【作者】 李克勇；

【导师】 孙凤艳； 王继江；

【作者基本信息】 复旦大学 ， 神经生物学， 2006， 博士

【摘要】 血管内皮生长因子(vascular endothelial growth factor,VEGF)最初发现于内皮细胞,以后又发现它也在脑内广泛表达。我们实验室以往研究结果揭示VEGF及其受体在幼年和成年大鼠脑内神经元均有表达,而且在急性分离的海马神经元,VEGF还可以通过钾通道的kv1.2蛋白酪氨酸磷酸化抑制延迟外向整合钾电流。最近,又有文献报导在海马脑片,VEGF可以抑制由电刺激兴奋性突触通路所诱导的突触后电位。为研究VEGF对中枢神经递质传递的直接调节作用,本实验采用全细胞膜片钳技术研究了VEGF对出生后14天大鼠海马脑片CA1区谷氨酸能兴奋性突触传递的作用以及γ-氨基丁酸能抑制性突触传递的作用及其机制。实验结果如下:一、VEGF对神经递质释放调节及其机制分析1.为了研究VEGF对突触传递的影响,我们首先观察了VEGF对自发的谷氨酸能兴奋性突触后电流(glutamatergic spontaneous excitatory postsynaptic currents,sEPSCs)和自发的γ-氨基丁酸能抑制性突触后电流(γ-amino-butylic acid ergicspontaneous inhibitory postsynaptic currents,GABAergic sIPSCs)的作用。研究记录了30个细胞,发现VEGF具有分别促进sEPSCs频率和抑制sIPSCs频率及幅度的作用,分别占记录细胞的30%和50%。该结果表明VEGF具有快速调节突触传递的作用。为了区分VEGF的这一作用究竟是突触前还是突触后的调节效应,我们从方法学的可行性角度出发,进一步研究了VEGF对γ-氨基丁酸能抑制性突触传递的作用并进行了深入探讨。2.为了明确VEGF是否对突触后GABA受体有直接调节作用,我们研究了VEGF对外源性GABA诱导的突触后电流的作用。外源性GABA所诱导的突触后电流的幅度可以作为突触后GABA受体反应性的指标。研究结果表明VEGF对外源性GABA诱导的突触后电流没有影响,提示VEGF对突触后GABA受体反应性没有直接调节作用。3.为了明确VEGF是否对突触前GABA的释放有抑制效应,我们以微小的γ-氨基丁酸能抑制性突触后电流(GABAergic miniature inhibitory postsynapticcurrents,GABAergic mIPSCs)的频率作为突触前轴突末梢GABA释放的指标,观察了VEGF对GABA释放的调节效应。研究观察到VEGF显著抑制了mIPSCs的频率,并有剂量依赖性。该结果提示VEGF通过突触前水平的调节,抑制了轴突末梢GABA的释放。4.为研究VEGF突触前抑制GABA释放的机制,我们分别用氯化钡和氯化隔阻断钾离子通道和高电压激活的钙离子通道(high-voRage-activated calciumchannels,HVA calcium channels),然后观察VEGF对mIPSCs频率的影响。研究观察到,当氯化钡存在时VEGF仍然显著抑制mIPSCs频率。而当氯化镉存在时,VEGF对mIPSCs频率的抑制作用被取消。这些结果提示VEGF是通过突触前轴突末梢电压依赖的钙离子通道(voltage-dependent calcium channels,VDCCs)抑制GABA的释放,同时也提示钾离子通道与VEGF的这一作用无关。二、VEGF对锥体神经元高电压激活的钙电流影响的研究以上结果提示VEGF参与了神经传递的调节,包括促进谷氨酸能兴奋性神经传递和抑制GABA能抑制性神经传递。机制分析表明VEGF通过VDCCs抑制突触前轴突末梢的GABA释放。VDCCs尤其是HVA钙离子通道不仅参与神经递质释放,而且在神经元的发育成熟和神经损伤中都起了重要作用。因此我们又研究了VEGF对HVA钙电流的直接作用。结果如下:1.VEGF可以快速可逆地、剂量依赖地抑制大鼠海马CA1区锥体神经元HVA钙电流。2.VEGF对HVA钙电流的电压依赖性没有影响。该结果提示VEGF对大鼠海马CA1区锥体神经元HVA钙电流具有直接快速的、可逆的和剂量依赖的抑制作用。结论:VEGF能快速调节突触前神经递质的释放以及抑制高电压激活的钙电流。机制分析揭示VEGF通过电压依赖的钙通道抑制突触前轴突末梢的GABA释放。创新点:1.首次提出VEGF具有促进谷氨酸能神经传递和抑制突触前GABA释放的作用。2.首次提出VEGF具有快速抑制神经元细胞膜高电压激活的钙电流的作用。3.首次提出VEGF通过抑制电压依赖的钙通道抑制突触前轴突末梢的GABA释放。更多还原

【Abstract】 Vascular endothelial growth factor (VEGF) was originally purified from endothelial cells. It has been demonstrated that VEGF widely expresses in the mammalian brain. Our previous studies have revealed that VEGF and its receptors locate at neurons in both immature and adult rat brains, and that VEGF inhibits the delayed outward potassium currents in hippocampal neurons via enhancing tyrosine phosphorylation of potassium channel kv 1.2 protein. In addition, a recent article reported that VEGF reduced the evoked postsynaptic potentials triggered by stimulating the excitatory pathways in hippocampal slices.To investigate whether VEGF regulates the neurotransmission, we used the whole-cell patch-clamp technique to record the glutamatergic spontaneous excitatory and GABAergic inhibitory postsynaptic currents (sEPSCs and sIPSCs) as well as the miniature inhibitory postsynaptic currents (mlPSCs) in the CAl pyramidal neurons of hippocampal slices from 14-day-old rats. The results are as follows:1. VEGF modulates the release of neurotransmitters in the hippocampus.(1) To study whether VEGF has any effect on neurotransmission, we separately recorded the glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) and the GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the hippocampal slices. We observed that VEGF significantly increased the frequency of sEPSCs and reduced the frequency and amplitude of sIPSCs. Among those recorded neurons, 30% and 50% of recorded neurons showed increase of sEPSCs and decrease of sIPSCs by VEGF, respectively. Then, we further analyzed whether the reduction of sIPSCs by VEGF was associated with the presynaptic or postsynaptic inhibition.(2) To study whether VEGF directly modulates the activities of postsynaptic GABA receptors, we recored the exogenous GABA-evoked postsynaptic chloride currents in hippocampal slices. The result showed that VEGF did not change the exogenous GABA-induced postsynaptic chloride currents, suggesting that VEGF-decreased sIPSCs does not relate to postsynaptic inhibition. Therefore, we further studied the effect of VEGF on the GABAergic miniature inhibitory postsynaptic currents (mlPSCs), of which frequency can reflect the release of GABA from presynaptic axonal terminals. We observed that VEGF dose-dependently reduced the mIPSC frequency. The results clearly demonstrated that the reduction of sIPSCs by VEGF was caused by inhibition of GABA release from the presynaptic axonal terminals.(3 ) To test the mechanism of the inhibition of GABA release by VEGF, we further studied the effect of VEGF on the release of GABA in the presence of barium, a general blocker of potassium channels, and cadmium, a nonspecific blocker of high-voltage-activated calcium channels (HVA calcium channels). The result showed that cadmium not barium prevented the reduction of the mIPSC frequency by VEGF. These results indicate that VEGF suppresses the release of GABA via HVA calcium channels. Furthermore, SU1498, a specific inhibitor of Flk-1, did not influence the efficacy of VEGF-inhibited mIPSCs, suggesting that Flk-1 is not involved in the VEGF’s inhibition.Putting together, the results suggest that VEGF enhances the gluatmatergic neurotransmission, and suppresses the GABA release from presynaptic neuronal terminals via VDCCs.2. VEGF inhibits HVA calcium currents in the CAl pyramidal neurons of hippocampal slices.As mentioned above, the inhibition of GABA release by VEGF is associated with the activities of VDCCs, especially the HVA calcium channels. Therefore, we next studied the effect of VEGF on the HVA calcium currents in the CAl pyramidal neurons of hippocampal slices from 14-day-old rats. The results showed that VEGF rapidly, reversibly, dose-dependently decreased the HVA calcium currents, and that VEGF did not influence the voltage-dependent property of HVA calcium currents.Collectively, VEGF can directly and specifically reduce the activities of HVA calcium channels in the hippocampal CAl pyramidal neurons.In the present thesis, we provide the first evidence that VEGF can directly modulate the release of neurotransmitters from the presynaptic axonal terminals via voltage-dependent calcium channels.更多还原