Communication between cortical neurons and astrocytes by extracellular ATP and glutamate

已经反复表明星形胶质细胞响应于ATP应用而释放谷氨酸。以下实验旨在研究这种释放的形态学基础和功能后果：1。大鼠皮层锥体神经元P2 Y和NMDA受体的相互作用(A)在大鼠前额叶皮层切片中，星形胶质细胞谷氨酸可能激活V层锥体神经元的代谢型谷氨酸受体（mGluR），从而增加邻近NMDA受体通道的电导。将通过膜片钳方法阐明哪组/类型的mGluR参与这种效应。(B)在脑切片中进行的实验将在大鼠大脑皮层的混合星形胶质细胞/神经元培养物中重复，以发现mGluR-NMDA受体相互作用是否仅发生在锥体神经元中。在急性分离的锥体神经元和星形胶质细胞的情况下，受体相互作用可能不会发生。(C)混合星形胶质细胞/神经元培养物中的Ca 2+成像将证实上述发现，并有助于将mGluR-NMDA受体相互作用的位点定位于细胞体或神经元过程。2. P2 Y受体在星形胶质细胞的细胞和亚细胞定位。我们将通过共聚焦激光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层的神经元和星形胶质细胞的突触前和/或突触后表达P2 Y免疫阳性。3. ATP诱导星形胶质细胞释放谷氨酸。(A)HPLC测定将有助于鉴定参与从富含星形胶质细胞的细胞培养物中通过ATP释放谷氨酸的P2 Y受体类型（P2 Y1，P2 Y2）。(B)自由活动大鼠前额叶皮层的微透析测量将用于澄清谷氨酸是否从神经元或星形胶质细胞释放。

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.