Clozapine-n-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called ‘Designer Receptors Exclusively Activated by Designer Drugs’ (DREADD) system. This system consists of synthetic G protein-coupled receptors which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N-desmythclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long-Evans rats. It was found that 1mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI) (a measure of sensorimotor gating). CNO (2 and 5mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist PCP or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1,2 and 5 mg/kg) had no effect on spontaneous locomotion, but 5mg/kg CNO pre-treatment significantly attenuated d-amphetamine-induced hyper-locomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5mg/kg CNO significantly attenuated the d-amphetamine induced increase in evoked-dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine and N-Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effects in vivo and is converted to clozapine and N-Des. Thus, emphasizing the need for a CNO-only-DREADD-free control group when designing DREADD based experiments.
Significance Statement: Recently, interest in clozapine-N-Oxide (CNO) has increased due to its exploitation as a ligand for the engineered G protein-coupled receptors (GPCRs) in the chemogenetic ‘Designer Receptors Exclusively Activated by Designer Drugs’ (DREADD) system. Our results highlight that in the experimental design there is a necessity for the inclusion of a group of animals which do not express DREADD receptors, but are given the same dose of CNO as the DREADD expressing animals. Currently, only a small minority of studies utilizing DREADDs employ this control. There needs to be careful consideration of the CNO dose being administered and of the possible biological effects of CNO.
Authors report no conflict of interest
The studies were funded by start-up funds provided to SDC by State University of New York at Buffalo and by NIH to RAE R01 DA025279.