CNO was the original DREADD ligan, but in rats and mice it doesn’t always stay as CNO. A small fraction (about 2 -7%) can convert into clozapine. Once clozapine shows up, it behaves a bit differently. After IV dosing it gets into the brain better than CNO, and it also binds DREADDs more strongly than CNO. A little can go a long way, and readout become hard to trust as “CNO only”. Clozapine also doesn’t keep to DREADDs; it shows up in the brain extracts, and it binds plenty of native receptors too (5-HT, dopamine, histamine, muscarine). That is why CNO is not the preferred DREADD ligan anymore.