Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay

Toshinari Mitsuoka; Kenji Hanamura; Noriko Koganezawa; Ruri Kikura-Hanajiri; Yuko Sekino; Tomoaki Shirao

doi:10.1016/j.vascn.2019.106583

Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay

J Pharmacol Toxicol Methods. 2019 Sep-Oct:99:106583. doi: 10.1016/j.vascn.2019.106583. Epub 2019 May 10.

Authors

Toshinari Mitsuoka¹, Kenji Hanamura², Noriko Koganezawa², Ruri Kikura-Hanajiri³, Yuko Sekino⁴, Tomoaki Shirao⁵

Affiliations

¹ Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan; Endowed Laboratory of Human Cell-Based Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
² Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan.
³ Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences, Kawasaki 210-9501, Japan.
⁴ Endowed Laboratory of Human Cell-Based Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
⁵ Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan. Electronic address: tshirao@gunma-u.ac.jp.

PMID: 31082488
DOI: 10.1016/j.vascn.2019.106583

Abstract

Introduction: In recent years, new psychoactive substances (NPS) have been widely distributed for abuse purposes. Effective measures to counter the spread of NPS are to promptly legislate them through the risk assessment. Phencyclidine analogues having inhibitory effects toward NMDA receptor (NMDAR) have recently emerged in Japan. Therefore, it is important to establish a high-throughput system for efficiently detecting NPS that can inhibit NMDAR activity.

Methods: Hippocampal neurons prepared from embryonic rats were incubated in 96-well microplates. After 3 weeks in vitro, cultured neurons were preincubated with phencyclidine (PCP) or PCP-analogues, including 3-methoxyphencyclidine (3-MeO-PCP) and 4-[1-(3-methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo), and then treated with 100 μM glutamate for 10 min. After fixation, cultured neurons were immunostained with anti-drebrin and anti-MAP2 antibodies. The linear cluster density of drebrin along the dendrites was automatically quantified using a protocol that was originally developed by us.

Results: The high-throughput immunocytochemical assay, measuring drebrin cluster density of cultured neurons, demonstrated that glutamate-induced reduction of drebrin cluster density in 96-well plates is competitively inhibited by NMDAR antagonist, APV. The reduction was also antagonized by PCP, 3-MeO-PCP and 3-MeO-PCMo. The inhibitory activity of 3-MeO-PCMo was lower than that of PCP or 3-MeO-PCP, with IC₅₀ values of 26.67 μM (3-MeO-PCMo), 2.02 μM (PCP) and 1.51 μM (3-MeO-PCP).

Discussion: The relative efficacy among PCP, 3-MeO-PCP and 3-MeO-PCMo calculated from IC₅₀ are similar to those from Ki values. This suggests that the high-throughput imaging analysis is useful to speculate the Ki values of new PCP analogues without performing the kinetic studies.

Keywords: Dendritic spine; Drebrin; High-throughput analysis; Immunocytochemistry; NMDA receptor antagonist; Phencyclidine; Primary neuronal culture; Psychoactive substances; Synapse.