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Open AccessArticle

Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses

1
Institute of Science and Technology (IST )Austria, 3400 Klosterneuburg, Austria
2
Department of Histological and Physiological Sciences, Faculty of Medical Science, University of Fukui, Yoshida, Fukui 910-1193, Japan
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(18), 6737; https://doi.org/10.3390/ijms21186737
Received: 10 August 2020 / Revised: 7 September 2020 / Accepted: 10 September 2020 / Published: 14 September 2020
(This article belongs to the Special Issue Molecular Structure and Function of Synapses)
The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography. View Full-Text
Keywords: electron microscopy; SDS-digested freeze–fracture replica labeling; image analysis; glutamate receptors; voltage-gated calcium channels; CA1; stratum oriens; stratum radiatum; deep learning electron microscopy; SDS-digested freeze–fracture replica labeling; image analysis; glutamate receptors; voltage-gated calcium channels; CA1; stratum oriens; stratum radiatum; deep learning
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MDPI and ACS Style

Kleindienst, D.; Montanaro, J.; Bhandari, P.; Case, M.J.; Fukazawa, Y.; Shigemoto, R. Deep Learning-Assisted High-Throughput Analysis of Freeze-Fracture Replica Images Applied to Glutamate Receptors and Calcium Channels at Hippocampal Synapses. Int. J. Mol. Sci. 2020, 21, 6737.

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