沉默视黄酸核受体α损伤大鼠原代海马神经元的钙兴奋性

目的:了解视黄酸核受体α(RARα)对大鼠神经元功能的必要性。方法:采用组织消化原代贴壁法分离培养大鼠原代海马神经元,利用腺病毒载体特异沉默RARα;利用Real-Time PCR分析沉默RARα对神经元视黄酸(RA)信号各受体以及神经细胞标志物的影响;利用活细胞钙影像分析沉默RARα对神经元钙兴奋性的影响。结果:免疫荧光显示,分离培养的细胞90%表达神经元标志神经元特异性烯醇化酶(NSE),腺病毒转染效率可达80%。PCR结果显示,RARα沉默后RARα表达降低75%(P<0.01),其他受体均显著降低(P<0.01),但RARβ显著上调(P<0.05)。活细胞钙影像显示,沉默组钙兴奋性显著降低(P<0.05),全反式视黄酸(ATRA)预处理24h能显著增强钙兴奋性(P<0.01)。结论:RARα的缺失能显著降低原代海马神经元的神经元标志物NSE的表达,并显著损伤神经元的钙兴奋性。

Mechanisms underlying low [Ca^(2+)]_o-induced increased excitability of hippocampal neurons

Objective Concentration of extracellular calcium （[Ca2＋]o） in the central nervous system decreases substantially in different conditions. It results in facilitating neuronal excitability. The goal of this study is to examine the mechanisms of enhanced neuronal excitation in low [Ca2＋]o in order to provide new clues to treat the hyperexcitability diseases in clinic. Methods Whole-cell patch-clamp technique and neuron culture were used in the study. Results The firing threshold of cultured hippocampal neurons decreased markedly in low [Ca2＋]o saline. Unexpectedly, apamine and isoprenaline, antagonists of medium afterhyperpolarization （mAHP） and slow AHP （sAHP） respectively, had no statistic significant effect on excitability of neurons. TTX at a low concentration was sufficient to inhibit/Nap, which blocked the increase of firing frequency in low [Ca2＋]o. It also reduced the number of spikes in normal [Ca2＋]o. Conclusion These results suggest that in cultured hippocampal neurons, modulation of spiking threshold but not AHP may cause the increased excitability in low [Ca2＋]o.

Regulating Neuronal Hyper-Excitability and Hyper-Synchrony in Epileptic Patients by Using PUFA, Calcium and ATP Buffering

Epilepsy is a severe neurological disorder clinically identified by hyper-excitability and/or hyper-synchrony in the cortex and other subcortical regions of the brain. To regulate such excitability and synchrony, Hodgkin and Huxley model has been deployed with either PUFA or calcium buffering coupled with ATP modulate neurotransmitter release. We formulate and analyze a system of differential equations that describe the effects of PUFA, ATP, and calcium buffering in regulating neuronal hyper-excitability and hyper-synchrony in epileptic patients. We observed that PUFA had diverse effects on the gating variables. Specifically, there was a significant reduction in the inhibitory potency of PUFA on the m-gates which may cause a direct inhibition of the voltage-gated Na＋ channels and thus reduce neuronal excitability in epileptic patients. Also, the activation of the potassium channels by PUFA directly limited the neuronal hyper-excitability, while a small change in voltage potential coupled with PUFA restraint activated the voltage dependent ion channels which aided in lowering epileptic excitability in patients. In addition, higher ATP buffer levels in the presence of PUFA caused a significant hyperpolarization which may decrease neuronal excitability while lower ATP level initiated neuron depolarization. These results clearly suggest that PUFA coupled with calcium and ATP buffering could be used to modulate neuronal excitability excessive synchrony in epileptic patients.

Excitotoxicity effects of glutamate on human neuroblastoma SH-SY5Y cells via oxidative damage

Objective To investigate the mechanisms of excitotoxic effects of glutamate on human neuroblastoma SH-SY5Y cells. Methods SH-SY5Y cell viability was measured by MTT assay. Other damaged profile was detected by lactate dehydrogenase （LDH） release and by 4＇, 6-diamidino-2-phenylindole （DAPI） staining. The cytosolic calcium concentration was tested by calcium influx assay. The glutamate-induced oxidative stress was analyzed by cytosolic glutathione assay, superoxide dismutase （SOD） assay and extracellular malondialdehyde （MDA） assay. Results Glutamate treatment caused damage in SH- SY5Y cells, including the decrease of cell viability, the increase of LDH release and the alterations of morphological structures. Furthermore, the concentration of cytoplasmic calcium in SH-SY5Y cells was not changed within 20 min following glutamate treatment, while cytosolic calcium concentration significantly increased within 24 h after glutamate treatment, which could not be inhibited by MK801, an antagonist of NMDA receptors, or by LY341495, an antagonist of metabotropic glutamate receptors. On the other hand, oxidative damage was observed in SH-SY5Y cells treated with glutamate, including decreases in glutathione content and SOD activity, and elevation of MDA level, all of which could be alleviated by an antioxidant Tanshinone IIA （Tan IIA, a major active ingredient from a Chinese plant Salvia Miltiorrhiza Bge）. Conclusion Glutamate exerts toxicity in human neuroblastoma SH-SY5Y cells possibly through oxidative damage, not through calcium homeostasis destruction mediated by NMDA receptors.