Figure 4. Quantification of non-operant measures of freely moving behavior across hunger states and AgRP neuron photostimulation protocols.

(A) Mice displayed major differences in their tendency to begin trials within the food port area, safety platform, or other areas (see schematic) across experimental conditions. Left: food-restricted mice, and fed mice receiving AgRP photostimulation beginning in the home cage (columns 1–2), were often located in the food port area prior to trial onset, but this was not the case for all other conditions (columns 3–6; see also Figure 2M–P). Right: By contrast, food-restricted mice, and fed mice receiving AgRP photostimulation beginning in the home cage (columns 1–3), were less likely to begin trials on the safety platform compared to fed mice in other conditions (columns 4–6). **p<0.01/n, *p<0.05/n; Bonferroni correction for multiple comparisons (n=6); Wilcoxon rank-sum test for independent samples. (B) Same data as in (A), represented as a probability matrix across all conditions. Top: Hierarchical clustering of the pre-cue starting probabilities revealed that the starting location of fed mice receiving AgRP neuron photostimulation prior to arena entry most closely resembles that of food-restricted mice, while fed mice receiving AgRP photostimulation within the arena more closely resembled fed mice. (C) Visual cue-evoked transitions between arena locations differed between food-restricted and fed mice. The thickness and shading of each arrow is proportional to the probability of mice making the indicated transition following visual cue onset. Food-restricted mice were very likely to move to, or remain at, the food port following food cue onset. Fed mice were most likely to transition to, or remain at the safety platform following food or shock cue onset. (D) The same data as in (C), represented as a probability matrix across all conditions. Top: Hierarchical clustering of food and shock cue-evoked transition probabilities between the food port and safety platform revealed that fed mice receiving AgRP neuron photostimulation prior to arena entry closely resembled food-restricted mice, while fed mice receiving AgRP photostimulation within the arena more closely resembled fed mice. See also Figure S3.