Off-target effects of DREADD ligands revealed by an anesthesia emergence paradigm in mice

Designer receptors exclusively activated by designer drugs (DREADDs) enable reversible control of specific neural circuits, but the pharmacological neutrality of their ligands is increasingly questioned. Here, we introduce an anesthesia emergence paradigm to systematically assess the off-target effects of DREADD ligands in DREADD-naive mice. We show that intraperitoneal administration of clozapine N-oxide (CNO), compound 21 (C21), or deschloroclozapine (DCZ) delays motor recovery from isoflurane anesthesia. CNO produced the largest delay, likely due to its back-conversion to clozapine. DCZ showed the smallest effect magnitude, although its difference from C21 remained inconclusive. We then show that subcutaneous administration, which should reduce clozapine back-conversion, reduces the CNO-induced recovery delay to levels comparable to those of C21. Finally, we provide a freely available, deep-learning-based automated behavioral pipeline that integrates the anesthesia emergence paradigm with a reproducible analysis tool for future studies. Together, these results underscore the importance of accounting for ligand off-target effects through careful dose selection and DREADD-free, ligand-treated controls in chemogenetic experiments.