title: Live-Cell, Label-Free Identification of GABAergic and Non-GABAergic Neurons in Primary Cortical Cultures Using Micropatterned Surface.
abstract: Excitatory and inhibitory neurons have distinct roles in cortical dynamics. Here we present a novel method for identifying inhibitory GABAergic neurons from non-GABAergic neurons, which are mostly excitatory glutamatergic neurons, in primary cortical cultures. This was achieved using an asymmetrically designed micropattern that directs an axonal process to the longest pathway. In the current work, we first modified the micropattern geometry to improve cell viability and then studied the axon length from 2 to 7 days in vitro (DIV). The cell types of neurons were evaluated retrospectively based on immunoreactivity against GAD67, a marker for inhibitory GABAergic neurons. We found that axons of non-GABAergic neurons grow significantly longer than those of GABAergic neurons in the early stages of development. The optimal threshold for identifying GABAergic and non-GABAergic neurons was evaluated to be 110 μm at 6 DIV. The method does not require any fluorescence labelling and can be carried out on live cells. The accuracy of identification was 98.2%. We confirmed that the high accuracy was due to the use of a micropattern, which standardized the development of cultured neurons. The method promises to be beneficial both for engineering neuronal networks in vitro and for basic cellular neuroscience research.
authors: Graduate School of Fundamental Science and Engineering, Waseda University, Tokyo, Japan.|Japan Society for the Promotion of Science (JSPS), Tokyo, Japan.
authors: Sho
authors: S
authors: Kono
authors: Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Japan.
authors: Hideaki
authors: http://orcid.org/0000-0003-3362-5376
authors: H
authors: Yamamoto
authors: Graduate School of Fundamental Science and Engineering, Waseda University, Tokyo, Japan.
authors: Takatoshi
authors: T
authors: Kushida
authors: Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan.
authors: Ayumi
authors: A
authors: Hirano-Iwata
authors: Research Institute of Electrical Communication, Tohoku University, Sendai, Japan.
authors: Michio
authors: M
authors: Niwano
authors: Graduate School of Fundamental Science and Engineering, Waseda University, Tokyo, Japan.
authors: Takashi
authors: T
authors: Tanii
mesh_terms: D000818:Animals; D001369:Axons / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D005260:Female; D059330:GABAergic Neurons / Q000166:cytology / Q000378:metabolism*; D005968:Glutamate Decarboxylase / Q000378:metabolism; D055616:Microtechnology / Q000295:instrumentation*; D009415:Nerve Net / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase