Extended Data Figure 5. LDRN^MOPR-mNAcSh projection activity is negatively modulated by sucrose consumption in an opioid receptor dependent manner.

a. Schematic of photometry and voluntary sucrose consumption paradigm. b. Expression of GCaMP6s in DRN^Penk (left, scale bar = 200 μm) and fiber placement in mNAcSh (scale bar = 200 μm). c. FD increases food intake (green) relative to ad libitum intake (white), and is reduced by systemic naloxone (blue, n = 8). d. Eating microstructure across ad libitum/saline (white), food deprived/saline (green) and food deprived/naloxone (blue) test days. After food deprivation, the majority of sucrose pellets consumed during multi-pellet bouts (left), and more pellets are eaten per multi-pellet bout (right). This shift in eating behavior is blunted by naloxone. e. Example of raw 405nm and 470nm channels from photometry experiments. f. Example of raw df/f trace highlighting specific single pellet (black) or multi-pellet (green) intake events during food deprived test day. Colored boxes on top expand color matched portions of the trace below. Orange lines indicate onset of pellet consumption. g. Average Z-scored trace aligned to onset of multi-pellet consumption of LDRN^Penk-mNAcSh terminals in FD/saline condition (green) and FD/naloxone condition (blue).

h. Average Z-scored trace (dark line) and error in on the ad libitum test day. When GCaMP activity is aligned to the onset of each pellet eaten in the ad libitum condition, no significant deviation in activity is observed (black, top). When aligned to only multi-pellet bout onset, there is no deviation from baseline activity (brown, bottom). i. Schematic of regulated food intake paradigm. Two pellets were non-contingently delivered every 90-150 seconds for 30 minutes. Mice were tested in either FD/saline or FD/naloxone conditions. j. Total number of sucrose pellets eaten in the regulated intake paradigm. Mice ate significantly more pellets on the food deprivation/saline test day (green), which was reduced to baseline levels after systemic naloxone (blue) (n = 10) k. Average Z-scored trace aligned to onset of multi-pellet consumption of LDRN^Penk-mNAcSh. Food deprived/saline trace (green) shows rapid and sustained inhibition. Food deprived/naloxone trace (blue) shows blunted response. l. Heatmap of individual trials across all tested mice in food deprived/saline (green) or food deprived/naloxone test days. Orange lines indicate onset of pellet consumption. m. Quantification of the average Z-score twenty seconds prior to the onset of multi-pellet pellet bouts versus twenty seconds after the onset. FD/saline traces (green) show significant reductions in GCaMP6s activity whereas FD/naloxone traces (blue) do not (n = 10). All error bars represent ± SEM and n = biologically independent mice. Post hoc p-values are derived from Two-way ANOVA with Sidak multiple comparisons (f, i).