Behavioral Effects of Acute Systemic Low-Dose Clozapine in Wild-Type Rats: Implications for the Use of DREADDs in Behavioral Neuroscience

Abstract

Designer receptors exclusively activated by designer drugs (DREADDs) are popular tools used to manipulate the activity of defined groups of neurons. Recent work has shown that DREADD effects in the brain are most likely not mediated by the proposed ligand clozapine-N-oxide (CNO) but its metabolite clozapine (CLOZ). However, it is not known whether low doses of CLOZ required to activate DREADDs already have DREADD-independent effects on behavior as described for higher CLOZ doses used in previous preclinical studies. To close this gap, we compared effects of acute systemic (i.p.) CLOZ treatment vs. vehicle (VEH) in a wide range of behavioral tests in male wild-type rats. We found that CLOZ doses as low as 0.05–0.1 mg/kg significantly affected locomotion, anxiety and cognitive flexibility but had no effect on working memory or social interaction. These results highlight the need for careful controls in future chemogenetic experiments and show that previous results in studies lacking CNO/CLOZ controls may require critical re-evaluation.

fMRI detects bilateral brain network activation following unilateral chemogenetic activation of direct striatal projection neurons

“…However, we should carefully consider the effective time window and possible unexpected DREADD-independent pharmacological effects of DREADD ligands. are reported to affect behavior in some conditioning (Ilg et al, 2018), we confirmed that CLZ (0.1 mg/kg, i.p.) did not affect the spontaneous locomotion in C57BL/6 mice (Supplementary Fig.…”

Long‐term chemogenetic suppression of spontaneous seizures in a mouse model for temporal lobe epilepsy

“…Seizure-suppressing effects could be obtained with two different hM4Di ligands: CNO or clozapine. Studies report effects of clozapine on anxiety-related behavior (both anxiolytic and anxiogenic) and locomotion (sedative effects) at doses of 0.05 mg/kg and higher in wild-type rats 21,22 and 0.5 mg/kg and higher in wildtype mice [23][24][25] . 17,18 Therefore novel ligands, such as compound 21, perlapine, JHU37160/152, and clozapine have been suggested for direct DREADD activation.…”

“…20 However, off-target effects may still occur given the interactions of clozapine with a broad range of receptors, including dopaminergic, adrenergic, serotonergic, histaminergic and muscarinergic receptors. Studies report effects of clozapine on anxiety-related behavior (both anxiolytic and anxiogenic) and locomotion (sedative effects) at doses of 0.05 mg/kg and higher in wild-type rats 21,22 and 0.5 mg/kg and higher in wildtype mice [23][24][25] . In this study such effects could not be demonstrated in the OFT after a single administration of 0.1 mg/kg clozapine or 10 mg/kg CNO to non-DREADD IHKA mice.…”

Chemogenetic inhibition of the amygdala modulates emotional behavior expression in infant rhesus monkeys

“…Second, we only conducted a pre-surgery test of CNO on the human intruder task and not the socioemotional attention eye tracking task. Although, it is possible that CNO/clozapine could have impacted social attention, this is unlikely considering a recent study demonstrating that low dose clozapine does not impact social behavior or working memory in naïve rats (Ilg et al, 2018).…”