A compartmentalized culture device for studying the axons of CNS neurons

• Main Authors: Cheng, Mei-Yun, 鄭美雲
• Other Authors: Chang, Yen-Chung
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/xr6dj3

博士 === 國立清華大學 === 分子醫學研究所 === 106 === The cell body of neuron integrates various signals from dendrites, and the integral signals are transmitted by axons and sent to appropriate targets via synapses. Devastating sequels resulting from axonal injury and degeneration are common in many acute and chronic neurological diseases. The research in axonal guidance and regeneration is important to develop effective clinical treatments. Here, I report a novel compartmentalized culture device for studying the molecular and cell biology and functions of the axons of central nervous system (CNS) neurons. The compartmentalized culture device allows the spatial separation of the somatodendrites and axons of CNS neurons. The device consists of two compartments separated by a septum constructed by attaching a porous polycarbonate track etch (PCTE) filter on top of a microchannel-filled polydimethylsiloxane (PDMS) membrane. The surface and microchannels of the septum are coated and filled, respectively, with materials that support neuron growth and neurite migration. When rat hippocampal neurons are cultured in the top compartment, both the axons and dendrites can migrate through the pores in PCTE filter and into the microchannels of the PDMS membrane. After 8-9 days in vitro, axons are the only processes that emerge from the septum and grow in the bottom compartment. Axons could be either studied by fluorescence immunocytochemistry after fixation or monitored in real-time. Axons containing ~3 µg protein can be isolated from each device for biochemical analyses. In addition, the septum also impedes the movement of small molecules between the top and bottom compartments. This feature allows the somatodendrites and axons of neurons, which occupy the top and bottom compartments of the device, respectively, to be manipulated independently. The potential applications of the device as a tool in diverse studies concerning neuronal axons and in screening reagents that regulate axonal functions have also been discussed. In conclusion, the compartmentalized culture device will be a valuable tool in separating axons from the central neurons for conducting in-depth molecular biology analyses of the axon and high-throughput screening for drugs regulating axonal functions.