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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 CO2) 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.
Highlights
The neural networks that underlie the respiratory chemosensory reflex are a primary target for understanding the etiology of several behavioral and physiological disorders
To confirm that NA neurons expressing the hM4D receptor were responsive to CNO, we performed recordings on locus coeruleus (LC) neurons, where we observed an inhibition of neuron firing upon CNO bath application (n = 3, Figure 1C)
In the whole-body plethysmography Designer Receptors Exclusively Activated by Designer Drugs (DREADD) protocol used in our lab and others, animals are handled, rectally probed for temperature, exposed to a novel environment, and intraperitoneally injected
Summary
The neural networks that underlie the respiratory chemosensory reflex are a primary target for understanding the etiology of several behavioral and physiological disorders. DREADD technology in combination with intersectional genetic deployment has been utilized to silence highly targeted neuronal populations while observing respiratory function in conscious and unrestrained mice by our lab and others, avoiding many confounds from earlier circuit mapping approaches (Ray et al, 2011, 2013; Brust et al, 2014; Hennessy et al, 2017; Sun and Ray, 2017; Sun et al, 2017) These and most other studies almost always included CNO only non-DREADD expressing sibling controls that showed no chemosensory or other respiratory effects, arguing that CNO had no off-target effects on breathing in conscious and unrestrained mice despite the high dose used, 10 mg/kg. It was shown that CNO and its metabolites are not equivalently distributed between the circulatory system and the brain (Gomez et al, 2017)
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