# Nonclinical human neural new approach methodologies (NAMs): Electrophysiological assessment of opioid agonist and antagonist combination

## TL;DR

This study uses human neural cells and electrophysiology to assess how opioids and their reversal agents affect brain activity patterns.

## Contribution

The study introduces a new method using hiPSC-derived neural cells and MEA to evaluate opioid effects and reversal in a human-relevant model.

## Key findings

- DAMGO modulated neural activity in a concentration-dependent manner, affecting multiple electrophysiological parameters.
- Naloxone reversed DAMGO-induced changes in neural activity and restored baseline patterns.
- Baseline network activity influenced divergent responses to opioids in subpopulations of neural cells.

## Abstract

New approach methodologies (NAMs) including microphysiological systems in combination with human induced pluripotent stem cell (hiPSC)-derived neural cells and multielectrode array (MEA) have demonstrated utility for evaluating electrophysiological effects of CNS active compounds including those with potential seizurogenic liability. Here, we extend a neural NAM assay to assessment of an opioid agonist and reversal agent.

An in vitro human NAM composed of hiPSC-neurons and hiPSC-astrocytes coupled with MEA platform was used to assess spontaneous neural electrophysiology. Neural cells were cultured on MEA plates then treated with varying concentrations of the μ-opioid receptor agonist DAMGO (D-Ala(2)-mephe(4)-gly-ol(5))enkephalin). Following DAMGO treatment, the opioid antagonist naloxone [10 μM] was added to each well to evaluate phenotypic reversal.

The opioid agonist DAMGO modulated neural electrophysiological activity in a concentration-dependent manner relative to vehicle control. These changes were observed across neural parameters relating to overall neural activity, single electrode burst, network burst, and synchronicity. These effects were reversed by the opioid antagonist naloxone. Moreover, DAMGO treatment disrupted higher order baseline neural patterns. Finally, subpopulation analysis revealed divergent opioid-induced higher order network response influenced by baseline network activity.

These findings demonstrate the hiPSC neural NAM can characterize human network electrical activity at baseline and following exposure to an opioid agonist and antagonist pair. Future studies will investigate additional opioids and reversal agents, as well as other MEA metrics and activity patterns that may best describe concentration- and time-dependent changes in neural activity.