血管内皮生长因子通过电压依赖的钙通道调节新生大鼠海马神经递质的释放

发布时间：2017-12-30 23:30

本文关键词：血管内皮生长因子通过电压依赖的钙通道调节新生大鼠海马神经递质的释放　出处：《复旦大学》2006年博士论文　论文类型：学位论文

【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。
[Abstract]:Vascular endothelial growth factor (vascular endothelial, growth factor, VEGF) originally found in endothelial cells, and later found that it also widely expressed in the brain. Our previous studies have revealed that VEGF and its receptor in juvenile and adult rat brain neurons and expressed in hippocampal neurons, VEGF can also inhibit the delayed outward the integration of potassium currents by Kv1.2 protein tyrosine phosphorylation of potassium channel. Recently, there are reports in hippocampal slices of VEGF can be inhibited by stimulation of excitatory synaptic pathways induced by postsynaptic potential.
Direct effect of VEGF on central neurotransmitter transmission, the effect of VEGF on the 14 day after birth of glutamate in rat hippocampal slices CA1 area of effect and mechanism of excitatory synaptic transmission and GABAergic inhibitory synaptic transmission. Using whole cell patch clamp technique, the experimental results are as follows:
The regulation of neurotransmitter release and its mechanism analysis by VEGF
1. in order to study the effect of VEGF on synaptic transmission, we first observed the VEGF excitatory postsynaptic currents on spontaneous glutamate (glutamatergic spontaneous excitatory postsynaptic currents, sEPSCs) and spontaneous GABAergic inhibitory postsynaptic currents (acid ergicspontaneous inhibitory postsynaptic currents y -amino-butylic, GABAergic sIPSCs) on record role. 30 cells, VEGF significantly increased the frequency of sEPSCs and reduced the frequency and amplitude of sIPSCs, respectively, were recorded 30% 50%. and the results show that VEGF has a fast regulating role in synaptic transmission. In order to distinguish whether this effect of VEGF is presynaptic or postsynaptic regulation effect, we learn from the view of feasibility and method according to the further study of the VEGF of GABAergic inhibitory synaptic transmission and the role of the deep Go into the discussion.
2. in order to clarify whether VEGF has direct effect on the postsynaptic GABA receptor, we studied VEGF exogenous GABA induced postsynaptic currents. Induced by exogenous GABA postsynaptic current amplitude can be used as indicators of the postsynaptic GABA receptor reaction. The results showed that VEGF induced by exogenous GABA postsynaptic currents had no effect, suggesting that VEGF of GABA receptor in the postsynaptic response no direct role.
3. in order to determine whether VEGF on the presynaptic GABA release has inhibitory effect, we inhibitory synapses in small GABAergic current (GABAergic miniature inhibitory postsynapticcurrents, GABAergic mIPSCs) as the frequency of presynaptic terminals GABA release of the index, the effects of VEGF on regulating effect of GABA release. It was observed that VEGF significantly inhibition of the frequency of mIPSCs, and in a dose dependent manner. The results suggest that VEGF by regulating the levels of presynaptic axon terminals, inhibited the release of GABA.
4. to study the VEGF presynaptic inhibition mechanism of GABA release, we were using barium chloride and chloride by blocking calcium channel and potassium channel high voltage activated (high-voRage-activated calciumchannels, HVA calcium channels), and then observe the effect of VEGF on the frequency of mIPSCs. The study observed that when barium chloride exists when VEGF still significantly inhibited mIPSCs frequency and when the cadmium chloride exists, the inhibition effect of VEGF on the mIPSCs frequency was cancelled. These results suggest that VEGF is a calcium channel by presynaptic axon terminals voltage dependent (voltage-dependent calcium channels, VDCCs) inhibited the release of GABA, and it has nothing to do the role of potassium channel and VEGF.
The study of the effect of two, VEGF on the high voltage activated calcium current of pyramidal neurons
The above results suggest that VEGF is involved in the regulation of neurotransmission, including promoting glutamatergic transmission and inhibition of GABA can inhibit nerve transfer nerve excitability. Mechanism analysis showed that the VEGF inhibitory presynaptic terminals through the VDCCs GABA release of.VDCCs especially HVA calcium channel not only involved in neurotransmitter release, but also in neuronal maturation and nerve injury have played an important role. So we study the direct effect of VEGF on HVA calcium current. The results are as follows:
1.VEGF can reversibly inhibit the HVA calcium current of the pyramidal neurons in the hippocampal CA1 region of the rat.
2.VEGF has no effect on the voltage dependence of HVA calcium current.
The results suggest that VEGF has a direct, reversible and dose-dependent inhibitory effect on the HVA calcium current of the pyramidal neurons in the hippocampal CA1 region of the rat hippocampus.
Conclusion: VEGF can quickly regulate the release of presynaptic neurotransmitters and inhibit the high voltage activated calcium currents. The mechanism analysis reveals that VEGF inhibits GABA release from presynaptic axonal terminals through voltage dependent calcium channels.
Innovation point:
1. it is the first time that VEGF has the effect of promoting glutamate neurotransmission and inhibiting pre - synapse GABA release.
2. it is the first time that VEGF has the effect of rapidly inhibiting the high voltage activation of calcium current in the cell membrane of neurons.
3. it is the first time that VEGF inhibits the GABA release of the presynaptic axon end by inhibiting the voltage dependent calcium channel.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R33

【共引文献】