Fig. 1: Identification of monosynaptic inputs to CeA-CRF neurons via rabies-based retrograde viral tracing.

a AAV helper viruses with Cre-dependent expression of the receptor of avian sarcoma leucosis virus envelope protein (TVA) and rabies glycoprotein G (RG) were used. Genetically modified rabies virus was pseudotyped with EnvA. The RG gene was replaced by EGFP. b Experimental timeline. AAV helper viruses were injected at day 1, and the rabies virus was injected at day 21 for the proper expression of the helper viruses. The infected mice were sacrificed on day 28. c Combination of the two-virus system and transgenic CRF-Cre mice allowed for brain-wide labeling of monosynaptic inputs (red) to CRF neurons (green) in the CeA. d Schematic of the input atlas of the CeA-CRF neurons at a whole-brain scale. e Tile-scan image shows the starter neurons in the CeA (scale bar: 1000 μm). Starter neurons were identified by the colocalization of EGFP (green) and DsRed (red) fluorescent proteins in the enlarged image (scale bar: 200 μm). f Proportion of starter neurons in the CeA. Only samples with more than 70% of the starters distributed within the CeA were used for further analysis. g Total number of starter neurons inside and outside of the CeA. h Total number of input neurons in the whole brain. i There was a linear relationship between starter and input neurons across all samples, indicating that the virus had the same transsynaptic efficiency in different samples. (N = 5).