Clozapine N-Oxide Administration Produces Behavioral Effects in Long-Evans Rats: Implications for Designing DREADD Experiments

Abstract

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 the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N-desmethylclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long-Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuated d-amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/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 emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

Keywords: CNO; DREADDs; behavior; voltammetry.

Figure 1.

A, B, Startle magnitude and PPI after treatment with CNO. CNO significantly reduced the startle response to 110 and 120 dB startle stimuli (A), but had no significant effect on PPI (B). *p < 0.05.

Figure 2.

Startle magnitude and PPI after treatment with CNO and PCP. A, B, PCP significantly increased the startle magnitude (A) and disrupted the PPI (B). CNO pretreatment had no effect on the disruptive effects of PCP.

Figure 3.

Startle magnitude and PPI after treatment with CNO and scopolamine. A, B, Scopolamine significantly increased the startle magnitude (A) and disrupted the PPI (B). CNO pretreatment had no effect on the disruptive effects of scopolamine.

Figure 4.

Effects of CNO on spontaneous locomotion and amphetamine-induced hyperlocomotion. Rats were pretreated with either vehicle or CNO, followed 20 min later by either vehicle or amphetamine. A–F, CNO at 1 mg/kg (A, B), 2 mg/kg (C, D), or 5 mg/kg (E, F) had no effect on spontaneous locomotion. Neither 1 nor 2 mg/kg CNO altered amphetamine-induced hyperlocomotion (B, D), but 5 mg/kg CNO significantly reduced the effects of amphetamine (F). *p < 0.05, **p < 0.01.

Figure 5.

Effects of CNO on baseline and amphetamine-induced DA signaling. A, B, Examples of 15 s DA overflow curves in vehicle-treated (A) and CNO-treated (B) rats before (solid) and after (dashed) a dose of 1.5 mg/kg, i.p., d-AMPH. C, D, CNO did not alter stimulated DA release (C) or uptake (D) under baseline conditions. E, F, CNO dose-dependently blunted the increased DA release in response to d-AMPH 30 min after systemic treatment (E); however, there were no significant effects on the magnitude of DA uptake inhibition (F). *p < 0.05

Figure 6.

A–C, Plasma levels of CNO (A), clozapine (B), and N-Des (C) at various time points after the administration of 5 mg/kg CNO.