Nutritive, Post-ingestive Signals Are the Primary Regulators of AgRP Neuron Activity.

Further details and an outline of resources used in this work can be found in Supplemental Experimental Procedures .

Animal Strains

All protocols were conducted according to National Institutes of Health guidelines for animal research and approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. Adult male and female mice (2 to 6 months old) were used. AgRP-IRES-Cre mice were used for monitoring AgRP neuron activity, and crossed with Ai32 mice to express channelrhodopsin-2 (ChR2) for photostimulation experiments. C57BL/6J mice were used for all other experiments. Dual-wavelength Fiber Photometry Two excitation wavelengths (490 nm and 405 nm) were modulated at different frequencies to detect calcium-dependent and calcium-independent GCaMP6s fluorescence signals, respectively. The emission lights were converted to electrical signals by a photoreceiver and demodulated by a real-time processor. Photostimulation 10 ms pulses of 450 nm light were provided at 20 Hz for 1 s, repeated every 4 s. The output power at the tip of the terminal fiber was set to ensure at least 2 mW/mm 2 irradiance on AgRP neurons.

Statistical Analysis

Data are presented as means ± SEMs. Comparisons between two groups were made with paired or unpaired two-tailed t-tests using Prism. One-way, two-way, and repeated measures ANOVAs were performed in Prism to make comparisons across multiple groups. Detailed statistical analyses are summarized in Table S1 .

Supplementary Material 1 2 3 Excel Table S1. Summary of Detailed Statistical Analyses.