To further determine whether the anxiety-like behavioral phenotype was exclusive for the activation of nNOS-expressing neurons, we also microinjected AAV-hSyn-DO-hM3Dq-eGFP into the vmPFC of nNOS-Cre mice to stimulate Cre-negative neurons (Supplementary Fig. 4a, b) and found no alterations in anxiety-like behaviors (Supplementary Fig. 4c–e), but there was a clear trend suggesting an analgesic effect (t24 = 2.040, p = 0.053, Student’s t-test). Here, NOS1 is linked to Anxiety.