A rapid Percoll gradient procedure for preparation of synaptosomes
A rapid Percoll gradient procedure for preparation of synaptosomes
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DOI：
10.1038/nprot.2008.171
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Robinson, Phillip J.
中科院分区：
文献类型：
--
作者：
Dunkley, Peter R.;Jarvie, Paula E.;Robinson, Phillip J.
新鲜脑组织在等渗介质中的均质化会剪切质膜，导致神经末梢与其轴突和突触后连接分离。然后，神经末梢膜重新封闭，形成突触体。这里描述的不连续Percoll梯度程序是为了在最短的时间内从脑匀浆中分离出突触体，以便进行功能实验。使用中速离心机分离突触体，同时保持等渗条件并将机械破坏的再悬浮步骤降至最低。这种方法在速度和产生相对均一的突触体方面比其他方法有优势，最大限度地减少了突触和神经胶质质膜以及突触体外线粒体的存在。纯化的突触体是有活性的，可以非常有效地吸收和释放神经递质。突触体的典型产量在每克大鼠大脑中含有2.5到4毫克的突触体蛋白。这个过程从大脑匀浆到从Percoll梯度收集突触体悬液需要类似1小时的时间。
Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes similar to 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.