Supplementary Figure 8: Tracing analysis identifies VGLUT2-positive afferent inputs to the ARC originating from the PVH, MHb, and Hpc regions.

(a) Western blot analysis of Cadm1 expression in total lysates from hippocampus and cortex after delivery of AAV-Cre to floxed Cadm1 mice (flox/flox) and control littermates (+/?). Total hippocampal lysates from Cadm1KO (KO) and Slc17a6-Cre, Cadm1^flox/flox (Vglut2) serve as positive controls for loss of Cadm1 expression. (b) Quantification of O₂ consumption, CO₂ production, energy expenditure, locomotor activity and RER in 9-week old floxed Cadm1 mice (flox/flox) (n=7) and control littermates (+/?) (n=6). (c) Representative confocal images of coronal sections through the brain of Slc17a6-Cre/POMC-eGFP transgenic mice stereotaxically injected with AAV2/EF1a-DIO-hChR2(H134R)-mCherry into the paraventricular hypothalamus (PVH), habenular nuclei (Hb), and hippocampus (Hpc). Nuclei have been visualized with DAPI staining. High magnification images are identified by white boxes. (d) Representative coronal brain section of Cadm1 floxed mice after stereotaxic rAAV8-CaMKIIa-mCherry-Cre injection to hypothalamic neurons in the paraventricaular hypothalamus (PVH), and ventromedial hypothalamus (VMH) regions to target Cre expression to excitatory neurons in these areas; confocal imaging of mCherry and the neuronal marker NeuN. (e) Representative western blot analysis showing AAV-Cre-mediated deletion of Cadm1 after stereotaxic injection of rAAV8-CaMKIIa-mCherry-Cre into the PVH region of the hypothalamus. (f) Quantification of locomotor activity, O₂ consumption, CO₂ production, RER, and energy expenditure from 9-week old floxed Cadm1 mice (n=9) and control littermates (n=8) after stereotaxic injection of rAAV8/CamKII-mCherry-Cre injection into the hypothalamus. (g) Glucose measurements during an ITT on 9-week floxed Cadm1 mice (n=6) and control littermates (n=6) after stereotaxic injection of rAAV8/CamKII-mCherry-Cre injection into the hypothalamus. (h) Representative confocal images of coronal sections through the brain of Slc17a6-Cre/POMC-eGFP transgenic mice stereotaxically injected with AAV2/EF1a-DIO-hChR2(H134R)-mCherry into the hippocampus (Hpc). High magnification image of the PVH is identified by dashed box. (i) Representative confocal images of coronal sections through the brain of Slc17a6-Cre/POMC-eGFP transgenic mice stereotaxically injected with AAV2/EF1a-DIO-hChR2(H134R)-mCherry into the paraventricular hypothalamus (PVH). High magnification image of the hippocampus is identified by dashed box. (j) Confocal image analysis after stereotaxic injection of AAV2/EF1a-DIO-hChR2(H134R)-mCherry to the habenular nuclei (Hb) of Slc17a6-Cre/POMC-eGFP transgenic mice. Axonal varicosities (red) and POMC-positive neurons (green) are visualized within the ARC region of the hypothalamus. Dotted white circles outline eGFP-POMC-positive cell body, used for 3D reconstruction analysis. (k) Surface rendering of Amira 3D reconstruction of eGFP-POMC-positive cell body receiving afferent inputs from the MHb. Cell is represented in red, while the synaptic input is color-coded, with the cold to warm colors spreading from 0 to 250nm distance between axonal varicosities and the soma (see color-coded horizontal bar for the distance definition). (l) Histogram shows the number of anterograde AAV-mCherry-labelled MHb axonal varicosities found within 250nm distance from the POMC cell body. (m) Double immunostaining of Cadm1 and mCherry in MHb region identify Cadm1-positive anterograde projections to the ARC region of hypothalamus. Results in panel g are presented as mean ± s.e.m. *P<0.05, and **P<0.01. Boxplots show median, lower and upper quartiles (box), and minimum and maximum (whiskers). Statistical analyses are described in the Methods and Supplementary Table 3.