Abstract
Chemogenetic studies with the ligand clozapine N-oxide (CNO) are predicated upon the assumption that CNO is devoid of actions at natural neuroreceptors. However, recent evidence shows that CNO may be converted back to clozapine (CLZ) in vivo, which could yield plasma concentrations that may be sufficient to occupy inter alia dopamine D2/3 and serotonin 5HT2A receptors in living brain. To test this phenomenon, we measured striatal dopamine D2/3 receptor occupancy with [18F]fallypride PET and serotonin 5HT2A occupancy ex vivo using [18F]MH.MZ. We found a CNO dose-dependent effect on the availability of both neuroreceptor sites. In parallel MR spectroscopy experiments, we found that CNO reduced creatine + phosphcreatine (Cr+PCr) and increased N-acetylaspartate + N-acetylaspartylglutamate (NAA+NAAG) signals in the prefrontal cortex, and also reduced the glutamate signal in dorsal striatum, with peak effect at 2 mg/kg. Thus, our findings suggest that conversion of CNO to CLZ in living rats imparts significant occupancy at endogenous neuroreceptors and significant changes to neurometabolite levels.
Highlights
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are bioengineered from the muscarinic G protein-coupled receptor and have no natural ligand, but possess high agonistic affinity for the exogenous ligand clozapine N-oxide (CNO) (Armbruster et al, 2007)
CNO is a major metabolite of clozapine (CLZ) (Baldessarini et al, 1993), which is a widely used atypical antipsychotic medication with binding to a broad range of neuroreceptors, namely dopamine D4, D2, D3, serotonin 5-HT2A, 5-HT2C, muscarinic M1, M2, M3, M4, adrenergic α1 and α2, as well as histamine H1 receptors
While CNO is considered devoid of any specific binding other than to DREADD, recent studies suggest that CNO can convert back to CLZ in living rats (MacLaren et al, 2016) and dogs (Meier, 1975)
Summary
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are bioengineered from the muscarinic G protein-coupled receptor and have no natural ligand, but possess high agonistic affinity for the exogenous ligand clozapine N-oxide (CNO) (Armbruster et al, 2007). Untransformed CNO has low permeability to the rat blood brain barrier (Gomez et al, 2017) and is mainly confined to the plasma compartment of CLZ-treated rats (Baldessarini et al, 1993) Given this background, it has been suggested that the action of CNO on DREADDs in the brain is obtained solely via its metabolite CLZ (Gomez et al, 2017). To test for effects of the treatment on neurometabolites, we examined neurochemical changes in the prefrontal cortex and striatum using Magnetic Resonance Spectroscopy (MRS) These experiments provide relevant information about dose-dependent effects of CNO at neuroreceptors in Long-Evans rats and may call for reinterpretation of some DREADD experiments published prior to the identification of CLZ as the in vivo activator after CNO administration
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