Alterations in Seizure Susceptibility and in Seizure-induced Plasticity after Pharmacologic and Genetic Manipulation of the Fibroblast Growth Factor-2 System

Summary:  Purpose: The adult brain undergoes activity-dependent plastic modifications during pathologic processes that are reminiscent of those observed during development. For example, seizures induce neuronal loss, neurogenesis, axonal and dendritic sprouting, gliosis, and circuit remodeling. Neurotrophic factors and fibroblast growth factor-2 (FGF-2), in particular, are well-known mediators in each of these cellular events. The aim of this minireview is to summarize and discuss the data supporting the idea that FGF-2 may be involved in seizure generation and in their sequelae.
Methods: We used epilepsy models of kainate and kindling, with FGF-2 knockout mice and FGF-2 overexpressing mice.
Results: Seizures increase FGF-2 mRNA and protein levels in specific brain areas and upregulate the expression of its receptor FGFR-1. Short-term intrahippocampal injection of FGF-2 cause seizures, whereas long-term i.c.v. infusion of low-dose FGF-2 does not affect kainate seizures but promotes behavioral recovery and reduces hippocampal damage. Kainate seizure severity is not altered in FGF-2 knockout mice, but is increased in FGF-2 overexpressing mice.
Conclusions: FGF-2 is implicated in seizure susceptibility and in seizure-induced plasticity.     

Non-NMDA receptors in frog retina: an immunocytochemical study

Glutamate is one of the main neurotransmitters in the retina. Its effects are mediated by a large number of ionotropic and metabotropic membrane receptors. The distribution of ionotropic AMPA receptor subunits GluR1-4, kainate receptor subunits GluR5-7 and KA2, delta receptors 1-2, as well as the metabotropic receptor mGluR6 were studied in the frog retina. Indirect immunofluorescence was used to localize the different receptor subunits. Results showed that all subunits, with the exception of GluR1 and GluR5, are widely distributed in the retina. They are mainly located in both plexiform layers: the outer (OPL) and the inner one (IPL), where punctate labelling, a sign of synaptic localization, is observed. The metabotropic receptor mGluR6 is localised only in the OPL. The AMPA receptor subunit GluR4 is localised on the glial Müller cells of the retina. The vast majority of the subunits possess specific patterns of labelling that indicate that they are involved with different retinal functions. The significance of the AMPA receptors and involvement of glia in modulation of synaptic transmission are discussed.     

迷走神经刺激对红藻氨酸致癫癎大鼠海马及齿状回NMDAR1的影响

目的:利用红藻氨酸(kainate,KA)致大鼠复杂部分性发作模型,观察海马及齿状回等易损脑区N-甲基-D-门冬氨酸受体1亚单位(NMDAR_1)的变化,以期进一步探明迷走神经刺激治疗癫(疒间)的作用机制。方法:免疫细胞化学法。结果:正常大鼠NMDAR_1阳性结构可见于海马各区及齿状回;KA给药后1h海马CA1、CA3、齿状回NMDAR_1的密度开始增高;3h达高峰,以后逐渐下降,24h后基本恢复正常;预先给予左侧迷走神经电刺激治疗的大鼠相应脑区NMDAR_1密度较KA致(疒间)组明显降低,具有统计学差异。结论:迷走神经刺激抑制癫(疒间)发作可能是通过降低易损脑区神经元NMDAR_1活性而发挥作用的。     

Neurochemical consequences of kainate-induced toxicity in brain: involvement of arachidonic acid release and prevention of toxicity by phospholipase A2 inhibitors

In kainate-induced neurotoxicity, the stimulation of kainate receptors results in the activation of phospholipase A₂ and a rapid release of arachidonic acid from neural membrane glycerophospholipids. This process raises arachidonic acid levels and produces alterations in membrane fluidity and permeability. These result in calcium influx and stimulation of lipolysis and proteolysis, production of lipid peroxides, depletion of ATP, and loss of reduced glutathione. As well as the above neurochemical changes, stimulation of ornithine decarboxylase, altered activities of protein kinase C isozymes, and expression of immediate early genes, cytokines, growth factors, and heat shock proteins have also been reported. Kainate-induced stimulation of arachidonic acid release, calcium influx, accumulation of lipid peroxides and products of their decomposition, especially 4-hydroxynonenal (4-HNE), along with alterations in cellular redox state and ATP depletion may play important roles in kainate-induced cell death. Thus the consequences of altered glycerophospholipid metabolism in kainate-induced neurotoxicity can lead to cell death. Kainate-induced neurotoxicity initiates apoptotic as well as necrotic cell death depending upon the intensity of oxidative stress and abnormality in mitochondrial function. Other neurochemical changes may be related to synaptic reorganization following kainate-induced seizures and may be involved in recapitulation of hippocampal development and synaptogenesis.     

尼莫地平对海马神经元保护作用的实验研究

目的:探讨尼莫地平对海藻酸(KA)处理海马神经元的保护作用。方法:在体外培养7d的海马神经元中加入不同浓度的尼莫地平(50、100、200μmol/L),30min后分别加入两种浓度的KA(50、100μmol/L),培养24h后,用MTT细胞活性测定法和免疫细胞化学染色评定尼莫地平对KA损伤海马神经元的保护作用。结果:50、100μmol/L的KA均能使海马神经元活性明显下降(P<0.05);50μmol/L的KA所致的神经元活性下降可被50、100、200μmol/L的尼莫地平阻断(P<0.05),仅200μmol/L的尼莫地平可阻断由100μmol/L的KA所致的神经元活性的下降(P<0.05)。免疫细胞化学染色所示的海马神经元形态学变化同MTT检测结果基本一致。结论:尼莫地平对KA所致的海马神经元损伤具有剂量依赖性保护作用。     

A pharmacological characterization of chloride- and potassium-dependent inhibitions in the CA3 region of the rat hippocampus in vitro

Summary Population spikes evoked in CA3 pyramidal cells of rat hippocampal slices by stimulation of the fimbria are subject to an early and a late inhibition following activation of the perforant path or the mossy fibres. The early inhibition is known to be GABA-mediated, and is blocked by addition of bicuculline to the superfusing medium; however the late inhibition is bicuculline-insensitive. Both inhibitions are reduced by the addition of (±)-baclofen or noradrenaline to the medium; the early inhibition only is blocked by D-Ala-D-Leu-enkephalinamide while the late inhibition is preferentially reduced by kainate. These data together with the results in the preceding paper suggest that both inhibitions are synaptically mediated, possibly by two distinct types of interneurone, one GABAergic and a second which may release an unidentified transmitter.     

The time course of NMDA-and kainate-induced cGMP elevation and glutamate release in cultured neuron

The levels of extracellular glutamate, intracellular Ca²⁺ ([Ca2+]_i) and cGMP were determined for 1 h with the excitatory amino acids, N-methyl-D-aspartate (NMDA) or kainate in cultured cerebellar granule cells. Both NMDA and kainate produced a time-dependent release of glutamate, and kainate was more potent than NMDA in glutamate elevation. The elevation of extracellular glutamate was not purely governed by intracellular Ca²⁺ concentration. However, in opposite to the time-dependent elevation of glutamate, the elevation of cGMP by NMDA and kainate were at maximum level in short-time (1 min) incubation then remarkably decreased with longer incubation times. Post-applications (30 min after agonist) of EAA antagonst did not block EAAs-induced glutamate elevation. However, NMDA antagonist, phencyclidine (PCP), blocked NMDA-induced cGMP elevation at pre- or post-application, but kainate antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), paradoxically augmented kainate-induced cGMP elevation for 1 h incubation. These results show that NMDA or kainate induces time-dependent elevations of extracellular glutamate, while the elevations of cGMP by these EAAs are remarkably decreased with longer incubation times. However, NMDA- and kainate-induced glutamate release was blocked by pre-application of each receptor antagonist but not by post-application while EAA-induced [Ca²⁺]_i was blocked by post-application of antagonist. These observations suggest that EAA-induced elevation of [Ca²⁺]_i is not parallel with elevation of glutamate release or cGMP.     

Epileptiform bursts elicited in CA3 hippocampal neurons by a variety of convulsants are not blocked by N-methyl-d-aspartate antagonists

Intracellular and extracellular recordings from CA₃ hippocampal neurons in vitro were used to study the ability of several NMDA (N-methyl-d-aspartate) receptor antagonists to suppress epileptiform bursts induced by NMDA and convulsants not thought to act at NMDA receptors. The antagonists, APV (d-2-amino-5-phosphonovalerate), AP-7 (d,l-2-amino-7-phosphonoheptanoate) and CPP (d,l-3-[(±)-2-car☐ypiperazin-4-yl-]-propyl-1-phosphonic acid), blocked the spontaneous and evoked bursts induced by NMDA. CPP, but not APV or AP-7, prevented the development of bursts induced by Mg-free medium. The NMDA antagonists failed to block bursting induced by kainate, 7 mM K⁺, mast cell degranulating peptide, anoxia or spontaneous bursting. In some cases the NMDA antagonists induced spontaneous bursts or enhanced burst frequency, a proconvulsant effect. It is concluded that activation of NMDA receptors is sufficient but not necessary for burst generation in the CA₃ region.     

Metabolic and pathological effects of temporal lobe epilepsy in rat brain detected by proton spectroscopy and imaging

The goal of these experiments was to test the hypothesis that in an animal model of temporal lobe epilepsy (TLE), magnetic resonance spectroscopic measurement of N-acetylaspartate (NAA) and other metabolites, together with magnetic resonance imaging, provides a sensitive in vivo method to localize and monitor the progression of neuronal cell death and gliosis. Seizures were induced in rats by unilateral hippocampal injection of kainate. Magnetic resonance measurements were made from 1 to 84 days using proton spectroscopic imaging (¹H-MRSI), T2-weighted imaging (T2WI) and diffusion-weighted imaging (DWI). The results were compared with findings on histological sections. Decreased NAA and creatine levels and increased apparent diffusion coefficient of water were found in the ipsilateral hippocampus after 14 days where neuronal loss and gliosis were observed. In the contralateral hippocampus a significant increase of choline level was observed. These results suggest that ¹H-MRSI is a useful in vivo method for localizing neuronal loss and may also indicate additional pathological and metabolic alterations. In addition, DWI may be a useful method for in vivo detection of tissue alterations due to TLE.     

Cytotoxic effects of kainate ligands on HEK cell lines expressing recombinant kainate receptors

Exposure of neurons either for prolonged periods of time or to high concentrations of excitatory amino acids (EAA), such as glutamate, results in neuronal death. Kainate also causes cell toxicity through the glutamate receptors. However, it is unclear whether the kainate receptor itself mediates any of the toxic responses. In the present study, HEK cells expressing the GluR6 ± KA2 receptor subunit(s) were studied for their susceptibility to toxicity through the kainate receptor by kainate ligands. The natural ligand, glutamate, did not result in toxicity to the recombinant cell lines over that observed with the untransfected HEK cells, whereas kainate produced a 2–3-fold increase in LDH in both the HEK/GluR6 (ANOVA, P = 0.0001) and HEK/GluR6 + KA2 (ANOVA, P = 0.0002) cell lines following treatment with various dosages, but did not affect the HEK cells. Similar 2–3-fold increases in LDH activity were detected in both recombinant cell lines following treatment with 100 nM of SYM2081 ((2S,4R)-4-methylglutamic acid), a dose at which agonistic activity is elicited. The rank order potencies for eliciting toxicity are consistent with the previously reported EC₅₀ values (SYM2081 > kainate >>> glutamate). Surprisingly, the kainate antagonist, NBQX, was the most toxic of the compounds tested although it had an affinity for the kainate receptor similar to glutamate. Treatment with as little as 10 nM elicited a dramatic increase in toxicity (6–10-fold) in the recombinant cell lines. At 1 μM, NBQX was significantly more toxic (Fisher PLSD, P < 0.05) than any of the other compounds tested. Thus, it appears that cell toxicity can be mediated via kainate receptor through two independent mechanisms: activation and blockage of the kainate receptor.