Supplementary Figure 1: Validation of hM3Dq expression and function in ARCPOMC neurons; generation and validation of Pomc-IRES-Cre mice.
From: A rapidly acting glutamatergic ARC→PVH satiety circuit postsynaptically regulated by α-MSH
a, Experimental schematics. b, Expression of AAV-DIO-hM3Dq-mCherry in ARCPOMC neurons in Pomc-Cre mice determined by mCherry immunolabeling (magenta). c, Representative trace of cell-attached recordings from a hM3Dq-expressing ARCPOMC neuron showing an increase in firing rate in response to CNO (10 μM). d, Expression of AAV-DIO-hM3Dq-mCherry (magenta) and Fos (green) in ARCPOMC neurons (right, overlay) in mice sacrificed 90 minutes after CNO administration. e, Effect of CNO/hM3Dq stimulation of ARCPOMC neurons on food intake. Mice from multiple litters, n = 5 animals. Paired two-tailed t-test: 24h: Saline (mean = 4.11, s.e.m. = 0.08843) versus CNO (mean = 3.354, s.e.m. = 0.2454): t(4) = 4.187, *P = 0.0138; 48h: Saline (mean = 7.89, s.e.m. = 0.2434) versus CNO (mean = 6.652, s.e.m. = 0.3109): t(4) = 6.420, **P = 0.0030. Data are presented as mean ± s.e.m. f, Schematic diagram of the Pomc-IRES-Cre allele. The IRES-Cre cassette was targeted just downstream of the stop codon in exon III of the Pomc gene. f, g, tdTomato expression in Pomc-IRES-Cre mice crossed with tdTomato reporter mice. h, tdTomato (magenta) and POMC expression (GFP) in the ARC (right, overlay). Yellow arrows indicate tdTomato-positive/POMC-negative neurons. i, Left, experimental schematic. Right, AAV-DIO-hM3Dq-mCherry expression in ARCPOMC neurons in Pomc-IRES-Cre mice as determined by mCherry immunolabeling. j, Expression of AAV-DIO-hM3Dq-mCherry (left) and Fos (middle) immunolabeling (right, overlay) in ARCPOMC neurons in Pomc-IRES-Cre mice sacrificed 90 minutes after CNO administration.
