Effects of clozapine-N-oxide and compound 21 on sleep in laboratory mice

Abstract

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are chemogenetic tools that modulate a targeted cell population using chemical ligands which bind specifically to modified receptors. Despite the common use of DREADDs in neuroscience and sleep research, potential effects of the DREADD actuator clozapine-N-oxide (CNO) on sleep, which could arise from back-metabolism to clozapine or binding to endogenous neurotransmitter receptors, have never been systematically tested. Here we show that intraperitoneal injections of commonly used doses of CNO alter sleep in wild-type male laboratory mice. Using electroencephalography (EEG) and electromyography (EMG) to analyse sleep, we found that CNO suppresses rapid eye-movement (REM) sleep, changes EEG spectral power during non-REM (NREM) sleep, and alters sleep architecture in a pattern previously reported for clozapine. Interestingly, we found that the novel DREADD actuator, compound 21 (C21), similarly modulates sleep despite not being able to back convert to clozapine. Our results demonstrate that CNO and C21 can affect the sleep of wild-type mice, which do not express DREADD receptors. This implies that back-metabolism to clozapine is not the sole mechanism underlying side effects of chemogenetic actuators and therefore any chemogenetic experiment should include a DREADD-free control group injected with the same CNO, C21 or newly developed actuator. We suggest that electrophysiological sleep assessment could serve as a sensitive tool to test the biological inertness of novel chemogenetic actuators.

A noradrenergic-hypothalamic neural substrate for stress-induced sleep disturbances

“…2 I and J ; P = 0.034). Previous studies reported that high doses of CNO have an effect on various sleep parameters ( 47 , 70 – 72 ), an effect likely resulting from the back metabolism of CNO to clozapine or off-target effects, and we therefore directly compared the effect of CNO between the mCherry and hM3DGq groups. The increase in the frequency of MAs in hM3DGq mice was also significant when compared with that in mCherry mice injected with CNO ( Fig.…”

Activation of basal forebrain astrocytes induces wakefulness without compensatory changes in sleep drive

“…We then verified that CNO had no effects on sleep architecture in Ctrl mice. This is a necessary control when using CNO as it has been shown to metabolize to compounds (e.g., clozapine) that can indirectly impact sleep [20][21][22][23][24]. The same dose delivered to Ctrl littermates had no impact on sleep architecture compared to vehicle (Figs 1 and 2, S1 Table ).…”

“…We found that activating BF astroglial Gq DREADDs in Gq +/mice induced long periods of continuous wakefulness (≥ 6 h) and an overall increase in wake time for the entire light phase ( We then verified that CNO had no effects on sleep architecture in Ctrl mice. This is a necessary control when using CNO as it has been shown to metabolize to compounds (e.g., clozapine) that can indirectly impact sleep [20][21][22][23][24]. The same dose delivered to Ctrl littermates had no impact on sleep architecture compared to vehicle (Figs 1 and 2, S1 Table ).…”