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Modification of seizure activity by electrical stimulation: II. Motor seizure
Abstract
Daily electrical stimulations of the amygdala and hippocampus at intensities sufficient to evoke after-discharges (ADs) resulted in the development of motor seizures, which could not initially be evoked by these stimulations. The triggering of ADs was critical for this development, as well as for the development of permanent changes in the characteristics of the AD. The wave form of the AD "spikes" became more complex. The frequency of these spikes and the duration of AD increased. The amplitude of the AD spikes increased in the structure stimulated as well as in secondary structures to which the AD was "projected". This increase in amplitude of "projected" spikes often correlated with the appearance of motor seizures. Other electrographic developments are discussed including the appearance of spontaneous "inter-ictal" spiking in the amygdala. It was found that the development of motor seizures by stimulation of the amygdala resulted in an increased ability of the contralateral amygdala, and the septal area, but not of the hippocampus, to drive motor seizures when stimulated ("transfer"). Motor seizure development in the hippocampus transferred to the contralateral hippocampus. These developments were shown, by means of control subjects, with lesions in the primary focus to involve changes outside the primary focus. The implications of these developments with respect to seizure development are discussed.
... In particular, EEG is invasive, expensive, and low throughput, requiring surgery to implant a monitoring device. However, convulsive seizures in animal models have behavioral manifestations that correlate with underlying brain activity, and therefore with seizure severity, although these correlates can depend on species and etiology [12,13,14]. Thus, there are several options for scoring seizure severity that differ in their precision and quantitative rigor. ...
... Thus, there are several options for scoring seizure severity that differ in their precision and quantitative rigor. The simplest approach is to observe seizure events, either in real time or on video, and score them according to a standard rubric, such as the Racine scale [12,13]. This is the lowest cost option but is not high-throughput enough for long-term monitoring and is partially subjective. ...
... In this study, we used a supervised learning approach to detect seizures in mouse behavioral videos. The community standard seizure severity score for pre-clinical models is the Racine scale [12,13,14], which is an ordinal scale from one to seven denoting progressively more pronounced behavioral manifestations of seizures, from whisker trembling (Racine score = 1) to a tonic-clonic seizure followed by tonic extension and possibly respiratory arrest or death (Racine score = 7) [13]. In order to enrich for seizure events we induced seizures using the convulsant pentylenetetrazole (PTZ), a gamma-aminobutyric acid (GABAA) receptor antagonist, has been commonly reported in the literature [33,13,34]. ...
Seizures are caused by abnormally synchronous brain activity that can result in changes in muscle tone, such as twitching, stiffness, limpness, or rhythmic jerking. These behavioral manifestations are clear on visual inspection and the most widely used seizure scoring systems in preclinical models, such as the Racine scale in rodents, use these behavioral patterns in semiquantitative seizure intensity scores. However, visual inspection is time-consuming, low-throughput, and partially subjective, and there is a need for rigorously quantitative approaches that are scalable. In this study, we used supervised machine learning approaches to develop automated classifiers to predict seizure severity directly from noninvasive video data. Using the PTZ-induced seizure model in mice, we trained video-only classifiers to predict ictal events, combined these events to predict an univariate seizure intensity for a recording session, as well as time-varying seizure intensity scores. Our results show, for the first time, that seizure events and overall intensity can be rigorously quantified directly from overhead video of mice in a standard open field using supervised approaches. These results enable high-throughput, noninvasive, and standardized seizure scoring for downstream applications such as neurogenetics and therapeutic discovery.
... Groups 4 and 5 rats were administered with stem bark ethanolic extract of BC orally at 125 mg/kg and 250 mg/kg daily, and on alternate days, they were co-administered 35 mg/kg of PTZ intraperitoneally after 30 min. The experiments were conducted for 26 days, and seizure activity was evaluated using the Racine scale [26] as follows: stage 0, no response; stage 1, restlessness and twitching; stage 2, head nodding, head clonus, and myoclonic jerks; stage 3, unilateral forelimb clonus; stage 4, rearing with bilateral forelimb clonus; stage 5, generalized tonic-colonic seizure with falling. Kindling was considered complete when the rats display seizure score of 4 or above for 3 consecutive administrations [26,27]. ...
... The experiments were conducted for 26 days, and seizure activity was evaluated using the Racine scale [26] as follows: stage 0, no response; stage 1, restlessness and twitching; stage 2, head nodding, head clonus, and myoclonic jerks; stage 3, unilateral forelimb clonus; stage 4, rearing with bilateral forelimb clonus; stage 5, generalized tonic-colonic seizure with falling. Kindling was considered complete when the rats display seizure score of 4 or above for 3 consecutive administrations [26,27]. At the end of the 26 days, the rats were subjected to behavioral tests, and after which they were euthanized, and their brain tissues harvested for histology and biochemical assays. ...
Objectives
Epilepsy is a neurological disorder resulting from excessive electrical discharge in the brain. Bombax costatum (BC) is an herb being used in African traditional medicine for the treatment of seizures. This study evaluated the possible anti-convulsant potential of stem bark ethanolic extract of BC on PTZ-induced kindling in rats.
Methods
Thirty-five Wistar rats were grouped into five ( n = 7) and received normal saline, 35 mg/kg of PTZ, 5 mg/kg diazepam followed by 35 mg/kg PTZ after 30 min and BC stem back ethanolic extract at 125 mg/kg and 250 mg/kg followed by 35 mg/kg of PTZ intraperitoneally after 30 min. BC was administered orally daily while normal saline and PTZ were given intraperitoneally every other day for 26 days. Seizure activity was evaluated using the Racine scale, cognitive abilities through modified elevated plus maze and anxiety through forced swimming test. Further, the levels of GABA and oxidative stress biomarkers were also evaluated from the rat’s brain homogenate.
Results
Pretreatment with BC significantly reduced ( p < .05) the seizure score and increased GABA level in BC treated rats when compared to PTZ alone treated rats. The first transfer latency of PTZ alone treated rats was significantly increased ( p < .05) relative to the control rats and rats pretreated with diazepam and BC extract. Pretreatment with BC extract at 250 mg/kg was shown to significantly increase ( p < .05) the activities of catalase, reduced glutathione, and superoxide dismutase compared to the PTZ alone treated rats.
Conclusions
Conclusively, BC was found to prevent seizure, avert neurodegeneration, and enhance cognition in PTZ-treated rats by regulating GABA level and enhancing antioxidant activity. Therefore, BC could be explored further for possible development of antiseizure agents.
... The animals were used in our previous study and a detailed description of the lithium-pilocarpine model of temporal lobe epilepsy can be found there [16]. Briefly, rats premedicated with LiCl (1 day before, 127 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO, USA) and (−)-scopolamine methyl bromide (1 h before, 1 mg/kg, i.p., Sigma-Aldrich) were intermittently injected with pilocarpine (10 mg/kg with 30 min intervals, i.p. up to 40 mg/kg, Sigma-Aldrich) to reach grade 4 on the Racine scale [17]. The severity of seizures was scored as described before according the modified Racine scale: facial automatism (1), head nodding (2), forelimb myoclonus (3), rearing (4), rearing and falling (5), wild running (6), and generalized clonictonic convulsions (7) [17,18]. ...
... Briefly, rats premedicated with LiCl (1 day before, 127 mg/kg, i.p., Sigma-Aldrich, St. Louis, MO, USA) and (−)-scopolamine methyl bromide (1 h before, 1 mg/kg, i.p., Sigma-Aldrich) were intermittently injected with pilocarpine (10 mg/kg with 30 min intervals, i.p. up to 40 mg/kg, Sigma-Aldrich) to reach grade 4 on the Racine scale [17]. The severity of seizures was scored as described before according the modified Racine scale: facial automatism (1), head nodding (2), forelimb myoclonus (3), rearing (4), rearing and falling (5), wild running (6), and generalized clonictonic convulsions (7) [17,18]. The seizures were stopped with diazepam (10 mg/kg, i.p., Sigma-Aldrich) after 75 min. ...
Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) is a commonly used tool for gene expression analysis. The selection of stably expressed reference genes is required for accurate normalization. The aim of this study was to identify the optimal reference genes for RT-qPCR normalization in various brain regions of rats at different stages of the lithium–pilocarpine model of acquired epilepsy. We tested the expression stability of nine housekeeping genes commonly used as reference genes in brain research: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on four standard algorithms (geNorm, NormFinder, BestKeeper, and comparative delta-Ct), we found that after pilocarpine-induced status epilepticus, the stability of the tested reference genes varied significantly between brain regions and depended on time after epileptogenesis induction (3 and 7 days in the latent phase, and 2 months in the chronic phase of the model). Pgk1 and Ywhaz were the most stable, while Actb, Sdha, and B2m demonstrated the lowest stability in the analyzed brain areas. We revealed time- and region-specific changes in the mRNA expression of the housekeeping genes B2m, Actb, Sdha, Rpl13a, Gapdh, Hprt1, and Sdha. These changes were more pronounced in the hippocampal region during the latent phase of the model and are thought to be related to epileptogenesis. Thus, RT-qPCR analysis of mRNA expression in acquired epilepsy models requires careful selection of reference genes depending on the brain region and time of analysis. For the time course study of epileptogenesis in the rat lithium–pilocarpine model, we recommend the use of the Pgk1 and Ywhaz genes.
... The dose and schedule of administration were chosen based on our pilot study and previous studies [29][30][31]. Additionally, seizure behavior was assessed for 3 h after KA administration using the established five stages of seizure activity in the Racine Scale (Racine, 1972) [32]: (1) Mouth and facial movements (orofacial movements), (2) Head nodding (head myoclonus and/or severe orofacial movements), (3) Forelimb clonus, (4) Forelimb clonus with rearing and, (5) Rearing, jumping, and falling with loss of postural control. ...
... The dose and schedule of administration were chosen based on our pilot study and previous studies [29][30][31]. Additionally, seizure behavior was assessed for 3 h after KA administration using the established five stages of seizure activity in the Racine Scale (Racine, 1972) [32]: (1) Mouth and facial movements (orofacial movements), (2) Head nodding (head myoclonus and/or severe orofacial movements), (3) Forelimb clonus, (4) Forelimb clonus with rearing and, (5) Rearing, jumping, and falling with loss of postural control. ...
Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 μM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.
... Briefly, intraperitoneal injections of 5 mg/kg of kainic acid (Abcam; Cat# ab120100) were repeated to reach and maintain SE for 3 hours (two rats received half doses, 2.5 mg/kg). We continuously monitored and scored seizures using the Racine scale (Racine 1972). After 3 hours of SE, KA-SE rats received a 8 mg/kg dose of diazepam (Patterson Veterinary, Inc.) to terminate SE. ...
... In addition, two experienced investigators blind to group identity visually scanned the prolonged immobility with staring. These progressed to alternating or bilateral clonus, rearing and falling (Racine 1972). Rats were considered epileptic if they had at least one documented seizure as defined above. ...
The mechanisms by which brain insults lead to subsequent epilepsy remain unclear. Insults including trauma, stroke, infections, and long seizures (status epilepticus, SE) increase the nuclear expression and chromatin binding of the neuron-restrictive silencing factor/RE-1 silencing transcription factor (NRSF/REST). REST/NRSF orchestrates major disruption of the expression of key neuronal genes, including ion channels and neurotransmitter receptors, potentially contributing to epileptogenesis. Accordingly, transient interference with REST/NRSF chromatin binding after an epilepsy-provoking SE suppressed spontaneous seizures for the 12 d duration of a prior study. However, whether the onset of epileptogenesis was suppressed or only delayed has remained unresolved. The current experiments determined if transient interference with REST/NRSF chromatin binding prevented epileptogenesis enduringly or, alternatively, slowed epilepsy onset. Epileptogenesis was elicited in adult male rats via systemic kainic acid-induced SE (KA-SE). We then determined if decoy, NRSF-binding–motif oligodeoxynucleotides (NRSE-ODNs), given twice following KA-SE (1) prevented REST/NRSF binding to chromatin, using chromatin immunoprecipitation, or (2) prevented the onset of spontaneous seizures, measured with chronic digital video-electroencephalogram. Blocking NRSF function transiently after KA-SE significantly lengthened the latent period to a first spontaneous seizure. Whereas this intervention did not influence the duration and severity of spontaneous seizures, total seizure number and seizure burden were lower in the NRSE-ODN compared with scrambled-ODN cohorts. Transient interference with REST/NRSF function after KA-SE delays and moderately attenuates insult-related hippocampal epilepsy, but does not abolish it. Thus, the anticonvulsant and antiepileptogenic actions of NRSF are but one of the multifactorial mechanisms generating epilepsy in the adult brain.
... For this group of individuals, other approaches need to be explored. One promising alternative is electric brain stimulation, which can be conducted through peripheral nerve stimulation, spinal cord stimulation or deep brain stimulation [12] [21]. Devices for electric brain stimulation can either perform continuous stimulation or responsive stimulation upon seizure detection, and in both cases patients do not recognize the stimulation [3] [12]. ...
Epilepsy is the most common, chronic, neurological disease worldwide and is typically accompanied by reoccurring seizures. Neuro implants can be used for effective treatment by suppressing an upcoming seizure upon detection. Due to the restricted size and limited battery lifetime of those medical devices, the employed approach also needs to be limited in size and have low energy requirements. We present an energy-efficient seizure detection approach involving a TC-ResNet and time-series analysis which is suitable for low-power edge devices. The presented approach allows for accurate seizure detection without preceding feature extraction while considering the stringent hardware requirements of neural implants. The approach is validated using the CHB-MIT Scalp EEG Database with a 32-bit floating point model and a hardware suitable 4-bit fixed point model. The presented method achieves an accuracy of 95.28%, a sensitivity of 92.34% and an AUC score of 0.9384 on this dataset with 4-bit fixed point representation. Furthermore, the power consumption of the model is measured with the low-power AI accelerator UltraTrail, which only requires 495 nW on average. Due to this low-power consumption this classification approach is suitable for real-time seizure detection on low-power wearable devices such as neural implants.
... To prevent peripheral effects of pilocarpine, (-)-scopolamine methylbromide (1 mg/kg, i.p.) was administered one hour before pilocarpine. Only rats with stage 4 or higher seizures on the Racine scale [70] lasting at least 90 minutes were included in the study. The control group received LiCl, scopolamine methylbromide, and saline. ...
Epilepsy is known to cause alterations in neural networks. However, many details of these changes remain poorly understood. The objective of this study was to investigate changes in the properties of hippocampal CA1 pyramidal neurons and their synaptic inputs in a rat lithium-pilocarpine model of epilepsy. In the chronic phase of the model, we found a marked loss of pyramidal neurons in the CA1 area. However, the membrane properties of the neurons remained essentially unal-tered. The results of the electrophysiological and morphological studies indicate that the direct pathway from the entorhinal cortex to CA1 neurons is reinforced in epileptic animals, whereas the inputs to them from CA3 are either unaltered or even diminished. In particular, the dendritic spine density in the str. lacunosum moleculare, where the direct pathway from the entorhinal cortex ter-minates, was found to be 2.5 times higher in epileptic rats than in control rats. Furthermore, the summation of responses upon stimulation of the temporoammonic pathway was enhanced by approximately twofold in epileptic rats. This enhancement is believed to be a significant con-tributing factor to the heightened epileptic activity observed in the entorhinal cortex of epileptic rats using an ex vivo 4-aminopyridine model.
... [51] Seizures were considered convulsive if the video record showed behaviors consistent with stages 3-5 on the Racine scale. [51,52] Acute Slice Preparation and Current-Clamp Recordings: Horizontal slices containing hippocampus were obtained from adult male C57BL/6 mice (6-to 8-week-old). All data were acquired and analyzed blind to viral treatment or genotype. ...
Intrinsic plasticity, a fundamental process enabling neurons to modify their intrinsic properties, plays a crucial role in shaping neuronal input‐output function and is implicated in various neurological and psychiatric disorders. Despite its importance, the underlying molecular mechanisms of intrinsic plasticity remain poorly understood. In this study, a new ubiquitin ligase adaptor, protein tyrosine phosphatase receptor type N (PTPRN), is identified as a regulator of intrinsic neuronal excitability in the context of temporal lobe epilepsy. PTPRN recruits the NEDD4 Like E3 Ubiquitin Protein Ligase (NEDD4L) to NaV1.2 sodium channels, facilitating NEDD4L‐mediated ubiquitination, and endocytosis of NaV1.2. Knockout of PTPRN in hippocampal granule cells leads to augmented NaV1.2‐mediated sodium currents and higher intrinsic excitability, resulting in increased seizure susceptibility in transgenic mice. Conversely, adeno‐associated virus‐mediated delivery of PTPRN in the dentate gyrus region decreases intrinsic excitability and reduces seizure susceptibility. Moreover, the present findings indicate that PTPRN exerts a selective modulation effect on voltage‐gated sodium channels. Collectively, PTPRN plays a significant role in regulating intrinsic excitability and seizure susceptibility, suggesting a potential strategy for precise modulation of NaV1.2 channels' function.
... After each KA injection, each rat was placed in a cage to record its seizure behavior for 3 h. The latency to tonic-clonic seizure onset (min) and seizure score were recorded based on Racine's scale as follows: state 0: no response; state 1: ear and facial twitching; state 2: myoclonic jerks; state 3: myoclonic jerks, rearing; state 4: turning over onto the side position, tonic-clonic seizures; state 5: turning over onto the back position, generalized tonic-clonic seizures [22]. On the 11th day, the rats were sacrificed for subsequent experiments ( Figure 1B). ...
The present study evaluated the antiseizure and neuroprotective effects of sodium houttuyfonate (SH), a derivative of Houttuynia cordata Thunb. (H. cordata), in a kainic acid (KA)- induced seizure rat model and its underlying mechanism. Sprague Dawley rats were administered normal saline, SH (50 or 100 mg/kg), or carbamazepine (300 mg/kg) by oral gavage for seven consecutive days before the intraperitoneal administration of KA (15 mg/kg). SH showed antiseizure effects at a dose of 100 mg/kg; it prolonged seizure latency and decreased seizure scores. SH also significantly decreased neuronal loss in the hippocampi of KA-treated rats, which was associated with the prevention of glutamate level increase, the upregulation of glutamate reuptake-associated proteins (excitatory amino acid transporters 1–3), glutamate metabolism enzyme glutamine synthetase, the downregulation of the glutamate synthesis enzyme glutaminase, and significant alterations in the expression of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor) and NMDA (N-methyl-D-aspartic acid receptor) receptor subunits in the hippocampus. Furthermore, the effects of SH were similar to those of the antiseizure drug carbamazepine. Therefore, the results of the present study suggest that SH has antiseizure effects on KA-induced seizures, possibly through the prevention of glutamatergic alterations. Our findings suggest that SH is a potential alternative treatment that may prevent seizures by preserving the normal glutamatergic system.
... The Racine scoring method was used to evaluate the epilepsy in the mice, with 4 or higher scores considered successful modeling. After 1 h of status epilepticus (SE), diazepam (4 mg/kg) was injected to terminate the SE [38,39]. The Racine score and latency of epilepsy (min) of mice in each group were recorded. ...
Epilepsy is a common neurological disorder, and the exploration of potential therapeutic drugs for its treatment is still ongoing. Vitamin D has emerged as a promising treatment due to its potential neuroprotective effects and anti-epileptic properties. This study aimed to investigate the effects of vitamin D on epilepsy and neuroinflammation in juvenile mice using network pharmacology and molecular docking, with a focus on the mammalian target of rapamycin (mTOR) signaling pathway. Experimental mouse models of epilepsy were established through intraperitoneal injection of pilocarpine, and in vitro injury models of hippocampal neurons were induced by glutamate (Glu) stimulation. The anti-epileptic effects of vitamin D were evaluated both in vivo and in vitro. Network pharmacology and molecular docking analysis were used to identify potential targets and regulatory pathways of vitamin D in epilepsy. The involvement of the mTOR signaling pathway in the regulation of mouse epilepsy by vitamin D was validated using rapamycin (RAPA). The levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were assessed by enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining was used to analyze the apoptosis of hippocampal neurons. In in vivo experiments, vitamin D reduced the Racine scores of epileptic mice, prolonged the latency of epilepsy, and inhibited the production of TNF-α, IL-1β, and IL-6 in the hippocampus. Furthermore, network pharmacology analysis identified RAF1 as a potential target of vitamin D in epilepsy, which was further confirmed by molecular docking analysis. Additionally, the mTOR signaling pathway was found to be involved in the regulation of mouse epilepsy by vitamin D. In in vitro experiments, Glu stimulation upregulated the expressions of RAF1 and LC3II/LC3I, inhibited mTOR phosphorylation, and induced neuronal apoptosis. Mechanistically, vitamin D activated the mTOR signaling pathway and alleviated mouse epilepsy via RAF1, while the use of the pathway inhibitor RAPA reversed this effect. Vitamin D alleviated epilepsy symptoms and neuroinflammation in juvenile mice by activating the mTOR signaling pathway via RAF1. These findings provided new insights into the molecular mechanisms underlying the anti-epileptic effects of vitamin D and further supported its use as an adjunctive therapy for existing anti-epileptic drugs.
... The control group rats were maintained at 30℃. All rats were individually observed for seizure behaviour during hyperthermia period using the five-stage scale [20]: ...
Background
Saiga antelope horn (SAH) is a traditional Chinese medicine for treating febrile seizure (FS) with precise efficacy, but its mechanism of action and functional substances are still unclear. Given the need for further research on SAH, our group conducted studies to elucidate its mechanisms and active substances.
Methods
An FS rat pup model was constructed through intraperitoneal injection of LPS and hyperthermia induction. Behavioural indicators of seizures, hippocampal histopathological alterations, serum levels of inflammatory cytokines and hippocampal levels of neurotransmitters were observed and measured to investigate the effects of SAH on FS model rats. Hippocampal metabolomics and network pharmacology analyses were conducted to reveal the differential metabolites, key peptides and pathways involved in the suppression of FS by SAH.
Results
SAH suppressed FS, decreased the inflammatory response and regulated the Glu-GABA balance. Metabolomic analysis revealed 13 biomarkers of FS, of which SAH improved the levels of 8 differential metabolites. Combined with network pharmacology, a “biomarker-core target-key peptide” network was constructed. The peptides of SAH, such as YGQL and LTGGF, could exert therapeutic effects via the arachidonic acid pathway. Molecular docking and ELISA results indicated that functional peptides of SAH could bind to PTGS2 target, inhibiting the generation of AA and its metabolites in hippocampal samples.
Conclusion
In summary, the functional peptides contained in SAH are the main material basis for the treatment of FS, potentially acting through neurotransmitter regulation and the arachidonic acid pathway.
Graphical abstract
... Mice were given intraperitoneal (i.p.) injections of KA, at doses ranging from 15 to 30 mg ⁄ kg body weight. Behavioural seizure activity was monitored for 2 h and classified according to a modified Racine scale [15]: stage 0, normal behavior; stage 1, immobility; stage 2, repetitive movements, myoclonic twitch, or head bobbing; stage 3, bilateral forelimb clonus and rearing; stage 4, continuous rearing and falling; and stage 5, generalized tonic-clonic seizure. ...
Background: The estriol which was evaluated for its effect on Kainic acid kindling model of epileptogenesis in mice followed by evaluation on kindling-induced changes in cognitive and motor functions. Material and methods: Kindling was induced on every alternate day for duration of 45 days and treatment with Kainic acid was given at dose ranging from 15 to 30 mg/kg body weight intraperitoneal (i.p) and estriol was also administered at dosage of 0.005 and 0.1 mg/kg through i.p. After induction of kindling the seizure severity was recorded and further percentage incidence of animals kindled at the end of 45 days was also recorded. Spatial learning and cognitive alterations were assessed by Morris water test (MWT) while motor function was assessed by grip strength meter. Results: Estriol increased the rate of kindling in both the sexes of mice at great scale. Percentage incidence of seizures was also intensified. A noticeable decline in the grip strength, Morris water was observed following KA- kindling in pre-treated estriol groups of mice both the sexes. Conclusion: Control animals developed a seizure score of 4 after the end of 5 weeks, mice treated with estriol exhibited kindling in first two weeks only Clomiphene at dose of 0.9 mg/kg i.p. exhibited anticonvulsant effects. The study displayed that estriol has powerful proconvulsant effect.
... 25 Synchronized behavioral videos were also analyzed simultaneously for the detected EEG-defined seizures to assess the level of epileptic behavioral seizures. The Racine seizure classification was used 26 ...
Objective
Methyl CpG‐binding protein 2 (MECP2) duplication syndrome is a rare X‐linked genomic disorder affecting predominantly males, which is usually manifested as epilepsy and autism spectrum disorder (ASD) comorbidity. The transgenic line MeCP2 Tg1 was used for mimicking MECP2 duplication syndrome and showed autism–epilepsy co‐occurrence. Previous works suggested that the excitatory/inhibitory (E/I) imbalance is a potential common mechanism for both epilepsy and ASD. The projection neurons and parvalbumin (PV) interneurons account for the majority of E/I balance in the hippocampus. Therefore, we explored how structural changes of projection and PV ⁺ neurons occur in the hippocampus of MeCP2 Tg1 mice and whether these morphological changes contribute to epilepsy susceptibility.
Methods
We used the interneuron Designer receptors exclusively activated by designer drugs mouse model to inhibit inhibitory neurons in the hippocampus to verify the epilepsy susceptibility of MeCP2 Tg1 (FVB, an inbred strain named as sensitivity to Friend leukemia virus) mice. Electroencephalograms were recorded for the definition of seizure. We performed retro‐orbital injection of virus in MeCP2 Tg1 (FVB):CaMKIIα‐Cre (C57BL/6) mice or MeCP2 Tg1 :PV‐Cre (C57BL/6) mice and their littermate controls to specifically label projection and PV ⁺ neurons for structural analysis.
Results
Epilepsy susceptibility was increased in MeCP2 Tg1 mice. There was a reduced number of PV neurons and reduced dendritic complexity in the hippocampus of MeCP2 Tg1 mice. The dendritic complexity in MeCP2 Tg1 mice was increased compared to wild‐type mice, and total dendritic spine density in dentate gyrus of MeCP2 Tg1 mice was also increased. Total dendritic spine density was increased in CA1 of MeCP2 Tg1 mice.
Significance
Overexpression of MeCP2 may disrupt crucial signaling pathways, resulting in decreased dendritic complexity of PV interneurons and increased dendritic spine density of projection neurons. This reciprocal modulation of excitatory and inhibitory neuronal structures associated with MeCP2 implies its significance as a potential target in the development of epilepsy and offers a novel perspective on the co‐occurrence of autism and epilepsy.
... Assessment of seizure behaviour Following PTZ injection, animals were monitored and seizure index recorded over 40 min based on a modified Racine scale [52] as listed in Table 2. Also, latency; time between PTZ injection and onset of seizures and duration; time interval from the onset to termination of seizures or death of the animal. ...
Apocynin (APO) is a plant derived antioxidant exerting specific NADPH oxidase inhibitory action substantiating its neuroprotective effects in various CNS disorders, including epilepsy. Due to rapid elimination and poor bioavailability, treatment with APO is challenging. Correspondingly, novel APO-loaded lipid nanocapsules (APO-LNC) were formulated and coated with lactoferrin (LF-APO-LNC) to improve br ain targetability and prolong residence time. Lavender oil (LAV) was incorporated into LNC as a bioactive ingredient to act synergistically with APO in alleviating pentylenetetrazol (PTZ)-induced seizures. The optimized LF-APO-LAV/LNC showed a particle size 59.7 ± 4.5 nm with narrow distribution and 6.07 ± 1.6mV zeta potential) with high entrapment efficiency 92 ± 2.4% and sustained release (35% in 72 h). Following subcutaneous administration, LF-APO-LAV/LNC brought about ⁓twofold increase in plasma AUC and MRT compared to APO. A Log BB value of 0.2 ± 0.14 at 90 min reflects increased brain accumulation. In a PTZ-induced seizures rat model, LF-APO-LAV/LNC showed a Modified Racine score of 0.67 ± 0.47 with a significant increase in seizures latency and decrease in duration. Moreover, oxidant/antioxidant capacity and inflammatory markers levels in brain tissue were significantly improved. Histopathological and immunohistochemical assessment of brain tissue sections further supported these findings. The results suggest APO/LAV combination in LF-coated LNC as a promising approach to counteract seizures.
Graphical Abstract
... Then, 20 methyl-scopolamine nitrate, status epilepticus was induced by the injection of a single dose of pilocarpine (400 mg/kg, i.p.) dissolved in 0.9% sterile saline. After pilocarpine injection, mice were observed for 6 hours to determine the severity and duration of acute seizures with reference to the Racine scale [20] ranging from 0 to 5 (level 0: no response; level 1: Hyperactivity and vibrissae clonus; level 2: head nodding, head clonus, and myoclonic jerks; level 3: unilateral forelimb clonus; level 4: pitching and bilateral forelimb clonus; level 5: tonic-clonic seizures with loss of motor control reflex). Twenty-four hours after induction of status epilepticus, the different substances were administered to the animals according to groups. ...
Epilepsy is a chronic neurological disorder that affects the central nervous system. Approximately 3% of the population at some point in their lives would be affected by epileptic disorders in the world, that is to say nearly 70 million people and therefore about 85% live in developing countries. The objective of this study is to determine the anticonvulsant effects of Lippia multiflora on the animal model of epileptogenesis induced by pilocarpine injection in mice during the acute phase. Mice were treated with different doses of the aqueous extract of L. multiflora or sodium valproate. The anticonvulsant effects were evaluated 24 after the injection of pilocarpine by referring to the Racine scale. Then a daily treatment was done for one week corresponding to the acute stage of the disease. Oxidative stress parameters were measured from brain samples taken on the last day of treatment. The injection of pilocarpine induced the Status epilepticus, which is translated by the appearance of convulsive seizures while the administration of different doses of the aqueous extract of L. multiflora led to a significant decrease of the seizures induced by pilocarpine in mice. In addition, L. multiflora extract restored endogenous levels of oxidative stress markers (MDA and NO) and increased the activity of oxidative stress enzymes (SOD, CAT and GSH). L. multiflora possesses anticonvulsant properties mediated by the involvement of GABAA receptors and antioxidants.
... The cannula was kept for an additional two mins after completion of infusion and withdrew slowly to minimize reflux along the injection tract. Seizure stages were classified according to the criteria described by Racine [54] 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 fullbody shaking, spasms; stage 4, chronic full-body spasms; stage 5, jumping, shrieking, falling over; stage 6, violent convulsions or death. Seizures at stage 4-6 that last for ≥ 30 min was defined as SE. ...
Background
Human patients often experience an episode of serious seizure activity, such as status epilepticus (SE), prior to the onset of temporal lobe epilepsy (TLE), suggesting that SE can trigger the development of epilepsy. Yet, the underlying mechanisms are not fully understood. The low-density lipoprotein receptor related protein (Lrp4), a receptor for proteoglycan-agrin, has been indicated to modulate seizure susceptibility. However, whether agrin-Lrp4 pathway also plays a role in the development of SE-induced TLE is not clear.
Methods
Lrp4 f/f mice were crossed with hGFAP-Cre and Nex-Cre mice to generate brain conditional Lrp4 knockout mice ( hGFAP-Lrp4 −/− ) and pyramidal neuron specific knockout mice ( Nex-Lrp4 −/− ). Lrp4 was specifically knocked down in hippocampal astrocytes by injecting AAV virus carrying hGFAP-Cre into the hippocampus. The effects of agrin-Lrp4 pathway on the development of SE-induced TLE were evaluated on the chronic seizure model generated by injecting kainic acid (KA) into the amygdala. The spontaneous recurrent seizures (SRS) in mice were video monitored.
Results
We found that Lrp4 deletion from the brain but not from the pyramidal neurons elevated the seizure threshold and reduced SRS numbers, with no change in the stage or duration of SRS. More importantly, knockdown of Lrp4 in the hippocampal astrocytes after SE induction decreased SRS numbers. In accord, direct injection of agrin into the lateral ventricle of control mice but not mice with Lrp4 deletion in hippocampal astrocytes also increased the SRS numbers. These results indicate a promoting effect of agrin-Lrp4 signaling in hippocampal astrocytes on the development of SE-induced TLE. Last, we observed that knockdown of Lrp4 in hippocampal astrocytes increased the extracellular adenosine levels in the hippocampus 2 weeks after SE induction. Blockade of adenosine A1 receptor in the hippocampus by DPCPX after SE induction diminished the effects of Lrp4 on the development of SE-induced TLE.
Conclusion
These results demonstrate a promoting role of agrin-Lrp4 signaling in hippocampal astrocytes in the development of SE-induced development of epilepsy through elevating adenosine levels. Targeting agrin-Lrp4 signaling may serve as a potential therapeutic intervention strategy to treat TLE.
... Rats were observed for 20 min for any convulsive behavior following each PTZ injection (Rajabzadeh et al. 2012). The classification of the seizures was based on the Racine score (Racine 1972) as follows: stage 0: no response, stage 1: ear and facial twitching, stage 2: myoclonic jerks without upright position, stage 3: myoclonic jerks, upright position with bilateral forelimb clonus, stage 4: tonic-clonic seizures, and stage 5: generalized tonic-clonic seizures, loss of postural control. ...
Epilepsy is a common neurological disorder that significantly affects the quality of life of patients. In this study, we aim to
evaluate the effectiveness of dental pulp stem cell (DPSC) transplantation in decreasing inflammation and cell death in brain
cells, thus reducing seizure damage. We induced seizures in rats using intraperitoneal injections of pentylenetetrazole (PTZ).
In the PTZ + DPSC group, we conducted bilateral hippocampal transplantation of DPSCs in PTZ-lesioned rat models. After
1 month, we performed post-graft analysis and measured some behavioral factors, such as working memory and long-term
memory, using a T-maze test and passive avoidance test, respectively. We investigated the immunohistopathology and distribution
of astrocyte cells through light microscopy and Sholl analysis. Additionally, we employed the Voronoi tessellation
method to estimate the spatial distribution of the cells in the hippocampus. Compared to the control group, we observed a
reduction in astrogliosis, astrocyte process length, the number of branches, and intersections distal to the soma in the hippocampus
of the PTZ + DPSC group. Further analysis indicated that the grafted DPSCs decreased the expression of caspase-3
in the hippocampus of rats with induced seizures. Moreover, the DPSCs transplant protected hippocampal pyramidal neurons
against PTZ toxicity and improved the spatial distribution of the hippocampal neurons. Our findings suggest that DPSCs
transplant can be an effective modifier of astrocyte reactivation and inflammatory responses.
... The copyright holder for this preprint (which this version posted May 15, 2024. ; https://doi.org/10.1101/2024.05.14.594246 doi: bioRxiv preprint extension; 4, forelimb clonus; 5, generalized clonic activity; 6, bouncing or running seizures; 7, full tonic extension; 8, death (Racine, 1972;Löscher et al., 1991). The latency to reach each stage and the maximum stage each mouse reached were recorded. ...
Tau reduction is a promising therapeutic strategy for Alzheimer’s disease. In numerous models, tau reduction via genetic knockout is beneficial, at least in part due to protection against hyperexcitability and seizures, but the underlying mechanisms are unclear. Here we describe the generation and initial study of a new conditional Tau flox model to address these mechanisms. Given the protective effects of tau reduction against hyperexcitability, we compared the effects of selective tau reduction in excitatory or inhibitory neurons. Tau reduction in excitatory neurons mimicked the protective effects of global tau reduction, while tau reduction in inhibitory neurons had the opposite effect and increased seizure susceptibility. Since most prior studies used knockout mice lacking tau throughout development, we crossed Tau flox mice with inducible Cre mice and found beneficial effects of tau reduction in adulthood. Our findings support the effectiveness of tau reduction in adulthood and indicate that excitatory neurons may be a key site for its excitoprotective effects.
SUMMARY
A new conditional tau knockout model was generated to study the protective effects of tau reduction against hyperexcitability. Conditional tau reduction in excitatory, but not inhibitory, neurons was excitoprotective, and induced tau reduction in adulthood was excitoprotective without adverse effects.
... Stage IV: Forelimb clonus and rearing. Stage V: Severe tonic-clonic seizures [25]. ...
Current treatments of brain arteriovenous malformation (BAVM) are associated with considerable risks and at times incomplete efficacy. Therefore, a clinically consistent animal model of BAVM is urgently needed to investigate its underlying biological mechanisms and develop innovative treatment strategies. Notably, existing mouse models have limited utility due to heterogenous and untypical phenotypes of AVM lesions. Here we developed a novel mouse model of sporadic BAVM that is consistent with clinical manifestations in humans. Mice with BrafV600E mutations in brain ECs developed BAVM closely resembled that of human lesions. This strategy successfully induced BAVMs in mice across different age groups and within various brain regions. Pathological features of BAVM were primarily dilated blood vessels with reduced vascular wall stability, accompanied by spontaneous hemorrhage and neuroinflammation. Single-cell sequencing revealed differentially expressed genes that were related to the cytoskeleton, cell motility, and intercellular junctions. Early administration of Dabrafenib was found to be effective in slowing the progression of BAVMs; however, its efficacy in treating established BAVM lesions remained uncertain. Taken together, our proposed approach successfully induced BAVM that closely resembled human BAVM lesions in mice, rendering the model suitable for investigating the pathogenesis of BAVM and assessing potential therapeutic strategies.
... Thirty minutes before the penicillin injection, the mice in the different groups received either a saline injection (i.p.) or CNO (1 mg/kg, i.p.), depending on the specific experiment being conducted. The severity of behavioral seizures was evaluated using Racine's criteria (Racine, 1972), which included the following: (1) facial movement, (2) head nodding, (3) unilateral forelimb clonus, (4) bilateral forelimb clonus and rearing, and (5) rearing and falling. Seizure stages 1-3 was classified as focal seizures (FSs), and stages 4-5 were classified as general seizures (GSs). ...
... Rats were divided into 4 groups, 1) saline +4 % PEG 4000, 2) saline + DMP, 3) KA + 4 % PEG 4000, 4) KA + DMP. All rats with KA treatment were visually monitored for 6-8 h and behavioral seizures were scored using a modified Racine scale [38,39]. Cytokine release was measured at 24 h post SE, and GSH, GSSG markers, Western blotting and neuropathology were assessed 48 h post SE. ...
Glutathione (GSH) is a major endogenous antioxidant, and its depletion has been observed in several brain diseases including epilepsy. Previous studies in our laboratory have shown that dimercaprol (DMP) can elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme and inhibit neuroinflammation in vitro. Here we determined 1) the role of cysteamine as a new mechanism by which DMP increases GSH biosynthesis and 2) its ability to inhibit neuroinflammation and neuronal injury in the rat kainate model of epilepsy. DMP depleted cysteamine in a time- and concentration-dependent manner in a cell free system. To guide the in vivo administration of DMP, its pharmacokinetic profile was determined in the plasma, liver, and brain. The results confirmed DMP's ability to cross the blood-brain-barrier. Treatment of rats with DMP (30 mg/kg) depleted cysteamine in the liver and hippocampus that was associated with increased GCL activity in these tissues. GSH levels were significantly increased (20 %) in the hippocampus 1 h after 30 mg/kg DMP administration. Following DMP (30 mg/kg) administration once daily, a marked attenuation of GSH depletion was seen in the SE model. SE-induced inflammatory markers including cytokine release, microglial activation, and neuronal death were significantly attenuated in the hippocampus with DMP treatment. Taken together, these results highlight the importance of restoring redox status with rescue of GSH depletion by DMP in post epileptogenic insults.
... Seizure intensity was assessed using the modified Racine limbic seizure scale [28]: 1 point -twitching of jaw and facial muscles; 2 points -increase in facial clonus progressing to the trunk muscles; 3 points -clonic abbreviation of forelegs; 4 points -rearing (standing on hind legs with support on paws and tail); 5 points -rearing and falling to the side with clonic convulsions of extremities, generalization of seizures, and progression to tonic convulsions with complete loss of posture. Only seizures with a score of 4 or more and lasting for more than 15 min were identified as SE. ...
Status epilepticus (SE) triggers many pathological changes in the nervous system that are not yet fully understood and may lead to the development of epilepsy. In this work, we studied the effects of SE on the properties of excitatory glutamatergic transmission in the hippocampus in a rat model of lithium-pilocarpine temporal lobe epilepsy. Studies were performed 1 day (acute phase of the model), 3 and 7 days (latent phase), and 30 to 80 days (chronic phase) after SE. Using real-time PCR, we found that in the latent phase there is a decrease in gene expression of GluA1 and GluA2 AMPA receptor subunits, which may also be accompanied by an increased proportion of calcium-permeable AMPA receptors, which play an essential role in the pathogenesis of many CNS diseases. In acute brain slices we found a decrease in the efficiency of excitatory synaptic neurotransmission in all phases of the model when recording field responses in the CA1 region of the hippocampus in response to stimulation of Schaffer collaterals by electric currents of different intensities. However, in the chronic phase we found an increase in the frequency of spontaneous excitatory postsynaptic potentials, indicating an increased background activity of the glutamatergic system in epilepsy. This is also supported by a decrease in the threshold of hind limb extension in the test of maximal electroshock seizure in rats with temporal lobe epilepsy compared to control animals. The results obtained indicate the presence of a number of functional alterations in the glutamatergic system related to epilepsy. These findings can be used to develop antiepileptogenic therapy.
... group, rats were given similar injections, but with a lower dose of pilocarpine hydrochloride (38.5 mg/kg). Animals were observed individually, and the severity of seizures was recorded using the Racine scale [18]. Animals exhibiting generalized tonic-clonic seizures (Racine scale grade III or higher) were included. ...
Epilepsy is a chronic neurological disorder characterized by recurrent seizures. Despite various treatment approaches, a significant number of patients continue to experience uncontrolled seizures, leading to refractory epilepsy. The emergence of novel anti-epileptic drugs, such as perampanel (PER), has provided promising options for effective epilepsy treatment. However, the specific mechanisms underlying the therapeutic effects of PER remain unclear. This study aimed to investigate the intrinsic molecular regulatory mechanisms involved in the downregulation of GluA2, a key subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, following epileptic seizures. Primary mouse hippocampal neurons were cultured and subjected to an epilepsy cell model. The expression levels of GluA2 and autophagy-related proteins were assessed using Western blotting and real-time fluorescent quantitative PCR. Immunofluorescence and immunohistochemistry techniques were employed to investigate the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Additionally, status epilepticus animal models were established to further validate the findings. The epilepsy cell model exhibited a significant decrease in GluA2 expression, accompanied by elevated levels of autophagy-related proteins. Immunofluorescence analysis revealed the nuclear translocation of CRTC1, which correlated with the expression of autophagy-related genes. Treatment with an autophagy inhibitor reversed the decreased expression of GluA2 in the epilepsy cell model. Furthermore, the calcium/calmodulin-dependent protein phosphatase inhibitor FK506 and CaN overexpression affected the dephosphorylation and nuclear translocation of CRTC1, consequently influencing GluA2 expression. Animal model results further supported the involvement of these molecular mechanisms in epilepsy. Our findings suggest that the downregulation of GluA2 following epileptic seizures involves the activation of autophagy and the regulation of CRTC1 nuclear translocation. These intrinsic molecular regulatory mechanisms provide potential targets for developing novel therapeutic strategies to alleviate refractory epilepsy and preserve cognitive functions in patients.
... /2024 Seven-week-old male and female mice were anesthetized under isoflurane and unilaterally injected with 100-150 nl of sterile 0.9% saline or kainate (20 mM in 0.9% saline; Tocris Biosciences) into the dorsal hippocampus using the following stereotaxic coordinates relative to the bregma (anterior-posterior, -2.0 mm; medial-lateral, -1.25 mm; dorsal-ventral, -1.60 mm) as previously described (Kang et al., 2021). In order to minimize loss of mice caused by status epilepticus (>30 minutes of Racine stage 3-5 seizure), diazepam (7.5 mg/kg) was injected intraperitoneally 1 hour after the first Racine stage 5 seizure, characterized by tonic-clonic convulsions, rearing, running, jumping, and falling (Racine, 1972). Saline-injected mice (i.e., sham controls) also received intraperitoneal injections of diazepam. ...
Parvalbumin-positive (PV+) GABAergic interneurons in the dentate gyrus provide powerful perisomatic inhibition of dentate granule cells (DGCs) to prevent overexcitation and maintain the stability of dentate gyrus circuits. Most dentate PV+ interneurons survive status epilepticus, but surviving PV+ interneuron mediated inhibition is compromised in the dentate gyrus shortly after status epilepticus, contributing to epileptogenesis in temporal lobe epilepsy. It is uncertain whether the impaired activity of dentate PV+ interneurons recovers at later times or if it continues for months following status epilepticus. The development of compensatory modifications related to PV+ interneuron circuits in the months following status epilepticus is unknown, although reduced dentate GABAergic inhibition persists long after status epilepticus. We employed PV immunostaining and whole-cell patch-clamp recordings from dentate PV+ interneurons and DGCs in slices from male and female sham controls and intrahippocampal kainate (IHK) treated mice that developed spontaneous seizures months after status epilepticus to study epilepsy-associated changes in dentate PV+ interneuron circuits. We found that the number of dentate PV+ cells was reduced in IHK treated mice. Electrical recordings showed that: 1) Action potential firing rates of dentate PV+ interneurons were reduced in IHK treated mice up to four months after status epilepticus; 2) Spontaneous inhibitory postsynaptic currents (sIPSCs) in DGCs exhibited reduced frequency but increased amplitude in IHK treated mice; and 3) The amplitude of evoked IPSCs in DGCs by optogenetic activation of dentate PV+ cells was upregulated without changes in short-term plasticity. Video-EEG recordings revealed that IHK treated mice showed spontaneous epileptiform activity in the dentate gyrus and that chemogenetic activation of PV+ interneurons abolished the epileptiform activity. Our results suggest not only that the compensatory changes in PV+ interneuron circuits develop after IHK treatment, but also that increased PV+ interneuron mediated inhibition in the dentate gyrus may compensate for cell loss and reduced intrinsic excitability of dentate PV+ interneurons to stop seizures in temporal lobe epilepsy.
Objective
Given its key homeostatic role affecting mitochondria, ionotropic and metabotropic receptors, and voltage‐gated ion channels, sigma‐1 receptor (Sig1R) represents an interesting target for epilepsy management. Antiseizure effects of the positive allosteric modulator E1R have already been reported in acute seizure models. Although modulation of serotonergic neurotransmission is considered the main mechanism of action of fenfluramine, its interaction with Sig1R may be of additional relevance.
Methods
To further explore the potential of Sig1R as a target, we assessed the efficacy and tolerability of E1R and fenfluramine in two chronic mouse models, including an amygdala kindling paradigm and the intrahippocampal kainate model. The relative contribution of the interaction with Sig1R was analyzed using combination experiments with the Sig1R antagonist NE‐100.
Results
Whereas E1R exerted pronounced dose‐dependent antiseizure effects at well‐tolerated doses in fully kindled mice, only limited effects were observed in response to fenfluramine, without a clear dose dependency. In the intrahippocampal kainate model, E1R failed to influence electrographic seizure activity. In contrast, fenfluramine significantly reduced the frequency of electrographic seizure events and their cumulative duration. Pretreatment with NE‐100 reduced the effects of E1R and fenfluramine in the kindling model. Surprisingly, pre‐exposure to NE‐100 in the intrahippocampal kainate model rather enhanced and prolonged fenfluramine's antiseizure effects.
Significance
In conclusion, the kindling data further support Sig1R as an interesting target for novel antiseizure medications. However, it is necessary to further explore the preclinical profile of E1R in chronic epilepsy models with spontaneous seizures. Despite the rather limited effects in the kindling paradigm, the findings from the intrahippocampal kainate model suggest that it is of interest to further assess a possible broad‐spectrum potential of fenfluramine.
Objective
Deep brain stimulation (DBS) is a promising approach for the treatment of epilepsy. However, the optimal target for DBS and underlying mechanisms are still not clear. Here, we compared the therapeutic effects of DBS on distinct septal subregions, aimed to find the precise targets of septal DBS and related mechanisms for the clinical treatment.
Methods
Assisted by behavioral test, electroencephalography (EEG) recording and analyzing, selectively neuronal manipulation and immunohistochemistry, we assessed the effects of DBS on the three septal subregions in kainic acid (KA)‐induced mouse seizure model.
Results
DBS in the medial septum (MS) not only delayed generalized seizure (GS) development, but reduced the severity; DBS in the vertical diagonal band of Broca (VDB) only reduced the severity of GS, while DBS in the horizontal diagonal band of Broca (HDB) subregion showed no anti‐seizure effect. Notably, DBS in the MS much more efficiently decreased abnormal activation of hippocampal neurons. EEG spectrum analysis indicated that DBS in the MS and VDB subregions mainly increased the basal hippocampal low‐frequency (delta and theta) rhythm. Furthermore, ablation of cholinergic neurons in the MS and VDB subregions blocked the anti‐seizure and EEG‐modulating effects of septal DBS, suggesting the seizure‐alleviating effect of DBS was dependent on local cholinergic neurons.
Significance
DBS in the MS and VDB, rather than HDB, attenuates hippocampal seizure by activation of cholinergic neurons‐augmented hippocampal delta/theta rhythm. This may be of great therapeutic significance for the clinical treatment of epilepsy with septal DBS.
Plain Language Summary
The optical target of deep brain stimulation in the septum is still not clear. This study demonstrated that stimulation in the medial septum and vertical diagonal band of Broca subregions, but not the horizontal diagonal band of Broca, could alleviate hippocampal seizure through cholinergic neurons‐augmented hippocampal delta/theta rhythm. This study may shed light on the importance of precise regulation of deep brain stimulation therapy in treating epileptic seizures.
Curcumin, a compound derived from turmeric, is traditionally utilized in East Asian medicine for treating various health conditions, including epilepsy. Despite its involvement in numerous cellular signaling pathways, the specific mechanisms and targets of curcumin in epilepsy treatment have remained unclear. Our study focused on identifying the primary targets and functional pathways of curcumin in the brains of epileptic mice. Using drug affinity responsive target stabilization (DARTS) and affinity chromatography, we identified key targets in the mouse brain, revealing 232 and 70 potential curcumin targets, respectively. Bioinformatics analysis revealed a strong association of these proteins with focal adhesions and cytoskeletal components. Further experiments using DARTS, along with immunofluorescence staining and cell migration assays, confirmed curcumin’s ability to regulate the dynamics of focal adhesions and influence cell migration. This study not only advances our understanding of curcumin’s role in epilepsy treatment but also serves as a model for identifying therapeutic targets in neurological disorders.
Objectives
Pathological forms of neural activity, such as epileptic seizures, modify the expression pattern of multiple proteins, leading to persistent changes in brain function. One such protein is activity‐regulated cytoskeleton‐associated protein (Arc), which is critically involved in protein‐synthesis–dependent synaptic plasticity underlying learning and memory. In the present study, we have investigated how the expression of ArcKR, a form of Arc in which the ubiquitination sites have been mutated, resulting in slowed Arc degradation, modifies group I metabotropic glutamate receptor–mediated long‐term depression (G1‐mGluR‐LTD) following seizures.
Methods
We used a knock‐in mice line that express ArcKR and two hyperexcitation models: an in vitro model, where hippocampal slices were exposed to zero Mg ²⁺ , 6 mM K ⁺ ; and an in vivo model, where kainic acid was injected unilaterally into the hippocampus. In both models, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 region of hippocampal slices in response to Schaffer collateral stimulation and G1‐mGluR‐LTD was induced chemically with the group 1 mGluR agonist DHPG.
Results
In the in vitro model, ArcKR expression enhanced the effects of seizure activity and increased the magnitude of G1‐mGluR LTD, an effect that could be blocked with the mGluR5 antagonist MTEP. In the in vivo model, fEPSPs were significantly smaller in slices from ArcKR mice and were less contaminated by population spikes. In this model, the amount of G1‐mGluR‐LTD was significantly less in epileptic slices from ArcKR mice as compared to wildtype (WT) mice.
Significance
We have shown that expression of ArcKR, a form of Arc in which degradation is reduced, significantly modulates the magnitude of G1‐mGluR‐LTD following epileptic seizures. However, the effect of ArcKR on LTD depends on the epileptic model used, with enhancement of LTD in an in vitro model and a reduction in the kainate mouse model.
Despite research illustrating the cerebellum may be a critical circuit element in the epilepsies, remarkably little is known about cerebellar engagement during seizures. We therefore implemented a novel method for repeated imaging of the cerebellum in awake, chronically epileptic animals. We found widespread changes in cerebellar calcium signals during behavioral seizures and during hippocampal seizures that remained electrographic only, arguing against cerebellar modulation simply reflecting motor components. Moreover, even brief interictal spikes produced widespread alterations in cerebellar activity. Changes were noted in the anterior and posterior cerebellum, along the midline, and both ipsilaterally and contralaterally to the seizure focus. Remarkably, changes in the cerebellum also occurred prior to any noticeable change in the hippocampal electrographic recordings, suggesting a special relationship between the cerebellum and hippocampal epileptiform activity. Together these results underscore the importance of the cerebellum in epilepsy, warranting a more consistent consideration of the cerebellum when evaluating epilepsy patients.
Objective
The objective of this study is to determine whether inhibition of mitophagy affects seizures through Clathrin‐mediated endocytosis (CME).
Methods
Pentylenetetrazol (PTZ) was intraperitoneally injected daily to establish a chronic PTZ‐kindled seizure. The Western blot (WB) was used to compare the differences in Parkin protein expression between the epilepsy group and the control group. Immunofluorescence was used to detect the expression of MitoTracker and LysoTracker. Transferrin‐Alexa488 (Tf‐A488) was injected into the hippocampus of mice. We evaluated the effect of 3‐methyladenine (3‐MA) on epilepsy behavior through observation in PTZ‐kindled models.
Results
The methylated derivative of adenine, known as 3‐MA, has been extensively utilized in the field of autophagy research. The transferrin protein is internalized from the extracellular environment into the intracellular space via the CME pathway. Tf‐A488 uses a fluorescent marker to track CME. Western blot showed that the expression of Parkin was significantly increased in the PTZ‐kindled model ( p < 0.05), while 3‐MA could reduce the expression ( p < 0.05). The fluorescence uptake of MitoTracker and LysoTracker was increased in the primary cultured neurons induced by magnesium‐free extracellular fluid ( p < 0.05); the fluorescence uptake of Tf‐A488 was significantly decreased in the 3‐MA group compared with the control group ( p < 0.05). Following hippocampal injection of Tf‐A488, both the epilepsy group and the 3‐MA group exhibited decreased fluorescence uptake, with a more pronounced effect observed in the 3‐MA group. Inhibition of mitophagy by 3‐MA from day 3 to day 9 progressively exacerbated seizure severity and shortened latency.
Significance
It is speculated that the aggravation of seizures by 3‐MA may be related to the failure to remove damaged mitochondria in time and effectively after inhibiting mitochondrial autophagy, affecting the vesicle endocytosis function of CME and increasing the susceptibility to epilepsy.
Summary
Abnormal mitophagy was observed in a chronic pentylenetetrazol‐induced seizure model and a Mg ²⁺ ‐free‐induced spontaneous recurrent epileptiform discharge model. A fluorescent transferrin marker was utilized to track clathrin‐mediated endocytosis. Using an autophagy inhibitor (3‐methyladenine) on primary cultured neurons, we discovered that inhibition of autophagy led to a reduction in fluorescent transferrin uptake, while impairing clathrin‐mediated endocytosis function mediated by mitophagy. Finally, we examined the effects of 3‐methyladenine in an animal model of seizures showing that it exacerbated seizure severity. Ultimately, this study provides insights into potential mechanisms through which mitophagy regulates clathrin‐mediated endocytosis in epilepsy.
Recent studies have shown that neuroinflammation plays an important role in the pathogenesis of many nervous and mental diseases, such as cortical ischemia, craniocerebral trauma, neurodegenerative diseases, epilepsy, etc. Therefore, when recording EEG in experimental models of these diseases, it is preferable to use noninvasive recording methods to exclude neuroinflammation. However, such approaches are rarely used, since it is difficult to perform reliable EEG recording in animals without the use of implanted electrodes. In the present work a new device for minimally invasive wireless EEG recording in rats is proposed. The electrodes are located on the surface of the skull and are attached to a platform, which is fixed to the skull with screws. This design avoids damage to brain tissue. The surgery is minimally traumatic, and EEG registration can be performed as early as 2–3 days after surgery. High reliability of electrode attachment allows long-term registration. This method of EEG registration has been tested on a lithium-pilocarpine model of temporal lobe epilepsy. EEG recordings in experimental and control rats were made under background conditions and with the use of functional loads – rhythmic photo- and phonostimulation, as well as sleep deprivation. It was shown that these functional loads allow increasing the severity of epileptiform manifestations on the EEG (spike frequencies), the maximum differences between the groups being manifested with a combination of the above loads. Thus, the main feature of the proposed EEG recording device is that it makes it possible to perform prolonged EEG studies on a free-moving rat without the development of possible neuroinflammation. This device can be used in experiments to study epileptogenesis and to test new antiepileptic drugs on experimental animals.
Hyperbaric oxygen (HBO2) inhibits GABAergic neurotransmission in the brain, which can lead to the development of a seizure disorders known as “oxygen epilepsy”. Deficiency in GABAergic transmission in HBO2, resulting from a decrease in the level of synaptic GABA, can be compensated by inhibition of neuronal and glial GABA transporters (GAT). The present study compared the anticonvulsant efficacy of two types of GABA transporters with tiagabine, a GAT-1 inhibitor, and SNAP 5114, a GAT-3 inhibitor. Anticonvulsant effects were assessed after administration of drugs into the lateral cerebral ventricle of rats 30 min before the start of hyperbaric oxygen exposure at 5 ATA. In separate experiments, the concentration of GABA in the striatum of rats was measured when breathing oxygen at a pressure of 5 ATA after GAT inhibition with tiagabine or SNAP 5114. New results obtained in the study were: (1) inhibition of GAT-1 or GAT-3 prevented the development of “oxygen epilepsy” in rats; (2) among the two inhibitors used, TGB was found to be more effective in preventing oxygen convulsions compared to SNAP 5114; (3) the combined use of TGB + SNAP 5114 caused an additive anticonvulsant effect; (4) oxygen convulsions appeared when GABA in the brain decreased by 30–40% of the initial level; (5) GAT-1 inhibition with tiagabine increased extracellular GABA 2.9-fold and 1.7-fold with SNAP 5114. GAT-1 and GAT-3 inhibition increased GABA to a level sufficient to restore impaired inhibitory neurotransmission in HBO2, and prevented the development of hyperbaric oxygen convulsions.
Neuronal pentraxin 2 (Nptx2), a member of the synaptic protein family linked to excitatory synaptic formation, is found to be upregulated in epileptic mice, yet its role in epilepsy has been unclear. In vivo, we constructed a mouse model of epilepsy by using kainic acid induction. In vitro experiments, a Mg ²⁺ ‐free medium was used to induce epileptiform discharges in neurons. The results showed that the Nptx2 was upregulated in epileptic mice. Moreover, Nptx2 knockdown reduced the number of seizures and seizure duration. Knocking down Nptx2 not only reduced the number and duration of seizures but also showed a decrease in electroencephalogram amplitude. Behavioral tests indicated improvements in learning and memory abilities after Nptx2 knockdown. The Nissl staining and Timms staining revealed that Nptx2 silencing mitigated epilepsy‐induced brain damage. The immunofluorescence staining revealed that Nptx2 absence resulted in a reduction of apoptosis. Nptx2 knockdown reduced Bax, cleaved caspase3, and cleaved caspase9 expression, while increased Bcl‐2 expression. Notably, Nptx2 knockdown inhibited GluA1 phosphorylation at the S831 site and reduced the GluA1 membrane expression. The PSD95 expression declined in the epilepsy model, while the Nptx2 knockdown reversed it. Collectively, our study indicated that Nptx2 silencing not only alleviated brain damage and neuron apoptosis but also improved learning and memory ability in epileptic mice, suggesting Nptx2 as a promising target for epilepsy treatment.
Brain disturbances during development can have a lasting impact on neural function and behavior. Seizures during this critical period are linked to significant long-term consequences such as neurodevelopmental disorders, cognitive impairments, and psychiatric symptoms, resulting in a complex spectrum of multimorbidity. The hippocampus-prefrontal cortex (HPC-PFC) circuit emerges as a potential common link between such disorders. However, the mechanisms underlying these outcomes and how they relate to specific behavioral alterations are unclear. We hypothesized that specific dysfunctions of hippocampal-cortical communication due to early-life seizure would be associated with distinct behavioral alterations observed in adulthood. Here, we performed a multilevel study to investigate behavioral, electrophysiological, histopathological, and neurochemical long-term consequences of early-life Status epilepticus in male rats. We show that adult animals submitted to early-life seizure (ELS) present working memory impairments and sensorimotor disturbances, such as hyperlocomotion, poor sensorimotor gating, and sensitivity to psychostimulants despite not exhibiting neuronal loss. Surprisingly, cognitive deficits were linked to an aberrant increase in the HPC-PFC long-term potentiation (LTP) in a U-shaped manner, while sensorimotor alterations were associated with heightened neuroinflammation, as verified by glial fibrillary acidic protein (GFAP) expression, and altered dopamine neurotransmission. Furthermore, ELS rats displayed impaired HPC-PFC theta-gamma coordination and an abnormal brain state during active behavior resembling rapid eye movement (REM) sleep oscillatory dynamics. Our results point to impaired HPC-PFC functional connectivity as a possible pathophysiological mechanism by which ELS can cause cognitive deficits and psychiatric-like manifestations even without neuronal loss, bearing translational implications for understanding the spectrum of multidimensional developmental disorders linked to early-life seizures.
Interneuron loss is a prominent feature of temporal lobe epilepsy in both animals and humans and is hypothesized to be critical for epileptogenesis. As loss occurs concurrently with numerous other potentially pro-epileptogenic changes, however, the impact of interneuron loss in isolation remains unclear. For the present study, we developed an intersectional genetic approach to induce bilateral diphtheria toxin-mediated deletion of Vgat-expressing interneurons from dorsal and ventral hippocampus. In a separate group of mice, the same population was targeted for transient neuronal silencing with DREADDs. Interneuron ablation produced dramatic seizure clusters and persistent epileptiform activity. Surprisingly, after one week seizure activity declined precipitously and persistent epileptiform activity disappeared. Occasional seizures (≈1/day) persisted to the end of the experiment at four weeks. In contrast to the dramatic impact of interneuron ablation, transient silencing produced large numbers of interictal spikes, a significant but modest increase in seizure occurrence and changes in EEG frequency band power. Taken together, findings suggest that the hippocampus regains relative homeostasis – with occasional breakthrough seizures – in the face of an extensive and abrupt loss of interneurons.
Significance Statement Interneuron loss is hypothesized to play a critical role in epileptogenesis, however, the co-occurrence of interneuron loss with other potentially epileptogenic changes has made assigning causal relationships challenging. Here, we utilized an intersectional genetic approach to delete hippocampal interneurons. Treatment produced robust – but transient – seizure clusters in the animals followed by a relative recovery with infrequent seizures. Findings support a critical role for interneuron loss in epileptogenesis, but also imply the existence of mechanisms that can rapidly restore excitatory/inhibitory balance in the brain.
Nuclear speckles, also known as interchromatin granule clusters (IGCs), are subnuclear domains highly enriched in proteins involved in transcription and mRNA metabolism and, until recently, have been regarded primarily as their storage and modification hubs. However, several recent studies on non-neuronal cell types indicate that speckles may directly contribute to gene expression as some of the active genes have been shown to associate with these structures.
Neuronal activity is one of the key transcriptional regulators and may lead to the rearrangement of some nuclear bodies. Notably, the impact of neuronal activation on IGC/nuclear speckles organization and function remains unexplored. To address this research gap, we examined whether and how neuronal stimulation affects the organization of these bodies in granular neurons from the rat hippocampal formation. Our findings demonstrate that neuronal stimulation induces morphological and proteomic remodelling of the speckles under both in vitro and in vivo conditions. Importantly, these changes are not associated with cellular stress or cell death but are dependent on transcription and splicing.
In this study, the role of adenosine A1 receptors of the hippocampal CA1 region in entorhinal cortex‐kindled seizures was investigated in rats. Animals were kindled by daily electrical stimulation of the entorhinal cortex. In fully kindled rats, N ⁶ ‐cyclohexyladenosine (CHA; a selective A1 receptor agonist) and 1, 3‐dimethyl‐8‐cyclopenthylxanthine (CPT; a selective A1 receptor antagonist) were microinfused bilaterally into the hippocampal CA1 region. Rats were stimulated and seizure parameters were measured. Results obtained showed that CHA (10 and 50 μ moles) decreased the afterdischarge duration (ADD) in the hippocampal CA1 region and entorhinal cortex, stage 5 seizure duration (S5D) and seizure duration (SD) only at the dose of 50 μ moles, and significantly increased the latency to stage 4 (S4L). Intrahippocampal CPT increased ADD and S5D, and significantly reduced the latency to stage 4 (S4L) at the dose of 10 lmoles. Pretreatment of rats with CPT (5 μ moles) before CHA (50 μ moles), significantly reduced the effect of CHA on seizure parameters. The results suggest that the CA1 region of the hippocampus plays an important role in spreading seizure spikes from the entorhinal cortex to other brain regions and activation of adenosine A1 receptors in this region participates in the anticonvulsant effects of adenosine agonists.
In unanesthetized cats, with permanent intracerebral electrodes, as many as 100 repeated hippocampal after-discharges (HAD's) were evoked during a period of 15–30 days. In addition, four cats were trained in conditioned avoidance, and the tests were repeated during HAD's. In other cats, after several HAD's had been evoked, ablations were made of the motor areas, fornix and amygdala, and then the HAD's were studied. Results were as follows: 1.1. Repetition of HAD's did not modify local thresholds, but considerably increased the behavioral disturbances according to a pattern which was repeated in different animals.2.2. Conditioned avoidance response was very little impaired during the first 10 sec of the evoked HAD, but was considerably disturbed at the height of the HAD. Repetition of the HAD's decreased the responses even during the first 10 sec of each HAD, with responses dropping to zero at the height of the HAD. Controls, however, showed that repetition of the HAD's did not produce lasting deficits in avoidance performance. Hippocampal stimulations which were not followed by HAD's did not modify the conditioned responses.3.3. During HAD's, thresholds and effects evoked by stimulation of the reticular mesencephalic substance were not modified. Hypothalamic thresholds were also kept constant. The septum excitability increased with a drop in threshold to about half of the control values, without modification of the pattern of response.4.4. Ablation of the motor areas had no effect on the electrical or clinical pattern of the HAD. Bilateral destruction of the fornix did not modify the clinical symptomatology, but affected the electrical pattern of the HAD's. After unilateral destruction of the amygdala, the ipsilateral facial motor manifestation which accompanied the HAD's disappeared, and did not return even during the generalized seizure produced after many repeated HAD's.5.5. Repetition of the seizures increased the duration of the HAD's two to five times, producing also an increase in the duration of “clonic” activity and short, irregular spikes. In general, however, the configuration of each pattern was not modified.6.6. A clear correlation was established between motor participation of the face and 4–6 c/sec activity of the amygdala. Other possible clinical EEG correlations are discussed in this paper.7.7. Two different classes of spread from the hippocampus to the amygdala are considered: (a) propagated: in which the pattern of the amygdala after-discharge corresponds to and seems to depend on the pattern of the hippocampal seizure; (b) reactive: in which the amygdala showed 4–6 c/sec waves, which were independent of, but triggered by HAD's.The propagated spread was asymptomatic while the reactive spread was associated with ipsilateral motor manifestations of the face. Similar examples have previously been recorded in other parts of the limbic system of the monkey, and the distinction between the two kinds of spread may have functional and perhaps diagnostic implications.
Rats with multiple recording and stimulating electrodes were stimulated electrically in the amygdala, hippocampus and reticular formation. Intensity of stimulation was varied in a systematic way to determine the threshold at which after-discharges (ADs) were produced in the vicinity of the stimulating electrode. AD thresholds were permanently reduced by 40–60% in the amygdala and 25% in the hippocampus by daily 1 sec bursts of stimulation. The reduction took place with subthreshold as well as suprathreshold stimulation, but not in the absence of stimulation.It was found that the reduction of AD thresholds in the amygdala had no effect on AD thresholds in the contralateral amygdala, septal area or hippocampus. Reduction of AD thresholds in the hippocampus, however, resulted in an increase in the AD threshold in the contralateral hippocampus.RésuméDes rats porteurs d'électrodes de stimulation et d'enregistrement multiples ont subi une stimulation électrique de l'amygdale, de l'hippocampe et de la formation réticulaire. L'intensité de la stimulation varie de façon systématique afin de déterminer le seuil auquel les post-décharges sont produites dans le voisinage de l'électrode de stimulation. Les seuils de post-décharge sont en permanence abaissés de 40 à 60% dans l'amygdale et de 25% dans l'hippocampe par des bouffées quotidiennes de stimulation durant une seconde. Cet abaissement survient aussi bien pour des stimulations sous-liminaires que sus-liminaires, mais non en l'absence de stimulation.L'auteur observe que la réduction des seuils de post-décharges dans l'amygdale n'a aucum effet sur les seuils de post-décharge dans l'amygdale controlatérale, la région septale ou l'hippocampe. L'abaissement des seuils de post-décharges dans l'hippocampe, par contre, provoque une augmentation du seuil de post-décharge dans l'hippocampe controlatéral.
Intracellular recordings were made from both pyramidal tract cells and non-pyramidal tract cells is the motor cortex of the cat. The responses elecited by single stimuli to the cortical surface or to the medullary pyramid were found to change during and after electrically induced seizures.Similar slow changes in the membrane potentials during and after seizures occurred in both types of cells but spikes superimposed on the smooth or oscillatory depolarization were more frequently observed in pyramidal tract elements.In these, antidromic spikes could be elicited during and after a seizure except when either marked depolarization occurred or when the stimulus fell in the refractory period.Responses characterized by a depolarizing wave (EPSP) could be generated by single stimuli during and after a seizure except towards the termination of the seizure.Responses characterized by a hyperpolarizing wave (IPSP) were never obtained by single shocks during and immediately after a seizure.The development, maintenance and termination of experimental epileptic seizure have been discussed on the basis of the above results.
The pattern of cortical after-discharge duration has been investigated in an acute, paralyzed cat preparation. In all animals the ectosylvian cortex was stimulated with 5 sec trains of 0.5 msec pulses of threshold voltage. Two frequencies of stimulation (25 and 50/sec) and two inter-period intervals (5 and 30 min) were used. However, in any given animal only one frequency and one inter-period interval was utilized. Thus, four groups of animals were studied. There was no statistically significant difference in the threshold voltage or the seizure duration of those animals stimulated at 25/sec as compared to those stimulated at 50/sec at either interval period. In each of the 4 groups, there was a significant difference among the seizure duration over the periods. There was considerable variability in the after-discharge duration both between animals and within the same animal.
Brief bursts of nonpolarizing electrical brain stimulation were presented once each day at constant intensity. At first the stimulation had little effect on behavior and did not cause electrographic afterdischarge. With repetition the response to stimulation progressively changed to include localized seizure discharge, behavioral automatisms and, eventually, bilateral clonic convulsions. Thereafter, the animal responded to each daily burst of stimulation with a complete convulsion. The effect was obtained from bipolar stimulation of loci associated with the limbic system, but not from stimulation of many other regions of the brain. Parametric studies and control observations revealed that the effect was due to electrical activation and not to tissue damage, poison, edema, or gliosis. The changes in brain function were shown to be both permanent and trans-synaptic in nature. Massed-trial stimulation, with short inter-burst intervals, rarely led to convulsions. The number of stimulation trials necessary to elicit the first convulsion decreased as the interval between trials approached 24 hours. Further increase in the inter-trial interval had little effect on the number of trials to first convulsion. High-intensity stimulation studies revealed that the development of convulsions was not based simply on threshold reduction, but involved complex reorganization of function. Experiments with two electrodes in separate parts of the limbic system revealed that previously established convulsions could facilitate the establishment of a second convulsive focus, but that the establishment of this second convulsive focus partially suppressed the otherwise permanent convulsive properties of the original focus.
After establishing an epileptogenic “focus” in the cat's cerebral cortex, an intracellular analysis from the involved elements was carried out during the development and course of the organized rhythmical electrographic seizures simultaneously monitored with surface electrodes. Neurons were classified on the basis of their behavior as active or passive. In the former, changes in membrane polarization were observed which characterized the various phases of the ictal episode. The previously present paroxysmal hyperpolarization shifts disappear at the onset and a prominent afterdepolarization develops. During the tonic phase the membrane potential markedly decreases and rhythmical oscillations with (or rerely without) action potentials appear above a sustained excessively depolarized membrane potential level. The clonic phase corresponds to a slow repolarization process and the end of the episode seems to be due to inactivation rather than to true membrane hyperpolarization. Some neurons appear to be activated only in the later phases of the seizure.