Abstract
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.
Highlights
The branched axonal morphology of neurons is important for information processing, and is constructed during development, and in the adult brain. [1,2] The mechanisms by which neurons locally control their axonal morphology remain to be elucidated
To investigate the effect of membrane depolarization on axonal arborisation, primary culture cerebellar granule neurons (CGNs) expressing enhanced green fluorescent protein (EGFP) were maintained in media containing 5 mM or 30 mM KCl
We found that membrane depolarization increases axonal branching of CGNs by increasing microtubule entry into the protrusion
Summary
The branched axonal morphology of neurons is important for information processing, and is constructed during development, and in the adult brain. [1,2] The mechanisms by which neurons locally control their axonal morphology remain to be elucidated. [5,6,7] For example, neuronal transcriptional factors, NeuroD1/2 respond to calcium via phosphorylation on their transcriptional activation domain, which is required for the depolarization-dependent dendritic growth of CGNs.[5,8,9] the effect of membrane depolarization on CGN axonal morphogenesis remains largely unknown. One approach is to use specific compounds called caged ligands, which are activated by light.[10] More recently, optogenetic approaches in which exogenous genes for light-sensitive molecules are introduced into cells have been developed.[11] This technique has been used to manipulate neuronal activity by using light-sensitive ion channels,[11] as well as intracellular signalling and transport by light-dependent dimerization molecules.[12,13] since light-induced approaches can cause phototoxicity, their application to investigate long-term events such as axonal morphogenesis has been limited
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