Extended Data Fig. 8: Synaptic connections within the ACCGlu–VTAGABA–VTADA–ACCGlu loop.
a, Schematic of virus injection for the functional connection of the ACCGlu–VTAGABA–VTADA–ACCGlu loop. The trans-monosynaptic anterograde-Cre/GFP virus is injected into the ACC to express Cre and GFP in the downstream VTAGABA neurons, which initiates the Cre-dependent retrograde trans-monosynaptic tracing rabies virus (DIO-TVA/RVG and RV-\(\Delta G\)-dsRed) and simultaniously turns on the Cre-dependent expression of WGA-Flpo (DIO-WGA-Flpo) that is trans-synaptically delivered into the downstream DA neurons to start the TH-driving Ch2R/mCherry expression. b, Immunofluorescence showing the co-localization of DsRed- and GFP-positive signals with GAD67 staining. The inset is enlarged on the right. Scale bar, 100 μm. c, Representative micrograph showing that TH-positive fibers densely surround the VTA-projecting ACCGlu neurons, which are labeled by the retrograde virus (DsRed). The inset is enlarged on the right. Scale bar, 40 μm. d, Schematic showing the effect of chemogenetic inhibition of VTADA terminals on VTA-projecting ACCGlu neurons in awake mice. e, Statistics showing the CNO effect on control virus-injected mice (n = 14 cells from 2 mice) as in (d). f, Statistics showing the effects of chemogenetic inhibition of VTADA terminals on VTA-projecting ACCGlu neurons in awake shame (n = 33 cells from 3 mice) vs SNI (n = 33 cells from 3 mice) mice. g, Schematic of virus injection for the functional connection of the ACCGlu–VTAGABA–VTADA–ACCGlu feedback loop. The Cre/GFP-carrying trans-monosynaptic anterograde virus is injected into the ACC to express Cre and GFP in the downstream VTAGABA neurons, which turns on the Cre-dependent expression of WGA-Flpo (DIO-WGA-Flpo) that is trans-synaptically delivered to the downstream DA neurons to start the Flpo-dependent Ch2R/mCherry expression driven by the TH-promoter. h, Statistics of cold/hot plate of the optogenetic activation group and control mice (Control, n = 8; ChR2, n = 8 mice). The data are presented as the mean ± s.e.m. Paired Student’s t-test for (e, f) and unpaired Student’s t-test for (h). n.s., P > 0.05, *P < 0.05, ***P < 0.001. Detailed statistics information can be found in the Source Data.
