Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts.
Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts.
复制标题
使用组织培养插入物进行长期胚胎大鼠脊髓神经元培养的突触发生和氨基酸释放。
DOI：
10.1016/j.jneumeth.2004.05.011
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Marsala,Silvia
中科院分区：
文献类型：
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作者：
Marsala,Martin;Kakinohana,Osamu;Hefferan,MichaelP;Cizkova,Dasa;Kinjoh,Kiyohiko;Marsala,Silvia
在本研究中，使用组织培养插入物（TCI）加上原代脊髓神经元培养，我们表征了一种新的灌注系统，它允许从培养的神经元连续收集灌注液。从Sprague-Dawley大鼠的E14脊髓的腰部分离原代脊髓神经元，铺在TCI上并用DMEM/B27/10%FBS喂养。在分离后1-4周，使用免疫荧光验证培养的神经元中突触和神经递质表型的发育。在接种后3-4周，观察到突触（Syn）的时间依赖性发育，并鉴定出密集的Syn阳性网络。接种的神经元亚群（35-40%）显示GABA免疫反应性并表达NMDAR 1受体。为了测量神经递质的释放，构建了一个容纳TCI的腔室，允许插入物穿过膜灌注。在灌流缓冲液中加入NMDA（10 mmol/l），诱导培养神经元氨基酸释放。在暴露后10 min内，NMDA刺激引起GABA（4050 ± 950%）和谷氨酸（130 ± 42%）释放。在对照非刺激细胞中，未测量到显著变化。这些数据表明，通过使用TCI，有可能维持胚胎脊髓神经元延长的时间，并且该系统可以代表一种简单的工具来鉴定与培养的脑和/或脊髓神经元的特定群体相关的神经递质和/或肽。
In the present study, using tissue culture inserts (TCI) coupled with a primary spinal cord neuronal culture, we characterize a new perfusion system, which permits continuous perfusate collection from cultured neurons. Primary spinal cord neurons were isolated from the lumbar portion of E14 spinal cords of Sprague–Dawley rats, plated on TCI and fed with DMEM/B27/10% FBS. At 1–4 weeks after isolation the development of synapses and neurotransmitter phenotype in cultured neurons was verified using immunofluorescence. A time-dependent development of synapses (Syn) was seen with a dense Syn-positive network identified at 3–4 weeks after plating. A sub-population of plated neurons (35–40%) showed GABA immunoreactivity and expressed NMDAR1 receptor. To measure neurotransmitter release, a chamber accommodating TCI was constructed permitting perfusion of the insert across the membrane. To evoke amino acid release from cultured neurons, NMDA (10mmol/l) was added into the perfusion buffer. Stimulation with NMDA evoked a significant GABA (4050 ± 950%) and glutamate release (130 ± 42%) during first 10min after exposure. In control non-stimulated cells no significant changes were measured. These data show that by using TCI it is possible to maintain embryonic spinal cord neurons for an extended period and that this system may represent a simple tool to identify neurotransmitter and/or peptides associated with a specific population of cultured brain and/or spinal cord neurons.