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.
Highlights
Microfabrication technologies have provided cellular neuroscience with a tool to manipulate cultured neurons at a single cell level, enabling us to engineer living neuronal networks of predetermined structure [1,2]
The axon-dendrite polarization axis of cultured hippocampal neurons can be controlled via the use of an asymmetric micropattern consisting of a 15 μm-diameter island for soma adhesion, a single 100 μm-pathway for axon growth, and three 20 μm-pathways for dendrite growth (Pattern #1; Fig 1A; 2 days in vitro (DIV)) [6]
When hippocampal neurons were cultured on the pattern for over 4 DIV, the majority of the neurons became morphologically abnormal and died (Fig 1B)
Summary
Microfabrication technologies have provided cellular neuroscience with a tool to manipulate cultured neurons at a single cell level, enabling us to engineer living neuronal networks of predetermined structure [1,2]. The major structure of a neuronal network is defined by the type of cells, their connectivity, and direction of the connections. Recent advancements have made it possible, for example, to control axon-dendrite polarity of single neurons [3,4,5,6] and to “wire”. Identification of GABAergic and Non-GABAergic Neurons on Micropatterns
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