Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Neuron. 2010 Apr 15;66(1):57-68. doi: 10.1016/j.neuron.2010.03.022.
The polarized nature of neurons and the size and density of synapses complicates the manipulation and visualization of cell biological processes that control synaptic function. Here we developed a microfluidic local perfusion (microLP) chamber to access and manipulate synaptic regions and presynaptic and postsynaptic compartments in vitro. This chamber directs the formation of synapses in >100 parallel rows connecting separate neuron populations. A perfusion channel transects the parallel rows, allowing access with high spatial and temporal resolution to synaptic regions. We used this chamber to investigate synapse-to-nucleus signaling. Using the calcium indicator dye Fluo-4 NW, we measured changes in calcium at dendrites and somata, following local perfusion of glutamate. Exploiting the high temporal resolution of the chamber, we exposed synapses to "spaced" or "massed" application of glutamate and then examined levels of pCREB in somata. Lastly, we applied the metabotropic receptor agonist DHPG to dendrites and observed increases in Arc transcription and Arc transcript localization.
神经元的极化特性以及突触的大小和密度使得控制突触功能的细胞生物学过程的操作和可视化变得复杂。在这里，我们开发了一种微流控局部灌流（microLP）室，用于体外访问和操作突触区域以及突触前和突触后隔室。该腔室指导在 >100 个平行行中形成连接分离神经元群体的突触。一个灌流通道横切平行行，允许以高空间和时间分辨率访问突触区域。我们使用该腔室研究突触到核的信号转导。使用钙指示剂 Fluo-4 NW，我们测量了局部灌流谷氨酸后树突和胞体中钙的变化。利用腔室的高时间分辨率，我们使突触暴露于“间隔”或“集中”应用谷氨酸，然后检查胞体中 pCREB 的水平。最后，我们将代谢型受体激动剂 DHPG 施加到树突上，观察到 Arc 转录和 Arc 转录物定位的增加。
