Off-Target Effects of Clozapine-N-Oxide on the Chemosensory Reflex Are Masked by High Stress Levels

Abstract

Respiratory chemosensory circuits are implicated in several physiological and behavioral disorders ranging from sudden infant death syndrome to panic disorder. Thus, a comprehensive map of the chemosensory network would be of significant value. To delineate chemosensory neuronal populations, we have utilized pharmacogenetic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) perturbations for acute neuronal perturbations in respiratory circuit mapping. Recent studies show that the biologically inert DREADD ligand clozapine-N-oxide (CNO) is back-metabolized into the bioactive compound clozapine in rodents, emphasizing the need for CNO-only DREADD-free controls, which have been carried out in several studies. However, we show that high CNO doses used in several chemosensory circuit mapping studies nonetheless affect the chemosensory ventilatory reflexes in control mice, which is unmasked by extensive habituation. Here, unhabituated control animals showed no differences in respiratory parameters after CNO administration, whereas habituated animals receiving the commonly used dose of 10 mg/kg of CNO show a deficit in the hypercapnic (high CO 2 ) chemosensory reflex, which is not present in 1 mg/kg CNO treated or saline control groups. Our findings indicate that even in appropriately controlled studies, additional masked CNO off-target effects may exist and underscore the importance of using minimal doses of activating ligand in combination with high levels of habituation.

Adipocyte Gs but not Gi signaling regulates whole-body glucose homeostasis

“…It is also plausible that chronic CNO administration potentiates the browning of iWAT and energy expenditure, which is beyond the scope of our acute GPCR stimulation studies. However, chronic treatment with high doses of CNO are associated with many adverse effects [62], mostly due to its back-transformation into clozapine [48], that could limit the attractiveness to such an approach. Nonetheless, these chemogenetic approaches will be invaluable tools for chronically or acutely controlling these pathways in a spatiotemporal manner.…”

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

“…A number of studies, including one of the authors' studies 7 , showed no significant non-specific response 39 , 40 , while higher doses (e.g., 10 mg/kg) can produce side effects, at least in some cases 41 . However, as with all behavioral experiments, proper controls 31 are essential due to potential off-targeted activity of CNO and its metabolites 42 . Such controls could include administration of CNO and saline controls to animals expressing DREADDs and administration of CNO to wild-type animals or in some specific cases a comparison of ipsi-and contralateral sites of the brain that respectively do and do not express chemogenetic receptors.…”

The Projection From Ventral CA1, Not Prefrontal Cortex, to Nucleus Accumbens Core Mediates Recent Memory Retrieval of Cocaine-Conditioned Place Preference

“…It was reported that 0.5 mg/kg CNO resulted in behavioral signs of seizure activity in approximately 20% of mice during fear conditioning (Garner et al, 2012 ). The high dose of CNO (10 mg/kg) also showed off-target effects (Martinez et al, 2019 ). A high dose of CNO may produce nonspecific side effects.…”