Supplementary Figure 4: Characterization of CRH neurons in the CeA and morphological assessment of cortical and hippocampal CRH neurons.

(a) Representative bright field photomicrographs of coronal wild-type brain sections covering the rostral to caudal extension of the CeA. Depicted are double ISHs of Crh mRNA (silver grains) together with CeA-specific markers (red staining) protein kinase C delta (Pkcδ) and somatostatin (Som). Black arrowheads indicate cells only expressing Crh. Red arrowheads indicate cells only expressing Pkcδ or Som, respectively. Double positive cells are indicated by black arrowheads enframed by a red line. In accordance with others^9,10, Crh neurons in the CeA appear to constitute a distinct GABAergic population as they show limited overlap with CeA markers Pkcδ or Som. (b) Quantification of Crh co-expression with Pkcδ at different CeA levels from bregma −0.82 to −1.82 (n = 3 mice from independent experiments). (c) Quantification of Crh co-expression with Som at different CeA levels from bregma −0.82 to −1.82 (n = 3 mice from independent experiments, 1-2 sections/mouse). (d-f) Overview of confocal images of the cortex (Ctx) and CA1 region of the hippocampus (Hip) in Crh-ires-Cre;Ai32 (d,e top) and Crh-ires-Cre;Ai9 (f, top) mice showing native ChR2-EYFP and tdTomato fluorescence (both in green) in CRH neurons. DAPI staining shown in blue. Higher power images illustrate that cortical (d, e middle) and hippocampal (f, middle) CRH neurons display bipolar and multipolar morphologies with aspiny dendrites (d-f, bottom) characteristic of GABAergic interneurons. Images in d-f are representatives from four independent experiments. Pyramidal cell layer (pyr), stratum radiatum (sr).