Live-Cell, Label-Free Identification of GABAergic and Non-GABAergic Neurons in Primary Cortical Cultures Using Micropatterned Surface

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2016 Aug 12

PMID 27513933

Citations 1

Authors

Sho Kono

Hideaki Yamamoto

Takatoshi Kushida

Ayumi Hirano-Iwata

Michio Niwano

Takashi Tanii

Affiliations

Soon will be listed here.

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.

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