile in PSD area and spine volume), based on prior studies of activity‐dependent synaptic structural enlargement. 28 , 29 To measure the distance between inhibitory and excitatory synapses, their locations were mapped onto the skeletonized dendrite. Dendritic shaft thickness was measured using maximal and minimal diameters, with a regularity index calculated by dividing the minimal by the maximal diameter. Presynaptic boutons were categorized by mitochondrial presence, and the number of mitochondria was counted per bouton. The presence of a spine apparatus (SA) was determined when at least three smooth endoplasmic reticulum stacks were visible. 2.3. Automated tape‐collecting ultramicrotome combined with SEM imaging and analysis For automated tape‐collecting ultramicrotome combined with SEM (ATUM‐SEM) imaging, tissue blocks were sectioned into 50‐nm ultrathin sections using an ultra Maxi knife (DiATOME). Serial sections were collected onto plasma‐hydrophilized carbon nanotube‐coated PET tapes (Boeckeler Instruments) with an ATUMtome (Boeckeler Instruments). The tapes were mounted on a silicon wafer, secured with conductive adhesive tape (Ted Pella), and carbon‐coated to prevent charging during SEM imaging. Imaging was performed using a Gemini 300 SEM (Carl Zeiss Microscopy) equipped with an in‐lens secondary electron detector or a backscattered electron detector (BSD). Large‐area imaging was conducted using Atlas 5 software (Fibics Incorporated) at 5‐kV beam voltage with BSD detection and a 7‐μs dwell time. High‐resolution images (5 nm per pixel) were obtained as 2 × 2 tiled images, stitched together to generate a composite 90 × 90 μm 2 image. Image stacks were aligned using the Fiji TrakEM2 plugin. Myelin sheath thickness was assessed by measuring the G‐ratio, calculated as the ratio of axonal radius to total nerve fiber radius including both axon and myelin sheath. 2.4. Electron tomography To visualize synaptic ultrastructure in cortical layer III, 250‐nm‐thick sections were collected on 200‐mesh grids (G200N, Gilder Grids) for electron tomography using a Bio‐HVEM (JEM‐1000BEF, JEOL) operating at 1000 kV (Korea Basic Science Institute). Excitatory synapses were randomly selected for imaging, and samples were tilted from +60° to −60° in 2° increments, producing a total of 61 tilt images per synapse captured with TEM Recorder software (JEOL System Technology). The tilt series was aligned and reconstructed into 3D tomograms using Composer and Visualizer‐Kai software ( TEMography.com , Frontiers). Virtual slices were extracted, and synaptic vesicles (SVs) were traced using reconstruction software ( https://synapseweb.clm.utexas.edu/software‐0 ). To prevent overcounting, only vesicles with clearly defined circular profiles were quantified in serial tilt images. Vesicle distribution and docked vesicle counts (within 50 nm of the active zone) were analyzed as correlates of synaptic strength. 2.5. Comparison of synapse distance to a random distribution To assess whether the spatial relationship between excitatory and inhibitory synapses deviated from a random distribution, we conducted computational simulations in Python comparing the control and epileptic groups. For each simulation iteration, a dendritic segment length was sampled either from the mean length of empirically reconstructed segments (15.94 μm) or from an artificially extended length (100 μm) to minimize edge effects. The number of excitatory and inhibitory synapses was determined by multiplying the corresponding measured densities by the sampled dendritic length for each group. Synapse positions were then randomly assigned along the dendrite based on a uniform distribution. For each inhibitory synapse, we calculated the shortest distance to the nearest excitatory synapse, generating a distribution of nearest‐neighbor distances per simulation. This procedure was repeated 1000 times per group, producing a dataset of simulated synaptic spatial distributions. We then compared the empirical cumulative distribution functions derived from experimental data with those from the simulated datasets using Kolmogorov–Smirnov tests to evaluate deviations from randomness. 2.6. Statistics All data are presented as mean ± standard error of the mean. Statistical analyses were performed using GraphPad Prism software (Research Resource Identifier: SCR_002798). Synaptic density measurements from SB‐SEM data were treated as single observations per dendrite. Normality was assessed, and comparisons between groups were conducted using either an unpaired Student t ‐test (for normally distributed data) or a Mann–Whitney U ‐test (for nonnormally distributed data). Statistical significance was set at p < .05. 3. RESULTS 3.1. Histological analysis Tissue samples were obtained from a patient who underwent epilepsy surgery (Figure S1A–D ). Compared to the control cortex, disorganized cortical lamination was observed in the epileptogenic region using H&E staining. Although cell density within cortical layer III (550–900 μm from the pial surface) was reduced in the epileptogenic area, dysmorphic neurons or balloon cells were not identified (Figure S1E ). Immunohistochemistry with an anti‐NeuN antibody also highlighted a characteristic radial microcolumnar arrangement (Figure S1F–I ), a predominant feature of FCD. 30 Based on these histopathological observations, the patient was diagnosed with FCD type Ia. 3.2. Ultrastructural synaptic analysis of cortical layer III pyramidal neurons Given the well‐established excitation–inhibition (E–I) imbalance in FCD, we investigated whether synaptic alterations favoring hyperexcitation might involve changes in excitatory and/or inhibitory synapses on cortical pyramidal neurons. Previous studies have reported a selective loss of parvalbumin‐expressing interneurons, which are key mediators of perisomatic inhibition, 31 along with a reduction in inhibitory postsynaptic currents in pyramidal neurons within FCD‐affected cortex. 4 , 11 , 16 This reduction in somatic inhibition likely contributes to increased cortical excitability. To further examine excitatory synaptic changes within FCD cortex, we first analyzed dendritic spines, which serve as the primary sites of excitatory synapses in pyramidal neurons. Using SB‐SEM, 3D reconstructions of dendritic segments were generated from both control and epileptogenic regions (Figure 1A,B ; Movie S1 ). Analysis of 20 reconstructed dendritic segments (mean segment length = 15.94 ± 3.56 μm) demonstrated a significant reduction in spine density in the epileptogenic region, affecting both spines with and without presynaptic partners (Figure 1C,D; total spines/10 μm: control, 11.8 ± 1.09; epileptogenic, 4.3 ± .31; p < .0001; synaptic spines/10 μm: control, 8.4 ± .95; epileptogenic, 2.9 ± .26; p < .0001; nonsynaptic spines/10 μm: control, 3.3 ± .65; epileptogenic, 1.3 ± .40; p = .016). Interestingly, despite this overall spine loss, some remaining spines in the epileptogenic region exhibited notably larger volumes, whereas spine length remained unchanged (Figure 1E,I , Figure S2A,B ; spine volume [μm 3 ]: control, .15 ± .01; epileptogenic, .35 ± .05; p = .048; spine length [μm]: control, 1.82 ± .06; epileptogenic, 1.70 ± .10; p = .26). Moreover, the PSD areas of the largest 20% of spines were significantly enlarged in the epileptogenic region (Figure S2C,D ; PSD area [μm 2 ]: control, .91 ± .06; epileptogenic, 1.40 ± .24; p = .013). FIGURE 1. Open in a new tab Reduced spine density and abnormal spine enlargement in the epileptic cortex. (A, B) Representative serial block‐face scanning electron microscopy (SB‐SEM) images and three‐dimensional (3D) reconstruction of dendritic segments with postsynaptic densities (PSDs) and presynaptic boutons in cortical layer III of the control and epileptogenic cortex. Note that all thin and thicker dendritic protrusions were classified as either synaptic or nonsynaptic spines based on the presence or absence of a presynaptic partner. Red, dendrite; yellow, PSD; light blue, presynaptic bouton. Scale bars = 1 μm in panel A and 2 μm in panel B. (C) 3D reconstruction images of distal dendritic segments, showing dendritic spines in the control (temporal cortex) and epileptogenic (uncus) regions. Note the presence of a subset of extra‐large spines in the epileptic area. Scale bar = 1 μm. (D) Quantification of spine density along the dendrite in the control and epileptogenic regions ( n = 10 dendrites). * p < .05; *** p < .001, **** p < .0001; unpaired Student t ‐test. (E) Morphological analysis of individual spine properties and representative SB‐SEM images of the corresponding spines. Yellow, PSD; blue line, spine length; red line, spine head diameter; green line, spine neck diameter. Scale bar = 1 μm. (F – I) Quantification of spine head diameter (F), spine neck diameter (G), the ratio of spine head diameter to neck diameter (H), and spine length (I; control, n = 156; epilepsy, n = 79). ** p < .01, **** p < .0001; NS, not significant; Mann–Whitney U ‐test. Data are represented as mean ± standard error of the mean. Further morphological analysis revealed increased diameters of both spine heads and necks in the epileptogenic region (Figure 1F,G ; spine head [μm]: control, .55 ± .02; epileptogenic, .72 ± .05; p = .007; spine neck [μm]: control, .15 ± .01; epileptogenic, .30 ± .02; p < .0001). The spine head‐to‐neck diameter ratio was significantly reduced in the dysplastic area (Figure 1H ; control, 3.92 ± .16; epileptogenic, 2.61 ± .21; p < .0001), suggesting that spine necks expanded disproportionately relative to their heads. This structural alteration may reduce the electrochemical compartmentalization of synaptic signals, facilitating the spread of excitatory activity and contributing to hyperexcitability. Additionally, we observed varicose‐type swelling in both epileptogenic and control dendrites, a feature commonly linked to excitotoxic damage or seizure‐related remodeling. 32 , 33 However, quantitative assessment of dendritic regularity showed no significant differences between groups (Figure S2E ) (control, .19 ± .02; epileptogenic, .21 ± .03; p = .60). To confirm the observed reduction in spine density and assess inhibitory synapses on distal dendrites, serial EM images were analyzed, quantifying excitatory (asymmetric) and inhibitory (symmetric) synapses on dendritic shafts and spines (Figure 2A ). Again, asymmetric synapse density was significantly lower in the epileptogenic region, whereas symmetric synapse density remained unchanged (Figure 2B,C ; Figure S3A ; asymmetric synapses/10 μm: control, 9.7 ± 1.04; epileptogenic, 4.4 ± .30; p = .0001; symmetric synapses/10 μm: control, .7 ± .26; epileptogenic, .7 ± .20; p = .86). FIGURE 2. Open in a new tab Spatial distribution of excitatory and inhibitory synapses along distal dendrites. (A) Representative electron microscopic images of asymmetric (excitatory) and symmetric (inhibitory) synapses formed on dendritic spines or directly onto the shaft. Yellow and red arrowheads, postsynaptic density (PSD). Scale bar = .5 μm. (B, C) Quantification of asymmetric (B) and symmetric (C) synaptic density ( n = 10 dendrites). *** p < .001; NS, not significant; unpaired Student t ‐test. (D) Schematic illustration of measuring the distance between excitatory and inhibitory synapses. Yellow circles, inhibitory synapse; red circles, excitatory synapse. (E) Representative image of skeletonized dendritic traces with annotated synapses. The distance from an inhibitory synapse (white arrows) to the nearest excitatory synapse was measured. Scale bar = 1 μm. (F) Cumulative distribution of distance measurements between inhibitory and excitatory synapses (control, n = 14; epilepsy, n = 23). NS, not significant; Kolmogorov–Smirnov test. (G) Average distance between inhibitory and excitatory synapses in the top 50% of the population in panel F (control, n = 8; epilepsy, n = 12). * p < .05; unpaired Student t ‐test. Data are represented as mean ± standard error of the mean. 3.3. Spatial distribution of excitatory and inhibitory synapses The precise spatial arrangement of excitatory and inhibitory synapses is crucial for shunting inhibition, which limits excessive excitatory input. 34 , 35 We hypothesized that increased separation between inhibitory and excitatory synapses in distal dendrites might impair local inhibition, thereby contributing to seizure activity. To test this, inhibitory synapse locations were mapped on skeletonized dendrite traces, and the distance to the nearest excitatory synapse was measured (Figure 2D,E ). In the epileptogenic region, cumulative distribution analysis revealed a greater proportion of excitatory synapses positioned ≥.6 μm from inhibitory synapses (Figure 2F,G ; cumulative distribution of distance between inhibitory and excitatory synapses [μm]: control, .86 ± .15; epileptogenic, 1.24 ± .24; p = .34; distance of top 50% [μm]: control, 1.23 ± .15; epileptogenic, 2.07 ± .29; p = .027). This suggests that inhibitory inputs in the epileptogenic region may be less effective in suppressing local excitatory activity, increasing the potential for hyperexcitability. To further assess whether the observed spatial relationship between excitatory and inhibitory synapses deviates from a random distribution, we performed computational simulations to produce the shortest distance distributions for each inhibitory synapse to the nearest excitatory synapse (Figure S3B,C ). In the control region, the actual distances between inhibitory and nearby excitatory synapses differed significantly from the random model ( p = .047), suggesting a nonrandom and precisely organized synaptic arrangement. 29 In contrast, this structured organization disappeared in the epileptogenic region ( p = .66), likely reflecting a disruption of synaptic spatial architecture due to the marked loss of excitatory synapses. 3.4. Intrasynaptic organelle analysis (vesicles, mitochondria, and spine apparatus) To explore whether SV organization was altered in enlarged excitatory synapses within the epileptogenic region, we quantified presynaptic bouton volume, total SV pool, and the number of docked vesicles using SB‐SEM datasets (Figure 3A ). Docked vesicles were defined as those found within 50 nm of the active zone membrane. Notably, the volume of presynaptic boutons, the number of docked SVs normalized to PSD surface area, and the total SV count normalized to bouton volume were all significantly greater in epileptogenic boutons compared to controls (Figure 3B–D ; bouton volume: control, .62 ± .06; epileptogenic, 1.32 ± .23; p < .0001; docked SVs/PSD area: control, 24.21 ± 1.74; epileptogenic, 34.40 ± 3.39; p = .004; total SVs/bouton volume: control, 686 ± 40.6; epileptogenic, 971 ± 90.9; p = .035). These findings suggest an expanded vesicle reserve in epileptogenic boutons, which may enhance neurotransmitter release and contribute to persistent hyperexcitability in the epileptogenic cortex. FIGURE 3. Open in a new tab Enlarged excitatory synapses with increased number of vesicles in the epileptogenic area. (A) Representative serial block‐face scanning electron microscopy images and three‐dimensional reconstruction of presynaptic boutons showing presynaptic vesicles including docked vesicles and mitochondria in the control and epileptogenic cortex. Scale bars = 1 μm. (B) Quantification of presynaptic bouton volume (control, n = 56 boutons; epileptic, n = 22 boutons). *** p < .001; unpaired Student's t ‐test. (C) Quantification of docked vesicle density normalized to postsynaptic density (PSD) surface area (control, n = 58; epileptic, n = 24). ** p < .01; unpaired Student t ‐test. (D) Quantification of total vesicle density normalized to presynaptic volume (control, n = 57; epileptic, n = 23). ** p < .01; unpaired Student t ‐test. Data are represented as mean ± standard error of the mean. To complement these findings, we conducted electron tomography using HVEM. Each synapse was imaged across 61 tilt angles (±60° in 2° increments; Figure S4A–C ). A total of 26 presynaptic boutons were partially reconstructed (12 control, 14 epileptogenic), enabling segmentation of key synaptic components, including mitochondria and SVs (Movie S2 ). In agreement with the SB‐SEM findings, the total number of SVs per bouton was significantly higher in the epileptogenic group (control: 173.8 ± 23.2; epileptogenic: 361.5 ± 67.5; p = .042; Figure S4D ). In contrast, the number of docked SVs per bouton showed a nonsignificant trend toward increase in a subset of boutons, which may reflect the limited statistical power due to the relatively small sample size (Figure S4E ). Presynaptic mitochondria are essential for supporting synaptic transmission, regulating calcium dynamics, and sustaining energy homeostasis. 36 , 37 We next examined whether presynaptic mitochondrial distribution was altered in the epileptogenic region (Figure 4A ). Although the proportion of boutons lacking or containing mitochondria did not differ between groups (Figure 4B ; boutons without mitochondria [%]: control, 31.50 ± 5.96; epileptogenic, 35.67 ± 4.33; boutons with mitochondria [%]: control, 68.50 ± 5.96; epileptogenic, 64.33 ± 4.33; p = .58), the individual number of mitochondria per bouton was significantly higher in the epileptogenic region compared to control (Figure 4C; mitochondria/bouton: control, 1.2 ± .06; epileptogenic, 1.7 ± .15; p = .03). We further assessed mitochondrial morphology to determine whether they are more spherical or elongated in the epileptogenic region (Figure S5A ). Intriguingly, both the individual volume and length of mitochondria were significantly greater in the epileptogenic cortex compared to the control (Figure 4D,E; volume [μm 3 ]: control, .08 ± .01; epileptogenic, .15 ± .01; p < .0001; length [μm]: control, .75 ± .05; epileptogenic, 1.49 ± .17; p < .0001). These findings may indicate compensatory mitochondrial remodeling in response to heightened synaptic activity and metabolic demand in the epileptogenic cortex. FIGURE 4. Open in a new tab Altered presynaptic mitochondria and spine apparatus (SA) in the epileptogenic cortex. (A) Representative electron microscopic images of synapses containing presynaptic mitochondria (left panels); three‐dimensional reconstructions of presynaptic mitochondria and synaptic components (middle and right panels, respectively). Light blue, presynaptic bouton; orange, mitochondria; yellow, PSD; red, spine. Scale bars = 1 μm. (B) Quantification of fraction of synapses containing or lacking presynaptic mitochondria in the control and epileptogenic cortical regions ( n = 10 dendrites); no significant difference by unpaired Student t ‐test. (C) Quantification of individual mitochondrial numbers per bouton (control, n = 64; epilepsy, n = 35). **** p < .0001; unpaired Student t ‐test. (D) Average length of individual presynaptic mitochondria (control, n = 82; epilepsy, n = 62). **** p < .0001; Mann–Whitney U ‐test. (E) Average volume of individual presynaptic mitochondria (control, n = 81; epilepsy, n = 63). **** p < .0001; unpaired Student t ‐test. (F) Representative electron microscopic images of postsynaptic protrusions with and without SA. Scale bar = 1 μm. (G) Percentage of postsynaptic protrusions containing or lacking SA ( n = 10 dendrites). **** p < .0001; unpaired Student t ‐test. Data are represented as mean ± standard error of the mean. The SA, a specialized organelle derived from the smooth endoplasmic reticulum, regulates synaptic calcium dynamics and plasticity. 38 , 39 Given the presence of abnormally large spines in the epileptogenic region, we examined whether these structures contained the SA (Figure 4F , Figure S5B ). Whereas a majority of spines in the control region exhibited an SA, a substantial proportion of enlarged spines in the epileptogenic region lacked this organelle (Figure 4G ; protrusions with SA [%]: control, 61.95 ± 3.52; epileptogenic, 18.65 ± 6.69; protrusions without SA [%]: control, 38.35 ± 3.52; epileptogenic, 81.35 ± 6.69; p < .0001). This suggests that these hypertrophic spines may be deficient in key organelles required for calcium signaling and synaptic plasticity. 3.5. Myelination changes in the epileptogenic cortex Neuronal activity modulates myelination, which is essential for network synchronization and efficient signal conduction. 40 , 41 A recent study indicates that aberrant neuronal activity can induce maladaptive myelination, potentially exacerbating seizure susceptibility and progression. 42 To evaluate axonal myelination changes in FCD, we measured the G‐ratio (the ratio of inner axonal diameter to total fiber diameter, including the myelin sheath) in control and epileptogenic regions (Figure 5A ). Our analysis revealed a significant reduction in G‐ratio values in the epileptogenic region, indicating increased myelin thickness (Figure 5B ; G‐ratio: control, .74 ± .01; epileptogenic, .68 ± .01; p < .0001). Notably, there was no statistically significant difference in axon diameter between the control and epileptogenic groups (Figure 5C; axon diameter [μm]: control, .71 ± .04; epileptogenic, .72 ± .04; p = .91). This confirms that the observed reduction in G‐ratio is not attributable to variations in axon size but rather reflects genuine alterations in myelin sheath thickness. This suggests that hyperactivity‐induced myelination changes may contribute to altered neuronal network properties in FCD. FIGURE 5. Open in a new tab Maladaptive myelination in the epileptogenic cortex. (A) Representative electron microscopic images of myelinated axons in the control and epileptogenic areas. Scale bar = .5 μm. (B, C) Quantification of G‐ratio values (B; inner axonal diameter divided by total fiber diameter, including the axon and myelin sheath) and axon diameter (C) in control and epileptogenic regions ( n = 70 per group). **** p < .0001; NS, not significant; unpaired Student t ‐test. Data are represented as mean ± standard error of the mean. 4. DISCUSSION FCD characterized by disrupted cortical structure is a leading cause of drug‐resistant epilepsy, particularly in pediatric and young adult populations. 1 , 2 , 3 Although genetic mutations affecting the mTOR signaling pathway, environmental influences, and somatic mosaic mutations have been associated with FCD, 43 , 44 , 45 the exact mechanisms underlying abnormal cortical network formation remain largely unclear. To investigate the neuroanatomical correlates of neuronal hyperexcitation, we utilized advanced volume electron microscopy techniques, including SB‐SEM, ATUM‐SEM, and electron tomography, to analyze ultrastructural synaptic alterations in the epileptogenic cortex of a patient with FCD type Ia. Our findings provide novel insights into local synaptic and axonal alterations that may contribute to cortical hyperexcitability and seizure propagation in FCD. The observed changes in inhibitory and excitatory synapses on layer III pyramidal neurons suggest that multiple mechanisms underlie seizure susceptibility in FCD (Figure S6 ). Histological examination confirmed characteristic features of FCD type I, such as disrupted cortical layering and a distinct radial microcolumnar arrangement, with no evidence of dysmorphic neurons or balloon cells (Figure S1 ). 30 Additionally, a reduction in cell density within cortical layer III was observed, which may reflect aberrant neuronal distribution or seizure‐related neuronal loss, ultimately influencing the E–I balance. It is important to acknowledge that diagnosing FCD type I, particularly subtype Ia, remains challenging for neuropathologists, as its features can be difficult to distinguish from normal variations in homotypic or heterotypic cortical architecture. The recent ILAE consensus update highlighted persistent challenges in the reproducibility of FCD I diagnoses, emphasizing that architectural abnormalities such as microcolumns and white matter heterotopia may be subtle and variably interpreted. 1 Histopathological agreement studies revealed that some cases initially considered FCD Ia may ultimately be reclassified as other subtypes of FCD following extended histopathological and genetic analyses. Therefore, although our specimen exhibited features supportive of FCD Ia including vertically aligned microcolumns, we recognize that definitive classification may evolve with the application of more comprehensive multilayered diagnostic strategies integrating immunohistochemical, molecular, and advanced imaging data. At the synaptic level, despite the hyperexcitable nature of the epileptogenic cortex, we observed a marked reduction in dendritic spine density in the distal dendrites of pyramidal neurons (Figure 1 ). This aligns with prior research demonstrating a decrease in excitatory synapses or dendritic spine loss in cortical regions of epileptic patients. 21 , 33 , 46 Further morphological analysis of the remaining dendritic spines in the epileptogenic cortex revealed that a subset displayed significantly enlarged spine heads and necks (Figure 1 ; Figure S2 ), along with an expanded PSD area. Notably, the constriction of spine necks was less pronounced in the epileptogenic cortex, potentially impairing electrochemical compartmentalization between spine heads and dendritic shafts. Such alterations may facilitate greater signal diffusion within the dendritic arbor, thus exacerbating neuronal excitability. This observation parallels recent findings in mouse models of schizophrenia, where an overabundance of extra‐large synapses resulted in supralinear dendritic and somatic integration, leading to an increase in neuronal firing. 47 Given that layer III pyramidal neurons serve as primary mediators of corticocortical communication, pathological amplification of excitatory synaptic activity in these neurons may enhance aberrant network dynamics and facilitate widespread seizure propagation through hyperactive corticocortical circuits. Analysis of excitatory and inhibitory synapse distribution on distal dendrites revealed a selective loss of asymmetric (excitatory) synapses, whereas symmetric (inhibitory) synapses—primarily innervated by somatostatin‐expressing interneurons—remained unaffected (Figure 2A–C ). Consequently, inhibitory and excitatory synapses were positioned farther apart on the same dendritic shafts in the epileptogenic region (Figure 2D–G ). Computational simulations also revealed that inhibitory–excitatory synapse spacing was nonrandom and highly organized in the control region, but this structured arrangement was disrupted in the epileptogenic cortex, likely due to the marked loss of excitatory synapses (Figure S3 ). Given the critical role of synaptic spatial arrangements in shunt inhibition, 34 , 35 this increased synapse spacing suggests that inhibitory synapses may be less effective at counteracting excitatory input, further contributing to cortical hyperexcitability. Because precise inhibitory–excitatory synapse positioning is essential for dendritic integration, neural computation, and overall network stability, 48 we propose that alterations in this spatial arrangement in pyramidal neuron dendrites contribute to both hyperexcitability and impaired synaptic plasticity in FCD patients. SV analysis using SB‐SEM and electron tomography confirmed that excitatory synapses in the epileptogenic region contained an increased number of total SVs as well as docked SVs within presynaptic terminals (Figure 3 , Figure S4 ). This accumulation of SVs suggests potential disruptions in vesicle cycling or prolonged neurotransmitter release machinery, which could enhance synaptic efficacy and promote excessive excitation. Additionally, the existing presynaptic boutons in the epileptogenic cortex exhibited a greater number of mitochondria per bouton, which also exhibited significantly enlarged and elongated morphology (Figure 4 , Figure S5 ). Elongated mitochondria are known to possess more interconnected cristae, increasing the surface area available for oxidative phosphorylation and thereby enhancing adenosine triphosphate production 49 —a critical adaptation for sustaining elevated synaptic activity, particularly in hyperexcitable or epileptogenic circuits. Moreover, the increased mitochondrial volume allows for greater calcium uptake capacity via the mitochondrial calcium uniporter, 49 improving the buffering of cytosolic calcium surges during repetitive neuronal firing or sustained synaptic transmission. Taken together, these morphological changes likely reflect a compensatory redistribution or remodeling of mitochondria aimed at maintaining calcium homeostasis and energy supply under conditions of heightened synaptic demand—an adaptive response similarly observed in other hyperexcitable neuronal states. 36 , 37 However, if sustained, such mitochondrial remodeling may become maladaptive, potentially leading to impaired calcium regulation or increased oxidative stress, thereby contributing to network instability and promoting seizure propagation. In parallel to presynaptic mitochondria, the SA in a subpopulation of spines is involved in calcium homeostasis and synaptic plasticity. 38 , 39 In our analysis, the prevalence of the SA in the control region is comparable to recent studies reporting SA presence in approximately 70%–88% of dendritic spines in the human neocortex. 50 , 51 Importantly, we revealed that the SA was notably absent in a significant proportion of enlarged spines within the epileptogenic cortex (Figure 4 ). This may further exacerbate synaptic dysfunction, potentially contributing to seizure‐related synaptic remodeling and impaired synaptic plasticity. Additionally, examination of axonal myelination revealed a significant reduction in G‐ratio values with no change in axonal diameter (Figure 5 ), indicative of increased myelin sheath thickness in the epileptogenic cortex. This observation contrasts with recent findings in FCD type II, which include reduced myelination or thinning of myelin sheaths. 52 , 53 These apparent discrepancies may reflect subtype‐specific patterns of myelin remodeling, in which milder forms of FCD exhibit compensatory or maladaptive myelin thickening in response to chronic hyperexcitability. Given the established role of activity‐dependent myelination in regulating network synchronization and seizure dynamics, 40 , 41 such alterations may modulate seizure propagation by affecting conduction velocity and circuit connectivity. 42 We propose that these divergent patterns of myelin remodeling across FCD subtypes may reflect differences in underlying cellular pathology and circuit excitability. This highlights the need for future comparative studies directly contrasting FCD type I and type II to clarify the broader spectrum of myelination abnormalities in epilepsy. Although our 3D EM data were obtained from a significantly larger cortical volume than in previous conventional transmission EM work, they are derived from a single case, limiting their extrapolation to the broader population of FCD patients with epilepsy. Further studies are required to clarify how synaptic connectivity is altered in specific neuron types and circuit levels, including interactions with glia cells, and whether these changes are consistently observed across different FCD cases. From a methodological standpoint, tissue fixation plays a critical role in preserving ultrastructure for high‐resolution electron microscopy, ensuring accurate morphological analysis at the synaptic and subcellular levels. Although perfusion fixation is widely regarded as the gold standard for brain tissue preservation due to its ability to rapidly and uniformly fix tissues in vivo, its application is not always feasible in clinical settings, particularly for human surgical specimens. In this study, immersion fixation was employed, and despite its potential limitations, the cortical tissue exhibited excellent ultrastructural preservation (Movies S1 and S2 ). This allowed for a detailed and quantitative assessment of synaptic morphology and intracellular organelles, underscoring the feasibility of utilizing surgically resected and immersion‐fixed human brain tissue for high‐resolution ultrastructural investigations, thereby enabling further research into epilepsy‐associated microstructural alterations and potentially broadening the scope of neuropathological studies on resected tissue samples. Although antiepileptic drugs could potentially influence synaptic architecture, their effects can be ruled out in this study, as both control and dysplastic cortical regions were obtained from the same patient undergoing identical treatment. Thus, the observed differences in synaptic organization are more likely due to FCD‐related pathological changes. However, it remains possible that some alterations in the epileptogenic cortex result from compensatory mechanisms or secondary responses aimed at mitigating epilepsy. Future studies integrating electrophysiological recordings and computational simulation modeling would be invaluable for determining the functional impact of these synaptic ultrastructural changes in the FCD cortex. 5. CONCLUSIONS This study using volume electron microscopy provides novel insights into the synaptic and axonal abnormalities in FCD, highlighting ultrastructural alterations that may contribute to cortical hyperexcitability. Key findings include the presence of extra‐large excitatory synapses with increased presynaptic vesicles, an elongated distance between inhibitory and excitatory synapses, and maladaptive myelination. Together, these structural alterations suggest that multiple mechanisms underlie hyperexcitability and seizure propagation in FCD. Further studies are needed to determine the functional impact of these changes and to explore potential therapeutic strategies for restoring synaptic balance in epilepsy linked to FCD. AUTHOR CONTRIBUTIONS Kea Joo Lee, Ji Yeoun Lee, and Yang Hoon Huh designed the experiments and supervised the project. Kea Joo Lee interpreted the results and finalized the paper. Gyu Hyun Kim, Na‐Young Seo, and Yang Hoon Huh performed the experiments and analyzed the data. Seung‐Ki Kim provided brain tissue samples resected during surgery. Jae‐Kyung Won and Ji Yeoun Lee contributed neuropathological assessments. All authors participated in manuscript preparation and approved the final version. FUNDING INFORMATION This work was supported by the KBRI Basic Research Programs (25‐BR‐01‐03 and 22‐BR‐02‐10 to K.J.L.), KBSI grants (C523400 and C512130 to Y.H.H.), and Korean NRF grants (RS‐2023‐00265524 to K.J.L., RS‐2022‐NR068424 to Y.H.H., and 2022R1A2C109210712 to J.Y.L.) funded by the Ministry of Science and ICT. CONFLICT OF INTEREST STATEMENT None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Supporting information Figure S1 . Histopathological assessment of the epileptogenic cortex. Figure S2 . Abnormal enlargement of dendritic spines in the epileptogenic region. Figure S3 . Disrupted spatial distribution of excitatory and inhibitory synapses in the epileptogenic region. Figure S4 . Increased presynaptic vesicle numbers per bouton in the epileptogenic region. Figure S5 . Morphological alterations in presynaptic mitochondria and postsynaptic spine apparatus. Figure S6 . Schematic model of synaptic alterations underlying hyperexcitability in the FCD cortex. EPI-67-2007-s003.docx (6.4MB, docx) Movie S1 . 3D reconstructions of dendritic segments using serial block‐face scanning electron microscopy. Download video file (109.6MB, mp4) Movie S2 . Tilting tomography of synaptic structures using high‐voltage electron microscopy. Download video file (69.1MB, mp4) ACKNOWLEDGMENTS The authors thank Namhee Kim (Rush University) for statistical advice, Sang Hoon Lee (KBRI imaging facility) and Hyo‐jeong Kim (SNU) for technical assistance with electron microscopy, and Chan Hee Lee, Ji Won Shin, Na Young Do, and Meeji Kim for image segmentation. Contributor Information Yang Hoon Huh, Email: [email protected]. Ji Yeoun Lee, Email: [email protected]. Kea Joo Lee, Email: [email protected]. 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EPI-67-2007-s003.docx (6.4MB, docx) Movie S1 . 3D reconstructions of dendritic segments using serial block‐face scanning electron microscopy. Download video file (109.6MB, mp4) Movie S2 . Tilting tomography of synaptic structures using high‐voltage electron microscopy. Download video file (69.1MB, mp4) Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. 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