Extended Data Fig. 6: Axonal projections of mPFC Pyr neurons and molecular identity of mPFC→LH neurons.
(a) Schematic of anterograde tracing experiment. AAV-CaMKII-Cre and AAV-DIO-tdTomato were injected into the mPFC of C57BL/6J mice to label axonal projections of mPFC Pyr neurons (n = 3 mice). (b) Injection site in the mPFC. Scale bar, 500 μm. Histology pictures in (b-g) are from the same animal. (c) tdTomato-labeled axon fibers in the thalamus. MD, mediodorsal thalamic nucleus; VA, ventral anterior thalamic nucleus; AM, anteromedial thalamic nucleus; Re, reuniens thalamic nucleus; Rt, reticular nucleus. Scale bar, 500 μm. (d) tdTomato-labeled axon fibers in the basolateral amygdala (BLA). Scale bar, 500 μm. (e) Axon fibers in the lateral hypothalamus (LH). Scale bar, 500 μm. (f) Axon fibers in the ventrolateral, lateral and dorsolateral periaqueductal gray (VLPAG, LPAG and DLPAG). Scale bar, 500 μm. (g) Axon fibers in the dorsal pons. LDTg, laterodorsal tegmental nucleus. Scale bar, 500 μm. (h) Top, schematic depicting injection of AAVrg-Cre into the LH of a C57BL/6J mouse. Bottom, fluorescence in situ hybridization (FISH) for Cre in the mPFC. Scale bar, 500 μm. (i) Double FISH was performed for Cre and Slc17A7 (gene encoding vesicular glutamate transporter 1), Slc32a1 (gene encoding vesicular GABA transporter), or Npr3. Top, fluorescence images showing Cre, Slc17A7 probe, and overlay of both channels. Middle, double FISH for Cre and Slc32a1. Bottom, double FISH for Cre and Npr3. Arrowheads indicate co-labeled cells. Scale bars, 25 μm. (j) Percentages of Cre-expressing neurons in the mPFC that also express Slc17A7, Slc32a1, or Npr3. Dots, data from single mice (Slc17A7, n = 4; Slc32a1, n = 4; Npr3, n = 2 mice). Mouse brain figures in (a,h) adapted with permission from ref. 61.
