CNO induced Ca2+ store and glutamate-dependent nonspecific Ca2+ signalling in DREADD-free brain slices

DREADD (design receptors exclusively activated by designer drugs) is a widely used powerful tool designed to study specific cellular functions. However, off-target effects of chemogenetic activators, including clozapine N-oxide (CNO) and deschloroclozapine (DCZ), have been reported. In our study, we demonstrated the direct off-target effects of CNO and DCZ on basal Ca²⁺ levels in the locus coeruleus nucleus in both neurons and astrocytes by combining viral microinjection, Ca²⁺ imaging and electrophysiology. We observed that CNO induced a Ca²⁺ store-dependent increase in basal Ca²⁺ in both DREADD-absent neurons and astrocytes; interestingly, CNO directly increased the frequency of spontaneous presynaptic glutamate release. Furthermore, ionotropic glutamate receptors contributed to the CNO-induced increase in Ca²⁺ in both DREADD-free neurons and astrocytes. Importantly, IP3R2-KO diminished CNO-induced Ca²⁺ raise in astrocytes but not neurons. Our results revealed direct Ca²⁺ store- and glutamate-dependent off-target effects of DREADD agonists during their cellular action, which may help elucidate the mechanisms underlying the off-target effects of chemogenetic tools.