Communication between cortical neurons and astrocytes by extracellular ATP and glutamate

研究表明，星形胶质细胞释放谷氨酸是对ATP作用的响应。本实验旨在研究这种释放的形态基础和功能后果：1.大鼠大脑皮层锥体神经元中P2Y和NMDA受体的相互作用。(A)在大鼠前额叶皮质脑片上，星形细胞谷氨酸可激活V层锥体神经元的代谢性谷氨酸受体(MGluR)，从而增加相邻NMDA受体通道的电导。膜片钳方法将阐明mGluR的哪一基团/类型参与了这一效应。(B)在脑片上进行的实验将在大鼠大脑皮层的星形细胞/神经元混合培养中重复，以确定mGluR-NMDA受体的相互作用是否只发生在锥体神经元中。在急性分离的锥体神经元中，在没有星形胶质细胞的情况下，可能不会发生受体相互作用。(C)星形胶质细胞/神经元混合培养中的钙成像将证实上述发现，并有助于确定mGluR-NMDA受体与细胞体或神经元突起相互作用的位置。2.P2Y受体在星形胶质细胞的细胞和亚细胞定位。我们将利用激光共聚焦扫描显微镜和电子显微镜结合免疫组织化学方法，在前额叶皮质的神经元和星形胶质细胞中寻找突触前和/或突触后的P2Y免疫阳性表达。3.ATP诱导星形胶质细胞释放谷氨酸。(A)高效液相色谱检测将有助于确定与ATP从富含星形胶质细胞的细胞培养中释放谷氨酸有关的P2Y受体类型(P2Y1、P2Y2)。(B)自由活动大鼠前额叶皮质的微透析测量将用于阐明谷氨酸是从神经元还是星形胶质细胞释放出来的。

It has repeatedly been shown that astrocytes release glutamate in response to ATP application. The following experiments are designed to investigate the morphological basis and the functional consequences of this release: 1. Interaction between P2Y and NMDA receptors in pyramidal neurons of the rat cortex. (A) In slices of the rat prefrontal cortex, astrocytic glutamate may activate metabotropic glutamate receptors (mGluR) of layer V pyramidal neurons and thereby increase the conductance of neighboring NMDA receptor-channels. It will be elucidated by the patch-clamp method, which group/type of mGluR is involved in this effect. (B) Experiments made in brain slices will be replicated in mixed astrocytic/neuronal cultures of the rat cerebral cortex to find out whether the mGluR-NMDA receptor interaction occurs in pyramidal neurons only. In acutely dissociated pyramidal neurons and in the absence of astrocytes a receptor-interaction will probably not take place. (C) Ca2+-imaging in the mixed astrocytic/neuronal cultures will confirm the above findings and help to localize the site of the mGluR-NMDA receptor interaction to cell bodies or neuronal processes. 2. Cellular and subcellular localization of P2Y receptors at astrocytes. We will search by confocal laser scanning microscopy and electron microscopy in conjunction with immunohistochemical methods for the pre- and/or postsynaptic expression of P2Y-immunopositivity at neurons and astrocytes in the prefrontal cortex. 3. ATP-induced release of glutamate from astrocytes. (A) HPLC determinations will help to identify the P2Y receptor-type (P2Y1, P2Y2) involved in the release of glutamate by ATP from cell cultures enriched in astrocytes. (B) Microdialysis measurements from the prefrontal cortex of freely moving rats will be used to clarify whether glutamate is released from neurons or astrocytes.