本文關鍵詞：血管內皮生長因子通過電壓依賴的鈣通道調節(jié)新生大鼠海馬神經(jīng)遞質的釋放　出處：《復旦大學》2006年博士論文　論文類型：學位論文

  更多相關文章： 血管內皮生長因子 血管內皮生長因子受體 全細胞膜片鉗 興奮性突觸傳遞 抑制性突觸傳遞 電壓依賴的鈣通道 海馬

【摘要】： 血管內皮生長因子(vascular endothelial growth factor,VEGF)最初發(fā)現(xiàn)于內皮細胞,以后又發(fā)現(xiàn)它也在腦內廣泛表達。我們實驗室以往研究結果揭示VEGF及其受體在幼年和成年大鼠腦內神經(jīng)元均有表達,而且在急性分離的海馬神經(jīng)元,VEGF還可以通過鉀通道的kv1.2蛋白酪氨酸磷酸化抑制延遲外向整合鉀電流。最近,又有文獻報導在海馬腦片,VEGF可以抑制由電刺激興奮性突觸通路所誘導的突觸后電位。 為研究VEGF對中樞神經(jīng)遞質傳遞的直接調節(jié)作用,本實驗采用全細胞膜片鉗技術研究了VEGF對出生后14天大鼠海馬腦片CA1區(qū)谷氨酸能興奮性突觸傳遞的作用以及γ-氨基丁酸能抑制性突觸傳遞的作用及其機制。實驗結果如下: 一、VEGF對神經(jīng)遞質釋放調節(jié)及其機制分析 1.為了研究VEGF對突觸傳遞的影響,我們首先觀察了VEGF對自發(fā)的谷氨酸能興奮性突觸后電流(glutamatergic spontaneous excitatory postsynaptic currents,sEPSCs)和自發(fā)的γ-氨基丁酸能抑制性突觸后電流(γ-amino-butylic acid ergicspontaneous inhibitory postsynaptic currents,GABAergic sIPSCs)的作用。研究記錄了30個細胞,發(fā)現(xiàn)VEGF具有分別促進sEPSCs頻率和抑制sIPSCs頻率及幅度的作用,分別占記錄細胞的30%和50%。該結果表明VEGF具有快速調節(jié)突觸傳遞的作用。為了區(qū)分VEGF的這一作用究竟是突觸前還是突觸后的調節(jié)效應,我們從方法學的可行性角度出發(fā),進一步研究了VEGF對γ-氨基丁酸能抑制性突觸傳遞的作用并進行了深入探討。 2.為了明確VEGF是否對突觸后GABA受體有直接調節(jié)作用,我們研究了VEGF對外源性GABA誘導的突觸后電流的作用。外源性GABA所誘導的突觸后電流的幅度可以作為突觸后GABA受體反應性的指標。研究結果表明VEGF對外源性GABA誘導的突觸后電流沒有影響,提示VEGF對突觸后GABA受體反應性沒有直接調節(jié)作用。 3.為了明確VEGF是否對突觸前GABA的釋放有抑制效應,我們以微小的γ-氨基丁酸能抑制性突觸后電流(GABAergic miniature inhibitory postsynapticcurrents,GABAergic mIPSCs)的頻率作為突觸前軸突末梢GABA釋放的指標,觀察了VEGF對GABA釋放的調節(jié)效應。研究觀察到VEGF顯著抑制了mIPSCs的頻率,并有劑量依賴性。該結果提示VEGF通過突觸前水平的調節(jié),抑制了軸突末梢GABA的釋放。 4.為研究VEGF突觸前抑制GABA釋放的機制,我們分別用氯化鋇和氯化隔阻斷鉀離子通道和高電壓激活的鈣離子通道(high-voRage-activated calciumchannels,HVA calcium channels),然后觀察VEGF對mIPSCs頻率的影響。研究觀察到,當氯化鋇存在時VEGF仍然顯著抑制mIPSCs頻率。而當氯化鎘存在時,VEGF對mIPSCs頻率的抑制作用被取消。這些結果提示VEGF是通過突觸前軸突末梢電壓依賴的鈣離子通道(voltage-dependent calcium channels,VDCCs)抑制GABA的釋放,同時也提示鉀離子通道與VEGF的這一作用無關。 二、VEGF對錐體神經(jīng)元高電壓激活的鈣電流影響的研究 以上結果提示VEGF參與了神經(jīng)傳遞的調節(jié),包括促進谷氨酸能興奮性神經(jīng)傳遞和抑制GABA能抑制性神經(jīng)傳遞。機制分析表明VEGF通過VDCCs抑制突觸前軸突末梢的GABA釋放。VDCCs尤其是HVA鈣離子通道不僅參與神經(jīng)遞質釋放,而且在神經(jīng)元的發(fā)育成熟和神經(jīng)損傷中都起了重要作用。因此我們又研究了VEGF對HVA鈣電流的直接作用。結果如下: 1.VEGF可以快速可逆地、劑量依賴地抑制大鼠海馬CA1區(qū)錐體神經(jīng)元HVA鈣電流。 2.VEGF對HVA鈣電流的電壓依賴性沒有影響。 該結果提示VEGF對大鼠海馬CA1區(qū)錐體神經(jīng)元HVA鈣電流具有直接快速的、可逆的和劑量依賴的抑制作用。 結論:VEGF能快速調節(jié)突觸前神經(jīng)遞質的釋放以及抑制高電壓激活的鈣電流。機制分析揭示VEGF通過電壓依賴的鈣通道抑制突觸前軸突末梢的GABA釋放。 創(chuàng)新點: 1.首次提出VEGF具有促進谷氨酸能神經(jīng)傳遞和抑制突觸前GABA釋放的作用。 2.首次提出VEGF具有快速抑制神經(jīng)元細胞膜高電壓激活的鈣電流的作用。 3.首次提出VEGF通過抑制電壓依賴的鈣通道抑制突觸前軸突末梢的GABA釋放。
[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.

【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R33

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