RESUMO
The establishment and maintenance of axonal patterning is crucial for neuronal function. To identify the molecular systems that operate locally to control axonal structure, it is important to manipulate molecular functions in restricted subcellular areas for a long period of time. Microfluidic devices can be powerful tools for such purposes. In this study, we demonstrate the application of a microfluidic device to clarify the function of local Ca2+ signals in axons. Membrane depolarization significantly induced axonal branch-extension in cultured cerebellar granule neurons (CGNs). Local application of nifedipine using a polydimethylsiloxane (PDMS)-based microfluidic device demonstrated that Ca2+ entry from the axonal region via L-type voltage-dependent calcium channels (L-VDCC) is required for branch extension. Furthermore, we developed a method for locally controlling protein levels by combining genetic techniques and use of a microfluidic culture system. A vector for enhanced green fluorescent protein (EGFP) fused to a destabilizing domain derived from E. coli dihydrofolate reductase (ecDHFR) is introduced in neurons by electroporation. By local application of the DHFR ligand, trimethoprim (TMP) using a microfluidic device, we were able to manipulate differentially the level of fusion protein between axons and somatodendrites. The present study revealed the effectiveness of microfluidic devices to address fundamental biological issues at subcellular levels, and the possibility of their development in combination with molecular techniques.
