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.