Fig. 1: A projection of CRH/GABA neurons in the medial BLA to the medial NAc shell.

a–c Retrograde tracing of CRH⁺ neuronal inputs to medial NAc shell of CRH-ires-CRE mice. a Schematic of construct and injection location of AAV2‐retro‐CAG‐FLEX‐tdTomato‐WPRE virus that permits retrograde access to projection neurons providing afferent inputs to NAc. b Example confocal micrograph of locally infected CRH⁺ axon terminals in medial NAc shell. c Retrograde tracing identifies the medial BLA as a robust source of CRH⁺ NAc inputs. d 3D image (z-stack; 0.5 μm steps) confirmed localization in the BLA of AAV-retro infected cells (red) that co-express endogenous CRH (green); dual labeled neurons = yellow. e–g Anterograde tracing of CRH⁺ axonal projections from BLA to medial NAc shell. e, The AAV1-DIO-tdTomato construct and the viral genetic experimental design. f Virus injection is confined to the BLA, g and absent from the central amygdala (CeA), shown by selective expression of tdTomato in BLA CRH⁺ neurons. h BLA-origin CRH⁺ axons and terminals in the medial NAc shell. i–k Virus injection into the medial NAc shell retrogradely infected somata in the BLA. i Combined fluorescence in situ hybridization (FISH) and immunostaining with GAD67 mRNA in CRH⁺ cells in the BLA. Arrowheads point to co-localized GAD67 mRNA and virus-reporter labeling. j a BLA → NAc cell (red) co-expresses endogenous CRH (green) and vGAT (magenta), but k does not co-express the glutamatergic marker CaMKII. ** = Major Island of Calleja, ac  anterior commissure, DB  diagonal band. Scale bars in i and k = 10 µm. To confirm findings, virus injections, projection assessment, and immunohistochemistry were assessed in mice from at least two independent litters.