The Chemogenetic Receptor Ligand Clozapine N-Oxide Induces in vivo Neuroreceptor Occupancy and Reduces Striatal Glutamate Levels

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 D_2/3 and serotonin 5HT_2A receptors in living brain. To test this phenomenon, we measured striatal dopamine D_2/3 receptor occupancy with [¹⁸F]fallypride PET and serotonin 5HT_2A 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.

Keywords: CNO; DREADD; clozapine; dopamine receptors; glutamate; serotonin receptors.

FIGURE 1

[¹⁸F]Fallypride binding in the rat prefrontal cortex and striatal regions (A), 5 mg/kg Clozapine N-oxide (CNO) induced a noticeable decline in receptor availability in the same rat (A). CNO induced occupancy on a group level at the dopamine D_2/3 receptors in the prefrontal cortex (B,C) as well as dorsal (D,E) and ventral (F,G) striatum. Horizontal lines and ^∗represents a significant effect of treatments, as measured with a one-way ANOVA (^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001).

FIGURE 2

Serotonin 5HT_2A ex vivo specific binding ration of [¹⁸F]MH.MZ and the CNO induced occupancy. Male rats are round and females are triangular. Horizontal line and ^∗represents a significant effect of treatments, as measured with a one-way ANOVA (^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001).

FIGURE 3

Neurometabolite concentrations as measured in vivo using MR spectroscopy. Cubic voxels 18 μL in the prefrontal cortex (A) and 27 μL dorsal striatum (B). A sample spectrum (C) from the voxel in the dorsal striatum and metabolite concentrations in the voxels following pretreatments with different doses of CNO in the prefrontal cortex (D) and in the dorsal striatum (E). ^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001 two-way ANOVA with Sidak’s multiple corrects test.