C57Bl/6J male mice (8 weeks old) were used for all EASI-FISH experiments with retrograde tracer labeling. The non-toxic retrograde tracers cholera toxin b (CTB) conjugated with different fluorophores (Alexa Fluor-488, Alexa Fluor-555, Alexa Fluor-594, Alexa Fluor-647; all Thermo Fisher, 0.5%) and FluoroGold (FG; Fluorochorome, 2%) were injected into the left hemisphere of five selected projection areas of the CEA: the bed nucleus of the stria terminalis (BNST; coordinates from bregma: AP 0.25 mm, ML 1.0 mm, DV 4.4 mm), the lateral part of the substantia nigra (lateral SN; AP –3.65 mm, ML 1.8 mm, DV 3.8 mm), the ventrolateral PAG (vlPAG; AP –4.65 mm, ML 0.5 mm, DV 2.35 mm), the parabrachial nucleus (PBN; AP –5.2 mm, ML 1.15 mm, DV 3.25 mm), and the parvocellular reticular nucleus (PCRt; AP –6.4 mm, ML 1.25 mm, DV 4.7 mm). The surgery was performed as described above. Animals received up to 0.5 ml 0.9% saline/0.5 ml 5% glucose (subcutaneously) during the surgery. 50 nl of retrograde tracer was injected into each region. For animal #1, BNST was injected with FG, lateral SN CTB-647, vlPAG CTB-594, PBN CTB-555, and PCRt CTB-488. For animal #2, BNST was injected with FG, lateral SN CTB-647, vlPAG CTB-488, PBN CTB-555, and PCRt CTB-594. For animal #3, BNST was injected with FG, lateral SN CTB-594, vlPAG CTB-647, PBN CTB-555, and PCRt CTB-488.
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