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Assays for measuring extracellular GABA levels and cell migration rate in acute slices.

机译:用于测定急性切片中细胞外GABA水平和细胞迁移速率的测定。

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The postnatal subventricular zone (SVZ) contains the largest pool of dividing and migrating neural precursors in the adult rodent brain. Neuronal precursors migrate throughout the SVZ and along the rostral migratory stream (RMS) towards the olfactory bulb where they differentiate into interneurons. To facilitate the investigation of cell migration in the SVZ and RMS, an inexpensive migration assay was developed for use in acute brain slices. Acute sagittal slices were kept at 37 degrees C in 5% O(2)/95% CO(2)-saturated solution and migrating cells in the SVZ and RMS were visualized using an upright infrared-differential interference contrast microscope. Time-lapse movies were acquired to identify the direction and measure the speed of cell migration. The neurotransmitter GABA and inhibitors of GABA receptors or transporters can be bath applied to determine the function of endogenous GABA on the direction and speed of cell migration. In parallel, the levels of endogenous GABA released from acute SVZ or RMS explants were measured with mass spectrometry. Additional techniques such as electrophysiology and immunohistochemistry confirmed the identity of cells as neuronal precursors and characterized the expression of GABA receptors and transporters. This report describes how modulations in the direction and speed of neuronal precursor migration can be accurately monitored and how changes in local GABA levels can be measured. The described techniques can be used to identify the endogenous factors that regulate cell migration. Identifying such factors is essential for the future therapeutic use of SVZ cells to replace damaged or lost cells.
机译:产后脑室下区域(SVZ)包含成年啮齿动物大脑中最大的分裂和迁移神经前体的库。神经元前体在整个SVZ中移动,并沿着鼻尖迁移流(RMS)移向嗅球,在那里它们分化为中间神经元。为便于研究SVZ和RMS中的细胞迁移,开发了一种廉价的迁移测定法,用于急性脑切片。急性矢状面切片在5%O(2)/ 95%CO(2)饱和溶液中保持在37摄氏度,并使用立式红外微分干涉对比显微镜观察SVZ和RMS中的迁移细胞。采集延时电影以识别方向并测量细胞迁移的速度。可将神经递质GABA和GABA受体或转运蛋白的抑制剂浸入水中,以确定内源性GABA在细胞迁移的方向和速度上的功能。同时,用质谱法测量了从急性SVZ或RMS外植体释放的内源GABA的水平。诸如电生理学和免疫组织化学等其他技术证实了细胞作为神经元前体的身份,并表征了GABA受体和转运蛋白的表达。该报告描述了如何精确监测神经元前体迁移的方向和速度的调制,以及如何测量局部GABA水平的变化。所描述的技术可用于识别调节细胞迁移的内源性因子。识别此类因素对于将来将SVZ细胞用于治疗受损或丢失的细胞至关重要。

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