DOI: 10.5061/dryad.0p2ngf208
title: Cold-induced hyperphagia requires AgRP-neuron activation in mice

description: To maintain energy homeostasis during cold exposure, the increased energy
 demands of thermogenesis must be counterbalanced by increased energy
 intake. To investigate the neurobiological mechanisms underlying this
 cold-induced hyperphagia, we asked whether agouti-related peptide (AgRP)
 neurons are activated when animals are placed in a cold environment and,
 if so, whether this response is required for the associated hyperphagia.
 We report that AgRP-neuron activation occurs rapidly upon acute cold
 exposure, as do increases of both energy expenditure and energy intake,
 suggesting the mere perception of cold is sufficient to engage each of
 these responses. We further report that silencing of AgRP neurons
 selectively blocks the effect of cold exposure to increase food intake but
 has no effect on energy expenditure. Together, these findings establish a
 physiologically important role for AgRP neurons in the hyperphagic
 response to cold exposure.
description: Mice expressing GCaMP6s in
 AgRP neurons were connected to a fiber-photometry system to enable
 fluorometric analysis of real-time neuronal activity. Briefly, for calcium
 recording<i> in vivo</i>, two excitation wavelengths (470 nm
 and 405 nm isosbestic) were used to indicate calcium-dependent and
 calcium-independent (i.e., due to bleaching and motion artifacts) GCaMP6s
 fluorescence, respectively. Light was delivered via fiber-coupled LEDs
 (LED lights: M470F3 and M405FP1, LED driver: DC4104, Thorlabs, Newton, NJ)
 and modulated by a real-time amplifier (RZ5P, Tucker-Davis Technology
 (TDT), Alachua, FL) at non-divisible frequencies (331 Hz and 231 Hz,
 respectively) to prevent signal interference between the channels.
 Excitation lights were bandpass filtered (475 ± 15nm , 405 ± 5nm; iFMC4,
 Doric Lenses, Quebec, QC, Canada) and the combined excitation light
 delivered through a fiberoptic patch cord (M75L01,Thorlabs) connected to a
 rotary joint (FRJ, 0.48 NA,
 Ø400<i>μ</i>
 m core; Doric Lenses) to prevent fiberoptic
 torsion during animal movement. A final connector patch cord (MFP, 0.48
 NA, Ø400<i> μ</i>
 m core; Doric Lenses) was connected to the
 implanted fiberoptic via a ceramic mating sleeve (ADAL1, Thorlabs).
 Emitted light was collected through the same patch cord, bandpass filtered
 (525 ± 25 nm; iFMC4) and transduced to digital signals by an integrated
 photodetector head. Electrical signals were sampled at a rate of 1017.25
 Hz and demodulated by the RZ5P real-time processor. Experiments were
 controlled by Synapse software (TDT). Custom MATLAB scripts were
 developed for analyzing fiber photometry data. The isosbestic 405-nm
 excitation control signal was subtracted from the 470-nm excitation signal
 to remove movement artifacts from intracellular calcium dependent GCaMP6s
 fluorescence. Baseline drift was evident in the signal due to slow
 photobleaching artifacts, particularly during the first several minutes of
 each recording session. A double exponential curve was fit to the raw
 trace of temperature-ramping experiments while a linear fit was applied to
 the raw trace of food presentation experiments and subtracted to correct
 for baseline drift. After baseline correction, dF/F (%) was calculated as
 individual fluorescence intensity measurements relative to median
 fluorescence of entire session for 470nm channel. Averaged dF/F (%) for
 each temperature (30 or 14°C) were limited to the 10-min period either
 before or after the 1-min ramp either between the 14°C to 30°C transition
 or the 30°C to 14°C transition.
description: Data is provided in .mat format.