Off-targets effects of CNO on somatosensory and anxiety-related behaviors in rats

Designer receptors exclusively activated by designer drugs (DREADDs) are a powerful chemogenetic tool for controlling targeted cell populations and manipulating brain functions. As a typical chemogenetic actuator in DREADD systems, clozapine-N-oxide (CNO) is widely utilized in neurobiological and pharmacological research. However, the acute off-target effects of CNO on somatosensory (e.g., itch, pain) stimulation-induced and anxiety-related behaviors in rodents have not been systematically investigated. Through classical behavioral paradigms, including evoked itch-scratching, noxious thermal pain assessment, and elevated plus maze testing, we demonstrated that intraperitoneal administration of standard CNO doses (2 and 4 mg/kg) modulates itch/pain perception and anxiety-like behaviors in Sprague-Dawley rats lacking DREADD expression. The key behavioral findings are: (1) High-dose CNO (4 mg/kg) significantly suppressed 5-HT-induced itch-scratching; (2) Both doses of CNO (2 and 4 mg/kg) reduced sensitivity to noxious thermal stimuli; (3) CNO induced an anxious phenotype without altering locomotor activity. Fiber photometry experiments further revealed that CNO enhanced glutamatergic neuronal activity in the prelimbic cortex but not in the central amygdala or the dorsal hippocampus. These results provide robust evidence that commonly employed doses of CNO—designed to activate DREADDs—elicit off-target effects on somatosensory and emotional behaviors in rats. The underlying mechanisms may involve CNO-induced modulation of glutamatergic signaling in key limbic regions, potentially mediated by interactions between the ligand or its metabolites and endogenous brain receptors. Verification of these off-target effects underscores the necessity of including ligand-only control groups in DREADD experiments and necessitates caution against overinterpreting behavioral outcomes attributed solely to chemogenetic manipulations.