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CNE_23127_sm_SuppFig1.tif9090KSupplementary Figure 1: (Magenta-green version of Figure 1 for the assistance of color-blind authors.) Specificity of probe binding. Tissue was treated with antisense and sense probes to control for nonspecific labeling of the probe. vGat (A), Gad65 (C), and Gad67 (E) labeling is shown in the rostral arcuate where the ventromedial hypothalamus (VMH) is apparent. The VMH contains primarily glutamatergic cells, so the lack of GABAergic markers in this nucleus is characteristic. Labeling can be seen in the arcuate nucleus and the anterior hypothalamic area (AHP). Pomc expression was present exclusively in the arcuate nucleus with robust labeling in the center of the nucleus (G). The vGlut2 probe displayed strong labeling in the VMH (I), with scattered cells throughout the arcuate nucleus. vGlut1 was not present in the arcuate nucleus. An image from cortex with strong vGlut1 labeling is shown (K). There was no labeling above background with any of the sense probes (B, D, F, H, J, L). AHP= Anterior hypothalamic area (posterior), ARC=arcuate nucleus, 3V= third ventricle, DMH=dorsal medial hypothalamus. Scale bar= 200 μm.
CNE_23127_sm_SuppFig2.tif11889KSupplementary Figure 2: (Magenta-green version of Figure 2 for the assistance of color-blind authors.) vGlut2 mRNA in POMC neurons. Example images of immunohistochemistry for POMC-EGFP (red) and fluorescent in situ hybridization for vGlut2 using a FITC-labeled probe and TSA-Biotin detection (green). Only a small portion of POMC-EGFP neurons expressed vGlut2 (A, white arrows). vGlut2 positive neurons lacking POMC-EGFP expression (A, right panel, green cells) and POMC-EGFP neurons without vGlut2 (A, right panel, red cells) are also present. Representative images of rostral (B, left panel), mid (B, center panel) and caudal (B, right panel) areas of the arcuate nucleus in coronal sections show vGlut2 present in POMC-EGFP neurons (white arrows), particularly in the rostral arcuate. 3V= Third ventricle. Scale bars= 50 μm. (C) Graph depicting the percentage of POMC-EGFP cells expressing vGlut2 throughout the rostral to caudal extent of the arcuate nucleus (n=7).
CNE_23127_sm_SuppFig3.tif17607KSupplementary Figure 3: (Magenta-green version of Figure 3 for the assistance of color-blind authors.) Gad65 and 67 mRNA in POMC neurons. Example images of POMC-EGFP detected by immunohistochemistry (A & B, red) and fluorescent in situ hybridization for Gad65 using a DIG-labeled probe and HNPP detection (A, green) or Gad67 using a FITC-labeled probe and TSA-Biotin detection (B, green). POMC-EGFP cells with (white arrows, yellow cells in right panels) and without Gad label, as well as Gad positive cells lacking POMC-EGFP (green cells in right panels) can be seen. The percentage of POMC cells expressing Gad65 (C, n=7) and Gad67 (D, n=3) throughout the arcuate nucleus is depicted in the bar graphs. Error bars represent the SEM. a= significantly different from sections 1-4; b= significantly different from section 1. 3V= Third ventricle. Scale bar= 50μm.
CNE_23127_sm_SuppFig4.tif12154KSupplementary Figure 4: (Magenta-green version of Figure 4 for the assistance of color-blind authors.) vGAT mRNA and vGAT-EGFP expression in POMC neurons. An example image of dual-in situ hybridization for Pomc using a DIG-labeled probe and HNPP detection (red) and vGat using a FITC-labeled probe and TSA-Biotin detection (green) is shown in (A). In situ hybridization for Pomc in tissue from vGAT-EGFP transgenic mice reveals that the transgene is not expressed in a notable number of POMC neurons (B). In situ hybridization for Gad67 in tissue from vGAT-EGFP mice reveals that vGAT-EGFP is expressed only in a portion of GAD67-expressing cells (C, yellow cells in right panel). 3V= Third ventricle. Scale bar= 50μm.

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