Mammalian cortex consists of excitatory glutamatergic neurons and inhibitory GABAergic neurons, which have specific gene expression patterns. These cells can feasibly be identified by immunostaining or using cells from transgenic animals, but these methods require chemical fixation or fluorescent labeling. Live-cell, label-free identification of excitatory-inhibitory neurons would serve as a novel system for studying single cell analysis and reconstruction of artificial neuronal circuit.
During cortical development, excitatory neurons first form immature network, which is then followed by integration of inhibitory neurons. Astonishingly, this time lag is conserved even when the cells are dissociated and cultured in vitro [1]. This finding led us to hypothesize that the two cells might be distinguishable based on morphological criteria. In this work, we investigated the axon growth rate of excitatory and inhibitory neurons on micropatterned surfaces and studied whether it could be used as a measure to identify the two cells types, without chemical fixation or fluorescent labeling.
To define process of axon extension, we fabricated micropatterned substrate by electron beam lithography, which have cell-permissive region (poly-D-lysine) and non-permissive region (2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane) [2]. Embryonic rat cortical neurons were cultured on the micropatterned surface, and axon length of each cell was monitored from 2 to 7 days in vitro (DIV). We found that axons of inhibitory neurons grow significantly slower than those of excitatory neurons. Setting a threshold length of 120 μm at 6 DIV enabled complete identification of the two cell types. Micropattern brings significant difference to define a differentiation of axon length rate in this research. This finding will allow in vitro construction of micropatterned neuronal circuit with defined cell types.
[1] K. Hayashi et al., J. Cell Sci. 116 (2003) 4419. [2] H. Yamamoto et al., J. Neurochem. 123 (2012) 904.