Table_1_The Chemogenetic Receptor Ligand Clozapine N-Oxide Induces in vivo Neuroreceptor Occupancy and Reduces Striatal Glutamate Levels.docx

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.

基于假设CNO（氯氮平N-氧化物）在天然神经受体上无作用，基于此假设进行的化学遗传学研究。然而，近期研究证据表明，CNO在体内可能转化为氯氮平（CLZ），从而产生足以占据活脑中的多巴胺D2/3和血清素5HT2A受体的血浆浓度。为验证此现象，我们利用[18F]fallypride正电子发射断层扫描（PET）测量了纹状体多巴胺D2/3受体占据率，并通过[18F]MH.MZ体外实验检测了血清素5HT2A受体占据率。我们发现CNO剂量依赖性地影响了神经受体位点可用性。在平行磁共振波谱实验中，我们发现CNO降低了前额叶皮层的磷酸肌酸（Cr+PCr）和N-乙酰天冬氨酸（NAA）及N-乙酰天冬氨酰谷氨酸（NAAG）信号，并在背侧纹状体中减少了谷氨酸信号，峰值效应出现在2 mg/kg剂量。因此，我们的研究结果表明，CNO在活鼠体内转化为CLZ能够显著占据内源性神经受体，并对神经代谢物水平产生显著影响。