Potentiation of glutamatergic agonist-induced inositol phosphate formation by basic fibroblast growth factor is related to developmental features in hippocampal cultures: neuronal survival and glial cell proliferation

We investigated the modulation by growth factors of phospholipase C (PLC)-linked glutamate receptors during in vitro development of hippocampal cultures. In defined medium, glial cells represent between 3 and 14% of total cell number. When we added basic fibroblast growth factor (bFGF) 2 h after plating, we found: (i) a neuroprotection from naturally occurring death for up to 5 days; (ii) a proliferation of glial cells from day 3; and (iii) a potentiation of quisqualate (QA)-induced inositol phosphate (IP) formation from 1 to 10 days in vitro (DIV) and 1S, 3R-amino-cyclopentane-1,3-dicarboxylate (ACPD) response from 3 to 10 DIV. The antimitotic cytosine-beta,D-arabinofuranoside (AraC) blocked glial cell proliferation induced by bFGF, but not neuroprotection. Under these conditions, the early potentiation of the QA response (1-3 DIV) was not changed, while the ACPD and late QA response potentiations were prevented (5-10 DIV). Epidermal growth factor was not neuroprotective but it induced both glial cell proliferation and late QA or ACPD potentiation. Surprisingly, the early bFGF-potentiated QA-induced IP response was blocked by 6, 7-dinitro-quinoxaline-2,3-dione (DNQX), suggesting the participation of ionotropic (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (KA) receptors. The delayed bFGF-potentiated ACPD-induced IP response is inhibited by (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), indicating possible activation of glial metabotropic receptors. These results suggest that, in hippocampal cultures, bFGF modulates AMPA and metabotropic glutamate receptors linked to the IP cascade, possibly in relation to the regulation of neuronal survival and glial cell proliferation, respectively.     

Effect of carbamazepine, oxcarbazepine and lamotrigine on the increase in extracellular glutamate elicited by veratridine in rat cortex and striatum

Lamotrigine, carbamazepine and oxcarbazepine inhibit veratrine-induced neurotransmitter release from rat brain slices in concentrations corresponding to those reached in plasma or brain in experimental animals or humans after anticonvulsant doses, presumably due to their sodium channel blocking properties. Microdialysis measurements of extracellular glutamate and aspartate were carried out in conscious rats in order to investigate whether corresponding effects occur in vivo. Veratridine (10 M) was applied via the perfusion medium to the cortex and the corpus striatum in the presence of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (1 mM in perfusion medium). Maximally effective anticonvulsant doses of carbamazepine (30 mg/kg), oxycarbazepine ( 60 mg/kg) and lamotrigine (15 mg/kg) were given orally.The uptake inhibitor increased extracellular glutamate and aspartate about 2-fold in striatum and about 7-fold and 3-fold, respectively, in cortex. Veratridine caused a further 2–3-fold increase in extracellular glutamate in striatum and cortex, respectively, but its effect on extracellular aspartate was less marked in both areas. None of the anticonvulsant compounds affected the veratridine-induced increases in extracellular glutamate or aspartate in the striatum which were, however, markedly inhibited by tetrodotoxin (1 M) and thus are sensitive to sodium channel blockade. In the cortex, the same drugs at the same doses did cause about 50% inhibition of the veratridine-induced increase in extracellular glutamate. Carbamazepine and to a lesser extent lamotrigine, but not oxcarbazepine, also inhibited the veratridine-induced increase in extracellular aspartate in the cortex.Although our results might seem to support the view that inhibition of glutamate and aspartate release is responsible for the anticonvulsant effects of lamotrigine, carbamazepine and oxcarbazepine, two complementary findings argue against this interpretation. First, as previously shown, inhibition of electrically induced release of glutamate requires 5 to 7 times higher concentrations of these compounds than release elicited by veratrine. Second, the present study indicates that doses totally suppressing convulsions caused no inhibition in the striatum and at best a 50% inhibition in the brain cortex. From this we conclude that the doses used here, although to some extent effective against veratridine, did not suppress the release of GLU and ASP elicited by the normal ongoing electrical activity of the glutamatergic and aspartatergic neurons and that the mechanism of the suppression of convulsions must be sought elsewhere.     

Interrelationship between retinal ischaemic damage and turnover and metabolism of putative amino acid neurotransmitters,glutamate and GABA

Conditions causing a reduction of oxygen availability (anoxia), such as stroke or diabetes, result in drastic changes in ion movements, levels of neurotransmitters and metabolites and subsequent neural death. Currently, there is no clinically available treatment for anoxia induced neural cell death resulting in drastic and permanent central nervous system dysfunction. However, there have been some exciting developments in experimentally induced anoxic conditions where several classes of drugs appear to significantly reduce neural cell death. This report aims to provide the foundations for understanding both the basic mechanisms involved in retinal ischaemic damage and experimental treatments used to prevent such damage. We discuss the normal release, actions and uptake of the fast retinal neurotransmitters, glutamate and GABA, in the vertebrate retina. Immunocytochemistry is used to demonstrate that both glutamate and GABA are found in the macaque retina. Following this is a discussion on how ischaemia may enhance neurotransmitter release or disrupt its uptake, thus causing an increase in extracellular concentration of these neurotransmitters and subsequent neuronal damage. The mechanisms involved in glutamate neurotoxicity are reviewed, because excess glutamate is the likely cause of retinal ischaemic damage. Finally, the mechanisms behind four possible modes of treatment of neurotransmitter toxicity and their advantages and disadvantages are discussed. Hopefully, further research in this area will lead to the development of a rational therapy for retinal, as well as cerebral ischaemia.Abbreviations -KG -ketoglutarate - AAT aspartate amino transferase - AC amacrine cell - ACL amacrine cell layer - BC bipolar cell - CNS central nervous system - EAA excitatory amino acids - G'ase glutaminase - GABA -amino butyric acid - GABA-T GABA transaminase - GAD glumatic acid decarboxylase - GC ganglion cell - GCL ganglion cell layer - GDH glutamate dehydrogenase - gj gap junction - GS glutamine synthetase - HC horizontal cell - ILM inner limiting membrane - INL inner nuclear layer - IPC inter-plexiform cell - IPL inner plexiform layer - IS inner segment of photoreceptor - NFL nerve fibre layer - NMDA N-methyl-D-aspartate - OLM outer limiting membrane - ONL outer nuclear layer - OPL outer plexiform layer - OS outer segment of photoreceptor - Ox acetateoxaloacetate - RL receptor layer - SSAD succinate semi-aldehyde decarboxylase - Succinate SA succinate semi aldehyde - TCA cycle tricarboxylic acid cycle     

Coexistence of GABA and glutamate in mossy fiber terminals of the primate hippocampus: an ultrastructural study.

One of the links in the trisynaptic circuit of the hippocampus is the synapse between the mossy fibre terminals of dentate granule cells and CA3 pyramidal cells of Ammon's horn. This synapse has been physiologically characterized as excitatory, and there is pharmacological and immunohistochemical evidence that mossy fibre terminals utilize glutamate as a neurotransmitter. This study demonstrates the presence of GABA-immunoreactivity in mossy fibre axons and terminals of the monkey at the electron microscopic level. We combined Golgi impregnation to identify CA3 pyramidal neurones, with postembedding immunocytochemistry to characterize the inputs to the identified cells. GABA immunoreactivity was present in mossy fibre terminals that made synaptic contact with complex embedded spines of identified Golgi-impregnated CA3 pyramidal neurones. GABA immunoreactivity could be demonstrated in serial sections of the same mossy fibre terminals by using 3 different antisera raised against GABA. In serial sections, the mossy fibre terminals were shown to be immunoreactive for both glutamate and GABA. In contrast, glutamate immunoreactivity but not GABA immunoreactivity was found in other terminals that did not have the morphological characteristics of mossy fibre terminals. GABA immunoreactivity in mossy fibre terminals was also demonstrated in a human surgical specimen of hippocampus. The coexistence of an "excitatory" amino acid and of an "inhibitory" amino acid in the same "excitatory" nerve terminal raises the possibility of corelease of the two transmitters, suggesting that the control of hippocampal neural activity is more complex than hitherto suspected.     

Participation of low-threshold calcium spikes in excitatory synaptic transmission in guinea pig medial frontal cortex

We studied the activation of low-threshold calcium spikes (LTS) by excitatory postsynaptic potentials in pyramidal neurons from guinea pig medial frontal cortex with intracellular recording. We used extracellular bicuculline and phaclofen and intracellular QX-314 to block inhibitory synaptic potentials and sodium currents. Postsynaptic potentials were evoked by stimulation of layer I. We found that large (> 10-15 mV) excitatory synaptic potentials evoked from membrane potentials more negative than -75 mV were able to trigger LTS. The activation of LTS resulted in an increase of the rising slope or amplitude of the synaptic potentials depending on the size of the excitatory postsynaptic potential (EPSP). We used 100 microM NiCl2 to confirm the presence of LTS as part of the EPSPs. The N-methyl-D-aspartate (NMDA) and non-NMDA components of the excitatory synaptic potentials were isolated using (+/-)2-amino-5-phosphonovaleric acid (APV; 50 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 microM); both components could, independently, trigger an LTS. With recordings made with K+ acetate-filled electrodes, we show that the activation of LTS was critical to allow excitatory synaptic potentials to reach the threshold of action potential firing; also, this amplification of synaptic responses produced the firing of more than a single action potential by the postsynaptic cell. These results demonstrate that in cortical pyramidal neurons the activation of low-threshold calcium spikes results in the amplification of synaptic responses.     

Regional and selective effects of oestradiol and progesterone on NMDA and AMPA receptors in the rat brain

We investigated the effect of 10 months ovariectomy and a correction therapy, 2 weeks before the rats were killed, of oestradiol, progesterone or their combination on NMDA and AMPA receptor binding in the hippocampus, dentate gyrus, striatum, nucleus accumbens and frontal cortex of the rat brain as well as on amino acid levels in frontal cortex. NMDA and AMPA binding densities were assayed by autoradiography using, respectively, L-[3H]glutamate and [3H]AMPA; amino acid concentrations were measured by high performance liquid chromatograhy (HPLC) coupled with UV detection. Ovariectomy was without effect on NMDA and AMPA binding density in all brain regions assayed except in the hippocampal CA1 region and dentate gyrus where it decreased NMDA binding density compared to intact rats values. Oestradiol restored and increased NMDA binding density in the CA1 subfield and the dentate gyrus of ovariectomized rats but, by contrast, it decreased binding density in the striatum and in the frontal cortex while having no effect in the CA2/3 subfield of the hippocampus and in the nucleus accumbens. Oestradiol was without effect on AMPA binding density in the hippocampus and the dentate gyrus but it reduced AMPA binding density in the striatum, the frontal cortex and the nucleus accumbens. Progesterone, and oestradiol combined with progesterone, decreased NMDA but not AMPA binding density in the frontal cortex of ovariectomized rats, and they were without effect on these receptors in the other brain regions assayed. Amino acid concentrations in the frontal cortex were unchanged after ovariectomy or steroid treatments. The effect of oestradiol in the hippocampus confirmed in the present study and our novel findings in the frontal cortex, striatum and nucleus accumbens may have functional significance for schizophrenia and neurodegenerative diseases.     

Generation and propagation of 4-AP-induced epileptiform activity in neonatal intact limbic structures in vitro

We examined the generation, propagation and pharmacology of 4-aminopyridine (4-AP)-induced epileptiform activity (EA) in the intact interconnected limbic structure of the newborn (P0-7) rat in vitro. Whole-cell recordings of CA3 pyramidal cells and multisite field potential recordings in CA3, CA1, dentate gyrus, and lateral and medial entorhinal cortex revealed 4-AP-induced EA as early as P0-1. At this age, EA was initiated in the CA3 region and propagated to CA1, but not to the entorhinal cortex. Starting from P3-4, EA propagated from CA3 to the entorhinal cortex. Along the CA3 septo-temporal axis, EA arose predominantly from the septal pole and spread towards the temporal site. Whereas the onset of 4-AP-induced EA decreased with age from 21.2 +/- 1.6 min at P0-1 to 4.7 +/- 0.63 min at P6-7, the seizure duration increased in the same age groups from 98 +/- 14 s to 269.4 +/- 85.9 s, respectively. The EA was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by DL-2-amino-5-phosphonovaleric acid (APV), (+)-MK-801 hydrogen maleate (MK-801) or (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG), suggesting that they were mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor activation. We conclude that: (i) the septal pole of the hippocampal CA3 region plays a central role in the generation of EA in the neonatal limbic system; and (ii) AMPA/kainate receptor-mediated EA can be generated in CA3 already at birth. Therefore, the recurrent collateral synapses and circuits required for the generation of EA are developed earlier than previously suggested on the basis of studies on hippocampal slices.     

银杏叶提取物对谷氨酸诱导的兔视网膜NO水平及细胞凋亡的影响

目的 :探讨银杏叶提取物 (extract of ginkgo biloba ,EGB)对谷氨酸诱导的兔视网膜NO水平及细胞凋亡的影响作用。方法 :利用谷氨酸兔玻璃体内注射 ,诱导视网膜细胞凋亡的模型 ,眼球后注射不同剂量的EGB ,采用分光光度法测定兔视网膜NO水平 ,琼脂糖凝胶电泳分析视网膜细胞DNA断裂。结果 :实验对照组视网膜的NO的水平与空白对照组相比显著增加 (P<0.001) ,EGB大剂量治疗组视网膜NO水平比实验对照组显著降低 (P<0.001) ,EGB小剂量治疗组视网膜的NO水平与实验组相比轻度降低 ,但无统计学差别。EGB大剂量治疗组和空白对照组视网膜细胞无DNA凋亡样断裂 ,而实验组和EGB小剂量治疗组视网膜细胞DNA凋亡样断裂。结论 :银杏叶提取物可能通过抑制谷氨酸诱导的兔视网膜NO自由基的产生而抑制细胞凋亡 ,对谷氨酸诱导的兔视网膜损伤具有重要保护作用。     

谷氨酸转运体、谷氨酸/胱氨酸转运体与谷氨酸神经细胞毒作用

谷氨酸转运体与谷氨酸／胱氨酸转运体在脑缺血疾病中起重要作用，谷氨酸转运体的结构或功能改变可使细胞间隙的谷氨酸浓度急剧升高，激活NMDA受体产生一系列的表现，同时抑制谷氨酸／胱氨酸转运体对胱氨酸的摄取，介导谷胱苷肽耗竭、氧自由基升高、胞内钙升高、线粒体损伤、细胞色素c释放等神经细胞毒环节，激活半胱天冬酶诱导凋亡。可进一步加重谷氨酸的神经细胞毒作用。     

藏药七十味珍珠丸对惊厥小鼠脑内氨基酸含量的影响

目的　观察七十味珍珠丸的抗惊厥作用及作用机制。方法　七十味珍珠丸ig给药 ,使用硫代氨基脲制成惊厥小鼠模型 ,测定惊厥开始时间、致死率 ;采用高压液相色谱仪测定小鼠脑内抑制性氨基酸γ 氨基丁酸 (GABA)、甘氨酸 (Gly)和兴奋性氨基酸谷氨酸 (Glu)、天门冬氨酸 (Asp)的含量。 结果　七十味珍珠丸对硫代氨基脲所致惊厥的开始时间 ,具有显著延迟作用 ,可明显减少小鼠致死率。有升高惊厥小鼠脑内GABA ,降低脑内Glu含量的作用趋势 ,但无显著差异 (P >0 .0 5 ) ,而对Glu/GABA的比值 ,却有明显的降低作用 (P <0 .0 5、P <0 .0 1) ,使Glu/GABA保持正常动物的水平 ,即维持中枢神经系统兴奋和抑制状态平衡的作用 ,阻止惊厥发生。结论　该药具有抗惊厥作用 ,其作用机制可能是通过调节脑内兴奋性和抑制性氨基酸的平衡来实现的