AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training

Abstract

Two intermingled hypothalamic neuron populations specified by expression of agouti-related peptide (AGRP) or pro-opiomelanocortin (POMC) positively and negatively influence feeding behavior, respectively, possibly by reciprocally regulating downstream melanocortin receptors. However, the sufficiency of these neurons to control behavior and the relationship of their activity to the magnitude and dynamics of feeding are unknown. To measure this, we used channelrhodopsin-2 for cell type-specific photostimulation. Activation of only 800 AGRP neurons in mice evoked voracious feeding within minutes. The behavioral response increased with photoexcitable neuron number, photostimulation frequency and stimulus duration. Conversely, POMC neuron stimulation reduced food intake and body weight, which required melanocortin receptor signaling. However, AGRP neuron-mediated feeding was not dependent on suppressing this melanocortin pathway, indicating that AGRP neurons directly engage feeding circuits. Furthermore, feeding was evoked selectively over drinking without training or prior photostimulus exposure, which suggests that AGRP neurons serve a dedicated role coordinating this complex behavior.

Figure 1

AGRP neurons are sufficient to evoke voracious food consumption in well-fed mice. (a) For light delivery, an optical fiber was implanted ~0.8 mm above the arcuate nucleus (ARC, red). (b) Experimental design. Food intake was recorded before, during and after photostimulation. The stimulus was applied repeatedly over 1 second followed by a 3 second break. Photostimulation frequency was 20 Hz with 10 ms light pulses. (c) Photostimulation in an AGRP-ChR2 mouse evoked voracious food consumption during the stimulation period (black trace) but not in a mouse without ChR2 expression (grey trace). (d) Food intake was dependent on the number of ChR2-expressing AGRP neurons. Circles represent 1 hour food intake for individual mice (n = 22). (e) Mice without ChR2 expression in AGRP neurons (n = 11) did not show a significant difference in food intake during photostimulation, whereas in mice with greater than 800 ChR2-expressing neurons (n = 16) the increase in food intake during photostimulation was significant. Circles connected by lines represent food intake for individual mice. (f) Food intake during photostimulation was significantly increased in mice with intermediate (300–700 neurons; n = 3) or high levels of ChR2 expression (>800 neurons; n = 16) relative to mice with low or no ChR2 (0–100 neurons; n = 14). Photostimulation-induced food consumption was nearly as great as the magnitude of re-feeding (n = 9) for 1 hour after 24 hours of food deprivation. n.s., not significant; ** P < 0.01; *** P < 0.001. Error bars represent s.e.m.

Figure 2

AGRP neuron-evoked feeding is dependent on the stimulation frequency. (a) Stimulus protocol 1. Bursts of light pulses were applied for 1 s followed by a 3 s break which repeated continuously for 1 hour. Within the burst, frequency (Hz) and number (pulses) was varied between 20, 10, 2, and 20 on successive days for AGRP-ChR2 mice. (b) Dependence of food intake on stimulation frequency (stimulus protocol 1) for mice with greater than 800 ChR2-expressing neurons (n = 8). As the stimulus frequency was reduced, feeding decreased. Grey dashed trace shows example of a mouse with intermediate ChR2 expression (600 neurons, not included in sample mean) which had a similar relationship. On the final day, food intake was measured again in response to a second 20 Hz stimulation trial and was found to be similar to the consumption from the first day. Circles represent food intake for individual mice. (c) Stimulus protocol 2. Bursts of 20 light pulses were delivered every 10 s such that 20 Hz and 2.5 Hz stimulation frequencies could be applied while maintaining the same number of stimuli over the 1 hour stimulation epoch. (d) Mice that received different stimulus frequencies (20 and 2.5 Hz) but the same total number of stimuli over 1 hour (stimulus protocol 2) also showed a reduction in food intake with decreasing stimulus frequency (n = 5). n.s., not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. Error bars represent s.e.m.

Figure 3

AGRP neuron-evoked feeding is rapidly initiated by stimulus onset and terminated after its offset. (a) Latency to food intake after photostimulation onset for each mouse (circles, n = 19). (b) For AGRP-ChR2 mice with greater than 800 ChR2-expressing neurons, raster plot of food intake aligned to the first pellet shows an initial feeding bout (red ticks) and more variable subsequent bouts (black ticks). Each row is from an individual mouse. (c) The duration of the first bout during continuous photostimulation (filled bar, n = 16) is reduced when stimulation is terminated 5 min after the first pellet (AFP) is taken (empty bar, n = 5). (d) Food intake for the first bout and over a 60 min period for mice continuously stimulated for 1 hour (filled circles) and mice in which stimulation is terminated 5 min AFP (empty circles). n.s., not significant; * P < 0.05; ** P < 0.01. Error bars represent s.e.m.

Figure 4

POMC neurons inhibit food intake and body weight through melanocortin receptors. (a,b) Photostimulation of POMC neurons (20 Hz, protocol 1 extended for 24 hours) leads to reduction of (a) food intake and (b) body weight (n = 9). (c) Pomc-cre mice without ChR2 expression in POMC neurons (n = 4) did not show a significant change in food intake over 24 hours of photostimulation. (d) In POMC-ChR2;A^y mice, hypophagia from POMC neuron activation was blocked by agouti antagonism of melanocortin signaling (n = 10). Baseline refers to average food intake or body weight for the two days prior to photostimulation. n.s., not significant; *** P < 0.001. Error bars represent s.e.m.

Figure 5

Evoked feeding does not require melanocortin suppression. Photostimulation of AGRP-ChR2;A^y mice evoked avid food intake, indicating that AGRP neurons can promote feeding independently of melanocortin receptor suppression (n = 7). n.s., not significant; *** P < 0.001. Error bars represent s.e.m.