Inhibition of Synaptic Glutamate Exocytosis and Prevention of Glutamate Neurotoxicity by Eupatilin from Artemisia argyi in the Rat Cortex.

The inhibition of synaptic glutamate release to maintain glutamate homeostasis contributes to the alleviation of neuronal cell injury, and accumulating evidence suggests that natural products can repress glutamate levels and associated excitotoxicity. In this study, we investigated whether eupatilin, a constituent of Artemisia argyi, affected glutamate release in rat cortical nerve terminals (synaptosomes). Additionally, we evaluated the effect of eupatilin in an animal model of kainic acid (KA) excitotoxicity, particularly on the levels of glutamate and N-methyl-D-aspartate (NMDA) receptor subunits (GluN2A and GluN2B). We found that eupatilin decreased depolarization-evoked glutamate release from rat cortical synaptosomes and that this effect was accompanied by a reduction in cytosolic Ca2+ elevation, inhibition of P/Q-type Ca2+ channels, decreased synapsin I Ca2+-dependent phosphorylation and no detectable effect on the membrane potential. In a KA-induced glutamate excitotoxicity rat model, the administration of eupatilin before KA administration prevented neuronal cell degeneration, glutamate elevation, glutamate-generating enzyme glutaminase increase, excitatory amino acid transporter (EAAT) decrease, GluN2A protein decrease and GluN2B protein increase in the rat cortex. Taken together, the results suggest that eupatilin depresses glutamate exocytosis from cerebrocortical synaptosomes by decreasing P/Q-type Ca2+ channels and synapsin I phosphorylation and alleviates glutamate excitotoxicity caused by KA by preventing glutamatergic alterations in the rat cortex. Thus, this study suggests that eupatilin can be considered a potential therapeutic agent in the treatment of brain impairment associated with glutamate excitotoxicity.

The Aerial Parts of Bupleurum Chinense DC. Aromatic Oil Attenuate Kainic Acid-Induced Epilepsy-Like Behavior and Its Potential Mechanisms.

The aerial parts of Bupleurum Chinense DC. aromatic oil (BAO) were a well-known Chinese herbal medicine plant extract used to treat epilepsy. This study aimed to explore the therapeutic effect of BAO on kainic acid- (KA-) induced epileptic rats and the possible mechanism of its antiepileptic effect. The composition and content of BAO were analyzed by GC-MS, and BAO was administered orally to alleviate the epileptic behavior induced by KA brain injection. The behavior of epileptic rats was determined by Racine grading criteria. And hematoxylin-eosin staining (HE), Nissl staining, immunohistochemistry, Elisa, Western blot, and other methods were used to study the antiepileptic mechanism of BAO, and the possible mechanism was verified by the epileptic cell model of hippocampal neurons induced by the low-Mg2+ extracellular fluid. BAO was mainly composed of terpenoids and aliphatic compounds. And BAO could improve KA-induced epilepsy-like behavior, neuroinflammation, and neurotransmitter abnormalities in the hippocampus. Furthermore, BAO could regulate the expression of GABA, NMDAR1, Notch1, and MAP2 to improve the symptoms of epilepsy. These results were also validated at the cellular level. These results indicated that BAO could alleviate the epilepsy-like behavior through the action of the Notch/NMDAR/GABA pathway.

Osthole inhibits proliferation of kainic acid­activated BV­2 cells by modulating the Notch signaling pathway.

Epilepsy is a syndrome involving chronic recurrent transient brain dysfunction. Activation and proliferation of microglia serve important roles in epilepsy pathogenesis and may be targets for treatment. Although osthole, an active constituent isolated from Cnidium monnieri (L.) Cusson, has been demonstrated to improve epilepsy in rats, its underlying mechanism remains to be elucidated. The present study investigated the effect of osthole on proliferation of kainic acid (KA)­activated BV­2 cells and explored the molecular mechanism by which it inhibited their proliferation. Using Cell Counting Kit­8, enzyme­linked immunosorbent assay, reverse transcription­quantitative PCR, western blot analysis and immunofluorescence staining, it was identified that following exposure of KA­activated BV­2 cells to 131.2 µM osthole for 24 h, cell proliferation and release of tumor necrosis factor α, interleukin 6 and nitric oxide synthase/induced nitric oxide synthase were significantly inhibited (P<0.05). Further experiments revealed that osthole significantly downregulated mRNA and protein levels of Notch signaling components in KA­activated BV­2 cells (P<0.05). Therefore, it was hypothesized that osthole inhibited the proliferation of microglia by modulating the Notch signaling pathway, which may be useful for the treatment of epilepsy and other neurodegenerative diseases characterized by Notch upregulation.

Anticonvulsive and neuroprotective effects of aqueous and methanolic extracts of Anacyclus pyrethrum root in kainic acid-induced-status epilepticus in mice.

PURPOSE: Epilepsy is a chronic neurological disorder characterized by spontaneous and recurrent seizures. The currently available synthetic antiepileptic drugs have a limited efficacy and are associated with a wide range of side effects. In Ayurveda, Anacyclus pyrethrum root (APR) has been used as a traditional antiepileptic remedy. The aim of the present study is to evaluate the anticonvulsive and neuroprotective effects of aqueous and methanol extracts of Anacyclus pyrethrum root (AEAPR and MEAPR) on experimental model of status epilepticus (SE). METHODS: Twenty four male mice were divided into four groups. The control and KA groups had free access to tap water for 5 days before the intraperitoneal injection of distillated water or kainic acid (KA; 30 mg/kg), respectively. In the treated groups, mice received extracts solutions MEAPR and AEAPR in drinking water at the concentration of 5 g/l for 5 days. At the fifth day, animals received intraperitoneal injection of KA. The behavioral changes latency of seizures, the number of wet dog shakes (WDS) and the mortality were observed over 6 h. Thereafter, the mice were sacrificed for immunohistochemical studies. RESULTS: Pretreatment with MEAPR and AEAPR decreases significantly the frequency of WDS (32.5% and 43.9%, p < 0.01; respectively), and increases considerably the latent period (77.9% and 91.9%, p<0.01; respectively) between the injection of the KA and the appearance of the SE as compared to the KA group. The duration and severity of seizure in the MEAPR or AEAPR-pretreated groups were significantly lower (p < 0.01 and p < 0.05 or p < 0.01; respectively) than those in the KA group. These behavioral results were confirmed by the immunohistochemical study at the level of the hippocampus, in which the c-FOS and GFAP expression of both MEAPR and AEAPR-treated animals largely reduced (p < 0.001) the number of labelled cells with respect to the group, which received the KA alone. CONCLUSION: Our results showed that the MEAPR and AEAPR have anticonvulsive effect and putative neuroprotective effect against seizures induced by KA. Further studies are required to identify its active ingredients responsible for the observed effects.

Magnolia officinalis reduces the long-term effects of the status epilepticus induced by kainic acid in immature rats.

During critical periods of neurodevelopment, the immature brain is susceptible to neuronal hyperexcitability, alterations such as hyperthermia, hypoxia, brain trauma or a preexisting neuroinflammatory condition can trigger, promote and prolong epileptiform activity and facilitate the development of epilepsy. The goal of the present study was to evaluate the long-term neuroprotective effects Magnolia officinalis extract, on a model of recurrent status epilepticus (SE) in immature rats. Sprague-Dawley rats were treated with kainic acid (KA) (3 mg/kg, dissolved in saline solution) beginning at day 10 P N every 24 h for five days (10 P N-14PN). Two experimental groups (KA) received two treatments for 10 days (14-24 P N): one group was treated with 300 mg/kg Magnolia Officinalis (MO) (KA-MO), and another was treated with 20 mg/kg of celecoxib (Clbx) (KA-Clbx) as a control drug. A SHAM control group at day 90 P N was established. Seizure susceptibility was analyzed through an after-discharge threshold (ADT) evaluation, and electroencephalographic activity was recorded. The results obtained from the ADT evaluation and the analysis of the electroencephalographic activity under basal conditions showed that the MO and Clbx treatments protected against epileptiform activity, and decreases long-term excitability. All rats in the KA-MO and KA-Clbx groups presented a phase I seizure on the Racine scale, corresponding to the shaking of a wet dog. In contrast, the KA group showed phase V convulsive activity on the Racine scale. Similarly, MO and Clbx exerted neuroprotective effects on hippocampal neurons and reduced gliosis in the same areas. Based on these results, early intervention with MO and Clbx treatments to prevent the inflammatory activity derived from SE in early phases of neurodevelopment exerts neuroprotective effects on epileptogenesis in adult stages.

Changes to gamma-aminobutyric acid levels during short-term epileptiform activity in a kainic acid-induced rat model of status epilepticus: A chemical exchange saturation transfer imaging study.

PURPOSE: To evaluate temporal changes in gamma-aminobutyric acid (GABA) signals in the hippocampus during epileptiform activity induced by kainic acid (KA) in a rat model of status epilepticus using chemical exchange saturation transfer (CEST) imaging technique. METHODS: CEST imaging and 1H magnetic resonance spectroscopy (1H MRS) were applied to a systemic KA-induced rat model to compare GABA signals. All data acquisition and analytical procedures were performed at three different time points (before KA injection, and 1 and 3 h after injection). The CEST signal was analyzed based on regions of interests (ROIs) in the hippocampus, while 1H MRS was analyzed within a 12.0 µL ROI in the left hippocampus. Signal correlations between the two methods were evaluated as a function of time change up to 3 h after KA injection. RESULTS: The measured GABA CEST-weighted signal intensities of the rat epileptic hippocampus before injection showed significant differences from those after (averaged signals from both hippocampi: 4.37% ±â€¯0.87% and 7.305 ±â€¯1.11%; P < 0.05), although the signal had increased slightly at both time points after KA injection, the differences were not significant (P > 0.05). In contrast, the correlation between the CEST imaging values and 1H MRS was significant (r ≥ 0.64; P < 0.05; in all cases). CONCLUSIONS: GABA signal changes during epileptiform activity in the rat hippocampus, as detected using CEST imaging, provided a significant contrast according to changes in metabolic activity. Our technical approach may serve as a potential supplemental option to provide biomarkers for brain disease.

Osthole inhibits proliferation and induces apoptosis in BV-2 microglia cells in kainic acid-induced epilepsy via modulating PI3K/AKt/mTOR signalling way.

CONTEXT: Osthole is a natural coumarin compound most frequently extracted from plants of the Apiaceae family such as Cnidium monnieri (L.) Cusson, Angelica pubescens Maxin.f., and Peucedanum ostruthium (L.). Osthole is considered to have potential therapeutic applications for the treatment of diseases including epilepsy. However, the mechanism of osthole induced-apoptosis in BV-2 microglia cells is not yet clear. OBJECTIVE: To investigate the molecular mechanisms underlying the effect of osthole on PI3K/AKt/mTOR expression in kainic acid (KA)-activated BV-2 microglia cells. MATERIALS AND METHODS: Optimal culture concentration and time of osthole were investigated by MTT assay. The concentration of osthole was tested from 10 to 400 µM and the culture time was tested from 2 to 72 h. Ultrastructure difference among control, KA and osthole group was analyzed under transmission electron microscope. The mRNA expression of PI3K/AKt/mTOR was investigated using reverse transcription (RT)-PCR and the protein expression was investigated using western blotting and immunofluorescence assay. Apoptosis rate of BV-2 cells between each group was measured by flow cytometry. RESULTS: IC50 for cell viability of BV-2 cells by osthole was 157.7 µM. Treated with osthole (140 µM) for 24 h significantly increased the inhibition rate. Pretreatment with osthole inhibited the KA-induced PI3K/AKt/mTOR mRNA and protein expression. The results of flow cytometry analysis showed that the apoptotic rate of osthole group was obviously higher than KA group. CONCLUSIONS: Date showed that osthole may be useful in the treatment of epilepsy and other neurodegenerative diseases that are characterized by over expression of PI3K/Akt/mTOR.

Influence of clove oil and eugenol on muscle contraction of silkworm (Bombyx mori).

Clove oil is used in fish anesthesia and expected to have a mechanism via glutamic receptor. The present study explores the activities of clove oil and its major compound, eugenol, in comparison with L-glutamic acid on glutamic receptor of silkworm muscle and fish anesthesia. It was found that clove oil and eugenol had similar effects to L-glutamic acid on inhibition of silkworm muscle contraction after treated with D-glutamic acid and kainic acid. Anesthetic activity of the test samples was investigated in goldfish. The results demonstrated that L-glutamic acid at 20 and 40 mM could induce the fish to stage 3 of anesthesia that the fish exhibited total loss of equilibrium and muscle tone, whereas clove oil and eugenol at 60 ppm could induce the fish to stage 4 of anesthesia that the reflex activity of the fish was lost. These results suggest that clove oil and eugenol have similar functional activities and mechanism to L-glutamic acid on muscle contraction and fish anesthesia.

Phase-amplitude coupled persistent theta and gamma oscillations in rat primary motor cortex in vitro.

In vivo, theta (4-7 Hz) and gamma (30-80 Hz) neuronal network oscillations are known to coexist and display phase-amplitude coupling (PAC). However, in vitro, these oscillations have for many years been studied in isolation. Using an improved brain slice preparation technique we have, using co-application of carbachol (10 µM) and kainic acid (150 nM), elicited simultaneous theta (6.6 ± 0.1 Hz) and gamma (36.6 ± 0.4 Hz) oscillations in rodent primary motor cortex (M1). Each oscillation showed greatest power in layer V. Using a variety of time series analyses we detected significant cross-frequency coupling in 74% of slice preparations. Differences were observed in the pharmacological profile of each oscillation. Thus, gamma oscillations were reduced by the GABAA receptor antagonists, gabazine (250 nM and 2 µM), and picrotoxin (50 µM) and augmented by AMPA receptor antagonism with SYM2206 (20 µM). In contrast, theta oscillatory power was increased by gabazine, picrotoxin and SYM2206. GABAB receptor blockade with CGP55845 (5 µM) increased both theta and gamma power, and similar effects were seen with diazepam, zolpidem, MK801 and a series of metabotropic glutamate receptor antagonists. Oscillatory activity at both frequencies was reduced by the gap junction blocker carbenoxolone (200 µM) and by atropine (5 µM). These data show theta and gamma oscillations in layer V of rat M1 in vitro are cross-frequency coupled, and are mechanistically distinct. The development of an in vitro model of phase-amplitude coupled oscillations will facilitate further mechanistic investigation of the generation and modulation of coupled activity in mammalian cortex.

Neuroprotective Effect of Electric Conduction Treatment on Hippocampus Cell Apoptosis in KA Induced Acute Temporal Lobe Epileptic Rats.

BACKGROUND: Electronic conduction, a new treatment approach for epilepsy, has been confirmed to reduce epileptiform discharge on EEG and convulsive behaviors, particularly epileptic discharge propagation and serious behavioral seizures, in rats with kainic acid (KA)-induced acute temporal lobe epilepsy (TLE). OBJECTIVE: Hippocampal cell apoptosis was examined to confirm the neuroprotective effect of electronic conduction therapy in rats with KA-induced acute TLE. METHODS: Rats were divided into four groups: control group (right CA3 injection of saline), KA group (right CA3 injection of KA), sham conduction group (KA rats with sham conduction), and conduction group (KA rats with electric conduction). Apoptotic cells were evaluated by flow cytometry, TUNEL staining, and mRNA expression levels of caspase-3, tumor necrosis factor-alpha, and glial fibrillary acidic protein measured by real-time quantitative PCR (qRT-PCR). RESULTS: The frequency of convulsive behaviors in the conduction group decreased significantly compared with the KA group and the sham conduction group. Significantly fewer apoptotic cells were detected in rats with conduction based on flow cytometry and TUNEL staining results. The qRT-PCR results indicated that KA-induced up-regulation of hippocampal caspase-3 mRNA expression was reduced 24 hours after KA injection in rats that received conduction treatment. CONCLUSION: Electronic conduction treatment can reduce seizure frequency and hippocampal cell apoptosis in rats with KA-induced acute TLE.

Neonatal domoic acid alters in vivo binding of [11C]yohimbine to α2-adrenoceptors in adult rat brain.

RATIONALE: Epilepsy is a debilitating seizure disorder that affects approximately 50 million people. Noradrenaline reduces neuronal excitability, has anticonvulsant effects and is protective against seizure onset. OBJECTIVE: We investigated the role of α2-adrenoceptors in vivo in a neonatal domoic acid (DOM) rat model of epilepsy. METHODS: We injected male Sprague-Dawley rats daily from postnatal day 8-14 with saline or one of two sub-convulsive doses, 20 µg/kg (DOM20) or 60 µg/kg (DOM60) DOM, an AMPA/kainate receptor agonist. The rats were observed in open field, social interaction and forced swim tests at day 50, 75 and 98, respectively. At ~120 days of age, four rats per group were injected and scanned with [11C]yohimbine, an α2-adrenoceptor antagonist, and scanned in a Mediso micro positron emission tomography (PET) scanner to measure α2-adrenoceptor binding. RESULTS: DOM60-treated rats spent more time in the periphery during the open field test and had a significant 26-33 % reduction in [11C]yohimbine binding in the hypothalamus, hippocampus and orbital prefrontal cortex compared to saline-treated rats. On the other hand, DOM20 rats had a significant 34-40 % increase in [11C]yohimbine binding in the hypothalamus, amygdala and entorhinal cortex compared to saline-treated rats, with no obvious behavioural differences. CONCLUSIONS: The current data clearly indicate that low concentrations of DOM given to rats in their second week of life induces long-term changes in α2-adrenoceptor binding in rat brain that may have relevance to the progression of an epilepsy phenotype.

Effects of a subconvulsive dose of kainic acid on the gene expressions of the arginine vasopressin, oxytocin and neuronal nitric oxide synthase in the rat hypothalamus.

Arginine vasopressin (AVP) synthesis in the hypothalamo-neurohypophysial system (HNS) is up-regulated by kainic acid (KA)-induced seizure in rats. However, it remains unknown whether a subconvulsive dose of KA affects the HNS. Here we examined the effects of subcutaneous (s.c.) administration of a low dose of KA (4 mg/kg) on the gene expressions of the AVP, oxytocin (OXT) and neuronal nitric oxide synthase (nNOS) in the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus, using in situ hybridization histochemistry. The expression of the AVP gene in the SON and PVN was judged to be up-regulated in KA-treated rats in comparison with saline-treated rats as controls. Next, the expression of the OXT gene was significantly increased in the SON at 6-24h and in the PVN at 6 and 12h after s.c. administration of KA. Finally, the expression of the nNOS gene was significantly increased in the SON and PVN at 3 and 6h after s.c. administration of KA. These results suggest that up-regulation of the gene expressions of the AVP, OXT and nNOS in the rat hypothalamus may be differentially affected by peripheral administration of a subconvulsive dose of KA.

Ceftriaxone modulates uptake activity of glial glutamate transporter-1 against global brain ischemia in rats.

Ceftriaxone(Cef) selectively increases the expression of glial glutamate transporter-1 (GLT-1), which was thought to be neuroprotective in some circumstances. However, the effect of Cef on glutamate uptake of GLT-1 was mostly assayed using in vitro studies such as primary neuron/astrocyte cultures or brain slices. In addition, the effect of Cef on neurons in different ischemic models was still discrepant. Therefore, this study was undertaken to observe the effect of Cef on neurons in global brain ischemia in rats, and especially to provide direct evidence of the up-regulation of GLT-1 uptake for glutamate contributing to the neuronal protection of Cef against brain ischemia. Neuropathological evaluation indicated that administration of Cef, especially pre-treatment protocols, significantly prevented delayed neuronal death in hippocampal CA1 subregion normally induced by global brain ischemia. Simultaneously, pre-administration of Cef significantly up-regulated the expression of GLT-1. Particularly, GLT-1 uptake assay with (3) H-glutamate in living cells from adult rats showed that up-regulation in glutamate uptake accompanied up-regulated GLT-1 expression. Inhibition of GLT-1 by antisense oligodeoxynucleotides or dihydrokainate significantly inhibited the Cef-induced up-regulation in GLT-1 uptake and the neuroprotective effect against global ischemia. Thus, we may conclude that Cef protects neurons against global brain ischemia via up-regulation of the expression and glutamate uptake of GLT-1. Glutamate uptake by glial glutamate transporter-1 (GLT-1) is the principal way to regulate extracellular glutamate homeostasis in central nervous system. Over-accumulation of glutamate results in excitotoxicity and injures neurons after cerebral ischemia. Ceftriaxone up-regulates GLT-1 expression and uptake of glutamate, diminishes the excitotoxicity of glutamate and then protects neurons against global brain ischemia.

GABA in nucleus tractus solitarius participates in electroacupuncture modulation of cardiopulmonary bradycardia reflex.

Phenylbiguanide (PBG) stimulates cardiopulmonary receptors and cardiovascular reflex responses, including decreases in blood pressure and heart rate mediated by the brain stem parasympathetic cardiac neurons in the nucleus ambiguus and nucleus tractus solitarius (NTS). Electroacupuncture (EA) at P5-6 stimulates sensory fibers in the median nerve and modulates these reflex responses. Stimulation of median nerves reverses bradycardia through action of γ-aminobutyric acid (GABA) in the nucleus ambiguus, important in the regulation of heart rate. We do not know whether the NTS or the neurotransmitter mechanisms in this nucleus participate in these modulatory actions by acupuncture. We hypothesized that somatic nerve stimulation during EA (P5-6) modulates cardiopulmonary inhibitory responses through a GABAergic mechanism in the NTS. Anesthetized and ventilated cats were examined during either PBG or direct vagal afferent stimulation while 30 min of EA was applied at P5-6. Reflex heart rate and blood pressure responses and NTS-evoked discharge were recorded. EA reduced the PBG-induced depressor and bradycardia reflexes by 67% and 60%, respectively. Blockade of GABAA receptors in the NTS reversed EA modulation of bradycardia but not the depressor response. During EA, gabazine reversed the vagally evoked discharge activity of cardiovascular NTS neurons. EA modulated the vagal-evoked cardiovascular NTS cellular activity for 60 min. Immunohistochemistry using triple labeling showed GABA immunoreactive fibers juxtaposed to glutamatergic nucleus ambiguus-projecting NTS neurons in rats. These glutamatergic neurons expressed GABAA receptors. These findings suggest that EA inhibits PBG-evoked bradycardia and vagally evoked NTS activity through a GABAergic mechanism, likely involving glutamatergic nucleus ambiguus-projecting NTS neurons.

Characterization of Human Hippocampal Neural Stem/Progenitor Cells and Their Application to Physiologically Relevant Assays for Multiple Ionotropic Glutamate Receptors.

The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors-namely,N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)-are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain region-specific human neural cells are necessary. Here, we characterized the functional differentiation of human fetal hippocampus-derived neural stem/progenitor cells-namely, HIP-009 cells. Calcium rise assay demonstrated that, after a 4-week differentiation, the cells responded to NMDA (EC50= 7.5 ± 0.4 µM; n= 4), AMPA (EC50= 2.5 ± 0.1 µM; n= 3), or KA (EC50= 33.5 ± 1.1 µM; n= 3) in a concentration-dependent manner. An AMPA-evoked calcium rise was observed in the absence of the desensitization inhibitor cyclothiazide. In addition, the calcium rise induced by these agonists was inhibited by antagonists for each receptor-namely, MK-801 for NMDA stimulation (IC50= 0.6 ± 0.1 µM; n= 4) and NBQX for AMPA and KA stimulation (IC50= 0.7 ± 0.1 and 0.7 ± 0.03 µM, respectively; n= 3). The gene expression profile of differentiated HIP-009 cells was distinct from that of undifferentiated cells and closely resembled that of the human adult hippocampus. Our results show that HIP-009 cells are a unique tool for obtaining human hippocampal neural cells and are applicable to systems for assay of ionotropic glutamate receptors as a physiologically relevant in vitro model.

Antiepileptogenic effect of curcumin on kainate-induced model of temporal lobe epilepsy.

CONTEXT: Temporal lobe epilepsy (TLE) is an intractable neurological disorder. Curcumin is the bioactive component of turmeric with anti-epileptic and neuroprotective potential. OBJECTIVE: The beneficial effect of curcumin on the intrahippocampal kainate-induced model of TLE was investigated. MATERIALS AND METHODS: Rats were divided into sham, curcumin-pretreated sham, kainate and curcumin-pretreated kainate groups. The rat model of TLE was induced by unilateral intrahippocampal injection of 4 µg of kainate. Rats received curcumin p.o. at a dose of 100 mg/kg/d starting 1 week before the surgery. Seizure activity (SE) and oxidative stress-related markers were measured. Furthermore, the Timm index for evaluation of mossy fiber sprouting (MFS) and number of Nissl-stained neurons were quantified. RESULTS: All rats in the kainate group had SE, while 28.5% of rats showed seizures in the curcumin-pretreated kainate group. Malondialdehyde and nitrite and nitrate levels significantly increased in the kainate group (p < 0.01 and p < 0.05, respectively), and curcumin significantly lowered these parameters (p < 0.05). Superoxide dismutase activity significantly decreased in the kainate group (p < 0.05) and curcumin did not improve it. Rats in the kainate group showed a significant reduction of neurons in Cornu Ammonis 1 (CA1) (p < 0.05), CA3 (p < 0.005) and hilar (p < 0.01) regions, and curcumin significantly prevented these changes (p < 0.05-0.005). The Timm index significantly increased in the kainate group (p < 0.005), and curcumin significantly lowered this index (p < 0.01). DISCUSSION AND CONCLUSION: Curcumin pretreatment can attenuate seizures, lower some oxidative stress markers, and prevent hippocampal neuronal loss and MFS in the kainate-induced model of TLE.

Pre-synaptic kainate receptor-mediated facilitation of glutamate release involves PKA and Ca(2+) -calmodulin at thalamocortical synapses.

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 µM) produced an increase in 4-aminopyridine (4-AP)-evoked glutamate release. In thalamocortical slices, KA (1 µM) produced an increase in the amplitude of evoked excitatory post-synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione, and persisted after pre-treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G-protein-independent regulation further in thalamocortical slices, the KAR-mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca(2+) permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca(2+) stores by thapsigargin, or inhibition of Ca(2+) -induced Ca(2+) -release by ryanodine. Thus, the KA-mediated modulation was contingent on both Ca(2+) entry through Ca(2+) -permeable KARs and liberation of intracellular Ca(2+) stores. Finally, sensitivity to W-7 indicated that the increased cytosolic [Ca(2+) ] underpinning KAR-mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre-synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca(2+) -calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G-protein involvement.

The synergic effect of regular exercise and resveratrol on kainate-induced oxidative stress and seizure activity in mice.

The synergic effect of regular exercise and resveratrol, a polyphenolic compound with potent antioxidant activity, was investigated against kainate-induced seizures and oxidative stress in mice. After 6 weeks of swimming training, the total body weight decreased and the blood concentration of lactate stabilized statistically in comparison with the sedentary mice, indicate that the training program increased the aerobic resistance of mice. Kainate (30 mg/kg) evoked seizure activity 5 min after injection, and seizure activity was measured seizure rating scores every 5 min up to 2 h. As previously well known experiments, regular exercise and resveratrol (40 mg/kg, daily supplementation for 6 weeks) have an inhibitory effect on kainate-induced seizure activity and oxidative stress. In particularly, a synergistic cooperation of regular exercise and resveratrol was observed in seizure activity, mortality and oxidative stress especially in SOD activity. These results suggest that regular exercise along with an anti-convulsant agent such as resveratrol could be a more efficient method for the prevention of seizure development than exercise alone.

Acupuncture suppresses kainic acid-induced neuronal death and inflammatory events in mouse hippocampus.

The administration of kainic acid (KA) causes seizures and produces neurodegeneration in hippocampal CA3 pyramidal cells. The present study investigated a possible role of acupuncture in reducing hippocampal cell death and inflammatory events, using a mouse model of kainic acid-induced epilepsy. Male C57BL/6 mice received acupuncture treatments at acupoint HT8 or in the tail area bilaterally once a day for 2 days and again immediately after an intraperitoneal injection of KA (30 mg/kg). HT8 is located on the palmar surface of the forelimbs, between the fourth and fifth metacarpal bones. Twenty-four hours after the KA injection, neuronal cell survival, the activations of microglia and astrocytes, and mRNA expression of two proinflammatory cytokines, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), were measured in the hippocampus. Acupuncture stimulation at HT8, but not in the tail area, significantly reduced the KA-induced seizure, neuron death, microglial and astrocyte activations, and IL-1ß mRNA expression in the hippocampus. The acupuncture stimulation also decreased the mRNA expression of TNF-α, but it was not significant. These results indicate that acupuncture at HT8 can inhibit hippocampal cell death and suppress KA-induced inflammatory events, suggesting a possible role for acupuncture in the treatment of epilepsy.

Electrophysiological monitoring of hearing function during cochlear perilymphatic perfusions.

CONCLUSION: The cochlear perilymphatic perfusion produces, by itself, significant effects in the cochlear physiology that could be associated with the surgical procedure. These effects need to be well characterized to allow a reliable quantification of the effects of the experimental agent being tested. OBJECTIVES: The study focused on the accurate description of the electrophysiological effects on the cochlear potential recordings of perilymphatic perfusions. METHODS: Two successive cochlear perilymphatic perfusions were carried out. The first used artificial perilymph. The second used artificial perilymph alone or a kainic acid (KA) solution in artificial perilymph. The compound action potential of the auditory nerve (CAP-AN) was recorded: (1) before the first perfusion, (2) after the first perfusion and (3) after the second perfusion, and compared between groups. RESULTS: The first intracochlear perfusion with artificial perilymph produced significant effects in the CAP-AN that could be related to the surgical procedure. These effects were analysed separately from the effects produced by the KA. In particular, the KA administered intracochlearly produced a significant increase in the latency and a decrease in the amplitude of the CAP-AN N1 wave compared with the controls that were perfused twice with artificial perilymph.