Negative feedback control of hypothalamic feeding circuits by the taste of food

Abstract

The rewarding taste of food is critical for motivating animals to eat, but whether taste has a parallel function in promoting meal termination is not well understood. Here, we show that hunger-promoting agouti-related peptide (AgRP) neurons are rapidly inhibited during each bout of ingestion by a signal linked to the taste of food. Blocking these transient dips in activity via closed-loop optogenetic stimulation increases food intake by selectively delaying the onset of satiety. We show that upstream leptin-receptor-expressing neurons in the dorsomedial hypothalamus (DMH^LepR) are tuned to respond to sweet or fatty tastes and exhibit time-locked activation during feeding that is the mirror image of downstream AgRP cells. These findings reveal an unexpected role for taste in the negative feedback control of ingestion. They also reveal a mechanism by which AgRP neurons, which are the primary cells that drive hunger, are able to influence the moment-by-moment dynamics of food consumption.

Keywords: arcuate nucleus; feeding; gustatory; hunger; hypothalamus; leptin receptor; microendoscopy; photometry; taste.

Figure 1 |. AgRP neurons track the taste of food

A, Experimental Setup. B, Example trace of AgRP activity aligned to licks. C, AgRP activity in fasted or fed mice drinking ensure. D, (left) PSTH of AgRP activity aligned to the first lick. (right) Minimum z-score in bout. E, Same as d but aligned to the last lick. F, Same as d but for the first or last 10 minutes of trial. G, Example whole-trial and zoomed-in traces of AgRP activity during licking. H-J, PSTH of AgRP activity aligned to the first lick. K, Minimum z-score during licking bouts of the solutions in h-j. L, Correlation coefficient between AgRP activity and true or shuffled lick data. M,N, Z-scored AgRP activity during consumption of sweet (m) or fatty (n) solutions. O, Mean z-score over ten minutes of consumption. P, Schematic illustrating how AgRP neurons differentially respond to food access and food ingestion. MDG = alpha-methyl-d-glucopyranoside. N.S. p>0.05, *p<0.05, **p<0.01 for direct comparisons; compared to water (k). #p<0.05, ##p<0.01, ###p<0.001 relative to 0 (k). #p<0.05 relative to empty (o). Dots represent individual mice. Data is presented as mean ± SEM. See also Figure S1.

Figure 2 |. Ingestion-triggered dips in AgRP neuron activity control meal duration

A, Schematic and table of stimulation protocols. B, Average licks per 2-min trial for ChR2 and control mice. C, Cumulative distribution function (CDF) of no laser trials preceded by either a closed-loop or no laser trial. D, Total licks for three different protocols. E, Total bouts for the interleaved closed-loop protocol shown in B (left) and tonic stimulation. F, Same as E but for bout length. G, Example feeding behavior during a closed-loop session, collapsed by trial type. H, Total bouts per block across all mice for closed-loop or no stimulation (left). Quantification of total bouts for first and second half of the session (right). I, Schematic of conditioned flavor preference protocol. J, Training data for paired and unpaired flavors. K, Preference for flavor (licks for paired flavor divided by total licks) before and after training. N.S. p>0.05, *p<0.05, **p<0.01. Dots represent individual mice. Data is presented as mean ± SEM.

Figure 3 |. DMH^LepR neurons are activated time-locked to ingestion

A, Inhibitory circuit schematic from DMH to ARC. B, Schematic and example of lens placement above GCaMP-expressing DMH^LepR neurons. Example field of view color-coded to responses during consumption. C, Schematic of single-cell calcium imaging during consumption. D, Heatmap of DMH^LepR responses (N=4–5) during consumption while fasted. E, Averaged traces of categories in D. F, Mean z-score of individual neurons over first ten minutes. G, Percentage of each category per mouse. H,I, Example T1 averaged trace during licking of Ensure (H) or water (I). Licks in gray. J,K, PSTH of T1 activity aligned the first or last lick of Ensure (J) or water (K). L, Peak z-score of individual neurons during licking. M, Correlation coefficient for neural activity against licks. N, Same as D but for chow. O, Same as E but for chow. P,Q, Same as L,M but for chow and object. R, Generation of pseudo-photometry trace during chow consumption. DMH = dorsomedial hypothalamus. ARC = arcuate nucleus. T1 = Type 1, T2 = Type 2, T3 = Type 3, NR = no response. N.S. p>0.05, *p<0.05, **p<0.01, ****p<0.0001. Dots represent individual mice unless otherwise noted. Data is presented as mean ± SEM. See also Figures S2 and S3.

Figure 4 |. DMH^LepR neurons are activated by the taste of food

A, Davis Rig used for brief access taste tests during imaging of DMH^LepR neurons. B, Example traces of neurons preferring sucrose, sucralose, or both, and corresponding quantification. C, Mean z-score of each neuron during sucrose ingestion plotted against either sucralose (red) or water (gray). P-value indicates significance relative to a slope of 0. D, Example responses to sucrose concentrations. E, Mean responses of activated neurons across all concentrations of sucrose. F, Peak z-score across sucrose concentrations. G, Licks per presentation. H, non-linear regression between peak z-score and licks. P-value indicates significance of fit relative to a linear regression. I-L, Same as D-H but for sucralose. N, Schematic of setup for taste panel experiment. O, K-means clustering of Ensure-activated neurons across all solutions, presented as one heatmap per cluster. P, Average traces for each cluster in O, aligned to solution access. Q, Noise-to-signal ratio R, entropy, and S, noise-to-signal ratio versus entropy plot of Ensure-activated neurons, colored by their preferred taste. Data is reported as the mean ±SEM. See also Figure S4.

Figure 5 |. Inhibiting DMH^LepR neurons increases food intake

A, Schematic and example of hM4Di expression in DMH^LepR neurons. B, Heatmap of hM4Di or mCherry expression across mice. C, Schematic of experimental design. D, Cumulative licks throughout the session. E, Total licks during Ensure access. F, Bout number during Ensure access. G, Bout length in licks during Ensure access. H-K, Same as D-G but during the start of the dark cycle. N.S. p>0.05, *p<0.05, **p<0.01, ***p<0.001 Data is presented as mean ± SEM. See also Figure S5.

Figure 6 |. Nutrients potentiate DMH^LepR neuron responses to gustatory signals

A, Schematic of intragastric (IG) infusion setup during calcium imaging. B, Heatmap of DMH^LepR neurons (N=5 mice) receiving an IG infusion of Ensure. C, Same as in B but with an osmolarity-matched saline control. D, Quantification of neural response types. E, Top, averaged trace of activated neurons. Bottom, mean z-score. F, Example overlay generated using CellReg to cross-register neurons. G, Heatmaps of aligned neurons, divided based on response patterns: activated by both, activated only by IG Ensure or licking Ensure, or not activated. H, (Top) Averaged traces from the categories in G. (Bottom) Percentage of all activated neurons that fall into one of three categories. I, Heatmaps during sucrose or sucralose consumption (N=7). J, Percentage of type 1 or 2 neurons per mouse. K, Averaged trace of type 1 neurons during consumption. L, Total licks per time bin. M, PSTH around licking bouts using a local baseline (N=4). N,O, Quantification of M without (N) and with (O) normalization to licks. P, Summary model of nutrients potentiating gustatory responses over time. Dots represent individual type 1 neurons. N.S. p>0.05, *p<0.05, **p<0.01, ****p<0.0001 Data is presented as mean ± SEM. See also Figure S6.