Inducible brain COX-2 facilitates the recurrence of hippocampal seizures in mouse rapid kindling

Brain cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, is rapidly and transiently induced by convulsions in hippocampal and cortical neurons. Therefore, we examined the effects of COX-2 on the 'rapid kindling' development in COX-2 knockout mice and in mice treated with nimesulide, a COX-2-selective inhibitor. Rapid kindling development was examined based on the incidence of hippocampal EEG seizures and behavioral seizures following repetitive electrical stimulation of the perforant path at an interval of 40 s, and on the total afterdischarge (AD) duration induced by 50 stimulations. In addition, we measured COX-2 mRNA expression by in situ hybridization and PGE2 concentration using enzyme immunoassay following rapid kindling stimulation. The results suggested that brain COX-2 mRNA levels were markedly increased in the hippocampal neurons and the concentration of PGE2 was elevated significantly, and that the incidence of AD and seizure behavior induction and the total AD duration were significantly decreased under conditions of COX-2 deficiency. Therefore, we concluded that inducible COX-2 facilitates the recurrence of hippocampal seizures.     

Synaptic degeneration and remodelling after fast kindling of the olfactory bulb

Kindling of the olfactory bulb using a novel fast protocol (within 24 h) was studied in rats. In target brain regions, the effects of kindling were measured on the concentration of glial fibrillary acidic protein (GFAP) by dot-blot and on the concentrations of neural cell adhesion molecule (NCAM) and the 25 kDa synaptosomal associated protein of the D3 immunoprecipitate (D3(SNAP-25)) by crossed immunoelectrophoresis. Bilateral increases in the levels of GFAP, indicating activation of astrocytes, were detected in primary olfactory cortical projection areas, including the piriform cortex, and also in the basolateral amygdala and dentate gyrus, suggesting that these regions may be functionally altered during the kindling process. In the piriform cortex and dentate gyrus increased NCAM/D3(SNAP-25) ratios found ipsilaterally at seven days after kindling probably reflect an elevated rate of synaptic remodelling. At this time, however, an overall pattern of ipsilateral decreases in the synaptic marker proteins NCAM and D3(SNAP-25) indicated that this remodelling occurred on a background of synaptic degeneration. These results confirm previous studies showing that kindling is associated with synaptic remodelling and neuronal degeneration in the hippocampal formation and extends the area of plasticity to include the piriform cortex which is believed to be central to the kindling process.     

Hippocampal Kindling in the Rat Is Associated with Time-Dependent Increases in the Concentration of Glial Fibrillary Acidic Protein

The effect of hippocampal kindling on the regional brain concentration of total glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, was studied in partially kindled rats, and in fully kindled rats after a post-kindling period of 24 h, 1 week, and 2 months. GFAP concentration was measured in arbitrary units by dot-blots. In the hippocampus, dentate gyrus, basolateral amygdala, pyriform cortex, and entorhinal cortex, limbic structures which are known to be involved in the kindling process, there was an increase in GFAP concentration which was maximal in the fully kindled animals studied after 24 h. In most brain areas, GFAP concentration was still elevated 1 week post-kindling, but had declined to control level 2 months post-kindling. A significant increase in GFAP was also found in septum, ventral pallidum/accumbens nucleus, and primary motor cortex of kindled rats with a post-kindling period of 24 h, whereas in several other brain regions GFAP was unchanged. These results suggest that astrocyte activation, indicative of degenerative changes in nearby neurons, is a transient and regional phenomenon in kindling occurring only during the development of the kindled state.     

Influence of experimental epileptogenesis on memory: the role of lipids in cognitive disorders

Learning and memory disorders accompanying epileptogenesis were studied in rats with the use of two experimental models of epilepsy, picrotoxin kindling and kainic treatment. Rise of exploratory activity and decrease in animal's capability for experimental extinction of a response were characteristic of the initial stage of epileptogenesis. It was suggested that a dysfunction of brain hippocampal system can be responsible for cognitive disorders. To reveal their mechanisms, lipid contents were determined in the neocortex and hippocampus in appropriate periods after exposure to epileptogenic factors. Long-term changes in hippocampal lipid spectrum were found five days after the exposure to kainic acid. In particular, after sodium valproate treatment (the compensation of kainic effects), the total content of phospholipids in hippocampus was decreased. The hippocampal sphingomyelin level dropped as a result of picrotoxin kindling. The sphingomyelin changes suggest some recovery processes in hippocampal cells and point to an adaptive role of membrane lipids in the mechanisms of the damaging epiptogenous effects.     

Effects of interleukin-10 on the epileptiform activity development in the hippocampus induced by brief hypoxia, bicuculline and electrical kindling

The comparative effects of antiinflammatory cytokine interleukin-10 on the epileptiform activity development in CA1 hippocampal neurons were studied in different functional models of epileptogenesis that are not accompanied the visible morphological disturbances in the brain cells: --in vitro hypoxic model in the rat hippocampal slices; 2--in vitro disinhibitory model with using GABAA antagonist, bicuculline, in the rat hippocampal slices; 3--partial hippocampal kindling model in freely moving rats. Interleukin-10 (1 ng/ml) depressed the posthypoxic hyperexcitability in CA1 pyramidal neurons of the rat hippocampal slices through a decrease of the effectiveness of hypoxia to depresses the functional neuronal activity in the rat hippocampal slices during hypoxic episode. On the other hand, interleukin-10 (1 ng/ml) did not affect an initiation of epileptiform activity in CA1 pyramidal neurons of the rat hippocampal slices induced by bicuculline. Interleukin-10 (1 ng/5 microl) applied to the dorsal hippocampus in awake rats depressed an initiation of focal seizures ("ictal"-like components of afterdischarges) induced by hippocampal kindling during the first six hours after an application. However, this cytokine did not affect neither the duration of "interictal"-like component of afterdischarges nor motor seizure development. Thus, our findings showed that antiinflammatory cytokine interleukin-10, in addition to its antihypoxic action, exert the neuroprotective effect on the initiation of "ictal"-like, but not "interictal"-like, epileptiform discharges.     

Characteristics of the electrical activity in hippocampal slices in mice with Corazol kindling

It has been shown in experiments on hippocampal slices of (CBA X C57BL/6)F1 mice with corazol kindling that the threshold of the appearance of the induced seizure discharge (ISD) in the area CA1 was decreased by stimulation of Schaffer collaterals. Diazepam provoked an increase in seizure susceptibility to corazol and penicillin and reduction of the ISD. The data suggest that alterations in neuronal reactivity, which follow kindling, can be found in an individual hippocampal segment, thus making it possible to investigate this phenomenon at the synaptic and molecular levels.     

Effect of the destruction and activation of the caudate nuclei on the convulsive action of corazol kindling

Chronic experiments on the rats have shown that the pharmacological destruction of caudate nuclei significantly elevates the general brain excitation and induces rapid development of the corazol kindling. The hippocampal destruction exerts an opposite effect. Regression analysis of this processes has shown that mechanism of the general brain excitation and those of epileptogenesis are different on the stage of the developing kindling. Caudate nucleus activation induces powerful inhibition of kindling behavioral convulsive reactions and its electrographic epileptic activity. These data suggest that the caudate nucleus is a significant structure of the antiepileptic brain system and confirm G. N. Kryzhanovsky's concept about the system-antisystem interrelationship in case of neuropathologic syndromes as a result of the system hyperactivity.     

Preferential increase in the hippocampal synaptic vesicle protein 2A (SV2A) by pentylenetetrazole kindling

The present study evaluated the expressional levels of synaptic vesicle protein 2A (SV2A) and other secretary machinery proteins (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, Munc18-1, N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)) in a pentylenetetrazole (PTZ) kindling model. Repeated administration of sub-convulsive PTZ (40 mg/kg, i.p.) progressively increased seizure susceptibility in mice and consistently induced clonic seizures in most animals tested at 15 days after the treatment. Western blot analysis revealed that, among the secretary machinery proteins examined, hippocampal SV2A was selectively elevated by PTZ kindling. PTZ kindling-induced SV2A expression appeared region-specific and the SV2A levels in the cerebral cortex or cerebellum were unaltered. In addition, SV2A expression by PTZ kindling was prominent in the hilar region of the dentate gyrus (DG) where GABAergic interneurons are located, but not in other hippocampal regions (e.g., the stratum lucidum of the CA3 and synaptic layers surrounding CA1 or CA3 pyramidal neurons). These findings suggest that PTZ kindling preferentially elevates SV2A expression in the hippocampus probably as a compensatory mechanism to activate the inhibitory neurotransmission.     

Bicuculline-induced blockade of neocortical rapid kindling suggesting facilitative GABAergic action on seizure development

Summary To investigate how GABAergic function affects seizure development, the effects of a GABA antagonist, bicuculline, on neocortical and hippocampal kindling were examined in chronically prepared rabbits. Kindling-inducing stimulations consisted of stimulus trains repeated at 5-min interstimulus intervals to produce so-called rapid kindling. The changes in after-discharge (AD) durations induced by each of 15 trials of stimulus trains per session were compared before and 30 min after i.p. injection of bicuculline solution (2 mg/kg) in each of three kindling groups consisting of 5 rabbits each, i.e. visual cortical, motor cortical and hippocampal kindling groups. In the visual cortex and to a less extent, the motor cortex kindling groups, the AD durations were shortened after bicuculline injection and did not show the progressive prolongation seen before the injection. In contrast, the hippocampal kindling group showed a further marked prolongation of the AD durations after the injection. The bicuculline-induced blockade of neocortical kindling suggests facilitative GABAergic action on seizure development, while the drug-induced enhancement of hippocampal kindling reflects the known inhibitory GABAergic action.     

MicroRNA-134/CREB/pCREB通路在癫痫中的表达及意义

目的探讨miR-134、CREB、pCREB在癫痫大鼠海马及难治性癫痫患者颞叶脑组织中的表达及意义。方法难治性癫痫患者及非癫痫对照组颞叶组织、氯化锂-匹罗卡品癫痫大鼠及空白对照组海马组织中,应用实时荧光定量PCR技术检测microRNA-134（miR-134）的表达,用Western blot方法检测CREB及p CREB的表达,用免疫组织化学方法检测人脑颞叶皮质及大鼠海马区CREB、p CREB的表达。结果与对照组相比miR-134表达在难治性癫痫患者中明显降低（P〈0.05）,在癫痫模型组中点燃后3、7、14、60 d明显降低（P〈0.05）,1 d与30 d表达降低较对照组差异无显著性（P〉0.05）;癫痫模型组CREB在3、7、14、60 d时间点明显升高（P〈0.05）、pCREB各时间点表达均高于空白对照组（P〈0.05）。结论难治性癫痫患者颞叶皮质及癫痫动物海马中miR-134表达下降,CREB、pCREB表达升高,提示其可能在癫痫发生发展机制中起重要作用。     

Effects of extracts of different parts of the brain of kindled animals on seizure activity of recipient rats

The peptide-containing fraction was emitted from the hippocampal and ventral mesencephalic region tissue of rats kindled with subconvulsant doses of corazol. Extracts were prepared by the help of hot acetic acid on the stage of generalized clonic-tonic seizure development. The intraventricular injection of VMR-extracts in relatively high dose increased seizure reactions which were induced in intact recipient rats by intraperitoneal corazol injection. The intraventricular injection of the extract in relatively low dose (100 times less) suppressed corazol-induced seizures in recipients. Data are discussed from the point of view of pathological epileptic system formation and the role played by peptides in supporting it's activity during pharmacological kindling.     

Amygdala Kindling Alters Protein Kinase C Activity in Dentate Gyrus

Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.     

Traumatic Brain Injury Increases β-Amyloid Peptide 1-42 in Cerebrospinal Fluid

Abstract: The β-amyloid peptides, Aβ1-42 and Aβ1-40, were quantified in ventricular CSF taken daily for up to 3 weeks from six individuals with severe traumatic brain injury (TBI). There was considerable interindividual variability in the levels of Aβ peptides, but in general Aβ1-42 levels equalled or exceeded those of Aβ1-40. Averaging the daily totals of our trauma cohort revealed that the levels of Aβ1-42 and Aβ1-40 rose after injury, peaking in the first week and then declining toward control levels over the next 2 weeks. Aβ1-42 levels were on average two to three times higher in the trauma cohort than in CSF from nontrauma samples. Compared with nontrauma samples, the Aβ1-40/Aβ1-42 ratio decreased about fivefold in the trauma patients, further indicative of increased Aβ1-42 levels. The ratio remained low at all time points studied. No change was measured in the levels of β-amyloid precursor protein during the same interval. These results suggest that Aβ1-42 becomes elevated in the CSF after severe brain trauma.     

Enduring Effects of Early Life Stress on Firing Patterns of Hippocampal and Thalamocortical Neurons in Rats: Implications for Limbic Epilepsy

Early life stress results in an enduring vulnerability to kindling-induced epileptogenesis in rats, but the underlying mechanisms are not well understood. Recent studies indicate the involvement of thalamocortical neuronal circuits in the progression of kindling epileptogenesis. Therefore, we sought to determine in vivo the effects of early life stress and amygdala kindling on the firing pattern of hippocampus as well as thalamic and cortical neurons. Eight week old male Wistar rats, previously exposed to maternal separation (MS) early life stress or early handling (EH), underwent amygdala kindling (or sham kindling). Once fully kindled, in vivo juxtacellular recordings in hippocampal, thalamic and cortical regions were performed under neuroleptic analgesia. In the thalamic reticular nucleus cells both kindling and MS independently lowered firing frequency and enhanced burst firing. Further, burst firing in the thalamic reticular nucleus was significantly increased in kindled MS rats compared to kindled EH rats (p<0.05). In addition, MS enhanced burst firing of hippocampal pyramidal neurons. Following a stimulation-induced seizure, somatosensory cortical neurons exhibited a more pronounced increase in burst firing in MS rats than in EH rats. These data demonstrate changes in firing patterns in thalamocortical and hippocampal regions resulting from both MS and amygdala kindling, which may reflect cellular changes underlying the enhanced vulnerability to kindling in rats that have been exposed to early life stress.     

行为训练与小鼠海马突触蛋白磷酸化水平的相关性

本文对梭箱回避行为训练是否能改变小鼠海马突触蛋白磷酸化水平进行了检定。在小鼠训练结束的不同时程制备突触体,以γ-~(32)P-ATP作为标记进行离体测定。结果如下:(1)正常小鼠各脑区突触蛋白磷酸化水平无显著差异;(2)学习训练刚结束时,海马突触蛋白磷酸化水平与学习训练成绩的优劣有相关性;(3)学习训练结束后24h,上述相关性更为明显,且成绩良好组右侧海马突触蛋白磷酸化水平显著高于左侧,而成绩低劣组相反。海马突触蛋白磷酸化的这种不对称现象的成因尚不清楚。这些结果表明,海马突触蛋白磷酸化反应可能是习得行为的分子机制。     

Organic calcium antagonists verapamil and ryodipine prevent an increase of free calcium in synaptosomes of the rat brain during corazol kindling]

In experiments on Wistar male rats it was shown that i.p. administration of verapamil or ryodipine (1,4-dihydropyridine) 15 min before each daily injection of pentylenetetrazol (PTZ) in a subconvulsive dose 30 mg/kg during 28 days significantly delayed the development of PTZ-induced kindling and attenuated kindled seizure reactions during 21-23 days as compared with control. One week after kindling an increase in free Ca2+ concentration in control rat brain synaptosomes by 60% was revealed; the treatment by verapamil and ryodipine prevented this increase. Nevertheless, Ca2+ antagonists studied at least under our experimental conditions inspite of a significant decrease in free Ca2+ concentration in synaptosomes would not completely prevent the development of kindling. Thus, an alteration in calcium homeostasis will not be the only mechanism of plasticity and long-term changes of neurons during kindling.     

Glutamate Binding to Brain Membranes Is Increased in Pentylenetetrazole-Kindled Rats

Abstract: The specific binding of L-[³H]glutamate to its receptors was investigated on crude membrane preparations from different brain regions of pentylenetetrazole-kindled rats using a binding assay technique. Pentylenetetrazole kindling induced by 10 intraperitoneal applications of 45 mg/kg over a period of 20 days resulted in a significant increase of both the convulsive susceptibility of animals to the convulsant and the specific L-[³H]glutamate binding in hippocampus and in motor, frontal, and inferiotemporal (acoustic) cortex tested with a L-[³H]glutamate concentration of 50 n M . No differences were observed in the other brain structures studied. Kinetic studies indicated that the enhanced L-[³H]glutamate binding to hippocampal membranes from kindled rats reflects changes in the density of the glutamate binding sites rather than an increase in receptor affinity. To study the effect of acute generalized convulsions on L-[³H]glutamate binding to synaptosomal membranes of hippocampus and visual cortex, rats were treated 24 h before the experiment with 60 mg/kg of pentylenetetrazole, i.p. Under these conditions, no differences between treated and control rats were observed. From these findings, it is concluded that the increase in glutamate receptor density demonstrated in hippocampus and several neocortical brain structures of pentylenetetrazole-kindled rats may be the expression of a specific enhancement of susceptibility of glutamatergic systems to this excitatory amino acid developing in the course of formation of pentylenetetrazole-induced kindling.     

Apelin, an endogenous neuronal peptide, protects hippocampal neurons against excitotoxic injury

Several G protein-coupled receptors (GPCRs) mediate neuronal cell migration and survival upon activation by their native peptide ligands but activate death-signaling pathways when activated by certain non-native ligands. In cultured neurons, we recently described expression of the unique seven-transmembrane (7TM) -G protein-coupled receptor, APJ, which is also strongly expressed in neurons in the brain and various cell types in other tissues. We now demonstrate that the endogenous APJ peptide ligand apelin activates signaling pathways in rat hippocampal neurons and modulates neuronal survival. We found that (i) both APJ and apelin are expressed in hippocampal neurons; (ii) apelin peptides induce phosphorylation of the cell survival kinases AKT and Raf/ERK-1/2 in hippocampal neurons; and (iii) apelin peptides protect hippocampal neurons against NMDA receptor-mediated excitotoxicity, including that induced by human immunodeficiency virus type 1. Thus, apelin/APJ signaling likely represents an endogenous hippocampal neuronal survival response, and therefore apelin should be further investigated as a potential neuroprotectant against hippocampal injury.