Hunger or thirst state uncertainty is resolved by outcome evaluation in medial prefrontal cortex to guide decision-making

Abstract

Physiological need states direct decision-making toward re-establishing homeostasis. Using a two-alternative forced choice task for mice that models elements of human decisions, we found that varying hunger and thirst states caused need-inappropriate choices, such as food seeking when thirsty. These results show limits on interoceptive knowledge of hunger and thirst states to guide decision-making. Instead, need states were identified after food and water consumption by outcome evaluation, which depended on the medial prefrontal cortex.

Extended Data Fig. 1. Recordings of consummatory responses to hydrated food and water.

a, Number of mPFC and M2 neurons recorded in hunger and thirst. b, Proportion of response types from all recorded mPFC neurons (1180). Need-state-appropriate and need-state-inappropriate selective neurons comprise 64% of recorded mPFC neurons. Other: neurons that respond to hydrated food and water but are not selective. c-d, Population mean firing rates of all recorded neurons (c) and only M2 neurons (d) that prefer hydrated food (left) or water (right) in hunger (upper row) or thirst (lower row). Responses are aligned to spout extension (dashed line). e-f, Mean firing rate of mPFC neurons (e) and all recorded neurons (f) for food-selective (left) and water-selective (right) neurons during hunger (top) and thirst (bottom) in trials with lower and higher lick rates (darker and lighter colors, respectively). Trials were sorted by number of licks and split into two equal portions (see Methods). Mean of licks in each subgroup of trials is shown in the insets. The mean firing rate for each trial-subgroup in hunger and thirst is shown for food- and water-preferring neurons. The response magnitudes and the response differences between food- and water-preferring neurons are similar in subgroups of trials with lower or higher numbers of licks, consistent with prior reports [ref: Takenouchi, K. et al. Emotional and behavioral correlates of the anterior cingulate cortex during associative learning in rats. Neuroscience 93, 1271–1287 (1999)]. g, Decoding accuracy of neural responses to food and water in hunger and thirst using all recorded neurons. Thick lines represent mean. Error bars represent SEM.

Extended Data Fig. 2. Characterization of prefrontal cortex silencing by optogenetic activation of inhibitory interneurons.

a, Experimental timeline of consumption and optogenetic silencing during pre-consumption period (red) or consumption period (orange). Grey bar indicates consumption window. ITI: inter-trial interval. b, Proportion of mPFC neurons activated (yellow), unmodulated (grey), or inhibited (yellow or red) by optogenetic stimulation of VGAT neurons during pre-consumption (red, upper panel) or consumption period (orange, lower panel) in hunger (left panel) or thirst (right panel) c, Firing rate of example mPFC neurons in hunger (upper panel) and thirst (lower panel) without VGAT neuron photostimulation (photoinhibition) (left, aligned to spout in), photoinhibition during pre-consumption period (middle, aligned to laser onset) and consumption period (right, aligned to laser onset). Grey bar indicates consumption period. d, Population responses in inhibited mPFC neurons to photoinhibition during the pre-consumption period (left, aligned to spout in) or consumption period (right, aligned to first lick) in hunger (upper panel) and thirst (lower panel). Responses are shown for preferred and non-preferred outcome with and without stimulation. Insets expand initial VGAT neuron photostimulation period, scale bar: 0.2 s. e, Mean firing rate for mPFC neurons activated by optogenetic VGAT neuron stimulation during pre-consumption period (left, aligned to spout in) and consumption period (right, aligned to first lick). Insets expand the end of the VGAT neuron photostimulation period aligned to stimulation offset (dashed line), scale bar: 0.2 s. f, Mean firing rate for all recorded neurons with VGAT neuron photostimulation during pre-consumption period (upper panel, aligned to spout in) and consumption period (lower panel, aligned to first lick) for activated and inhibited neurons. Insets expand the end of the VGAT neuron photostimulation period aligned to stimulation offset (dashed line), scale bar: 0.2 s. Grey bar indicates consumption period. g, For successive optogenetic inhibition trials, no effect of cumulative optogenetic inhibition on firing rate for inhibited neurons during pre-consumption (left) and consumption (right) period for laser trials (see Methods). h, In freely moving mice, lick-triggered consumption of food and water without (grey) or paired with lick-triggered mPFC silencing (red) in hunger (n=12) and thirst (n=9). Thick lines represent mean. Error bars represent SEM. ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Extended Data Fig. 3. Behavioral apparatus and training.

a, Diagram of behavioral apparatus. b, (Left) Individual sessions (green: hunger, blue: thirst) required to reach training criteria for lever presses in constant need state (grey shading) and alternating need state (brown shading). (Right) Mean training sessions in constant need state (grey, n=9 mice in hunger, n=9 mice in thirst) and after need state was switched (brown, n=27 mice). Box: IQR, red horizontal lines: median, whiskers: closest data points>1.5*IQR. c, Total lever presses for water (blue) and food (green) in constant need state (n = 7). d, Breakpoint ratio in hunger (green) and thirst (blue) for constant need state animals. e, Examples of training performance of need state switching animals with low (top) or high (bottom) food bias in initial training. Testing after each need state switch was separated by 3–4 days. Sessions in the same need state were either consecutive or every other day. Thick lines represent mean. Error bars represent SEM. ***P<0.001; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Extended Data Fig. 4. mPFC is not required for constant need state decision-making.

a-c, Effect of mPFC silencing during pre-choice phase in constant need state animals on error rate and preference index (a), reaction time of correct presses (b) and lick count (c) in hunger and thirst with (red) or without (grey) mPFC silencing. d-f, Effect of mPFC silencing during the outcome evaluation period in constant need state animals on error rate and preference index (d), reaction time of correct presses (e) and lick count (f) in hunger and thirst with (orange) or without (grey) mPFC silencing (regular n = 13 mice, stimulation n = 9 mice each during hunger and thirst). g, Anatomical location of optical fiber placement for constant need state animals. Gray circles indicate tip of optical fiber. Cg1: cingulate cortex area 1, Cg2: cingulate cortex area 2, DP: dorsal peduncular cortex, D3V: dorsal 3rd ventricle, fmi: forceps minor of the corpus callosum, IL: infralimbic cortex, M1: primary motor cortex, M2: secondary motor cortex, MO: medial orbital cortex, PrL: prelimbic cortex, VO: ventral orbital cortex. Thick lines represent mean. Error bars represent SEM. ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Extended Data Fig. 5. Breakpoint analysis for food and water reward in hunger and thirst.

a, Cumulative presses for food (green) and water (blue) for one example animal throughout one breakpoint session in hunger (left) and thirst (right) b, Total lever presses for water (blue) and food (green) in a group of mice switching between hunger and thirst (n = 7). c, Breakpoint ratio in hunger (green) and thirst (blue) for switching need state animals. d, Post-reinforcer pausing in seconds as a measure of motivation during the early, middle (mid), or late part of the breakpoint session for switching animals. e, Total lever presses for water (blue) and food (green) in switching and constant need state conditions with pre-exposure to food and water in home cage. f, Breakpoint ratio in hunger (green) and thirst (blue) for switching and constant need state animals with pre-exposure to food and water in home cage (n = 7 mice for each group in hunger and thirst). g, Breakpoint ratio for switching and constant animals in regular or pre-exposure conditions separately for hunger (left) and thirst (right). Thick lines represent mean. Error bars represent SEM. *P<0.05; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Extended Data Fig. 6. Food bias and learning rates for decision-making in thirst and hunger.

a, Error rates for decisions in hunger and thirst with need state switching (n = 27) for mice with intermediate experience on the task under need state-switching conditions as well as for mice during constant need state (n = 18). b, Error rates during early decision-making sessions under need state-switching conditions just after the task had been learned (left) and after extensive experience with task in late sessions (right) (early n = 27 mice, late n = 22 mice). c, Transition trial in hunger and thirst when correct performance exceeded 80% correct during early, intermediate, and late sessions shows lag in correct performance in thirst due to initial food-seeking bias. Boxplot’s central mark indicates the median, bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers, and the outliers are plotted individually using the ‘+’ symbol. d, Example of Weibull fit to correct responses for one session in hunger (left) and thirst (right). e, Cumulative density function for the three Weibull fit parameters (n = 27 mice). (Left) offset a (*P<0.05), (middle) onset Latency L, (right) shape/steepness of function S. f, Comparison of initial food bias during regular switching (green, n=22) and after mice were held in constant thirst and switched back to hunger (black, n=7) matched for equal number of training sessions (see Methods for more details). g, To test if mice determine which lever gives the greatest reward for their current need in each session, we reversed the lever contingencies (‘lever reversal’, n = 14). In both hunger and thirst, the initial performance (first block of 10 trials) was opposite for the reversed contingencies with mice pressing the lever previously associated with the need-appropriate outcome. Boxplot’s central mark indicates the median, bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers, and the outliers are plotted individually using the ‘+’ symbol. Error bars represent SEM. *P<0.05; **P<0.01; ***P<0.001; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Extended Data Fig. 7. Body weight and decision-performance.

Mean (n = 17 mice) preference index (black) and body weight (red) over multiple behavioral sessions switching animals between food restriction (hunger, green shading) and water restriction (thirst, blue shading). Error bars represent SEM.

Extended Data Fig. 8. mPFC silencing during pre-choice and outcome evaluation

a, Error rates (left) and reaction times (right) in the pre-choice stimulation period (n = 17 mice). b, Error rates (left) and reaction times (right) in the outcome evaluation period (n = 17 mice). c, Conditioned place preference test (n=12). Black horizontal lines: median, box: interquartile (25^th-75^th percentile) range (IQR), whiskers: closest data points>1.5*IQR. d, Venn Diagram showing animals affected by mPFC silencing during the pre-choice (n = 13 mice) and outcome evaluation period (n = 12 mice), with one animal being unaffected in both periods. Mice affected by optogenetic stimulation (responders) showed an effect on performance greater than 2 standard deviations above or below the mean error rate of 3 independent non-stimulation sessions. The same mice were used in both hunger and thirst conditions and thus differences in sensitivity to mPFC silencing in hunger and thirst are measured across the same animals. e, Number of animals showing an effect in pre-choice and outcome evaluation stimulation depending on unilateral vs. bilateral fiber implant. f, Anatomical location of non-responding animals during outcome evaluation stimulation. Thick lines represent mean. Error bars represent SEM. *P<0.05; **P<0.01; ***P<0.001; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Extended Data Fig. 9. Laser stimulation control experiments using C57BL/6J mice with bilateral fiber placement.

a, Within-session learning curve in hunger (green) and thirst (blue) during need state switching with no laser stimulation (n=16). b, Within-session learning curve in hunger (green) and thirst (blue) during laser stimulation on all trials during the outcome evaluation phase. c, Within-session learning curve in hunger (green) and thirst (blue) during laser stimulation during trials 25–50 and 75–100 during the pre-choice phase. d, Preference Index in hunger and thirst in session with no laser stimulation (grey), with laser stimulation during the outcome evaluation phase (orange), and with laser stimulation during pre-choice phase (red). e, Error rate in hunger and thirst in session with no laser stimulation (grey), with laser stimulation during the outcome evaluation phase (orange), and with laser stimulation during pre-choice phase (red). f, Comparison of reaction times in all three conditions in hunger and thirst for correct trials (left) and error trials (right). g, Bilateral fiber placement of C57BL/6J mice (n = 16). Thick lines represent mean. Error bars represent SEM. *P<0.05; **P<0.01; ***P<0.001; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Extended Data Fig. 10. mPFC controls evaluative decision-making in both hunger and thirst.

a, Reaction time and licks per trial in thirst during regular need state switching (blue) and with mPFC silencing during outcome evaluation after holding thirst state constant for several sessions (orange) (n = 6 mice). b, Reaction time and licks per trial in hunger after being in constant thirst for several sessions without (green) and with mPFC silencing during outcome evaluation (orange) (n = 7 mice). Thick lines represent mean. Error bars represent SEM. *P<0.05; **P<0.01; ***P<0.001; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Figure 1:. Robust discrimination between hydrated food and water.

a, Ad libitum consumption of different gelled food formulations in hunger and thirst (n=5). Best contrast: Formulation-3. b, Lick-triggered (FR-10 licks) food and water delivery preference test showing average licks/trial in hunger (n = 9) and thirst (n = 9) during constant need states. c, Coronal section of mPFC from Vgat::ChR2-EYFP mouse showing placement of Neuropixel probes (colors indicate different animals, n = 5 mice). M2: secondary motor cortex, Cg1: cingulate cortex area 1, PrL: prelimbic cortex, IL: infralimbic cortex. Scale bar, 1 mm. d, Proportion of food-selective and water-selective mPFC neurons in hunger and thirst. Error bars: SEM from Bernoulli distribution. e, Example mPFC neurons that prefer food or water in hunger (upper panels) and thirst (lower panels). Top, neuronal response spike-rasters for water (blue) and food (green) trials. Bottom, trial-averaged spike rate for food and water trials. Dashed lines: consumption period starting at first lick (5 s). Only trials with >1 lick included. f, Population mean firing rates of neurons that prefer food (left) or water (right) in hunger (top) or thirst (bottom). Responses aligned to spout extension (dashed line). g, Mean firing rate increase (relative to pre-consumption baseline) during consumption window for the preferred outcome for food- and water-selective neurons in hunger and thirst. h, Decoding accuracy of mPFC neural responses to food and water in hunger and thirst. i, Baseline firing rate for food- and water-selective neurons in hunger and thirst. Thick lines represent mean. Error bars represent SEM. *P<0.05; **P<0.01; ***P<0.001; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Figure 2:. Decision-making about hunger or thirst.

a, Schematic of behavioral task and timing for optogenetic silencing. b, Preference index (left) and error rates (right) of lever press choices in constant need states (n=9 mice in each). c, Preference index during mPFC silencing in constant need state (regular n=13, stimulation during pre-choice and outcome evaluation n=9, for both hunger and thirst). d, Behavioral performance for one animal in hunger (left) and thirst (right), including lick raster. e, Decision-making performance of need state switching animals (n=27) in hunger and thirst. f, Learning curves in switching (n=27) or constant (n=18) need state conditions. g, Preference index (left) and error rate (right) for need state switching animals. h,i, Reaction time in hunger and thirst (h) and for correct or incorrect responses (i) (n=27). j, Preference index during the first 10 trials in hunger and thirst (n=27). Box: Interquartile range (IQR), red horizontal lines: median, whiskers: closest data points>1.5*IQR. k, Maximum consecutive error bouts in hunger (green) and thirst (blue) in first 10 trials for early (n=27), intermediate (n=27) or late sessions (n=22). l, Development of the food bias (first 10 trials) during early (n=27), intermediate (n=27) or late sessions (n=22) in comparison with the preference index for constant animals (n=18). m, Initial food bias (first 10 trials) during regular switching (green, n=27) and after mice were held in constant thirst and switched back to hunger (black, n=7) (see Methods). n, Preference index of regular sessions and sessions with pre-exposure to water and food in thirst (left, n=7) or hunger (right, n=6). Thick lines represent mean. Error bars represent SEM. ***P<0.001; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.

Figure 3. mPFC is required for evaluating action-outcome relationships to inform decision-making when need states are uncertain.

a, Example session performance in thirst with pre-choice mPFC inhibition. b, Mean performance in hunger and thirst for all animals during three separate sessions: before, during and after pre-choice mPFC inhibition animals (n = 17). c, Preference index comparison between pre-choice mPFC inhibition (red) and regular responses (grey) in hunger and thirst in the same trials (trial 25–50 and 75–100). d, Effect of mPFC inhibition during pre-choice period on licks per trial. e, Example session in thirst with mPFC silencing during outcome evaluation period. f, Mean performance in hunger and thirst during three separate sessions: before, during and after outcome evaluation mPFC inhibition (n = 17, Supplementary Fig. 1 for individual subject data). g, Preference index comparison between outcome evaluation mPFC inhibition (orange) and regular responses (grey) in hunger and thirst during the whole session (all 100 trials). h, Effect of mPFC inhibition during outcome evaluation period on licks per trial. i, Anatomical location of optical fiber placement. Colored sites are locations that elicited a deviation from the mean of regular performance by at least 2 standard deviations with inhibition during the pre-choice period (red) and outcome evaluation period (orange). Cg1: cingulate cortex area 1, Cg2: cingulate cortex area 2, DP: dorsal peduncular cortex, D3V: dorsal 3rd ventricle, fmi: forceps minor of the corpus callosum, IL: infralimbic cortex, M1: primary motor cortex, M2: secondary motor cortex, MO: medial orbital cortex, PrL: prelimbic cortex, VO: ventral orbital cortex. j, Example lever press performance for one animal during need state switching with mPFC silencing during outcome evaluation in hunger and thirst before and after constant thirst state. k, Preference index in thirst during regular need state switching (blue) and with mPFC silencing during outcome evaluation after holding thirst state constant for several sessions (orange) (n = 6). l, Preference index in hunger after being in constant thirst for several sessions without (green) and with mPFC silencing during outcome evaluation (orange) (n = 7). Thick lines represent mean. Error bars represent SEM. *P<0.05; **P<0.01; ***P<0.001; ns, P>0.05. Detailed information about the exact test statistics, sidedness, and values are provided in Supplementary Table 1.