A low-density culture method of cerebellar granule neurons with paracrine support applicable for the study of neuronal morphogenesis
A low-density culture method of cerebellar granule neurons with paracrine support applicable for the study of neuronal morphogenesis
复制标题
DOI：
10.1016/j.brainres.2013.09.046
复制
发表时间：
2013-11-20
期刊：
影响因子：
2.9
通讯作者：
Konishi, Yoshiyuki
中科院分区：
文献类型：
--
作者：
Kubota, Kenta;Seno, Takeshi;Konishi, Yoshiyuki
小脑颗粒神经元培养已被用于研究神经元功能的分子机制，包括神经元的形态发生。然而，该系统的一个局限性是难以分析孤立的神经元，因为这些神经元需要保持高密度。因此，在本研究中，我们旨在开发一种简单、经济的培养低密度小脑颗粒神经元的方法。将两种不同密度的小脑颗粒细胞(低密度和高密度)共培养，以使低密度培养得到高密度培养的旁分泌信号的支持。这种方法能够在没有星形细胞饲养层培养或补充如B27的情况下对分离的小脑颗粒神经元进行形态分析。利用这种方法，我们研究了一个极性因子的作用。利用海马神经元的研究表明，糖原合成酶-3(GSK-3)是神经元极性的重要调节因子，抑制GSK-3会导致多个轴突的形成。GSK-3的药物抑制剂(6-溴-靛玉红-3‘-肟和氯化锂)不能引起小脑颗粒神经元多个轴突的形成，但显著缩短了它们的长度。通过引入激酶死亡形式的GSK-3β(K85A)获得了一致的结果。这些结果表明，GSK-3不直接参与小脑颗粒神经元的神经元极性调控。总之，本研究为低密度小脑颗粒神经元的培养提供了一种简单的方法，并对GSK-3在神经元形态发生中的神经元类型依赖功能提供了见解。(C)2013爱思唯尔B.V.保留所有权利。
Cerebellar granule neuronal cultures have been used to study the molecular mechanisms underlying neuronal functions, including neuronal morphogenesis. However, a limitation of this system is the difficulty to analyze isolated neurons because these are required to be maintained at a high density. Therefore, in the present study, we aimed to develop a simple and cost-effective method for culturing low-density cerebellar granule neurons. Cerebellar granule cells at two different densities (low- and high-density) were co-cultivated in order for the low-density culture to be supported by the paracrine signals from the high-density culture. This method enabled morphology analysis of isolated cerebellar granule neurons without astrocytic feeder cultures or supplements such as B27. Using this method, we investigated the function of a polarity factor. Studies using hippocampal neurons suggested that glycogen synthase kinase-3 (GSK-3) is an essential regulator of neuronal polarity, and inhibition of GSK-3 results in the formation of multiple axons. Pharmacological inhibitors for GSK-3 (6-bromoindirubin-3'-oxime and lithium chloride) did not cause the formation of multiple axons of cerebellar granule neurons but significantly reduced their length. Consistent results were obtained by introducing kinase-dead form of GSK-3 beta (K85A). These results indicated that GSK-3 is not directly involved in the control of neuronal polarity in cerebellar granule neurons. Overall, this study provides a simple method for culturing low-density cerebellar granule neurons and insights in to the neuronal-type dependent function of GSK-3 in neuronal morphogenesis. (C) 2013 Elsevier B.V. All rights reserved.