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

A high-throughput immunocytochemical assay using cultured rat hippocampal neurons can detect whether new psychoactive substances (NPS) inhibit NMDA receptor activity. Glutamate-induced reduction of drebrin cluster density along dendrites was competitively blocked by the NMDA receptor antagonist APV. The phencyclidine analogues PCP, 3-MeO-PCP, and 3-MeO-PCMo also antagonized the reduction, with IC50 values of 2.02 μM, 1.51 μM, and 26.67 μM respectively, indicating that 3-MeO-PCMo is less potent. The relative inhibitory potencies from IC50 values matched those from Ki values, suggesting the assay can estimate Ki for new PCP analogues without kinetic studies.

Study at a glance

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.