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J Pharmacol Toxicol Methods. 2019 Sep - Oct;99:106607. doi: 10.1016/j.vascn.2019.106607. Epub 2019 Jul 02.

High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons.

Journal of pharmacological and toxicological methods

Kenji Hanamura, Noriko Koganezawa, Kazumi Kamiyama, Natsume Tanaka, Takero Oka, Mai Yamamura, Yuko Sekino, Tomoaki Shirao


  1. Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan.
  2. Endowed Laboratory of Human Cell-Based Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
  3. Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan. Electronic address: [email protected].

PMID: 31271780 DOI: 10.1016/j.vascn.2019.106607


INTRODUCTION: Detection of drug effects on neuronal synapses is important for predicting their adverse effects. We have used drebrin as a marker to detect the synaptic changes in cultured neurons. High concentration of glutamate decreases the amount of drebrin in synapses. To increase the availability of this method for high throughput analysis, we applied the drebrin-based evaluation of synapses to high-content imaging analysis using microplates.

METHODS: Three weeks old cultured neurons were fixed and processed for immunocytochemistry to visualize drebrin clusters, dendrites and neuronal cell bodies. After automated image acquisition, total number of drebrin clusters per fields, linear density of drebrin cluster along dendrites, dendrite length and neuron number were automatically measured by a custom-designed protocol.

RESULTS: Automated image acquisition and analysis showed that dendrite length and drebrin cluster density along dendrites are measured consistently and reproducibly. In addition, application of 10-100 μM glutamate for 10 min or 0.5-50 μM latrunculin A for 5 min significantly decreased drebrin cluster density without affecting neuron number. These results were consistent with our previous results using manual image acquisition and analysis with regular fluorescence microscope and image analysis software. Furthermore, 0.3 or 1.0 μM staurosporine for 24 h significantly decreased neuron number.

DISCUSSION: The present study demonstrates that this high-throughput imaging analysis of drebrin cluster density along dendrites for detecting the effects of substances on synapses is sensitive enough to detect the effects of glutamate receptor activation and latrunculin A treatment, and indicates that this analysis will be useful for safety pharmacology study.

Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

Keywords: Dendritic spine; Drebrin; High-content imaging analysis; High-throughput analysis; Immunocytochemistry; Primary neuronal culture; Synapse

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