CNO Administration Increases Dopamine and Glutamate in the Medial Prefrontal Cortex of Wistar Rats: Further Concerns for the Validity of the CNO-activated DREADD Procedure.

The chemogenetic procedure DREADD (designer receptor exclusively activated by designer drugs) is an inventive way to selectively affect g-coupled protein receptors. In theory, DREADD receptors are only activated by administering inert compounds, primarily clozapine N-oxide (CNO). Research has shown that CNO does not cross the blood-brain barrier, and CNO is converted back to clozapine and N-desmethylclozapine (N-Des) in the brain. Clozapine and N-Des have many neurological effects including alterations in glutamate and dopamine (DA) levels in multiple brain regions. The current study examined the effects of peripheral administration of CNO on glutamate and DA levels in the medial prefrontal cortex (mPFC). Wistar rats were administered CNO, and microdialysis samples were collected from the mPFC. Administration of CNO significantly increased glutamate (31-87%) and DA (65-126%), CNO-induced increases in DA occurred for a longer duration than glutamate, and that for the two highest doses of CNO there was a significant correlation between the increase in glutamate and DA in the mPFC. In the mPFC, CNO-induced increases in DA occurred at 0.5 mg/kg, while increases in glutamate were observed at doses greater than 1.0 mg/kg. The source of the DA and glutamate could be caused by activation of projection neurons or local effects. The data replicate findings that CNO is not an inert compound and that interpretation of CNO-activated DREADD findings should be done with caution. The data indicate that low ('safe') doses of CNO still have neurochemical effects and that controlling for the actions of clozapine/N-Des in CNO-DREADD studies has many concerns.