Fig. 1. Chemogenetic double virus transduction and injection sites in the LPFC and CdN.
a Illustration of the chemogenetic double virus transduction method. The locally transducing virus (AAV5-hSyn-DIO-hM4Di-mCherry) was injected into the LPFC, while the retrograde virus (FuG-E(NeuRet)-nls/Cre-2A-eGFP) was injected into the CdN. Only doubly-transduced neurons whose cell bodies were in the LPFC and axon terminals in the CdN expressed the hM4Di-mCherry gene. The activity of these doubly-transduced neurons could be suppressed by CNO administration. b Injection sites of the two viruses. Above: View of virus injection tracks from the diagonal direction for Monkey W (left) and Monkey S (right). Orange dots indicate AAV injection tracks in the LPFC. Green dots indicate NeuRet injection tracks in the CdN. Below: Representative 2D coronal reconstructions of injection positions (white dots) in the LPFC and CdN, corresponding to the tracks boxed in the upper panels. Scale bar: 5 mm. c Doubly-transduced, mCherry-positive neurons (magenta) in the right LPFC as observed using a WIG filter cube and d the micrograph of the same area as (c) as observed using a NIBA filter cube. e The superimposed image of (c) and (d). Scale bar: 100 µm. f eGFP-positive neurons (green) in the ipsilateral CdN of Monkey S. g Micrograph of the same area as in (f). h The superimposed image of (f) and (g). Scale bar: 100 µm. AAV adeno-associated virus, LPFC lateral prefrontal cortex, PS principal sulcus, AS arcuate sulcus, CdN caudate nucleus, Pt putamen, DREADD Designer Receptors Exclusively Activated by Designer Drugs.