Microelectrode array scaled for human hippocampal slices.

GSDMD knockdown exacerbates hippocampal damage and seizure susceptibility by crosstalk between pyroptosis and apoptosis in kainic acid-induced temporal lobe epilepsy

BackgroundA consensus has formed that neural circuits in the brain underlie the pathogenesis of temporal lobe epilepsy (TLE). In particular, the synaptic excitation/inhibition balance (E/I balance) has been implicated in shifting towards elevated excitation during the development of TLE.MethodsSprague Dawley (SD) rats were intraperitoneally subjected to kainic acid (KA) to generate a model of TLE. Next, electroencephalography (EEG) recording was applied to verify the stability and detectability of spontaneous recurrent seizures (SRS) in rats. Moreover, hippocampal slices from rats and patients with mesial temporal lobe epilepsy (mTLE) were assessed using immunofluorescence to determine the alterations of excitatory and inhibitory synapses and microglial phagocytosis.ResultsWe found that KA induced stable SRSs 14 days after status epilepticus (SE) onset. Furthermore, we discovered a continuous increase in excitatory synapses during epileptogenesis, where the total area of vesicular glutamate transporter 1 (vGluT1) rose considerably in the stratum radiatum (SR) of cornu ammonis 1 (CA1), the stratum lucidum (SL) of CA3, and the polymorphic layer (PML) of the dentate gyrus (DG). In contrast, inhibitory synapses decreased significantly, with the total area of glutamate decarboxylase 65 (GAD65) in the SL and PML diminishing enormously. Moreover, microglia conducted active synaptic phagocytosis after the formation of SRSs, especially in the SL and PML. Finally, microglia preferentially pruned inhibitory synapses during recurrent seizures in both rat and human hippocampal slices, which contributed to the synaptic alteration in hippocampal subregions.ConclusionsOur findings elaborately characterize the alteration of neural circuits and demonstrate the selectivity of synaptic phagocytosis mediated by microglia in TLE, which could strengthen the comprehension of the pathogenesis of TLE and inspire potential therapeutic targets for epilepsy treatment.

Temporal lobe epilepsy (TLE) is a chronic neurological disorder that is often resistant to antiepileptic drugs. The pathogenesis of TLE is extremely complicated and remains elusive. Understanding the molecular mechanisms underlying TLE is crucial for its diagnosis and treatment. In the present study, a lithium-pilocarpine-induced TLE model was employed to reveal the pathological changes of hippocampus in rats. Hippocampal samples were taken for proteomic analysis at 2 weeks after the onset of spontaneous seizure (a chronic stage of epileptogenesis). Isobaric tag for relative and absolute quantization (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry (LC–MS/MS) technique was applied for proteomic analysis of hippocampus. A total of 4173 proteins were identified from the hippocampi of epileptic rats and its control, of which 27 differentially expressed proteins (DEPs) were obtained with a fold change > 1.5 and P < 0.05. Bioinformatics analysis indicated 27 DEPs were mainly enriched in “regulation of synaptic plasticity and structure” and “calmodulin-dependent protein kinase activity,” which implicate synaptic remodeling may play a vital role in the pathogenesis of TLE. Consequently, the synaptic plasticity-related proteins and synaptic structure were investigated to verify it. It has been demonstrated that CaMKII-α, CaMKII-β, and GFAP were significant upregulated coincidently with proteomic analysis in the hippocampus of TLE rats. Moreover, the increased dendritic spines and hippocampal sclerosis further proved that synaptic plasticity involves in the development of TLE. The present study may help to understand the molecular mechanisms underlying epileptogenesis and provide a basis for further studies on synaptic plasticity in TLE.

Neuronal firing patterning in the dentate gyrus of patients with epilepsy remains unknown at the microcircuit level. Advancements in high-density CMOS-based microelectrode arrays can be harnessed to study network activity with unprecedented spatial and temporal resolution. We use novel computational methods with high-density electrophysiology recordings to spatially map network activity of human hippocampal brain slices from six patients with mesial temporal lobe epilepsy. Two slices from the dentate gyrus exhibited synchronous bursting activity in the presence of low magnesium media with kainic acid, representative of seizure-like behavior. We bridged microscale circuit dynamics with alterations in theta oscillations at the network scale. Future studies may apply this approach to spatially elucidate functional networks and their possible role in seizures.NEW & NOTEWORTHY We apply high-density CMOS-based microelectrode arrays to excised patient brain slices, mapping the communication patterns of hundreds of neurons at unprecedented resolution. We developed novel computational techniques to spatially map neuronal dynamics. In patient slices, our findings suggest that recurrent feedback localized within the dentate gyrus of the hippocampus is linked to a previously unreported phenomenon of theta propagations. This bridges microscale circuit dynamics with alterations in theta oscillations.

Objective To observe the effects of cyclooxygenase⁃2 inhibitor, celecoxib on expression of nuclear factor⁃κBp65 (NF⁃κBp65) and P⁃glycoprotein (P⁃gp) in the hippocampus of rats with chronic temporal lobe epilepsy (TLE), to investigate the relationship between NF⁃κBp65, P⁃gp and the pathogenesis of TLE, and to explore the potential of cyclooxygenase⁃2 inhibitor as an adjunctive therapy of anti⁃epileptic drug. Methods Thirty male Sprague⁃Dawley (SD) rats were divided into normal saline control group, TLE model group and celecoxib treatment group (n = 10 in each group). TLE model was induced by injection of kainic acid into the CA3 area of hippocampus using a stereotaxic apparatus. Eight weeks after status epilepticus, the rats in celecoxib treatment group received intraperitoneal injection of celecoxib (10 mg/kg) once daily for 10 d. The expression of NF ⁃ κ Bp65 and P ⁃ gp in hippocampus of rats was detected by immunohistochemical technique and Western blotting. Results Compared with normal saline control rats, the expression of NF⁃κBp65 and P⁃gp, and NF⁃κBp65 nuclear translocation in hippocampus of rats with TLE increased significantly (P < 0.05, for all). Celecoxib administration down⁃regulated the expression of NF⁃ κ Bp65 and P ⁃gp, and prevented NF⁃κ Bp65 translocation into nucleus in the hippocampus of TLE rats significantly (P < 0.05, for all). Conclusion These findings suggest that the pathogenesis of TLE is accompanied by an increase in NF⁃κBp65 and P⁃gp expression and NF⁃κBp65 nuclear translocation during chronic epilepsy period, and the administration of celecoxib may provide anti⁃epilepsy against inflammatory response and multi⁃drug resistance. DOI：10.3969/j.issn.1672-6731.2011.02.022

Hippocampal tissue of patients with refractory temporal lobe epilepsy is associated with astrocyte activation, inflammation, and altered expression of channels and receptors

AimsIt has been reported that the G‐alpha interacting protein (GAIP) interacting protein, C terminus 1 (GIPC1/GIPC) engages in vesicular trafficking, receptor transport and expression, and endocytosis. However, its role in epilepsy is unclear. Therefore, in this study, we aimed to explore the role of GIPC1 in epilepsy and its possible underlying mechanism.MethodsThe expression patterns of GIPC1 in patients with temporal lobe epilepsy (TLE) and in mice with kainic acid (KA)‐induced epilepsy were detected. Behavioral video monitoring and hippocampal local field potential (LFP) recordings were carried out to determine the role of GIPC1 in epileptogenesis after overexpression of GIPC1. Coimmunoprecipitation (Co‐IP) assay and high‐resolution immunofluorescence staining were conducted to investigate the relationship between GIPC1 and metabotropic glutamate receptor 7 (mGluR7). In addition, the expression of mGluR7 after overexpression of GIPC1 was measured, and behavioral video monitoring and LFP recordings after antagonism of mGluR7 were performed to explore the possible mechanism mediated by GIPC1.ResultsGIPC1 was downregulated in the brain tissues of patients with TLE and mice with KA‐induced epilepsy. After overexpression of GIPC1, prolonged latency period, decreased epileptic seizures and reduced seizure severity in behavioral analyses, and fewer and shorter abnormal brain discharges in LFP recordings of KA‐induced epileptic mice were observed. The result of the Co‐IP assay showed the interaction between GIPC1 and mGluR7, and the high‐resolution immunofluorescence staining also showed the colocalization of these two proteins. Additionally, along with GIPC1 overexpression, the total and cell membrane expression levels of mGluR7 were also increased. And after antagonism of mGluR7, increased epileptic seizures and aggravated seizure severity in behavioral analyses and more and longer abnormal brain discharges in LFP recordings were observed.ConclusionGIPC1 regulates epileptogenesis by interacting with mGluR7 and increasing its expression.

Temporal lobe epilepsy (TLE) in children is one of the most common refractory epilepsies. MicroRNAs (miRNAs) show abnormal expression in neurological disorders. The objective of this study was to determine changes in expression and the role of miR-29a in children with TLE. Sixty-five TLE patients and 70 normal controls were recruited. The levels of miR-29a were quantified using qRT-PCR. An in vitro TLE cell model was established using primary hippocampal cells cultured in magnesium-free medium. Cell viability, cell apoptosis and inflammatory cytokine concentrations were evaluated. The luciferase reporter assay was applied to confirm the target gene, HMGB1. A low level of MiR-29a expression was observed in the serum of children with TLE, which demonstrated a negative association with the concentration of serum TNF-α, IL-6, and IFN-γ. The level of MiR-29a demonstrated high specificity and sensitivity in children with TLE. A low level of expression of miR-29a was also detected in the TLE cell model. MiR-29a over-expression reversed the decreased cell viability induced by TLE, and alleviated cell apoptosis. Release of TNF-α, IL-6, and IFN-γ induced by TLE was also inhibited by miR-29a over-expression. HMGB1, which was downregulated in the serum of TLE patients, was shown to be a target gene of miR-29a, and negatively correlated with miR-29a level. The downregulation of serum miR-29a may serve as a non-invasive diagnostic biomarker for children with TLE. MiR-29a may be involved in the pathogenesis of TLE through regulation of neuronal apoptosis and neuroinflammation via targeting HMGB1.

Surgery: All Ages

Up-regulated ephrinB3/EphB3 expression in intractable temporal lobe epilepsy patients and pilocarpine induced experimental epilepsy rat model

The dentate gyrus, a region of the hippocampal formation, displays the highest level of plasticity in the brain and exhibits neurogenesis all through life. Dentate neurogenesis, believed to be essential for learning and memory function, responds to physiological stimuli as well as pathological situations. The role of dentate neurogenesis in the pathophysiology of temporal lobe epilepsy (TLE) has received increased attention lately because of its disparate response in the early and chronic stages of the disease. Acute seizures or status epilepticus immensely enhance dentate neurogenesis and lead to an aberrant migration of newly born neurons into the dentate hilus and the formation of epileptogenic circuitry in the injured hippocampus. Conversely, spontaneous recurrent seizures that arise during chronic TLE are associated with dramatically reduced dentate neurogenesis. In this review, we discuss the potential significance of enhanced but abnormal neurogenesis taking place shortly after brain injury or the status epilepticus towards the development of chronic epilepsy, and prospective implications of dramatically waned dentate neurogenesis occurring during chronic epilepsy for learning and memory function and depression in TLE. Furthermore, we confer whether hippocampal neurogenesis is a possible drug target for preventing TLE after brain injury or the status epilepticus, and for easing learning and memory impairments during chronic epileptic conditions. Additionally, we discuss some possible drugs and approaches that need to be evaluated in future in animal models of TLE to further understand the role of neurogenesis in the pathogenesis of TLE and whether modulation of neurogenesis is useful for treating TLE.

Converging evidence suggests that abnormalities of brain development may play a role in the pathogenesis of temporal lobe epilepsy (TLE). As sulco-gyral patterns are thought to be a footprint of cortical development, we set out to quantitatively map folding complexity across the neocortex in TLE. Additionally, we tested whether there was a relationship between cortical complexity and features of hippocampal maldevelopment, commonly referred to as malrotation. To quantify folding complexity, we obtained whole-brain surface-based measures of absolute mean cortical curvature from MRI scans acquired in 43 drug-resistant patients with TLE with unilateral hippocampal atrophy, and 40 age- and sex-matched healthy controls. In patients, we correlated changes in cortical curvature with 3-dimensional measures of hippocampal positioning. We found increased folding complexity in the temporolimbic cortices encompassing parahippocampal, temporopolar, insular, and fronto-opercular regions. Increased complexity was observed ipsilateral to the seizure focus in patients with left TLE (LTLE), whereas these changes were bilateral in patients with right TLE (RTLE). In both TLE groups, increased temporolimbic complexity was associated with increased hippocampal malrotation. We found tendencies for increased complexity in bilateral posterior temporal cortices in LTLE and contralateral parahippocampal cortices in RTLE to be predictive of unfavorable seizure outcome after surgery. The anatomic distribution of increased cortical complexity overlapping with limbic seizure networks in TLE and its association with hippocampal maldevelopment further imply that neurodevelopmental factors may play a role in the epileptogenic process of TLE.

Temporal lobe epilepsy is a multifactorial neurological dysfunction syndrome that is refractory, resistant to antiepileptic drugs, and has a high recurrence rate. The pathogenesis of temporal lobe epilepsy is complex and is not fully understood. Intracellular calcium dynamics have been implicated in temporal lobe epilepsy. However, the effect of fluctuating calcium activity in CA1 pyramidal neurons on temporal lobe epilepsy is unknown, and no longitudinal studies have investigated calcium activity in pyramidal neurons in the hippocampal CA1 and primary motor cortex M1 of freely moving mice. In this study, we used a multi-channel fiber photometry system to continuously record calcium signals in CA1 and M1 during the temporal lobe epilepsy process. We found that calcium signals varied according to the grade of temporal lobe epilepsy episodes. In particular, cortical spreading depression, which has recently been frequently used to represent the continuously and substantially increased calcium signals, was found to correspond to complex and severe behavioral characteristics of temporal lobe epilepsy ranging from grade II to grade V. However, vigorous calcium oscillations and highly synchronized calcium signals in CA1 and M1 were strongly related to convulsive motor seizures. Chemogenetic inhibition of pyramidal neurons in CA1 significantly attenuated the amplitudes of the calcium signals corresponding to grade I episodes. In addition, the latency of cortical spreading depression was prolonged, and the above-mentioned abnormal calcium signals in CA1 and M1 were also significantly reduced. Intriguingly, it was possible to rescue the altered intracellular calcium dynamics. Via simultaneous analysis of calcium signals and epileptic behaviors, we found that the progression of temporal lobe epilepsy was alleviated when specific calcium signals were reduced, and that the end-point behaviors of temporal lobe epilepsy were improved. Our results indicate that the calcium dynamic between CA1 and M1 may reflect specific epileptic behaviors corresponding to different grades. Furthermore, the selective regulation of abnormal calcium signals in CA1 pyramidal neurons appears to effectively alleviate temporal lobe epilepsy, thereby providing a potential molecular mechanism for a new temporal lobe epilepsy diagnosis and treatment strategy.

Local field potential (LFP) recordings within the brain have become an important tool used by neuroscientists to make inferences about the activity of a population of cells near an electrode. Each passing year analysis of LFPs in neuroscience seems to bring important new insights on the possible workings of networks in the brain to produce behavior (Buschman and Miller 2007; Canolty et al. 2006; Gregoriou et al. 2009; Liu and Newsome 2006; Lubenov and Siapas 2009; Pesaran et al. 2008; Womelsdorf et al. 2006). Indeed LFPs have become a near-ubiquitous tool in neurophysiology seemingly in use anywhere extracellular spikes are also recorded.

Epilepsy is a highly prevalent and drug-refractory neurological disorder characterized by spontaneous recurrent seizures. Estrogen is identified to be proconvulsant and lowers the seizure threshold of female epilepsy. Estrogen receptor β (ERβ) has been proposed to mediate neuroprotection in epilepsy, although the underlying mechanism remains unknown.Rationale: In this study, we investigated the role of ERβ in the epileptogenesis of female temporal lobe epilepsy (TLE).Methods: Immunohistochemistry, immunofluorescence, western blots, Golgi staining, 1H MRS and whole-cell patch-clamp were used to evaluate ERβ expression, pathological changes, and synaptic excitation /inhibition (E/I) balance in female TLE patients and ovariectomized (OVX) chronic epileptic mice. Electroencephalogram (EEG) recordings were recorded to evaluate the epileptic susceptibility in OVX WT and ERβ-/- mice. And high-throughput RNA-sequence was performed to identify differential expression genes (DEGs) which can elucidate the potential mechanism of ERβ regulating the seizure susceptibility.Results: ERβ expression was decreased in the brains of female TLE patients and OVX chronic epileptic mice. ERβ deletion enhanced seizure susceptibility and exacerbated the imbalance of synaptic E/I in hippocampal CA1 area of OVX epileptic mice. In line with these observations, RNA-sequence data further identified glutamine ligase (GLUL) as the target of ERβ involved in regulating synaptic E/I in CA1. Furthermore, ERβ agonist WAY-200070 markedly suppressed epileptic phenotypes and normalized GLUL expression in CA1 region of kainic acid (KA) induced OVX chronic epileptic model.Conclusions: Our data provide novel insight into the pathogenesis of female TLE, and indicate ERβ provides a new therapeutic strategy for female TLE patients.