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Title: 胶质细胞释放的ATP对海马突触传递的抑制作用
Author: 张景明
Degree Level: 博士
Issued Date: 2003
Degree Grantor: 中国科学院上海生命科学院,神经科学研究所
Place of Degree Grantor: 中国科学院上海生命科学院,神经科学研究所
Supervisor: 蒲慕明
Keyword: 突触传递 ; 腺苷 ; 胶质细胞 ; 活动依赖性
Alternative Title: ATP Secreted by Glias Inhibits the Synaptic Transmission in Hippocampus
Abstract: ATP(adenosine-5'-triphosphate)是一种在中枢神经系统中广泛存在的物质,它不仅是细胞内携带能量的分子,也是重要的信号分子。在脊髓背角、上丘脑等部位存在着以ATP作为递质的突触联系,ATP也被证明参与了星型胶质细胞之间的信息传递过程。ATP还可能与其它递质一起从突触末梢的囊泡共同释放出来,对突触传递起到调控作用。我们在离体培养的大鼠海马神经元和急性分离海马脑片上,主要利用电生理的方法,证明了胶质细胞是内源性ATP的主要来源。胶质细胞释放ATP的过程是依赖于兴奋性神经元的活动的,所释放的ATP反过来又对突触传递起到调控作用,并且随着兴奋性神经元活动状态的升高,内源性ATP还可以介导突触间抑制效应。根据上述结果我们提出了神经元和胶质细胞间通过神经递质和腺嘿吟核普酸进行双向的信号传递的模型。我们的结果显示:(一)外加0.5-100μM的ATP可剂量依赖性地抑制海马神经元间兴奋性突触后电流(excitatory post-synaptic current,EPSC)和抑伟(性突触后电流(inhibitory post-synaptic current,IPSC)。(二)对不同的拮抗剂进行的研究表明,ATP的抑制作用主要是通过PZY受体介导的,腺普(adenosine)受体在这个过程中也起着部分作用;至少有相当大一部分的抑制作用是由ATP直接产生的。(三)观察了ATP对微小兴奋性突触后电流(miiliature EPSC,mEPSC)及微小抑制性突触后电流(minioture IPSC,mIPSC)幅度和频率的影响,所引起的paired-pulse ratio的改变,对外加谷氨酸(glutamate,Glu)或y-氨基丁酸(γ-amino butyric acid,GABA)直接诱发的电流的影响,以及对电压依赖性钙通道(voltage-dependent calcium channel)的作用,结果均提示ATP的抑制作用是通过突触前机制完成的。(四)PZY受体阻断剂reoctive blue 2(RB-2)能够使EPSC增大至给药前的117士8.4%;而经过细胞外核普酸酶(ecto-nucleotidase)抑制剂dipyridomole(DPM)或细胞外ATP酶(ecto-ATPase)抑制剂ARL 67156处理后,神经元间的EPSC和IPSC都有明显的降低,提示内源性ATP的作用。(五)将10Hz,1秒钟的闭上刺激施加于突触前神经元,所引起的EPSC产生了明显的短时程抑制现象,表现为1/I1值下降。外加RB-2可使该作用加强,而外加ATP则作用相反。串刺激产生的IPSC也有类似的短时程抑制现象,外加ATP同样可减弱此抑制,但外加RB-2则没有明显的影响,提示内源性ATP的释放可能与神经元的种类有关。(六)在上述串刺激结束后,对照组神经元EPSC恢复的时间常数是589ms,而经ATP处理后恢复时间常数延长为876ms;(七)在有交互突触的两个神经元上我们看到,用上述高频串刺激引起一个方向上兴奋性突触的活动,可在另一方向的突触上观察到突触传递减弱的现象,RB-2能够阻断这一现象,提示一个兴奋性突触的连续活动可能使大量的内源性ATP释放出来而作用于邻近的突触。(八)上述的突触间抑制效应可被DNQx所阻断,表明ATP的释放是依赖于non-NMDA受体的活动的;同时这种突触间抑制效应也能够被胶质细胞代谢阻断剂Fluoroacetate(FAC)或缝隙连接 (gap junction)阻断剂octanol阻断,提示内源性ATP释放和胶质细胞的功能密切相关。(九)进一步比较了ATP和RB-2对两种培养神经元作用的差异:一种是神经元和胶质细胞共培养,另一种是不含胶质细胞的培养神经元。ATP对两种培养状态下神经元的突触传递都有抑制作用,而RB-2的增强作用仅见于共培养的神经元。并且,在不含胶质细胞的情况下,我们没有观察到突触间抑制的现象,再次说明ATP的释放与胶质细胞的功能密切相关。(十)培养的海马胶质细胞经Glu处理后能够释放ATP到细胞外液中,这个现象可以被DNQX阻断,证明兴奋性神经元的活动能够通过non-NMDA受体诱导胶质细胞释放ATP;而同样的处理只能引起神经元释放少量的ATP,且不能被DNQX阻断;GABA不能引起胶质细胞释放ATP。(十一)在急性分离的海马脑片上,内源性腺普起着对兴奋性突触传递的紧张性调控作用,支配CAI区锥体神经元的Schoffer coHoterols也存在明显的由腺普介导的突触间抑制现象,而这些腺普至少大部分是来源于ATP的降解。(十二)急性海马脑片中,阻断胶质细胞的功能后,腺营不再参与突触间抑制现象。细胞外核普酸酶活性被抑制后表现出来的内源性ATP对突触传递的调控作用也消失了。我们以上的工作,不仅阐明了细胞外ATP对中枢神经系统神经元间突触传递的调控作用的机理,而且证明存在内源性ATP的紧张性调控作用。这些内源性ATP主要是由胶质细胞释放的,而释放的过程依赖于兴奋性神经元的活动。这些结果提示神经元和胶质细胞间双向的信息交换对神经系统信号传递过程有非常重要的调控作用.
English Abstract: Extracellular ATP is currently recognized as an important signal molecule in Central Nervous System (CNS). Although purine receptors are widely distributed throughout the CNS, direct ATP neurotransmission has only been reported in a few cases, suggesting that extracellular ATP may mainly function as a modulator rather than direct neurotransmitter. Indeed, both inhibiting and potentiating effects of ATP on synaptic transmission have been frequently reported, although there are still debates as for whether ATP can modulate synaptic transmission directly by activating P2 receptors or indirectly by generating adenosine through its extracellular catabolism by the ecto-nucleotidase cascade.Multiple sources and mechanisms of endogenous ATP release have been suggested. Vesicular co-release of ATP with Ach and NE has been reported in sympathetic neurons, but the clear evidence of such co-release mechanism with glutamate and GABA, the most important excitatory and inhibitory transmitter in CNS, is still missing. Non-vesicular neuronal release of ATP has been also suggested. ATP released from astrocyte has been reported as an important messenger underlying inter-glia communications. Despite the facts that both ATP release and ATP mediated modulation of synaptic transmission have been reported frequently, modulation of synaptic activity by endogenous ATP with clear release source and mechanism has been rarely elucidated.In the present study we showed that in cultured hippocampal neurons,ATP (0.5-100μM) inhibited both glutamatergic and GABAergic synaptic transmission, and this inhibitory effect was dose-dependent.ATP increased the frequency of mEPSC, but did not change their amplitude. ATP augmented the paired-pulse ratio (I_2 / I_1) of two successive EPSC, repressed the voltage-gated calcium channel, but did not affect Glu- or GABA-induced current. All these suggest a pre-synaptic site of ATP action.The results of application of ATP and adenosine antagonists indicate that the effects of ATP were mediated by P2Y receptor, adenosine receptor was also involved.Reactive blue 2 (RB-2), a P2Y receptor antagonist, increased the amplitude ofeEPSC (117+8.4%), suggesting there was endogenous ATP release.Repeated activation of pre-synaptic neuron with a 10 Hz, Is pulse train caused a short term depression (I/I_1 value decreased) of eEPSC. RB-2 enhanced the depression and prolonged the recovery time constant of eEPSCs from 589 ms to 876 ms, whereas application of ATP reduced the depression.On pairs of neurons which had reciprocal synapses, we found intense activities of glutamatergic synapse caused hetero-synaptic inhibition. This inhibition was blocked by RB-2 or Glu receptor antagonist DNQX.The effects of application of ATP and RB-2 on synaptic activity in pure neuron culture and mixed culture of neurons and glia cells were different, suggesting there was endogenous ATP release from glial cells in the mixed culture. Glu substantially induced ATP release from pure astrocytes, this can be blocked by DNQX. Although Glu induced mild ATP release from neurons, this is insensitive to DNQX. GABA did not induce ATP release from astrocytes. In acutely dissociated hippocampal slices, ATP still had dramatic inhibitory effect on the excitatory synaptic transmission (EPSP) . CPT but not RB-2increased EPSPs in slices, suggesting the effect of endogenous adenosine but not ATP. When the activity of ecto-nucleotidase was inhibited, RB-2; substantially facilitated the EPSP. After the function of glia was blocked byfluoroacetate (FAC), RB-2's facilitating effect on EPSP disappeared, suggesting that ATP comes from glial cells.10. Heterosynaptic suppression was also observed on Schaffer Collaterals. This phenomenon was mediated by adenosine. The suppression could be mediated by endogenous ATP after the activity of ecto-ATPase had beeninhibited by ARL 67156. When glial cells were inhibited by FAC, CPT did not alter the heterosynaptic suppression any longer. These results suggest that endogenous adenosine, which mediated the heterosynaptic suppression, come from the breakdown of endogenous ATP. The source of endogenous ATP was glial cells.Our data has elucidated the mechanism of the modulatory effect of extracellular ATP (and its catabolic product adenosine) on synaptic transmission in the central nervous system. Also, we have demonstrated that endogenous ATP released from glial cells had a strong tonic inhibitoiy effect on synaptic transmissions and the release of ATP was dependent on the activity of glutamatergic neurons. This communication between neurons and glias may have remarkable physiological significance to the signal transduction in the nervous system.
Language: 中文
Content Type: 学位论文
URI: http://ir.sibs.ac.cn/handle/331001/2241
Appears in Collections:神经所(总)_学位论文

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胶质细胞释放的ATP对海马突触传递的抑制作用.张景明[d].中国科学院上海生命科学院,神经科学研究所,2003.20-25
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