Inhibitory effects of (synthetic) cannabinoids and (designer) benzodiazepines on spontaneously active neuronal networks of primary rat cortical cultures in vitro

The use of novel psychoactive substances (NPS) has been increasing over the last 20 years. Given the coinciding increasing number of health incidents, it is of paramount importance to comprehend the neurotoxic hazards associated with these often potent but poorly characterized designer drugs. The objective of this study was therefore to investigate the neuromodulatory effects of various (designer) drugs, including cannabidiol (CBD), a synthetic cannabinoid receptor agonist (RCS-4), three canonical benzodiazepines (diazepam, oxazepam, and lorazepam) and two designer benzodiazepines (flunitrazolam and fluetizolam). To that aim, we measured changes in spontaneous neuronal activity following acute drug exposure in primary rat cortical cultures grown on microelectrode arrays (MEAs). Acute exposure to ≥ 3 µM CBD resulted in a concentration-dependent decrease of all major activity parameters, such as the number of spikes, the number of bursts, the number of spikes in a burst, burst duration, the number of network bursts, the number of spikes in a network burst, and network burst duration. These effects were comparable to those observed following acute exposure to the synthetic cannabinoid receptor agonist RCS-4, which inhibited neuronal activity at ≥ 1 µM. At the highest concentration, the decrease in neuronal activity was paralleled by an increase in burst duration. The three canonical benzodiazepines (diazepam, oxazepam, and lorazepam) concentration-dependently decreased neuronal activity (number of spikes, number of bursts and number of network bursts) with lowest observed effect concentrations (LOECs) of 0.1 µM, 1 µM and 0.1 µM, respectively. The decrease in neuronal activity was paralleled by an increase in burst duration, which was particularly profound for diazepam. The two designer benzodiazepines (flunitrazolam, and fluetizolam) potently decreased neuronal activity with LOECs of 0.01 µM and 0.03 µM, respectively. Comparable to the canonical benzodiazepines, the decrease in neuronal activity was paralleled by a marked increase in burst duration. Our findings demonstrate the applicability of MEA recordings for neurotoxicity assessment and potency ranking of diverse (designer) drugs. The strong potency of some of these drugs is particularly concerning and underscores the urgent need for better regulation and control of these substances to safeguard public health.