A patient-derived mutation of epilepsy-linked LGI1 increases seizure susceptibility through regulating Kv1.1.

BACKGROUND: Autosomal dominant lateral temporal epilepsy (ADLTE) is an inherited syndrome caused by mutations in the leucine-rich glioma inactivated 1 (LGI1) gene. It is known that functional LGI1 is secreted by excitatory neurons, GABAergic interneurons, and astrocytes, and regulates AMPA-type glutamate receptor-mediated synaptic transmission by binding ADAM22 and ADAM23. However, > 40 LGI1 mutations have been reported in familial ADLTE patients, more than half of which are secretion-defective. How these secretion-defective LGI1 mutations lead to epilepsy is unknown. RESULTS: We identified a novel secretion-defective LGI1 mutation from a Chinese ADLTE family, LGI1-W183R. We specifically expressed mutant LGI1W183R in excitatory neurons lacking natural LGI1, and found that this mutation downregulated Kv1.1 activity, led to neuronal hyperexcitability and irregular spiking, and increased epilepsy susceptibility in mice. Further analysis revealed that restoring Kv1.1 in excitatory neurons rescued the defect of spiking capacity, improved epilepsy susceptibility, and prolonged the life-span of mice. CONCLUSIONS: These results describe a role of secretion-defective LGI1 in maintaining neuronal excitability and reveal a new mechanism in the pathology of LGI1 mutation-related epilepsy.

Prolonged febrile seizures induce inheritable memory deficits in rats through DNA methylation.

AIMS: Febrile seizures (FSs) are the most common types of seizures in young children. However, little is known whether the memory deficits induced by early-life FSs could transmit across generations or not. METHODS: The memory functions of different generations of FS rats were behaviorally evaluated by morris water maze, inhibitory avoidance task, and contextual fear conditioning task. Meanwhile, molecular biology and pharmacological methods were used to investigate the role of DNA methylation in transgenerational transmission of memory defects. RESULTS: Prolonged FSs in infant rats resulted in memory deficits in adult and transgenerationally transmitted to next generation, which was mainly through mothers. For these two generations, DNA methyltransferase (DNMT) 1 was upregulated, leading to transcriptional inhibition of the synaptic plasticity protein reelin but not the memory suppressor protein phosphatase 1. DNMT inhibitors prevented the high expression of DNMT1 and hypermethylation of reelin gene and reversed the transgenerationally memory deficits. In addition, enriched environment in juvenile rats rescued memory deficits induced by prolonged FSs. CONCLUSIONS: Our study demonstrated early experience of prolonged FSs led to memory deficits in adult rats and their unaffected offspring, which involved epigenetic mechanisms, suggesting early environmental experiences had a significant impact on the transgenerational transmission of neurological diseases.

Correction to: Gender difference in acquired seizure susceptibility in adult rats after early complex febrile seizures.

In the original publication of the article, the representative EEG of female rat pups with FS in Figure 1 C and D was incorrectly intercepted from that of male rat pups. This correction does not affect the conclusions of the paper. Figure 1 has been corrected on the online PDF version and displayed below.

Early hypoactivity of hippocampal rhythms during epileptogenesis after prolonged febrile seizures in freely-moving rats.

Prospective and experimental studies have shown that individuals with early-life complex/prolonged febrile seizures (FSs) have a high incidence of temporal lobe epilepsy during adulthood, revealing a close relationship between FSs and epilepsy. However, little is known about how epileptogenesis develops after FSs. The present study was designed to investigate acquired seizure susceptibility and analyze local field potentials during the latent period after FSs. We found that the seizure susceptibility decreased in 35-day-old (P35) FS rats but increased in P60 FS rats. Consistently, hippocampal electroencephalogram (EEG) power in every band was decreased at P35 but increased at P60 in FS rats. Our results provide direct evidence for hypoactivity but not hyperactivity during the early phase of the latent period, displaying a broad decrease in hippocampal rhythms. These characteristic EEG changes can be a useful biomarker for the early diagnosis of epileptogenesis induced by FSs.

Protective effect of carnosine on febrile seizures in immature mice.

Febrile seizures (FSs) are the most common type of convulsions in childhood and complex FSs represent an increased risk for development of temporal lobe epilepsy. The aim of this study was to analyze the anticonvulsant effects of carnosine, an endogenous dipeptide composed of alanine and histidine, on hyperthermia induced seizure in immature mice. Injection of carnosine significantly increased the latency and decreased the duration of FSs in a dose-dependent manner. In addition, histidine had similar effects on FSs as carnosine. The protective effect of carnosine or histidine was completely abolished by α-fluoromethylhistidine (α-FMH), a selective and irreversible histidine decarboxylase inhibitor, or in histidine decarboxylase deficient (HDC-KO) mice. Peripheral carnosine administration increased the level of carnosine, histidine and histamine in the cortex and hippocampus of mice pups, but decreased glutamate contents in the cortex and hippocampus. These results indicate that carnosine can protect against FSs in mice pups through its conversion to histamine, suggesting that it may serve as an efficient anti-FSs drug in the future.

Gender difference in acquired seizure susceptibility in adult rats after early complex febrile seizures.

Gender differences are involved in many neurological disorders including epilepsy. However, little is known about the effect of gender difference on the risk of epilepsy in adults with a specific early pathological state such as complex febrile seizures (FSs) in infancy. Here we used a well-established complex FS model in rats and showed that: (1) the susceptibility to seizures induced by hyperthermia, pentylenetetrazol (PTZ), and maximal electroshock (MES) was similar in male and female rat pups, while males were more susceptible to PTZ- and MES-induced seizures than age-matched females in normal adult rats; (2) adult rats with complex FSs in infancy acquired higher seizure susceptibility than normal rats; importantly, female FS rats were more susceptible to PTZ and MES than male FS rats; and (3) the protein expression of interleukin-1ß, an inflammatory factor associated with seizure susceptibility, was higher in adult FS females than in males, which may reflect a gender-difference phenomenon of seizure susceptibility. Our results provide direct evidence that the acquired seizure susceptibility after complex FSs is gender-dependent.

Apigenin has anti-atrophic gastritis and anti-gastric cancer progression effects in Helicobacter pylori-infected Mongolian gerbils.

ETHNOPHARMACOLOGICAL RELEVANCE: Apigenin, one of the most common flavonoids, is abundant in celery, parsley, chamomile, passionflower, and other vegetables and fruits. Celery is recognized as a medicinal vegetable in Oriental countries to traditionally treat inflammation, swelling, blood pressure, serum lipid, and toothache. In this study, we investigated apigenin treatment effects on Helicobacter pylori-induced atrophic gastritis and gastric cancer progression in Mongolian gerbils. MATERIALS AND METHODS: Five to eight-week-old Mongolian gerbils were inoculated with Helicobacter pylori for four weeks without (atrophic gastritis group) or with N'-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (gastric cancer group) in drinking water, and were then rested for two weeks. During the 7th-32th (atrophic gastritis group) or the 7th-52th (gastric cancer group) weeks, they were given various doses (0-60 mg/kgbw/day) of apigenin. At the end of the 32th (atrophic gastritis group) or the 52th (atrophic gastritis group) week, all Mongolian gerbils were sacrificed using the CO2 asphyxia method. The histological changes of Helicobacter pylori colonization, neutrophil and monocyte infiltrations, and atrophic gastritis in both atrophic gastritis and gastric cancer Mongolian gerbils were examined using immunohistochemistry stain and Sydney System scoring. RESULTS: Apigenin treatments (30-60 mg/kgbw/day) effectively decreased atrophic gastritis (atrophic gastritis group) and dysplasia/gastric cancer (gastric cancer group) rates in Mongolian gerbils. Apigenin treatment (60 mg/kgbw/day) significantly decreased Helicobacter pylori colonization and Helicobacter pylori-induced histological changes of neutrophil and monocyte infiltrations and atrophic gastritis in both atrophic gastritis and gastric cancer Mongolian gerbils. CONCLUSIONS: Apigenin has the remarkable ability to inhibit Helicobacter pylori-induced atrophic gastritis and gastric cancer progression as well as possessing potent anti-gastric cancer activity.

A Transient Upregulation of Glutamine Synthetase in the Dentate Gyrus Is Involved in Epileptogenesis Induced by Amygdala Kindling in the Rat.

Reduction of glutamine synthetase (GS) function is closely related to established epilepsy, but little is known regarding its role in epileptogenesis. The present study aimed to elucidate the functional changes of GS in the brain and its involvement in epileptogenesis using the amygdala kindling model of epilepsy induced by daily electrical stimulation of basolateral amygdala in rats. Both expression and activity of GS in the ipsilateral dentate gyrus (DG) were upregulated when kindled seizures progressed to stage 4. A single dose of L-methionine sulfoximine (MSO, in 2 µl), a selective GS inhibitor, was administered into the ipsilateral DG on the third day following the first stage 3 seizure (just before GS was upregulated). It was found that low doses of MSO (5 or 10 µg) significantly and dose-dependently reduced the severity of and susceptibility to evoked seizures, whereas MSO at a high dose (20 µg) aggravated kindled seizures. In animals that seizure acquisition had been successfully suppressed with 10 µg MSO, GS upregulation reoccurred when seizures re-progressed to stage 4 and re-administration of 10 µg MSO consistently reduced the seizures. GLN at a dose of 1.5 µg abolished the alleviative effect of 10 µg MSO and deleterious effect of 20 µg MSO on kindled seizures. Moreover, appropriate artificial microRNA interference (1 and 1.5×10(6) TU/2 µl) of GS expression in the ipsilateral DG also inhibited seizure progression. In addition, a transient increase of GS expression and activity in the cortex was also observed during epileptogenesis evoked by pentylenetetrazole kindling. These results strongly suggest that a transient and region-specific upregulation of GS function occurs when epilepsy develops into a certain stage and eventually promotes the process of epileptogenesis. Inhibition of GS to an adequate degree and at an appropriate timing may be a potential therapeutic approach to interrupting epileptogenesis.

Wide therapeutic time-window of low-frequency stimulation at the subiculum for temporal lobe epilepsy treatment in rats.

Low-frequency stimulation (LFS) has been considered as an option for the treatment of intractable epilepsy. However, previous data showed that LFS of certain brain regions only exerts its effect within a very narrow therapeutic time window, which lasts from seconds to tens of seconds, thus restricting its clinical application. The present study was designed to determine whether there exists a target with a wider therapeutic window for LFS treatment. Therefore, evoked seizures in the rat were induced by amygdala kindling and spontaneous seizures were induced by pilocarpine. The effects of different modes of LFS at the subiculum on the progression and severity of evoked seizures and the frequency of spontaneous seizure were evaluated. We found that (i) LFS at 1Hz delivered to the subiculum before and immediately after the kindling stimulations, or after the cessation of afterdischarge (afterdischarge duration, ADD) decreased the seizure stages and shortened the ADD both in seizure acquisition and expression in amygdaloid-kindled seizures. In addition, even LFS delivered after duration of double the ADD prolonged the kindling progression. (ii) LFS delivered at 1Hz, but not 0.5, 3 or 130Hz, immediately after the cessation of kindling stimulations retarded the progression of kindling seizures. (iii) Pilocarpine-induced spontaneous seizures were completely inhibited by 1Hz LFS. Thus, these results demonstrated that LFS of the subiculum has a wide therapeutic time-window for temporal lobe epilepsy treatment in rats, suggesting that the subiculum may be a promising and suitable target for clinical application.

Therapeutic time window of low-frequency stimulation at entorhinal cortex for amygdaloid-kindling seizures in rats.

The present study was designed to determine whether low-frequency stimulation (LFS) of the entorhinal cortex(EC) has an anticonvulsive effect, and whether LFS delivered at different times plays different roles. We found that LFS of the EC immediately or 4 s after kindling stimulation had an anticonvulsive effect, and that the latter had a better effect on both kindling and kindled seizures. However, LFS delivered after the cessation of afterdischarge or 10 s after the kindling stimulation, augmented the epileptic activity. So the EC is a potential target for LFS to interfere with epilepsy. Our findings suggest that even in the duration of afterdischarge, there exists a "time window" for LFS treatment, indicating that the time delay of closed-loop stimulation is crucial for LFS treatment.

Mediodorsal thalamic stimulation is not protective against seizures induced by amygdaloid kindling in rats.

Deep brain stimulation (DBS) is now emerging as a new option for treating intractable epilepsy. Cumulative studies suggest that the mediodorsal thalamic nucleus (MD) is involved in limbic seizure activity. This study aims to investigate whether DBS of the MD can protect against seizures induced by amygdaloid kindling. We studied the effect of low-frequency stimulation (LFS, 1 Hz) or high-frequency stimulation (HFS, 100 Hz) in the MD on amygdaloid kindling seizures. During the kindling acquisition, DBS in the MD was daily administered immediately after the kindling stimulus or before the kindling stimulus (preemptive DBS). The effects of both post-treatment of DBS and preemptive DBS in the MD on the expression of amygdaloid kindling seizures were evaluated. We found the DBS or preemptive DBS in the MD, either LFS or HFS, did not significantly change the rate of amygdaloid kindling. Similarly, DBS or preemptive DBS in the MD did not significantly change any parameters representing the expression of amygdaloid kindling. Our study suggests that DBS in the MD may have no significant effect on limbic seizures.

Mode-dependent effect of low-frequency stimulation targeting the hippocampal CA3 subfield on amygdala-kindled seizures in rats.

Brain stimulation with low-frequency stimulation (LFS) is emerging as an alternative treatment for refractory epilepsy. The present study aimed to investigate the effects of LFS targeting the hippocampal CA3 subfield in different modes on amygdala-kindled seizures in Sprague-Dawley rats. When fully kindled seizures were achieved by daily electrical stimulation of the amygdala, LFS (15 min train of 0.1 ms pulses at 1 Hz and 100 microA) of the CA3 was applied in several modes. Post-treatment with LFS significantly reduced the severity of and susceptibility to evoked seizures, whereas pre-treatment with LFS resulted in a similar but much weaker inhibition of seizures. Interestingly, prior consecutive daily application of LFS in the absence of kindling stimulation did not reduce subsequent evoked seizures, but abolished the anti-epileptic effect of post-treatment. These results indicated that LFS of the CA3 is able to reduce kindled seizures in a mode-dependent manner without cumulative feature. The hippocampal CA3 subfield could be considered as a potential target for epilepsy treatment using LFS, and should be delivered in an appropriate stimulation mode.

In vitro activity of Impatiens balsamina L. against multiple antibiotic-resistant Helicobacter pylori.

Impatiens balsamina L. has been used as indigenous medicine in Asia for the treatment of rheumatism, fractures, and fingernail inflammation. In this study, anti-H. pylori activity of I. balsamina L. was investigated. The MICs, MBCs, time-kill assay, and effect of environmental pH for the plant extracts were determined. The test H. pylori strains have resistance to clarithromycin (CLR), metronidazole (MTZ), and levofloxacin (LVX). From our results, all part (root/stem/leaf, seed, and pod) extracts of I. balsamina L. exhibited bactericidal H. pylori activity. Specifically, the pod extract had significantly lower MICs and MBCs (1.25-2.5 and 1.25-5.0 microg/ml, respectively). Of the five pod-extraction solvents, both ethyl acetate and acetone were the most efficient for the anti-H. pylori compounds of the pod extraction. The dose-dependency of the pod extract's bactericidal activity was H. pylori strain-dependent. Bactericidal H. pylori activity of the pod extract was not affected by the environmental pH (2-8). In summary, the acetone and ethyl acetate pod extracts of I. balsamina L. exhibited very strong anti-H. pylori activity. This activity exceeded that of MTZ and approximated to that of AMX.

Low-frequency stimulation of the hippocampal CA3 subfield is anti-epileptogenic and anti-ictogenic in rat amygdaloid kindling model of epilepsy.

Neuromodulation with low-frequency stimulation (LFS), of brain structures other than epileptic foci, is effective in inhibiting seizures in animals and patients, whereas selection of targets for LFS requires further investigation. The hippocampal CA(3) subfield is a key site in the circuit of seizure generation and propagation. The present study aimed to illustrate the effects of LFS of the CA(3) region on seizure acquisition and generalization in the rat amygdaloid kindling model of epilepsy. We found that LFS (monophasic square-wave pulses, 1Hz, 100 microA and 0.1ms per pulse) of the CA(3) region significantly depressed the duration of epileptiform activity and seizure acquisition by retarding progression from focal to generalized seizures (GS). Moreover, GS duration was significantly shortened and its latency was significantly increased in the LFS group demonstrating an inhibition of the severity of GS and the spread of epileptiform activity. Furthermore, LFS prevented the decline of afterdischarge threshold (ADT) and elevated GS threshold indicating an inhibition of susceptibility to GS. These results suggest that LFS of the hippocampal CA(3) subfield is anti-epileptogenic and anti-ictogenic. Neuromodulation of CA(3) activity using LFS may be an alternative potential approach for temporal lobe epilepsy treatment.

Low-frequency stimulation of the tuberomammillary nucleus facilitates electrical amygdaloid-kindling acquisition in Sprague-Dawley rats.

Histamine plays a suppressive role in seizure. The tuberomammillary nucleus (TM) is the only locus of histaminergic neurons in the brain. To determine whether deep brain stimulation (DBS) of the TM provides protection against seizures, we tested the effects of low-frequency stimulation (LFS, 1 Hz), high frequency stimulation (HFS, 100 Hz), and electrolytic lesions of the TM on seizures generated by amygdaloid kindling, pentylenetetrazol (PTZ) and maximal electroshock (MES) in rats. LFS of TM accelerated the progression of behavioral seizure stage and increased the mean afterdischarge duration (ADD) during acquisition of amygdaloid-kindling seizures, but had no considerable anticonvulsive effect in fully kindled animals. It augmented the MES-induced seizures as well, but had no appreciable effects on PTZ-kindled seizures. In addition, both HFS and bilateral lesions of the TM exacerbated the progression of amygdaloid-kindling seizures. These results suggest that specific negative sites for DBS exist in the brain, such as the TM. This study indicates that it is crucial to choose a suitable target for DBS in the clinical treatment of epilepsy.

Time-dependent effect of low-frequency stimulation on amygdaloid-kindling seizures in rats.

Low-frequency stimulation (LFS) has been considered as a new option for the treatment of intractable epilepsy. The present study was designed to determine whether LFS of the kindling focus given at different time points after seizures exert different roles on kindling seizures. Our results showed that: (i) In kindling animals, LFS delivered immediately after cessation of the kindling stimulus inhibited the seizure stage during kindling acquisition, whereas LFS delivered after the cessation of afterdischarge accelerated the kindling progression to stages 1 and 2. (ii) In fully kindled animals, when using the generalized seizure threshold current as the kindling stimulus, immediate LFS decreased the incidence of generalized seizures and the average seizure stage as well as shortened the cumulative generalized seizure duration (GSD). However, delayed LFS prolonged the cumulative GSD and afterdischarge duration. Our study indicates that there is a time-dependent aspect of LFS treatment, and immediate LFS has anti-epileptogenic action.

Low-frequency stimulation of cerebellar fastigial nucleus inhibits amygdaloid kindling acquisition in Sprague-Dawley rats.

Low-frequency stimulation (LFS) of the kindling focus or the piriform cortex inhibits kindling epileptogenesis, but whether LFS of brain targets outside the limbic system has anticonvulsive actions remain unknown. The current study was designed to investigate the effect of LFS of the cerebellar fastigial nucleus (FN) on seizure progression induced by amygdaloid kindling. Stimulation at 1 Hz (15-min train of 0.1-ms pulses, 100 muA), but not at 3 Hz, in the ipsilateral FN immediately after the daily kindling stimulus (1-s train of 1-ms pulses at 60 Hz and 100-300 muA) significantly inhibited the seizure stage and afterdischarge duration in kindling acquisition. Neither 1 Hz nor 3 Hz stimulation of the contralateral FH had any significant effect. It is interesting that delaying delivery (immediately after the cessation of afterdischarge) of LFS in the ipsilateral FN accelerated the rate of kindling acquisition compared to controls. Our study suggests that LFS of targets outside the limbic system, such as the FN, may have a significant anti-epileptogenic action, and the effects of LFS depend on the frequency and timing of stimulation.

[Involvement of endogenous histamine in modulatory effect of morphine on seizure susceptibility in mice].

OBJECTIVE: To investigate the modulatory effects of morphine on the susceptibility to pentylenetetrazole-induced seizures, and the involvement of endogenous histamine in this process. METHODS: Both the wild-type (WT) mice and histidine decarboxylase (a key enzyme for histamine biosynthesis) deficient (HDC-KO) mice were subcutaneously injected with different doses of morphine, and 1 hour later the pentylenetetrazole solution (1.5 %) was infused into the tail vein at a constant rate of 0.3 ml/min. The minimal dose of pentylenetetrazole (mg/kg) needed to induce myoclonic jerks and clonus convulsion was recorded as the thresholds of seizures. RESULT: In WT mice, morphine dose-dependently decreased the thresholds of both myoclonic jerks and clonus convulsion. In HDC-KO mice, morphine at 10 mg/kg only significantly decreased the threshold of myoclonic jerks from (38.6 +/-2.9)mg/kg to (32.5 +/-0.7)mg/kg, but had no significant effect on the threshold of clonus convulsion [from (51.8 +/-2.1)mg/kg to (47.6 +/-1.2)mg/kg]. In addition, the value of decreased myoclonic jerks (15.8 +/-1.4)% and clonus convulsion (8.3 +/-0.9)% thresholds were much lower in HDC-KO mice than in WT mice [(26.1 +/-2.5)% and (20.8 +/-2.4)%, respectively]. CONCLUSION: Morphine can decrease the thresholds of pentylenetetrazole in induction of seizure, and the endogenous histamine may be involved in this process.

[Effects of acute maximal electroshock and chronic transauricular kindled seizures on learning abilities in Sprague-Dawley rats].

OBJECTIVE: To investigate effects of acute maximal electroshock (MES) and chronic transauricular kindled seizures on learning abilities in Sprague-Dawley rats. METHODS: An acute MES was induced by giving an alternating current (150 mA, 0.2 s) through ear-clip electrodes. Chronic transauricular kindled seizure was induced by repeated application of initially subconvulsive electrical stimulation (40 mA, 0.2 s) through ear-clip electrodes once every 24 h. An 8-arm radial maze (4 arms baited) was used to measure learning abilities. Histamine, glutamate and gamma-aminobutyric acid (GABA) were measured by high-performance liquid chromatography (HPLC). RESULT: In the acquisition learning process, an acute MES increased reference memory errors but not working memory errors. In addition, it increased GABA levels in the hippocampus. On the other hand, chronic transauricular kindled seizures increased both working and reference memory errors in retrieval memory process, and this lasted for at least 3 weeks. Chronic transauricular kindled seizures induced CA1 neuron damage and a decrease in histamine levels in the hippocampus. CONCLUSION: Different types of kindling seizure produce different effects on cognitive behavior: (1) an acute MES impairs learning ability, which may be associated with an abnormal plasticity and an increase of GABA in the hippocampus; (2) the chronic transauricular kindled seizure impairs retrieval memory mainly, which may be related to CA1 neuron damage and a decrease in histaminergic activity in the hippocampus.

Carnosine inhibits pentylenetetrazol-induced seizures by histaminergic mechanisms in histidine decarboxylase knock-out mice.

In the present study, we used both histidine decarboxylase-deficient (HDC-KO) mice and wild-type (WT) mice to elucidate the possible role of carnosine in pentylenetetrazol (PTZ)-induced seizures. In the acute PTZ challenge study, PTZ (75 mg/kg) was injected intraperitoneally (i.p.) to induce seizures. Carnosine (200, 500 or 1000 mg/kg, i.p.) significantly decreased seizure stage, and prolonged the latency for myoclonic jerks in WT mice in a dose-dependent manner. The effects of carnosine (500 mg/kg) were time-dependent and reached a peak at 1h. However, it had no significant effect on HDC-KO mice. Carnosine (500 mg/kg) also significantly elevated the thresholds in WT mice but not HDC-KO mice following intravenous (tail vein) administration of PTZ. We also found that alpha-fluoromethylhistidine substantially reversed the protective effects of carnosine in WT mice. In addition, carnosine pretreatment reduced the cortical EEG activity induced by PTZ (75 mg/kg, i.p.). These results indicate that carnosine can protect against PTZ-induced seizures and its action is mainly through the carnosine-histidine-histamine metabolic pathway. This suggests that carnosine may be an endogenous anticonvulsant factor in the brain and may be used as a new antiepileptic drug in the future.