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 for remote control of targeted cell populations using chemical actuators that bind to modified receptors. Despite the popularity of DREADDs in neuroscience and sleep research, potential effects of the DREADD actuator clozapine-N-oxide (CNO) on sleep have never been systematically tested. Here, we show that intraperitoneal injections of commonly used CNO doses (1, 5, and 10 mg/kg) alter sleep in wild-type male laboratory mice. Using electroencephalography (EEG) and electromyography (EMG) to analyse sleep, we found a dose-dependent suppression of rapid eye movement (REM) sleep, changes in EEG spectral power during non-REM (NREM) sleep, and altered sleep architecture in a pattern previously reported for clozapine. Effects of CNO on sleep could arise from back-metabolism to clozapine or binding to endogenous neurotransmitter receptors. Interestingly, we found that the novel DREADD actuator, compound 21 (C21, 3 mg/kg), similarly modulates sleep despite a lack of back-metabolism to clozapine. Our results demonstrate that both CNO and C21 can modulate sleep of mice not expressing DREADD receptors. This implies that back-metabolism to clozapine is not the sole mechanism underlying side effects of chemogenetic actuators. 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.

Parvalbumin neurons in the anterior nucleus of thalamus control absence seizures

“…They observed that injections of the medium (5 mg/kg) or high dose (10 mg/kg) of CNO could significantly increase in spectral power in the range between 0.5 and 1.25 Hz during NREM sleep. However, the low dose of CNO (1 mg/kg) has no systematic effects on sleep spectrogram 39 . It suggested that the influence of CNO (1 mg/kg) alone on delta rhythms in our study is low.…”

“…However, the low dose of CNO (1 mg/ kg) has no systematic effects on sleep spectrogram. 39 It suggested that the influence of CNO (1 mg/kg) alone on delta rhythms in our study is low. Recent studies revealed that the mechanism of SWD, delta rhythm, and sleep spindles involve the same thalamocortical circuitry.…”

Neuronal PAS domain 1 identifies a major subpopulation of wakefulness-promoting GABAergic neurons in basal forebrain

“…Npas1 + neuron-mediated suppression of REM sleep was due to a reduced number of entries into REM. Control experiments in a separate group of mice confirmed that 0.3 mg/kg CNO administered without hM3d(Gq) transduction did not affect the amount of sleep or sleep architecture, consistent with other published findings that low doses of CNO do not have marked effects on sleep or sleep physiology (Anaclet et al, 2015; Traut et al, 2023). A previous study confirmed that CNO depolarizes and increases action potential discharge of BF Npas1 + neurons in the VP subregion transduced with hM3d(Gq)-mCherry (Morais-Silva et al, 2023), promoting activation of the immediate early gene product, cFOS.…”

Deschloroclozapine exhibits an exquisite agonistic effect at lower concentration compared to clozapine-N-oxide in hM3Dq expressing chemogenetically modified rats

“…This suggests that DCZ exhibits comparable effects to CNO at significantly lower dose. In addition, CNO at a dose of 1 mg/kg may not raise significant concerns regarding off-target effects ( Traut et al, 2023 ). The lack of differences between CNO and DCZ in in vivo Fos observations can be considered as a significant finding of this study.…”