A subcortical switchboard for perseverative, exploratory and disengaged states

Abstract

To survive in dynamic environments with uncertain resources, animals must adapt their behaviour flexibly, choosing strategies such as persevering with a current choice, exploring alternatives or disengaging altogether. Previous studies have mainly investigated how forebrain regions represent choice costs and values as well as optimal strategies during such decisions^1-5. However, the neural mechanisms by which the brain implements alternative behavioural strategies such as persevering, exploring or disengaging remain poorly understood. Here we identify a neural hub that is critical for flexible switching between behavioural strategies, the median raphe nucleus (MRN). Using cell-type-specific optogenetic manipulations, fibre photometry and circuit tracing in mice performing diverse instinctive and learnt behaviours, we found that the main cell types of the MRN-GABAergic (γ-aminobutyric acid-expressing), glutamatergic (VGluT2⁺) and serotonergic neurons-have complementary functions and regulate perseverance, exploration and disengagement, respectively. Suppression of MRN GABAergic neurons-for instance, through inhibitory input from lateral hypothalamus, which conveys strong positive valence to the MRN-leads to perseverative behaviour. By contrast, activation of MRN VGluT2⁺ neurons drives exploration. Activity of serotonergic MRN neurons is necessary for general task engagement. Input from the lateral habenula that conveys negative valence suppresses serotonergic MRN neurons, leading to disengagement. These findings establish the MRN as a central behavioural switchboard that is uniquely positioned to flexibly control behavioural strategies. These circuits thus may also have an important role in the aetiology of major mental pathologies such as depressive or obsessive-compulsive disorders.

Fig. 1. VGAT-expressing MRN neurons control perseverative state.

a, Schematic of the MNOI test (left) and an example sequence of annotated actions (top right), with three interaction states extracted by a HMM (bottom right). b, Left, average duration of interaction with each object plotted against the frequency of interactions with different objects in each HMM state in control mice (median ± bootstrap standard error, n = 20 experiments from 10 mice). Right, median fraction of time spent in each state. c, Left, schematic of optogenetically activating or suppressing VGAT⁺ MRN neurons. Right, example image of virus expression in VGAT⁺ MRN neurons with optic fibre position. PAG, periaqueductal grey; PnO, pontine reticular formation; RtTg, reticulotegmental nucleus. Scale bar, 0.5 mm. d, Median fraction of time spent in the perseverative (blue), exploratory (green) and disengaged state (orange) during the MNOI test in mice with activation (Act; left) or suppression (Supp; right) of VGAT⁺ MRN neurons. e, Fraction of time spent in each interaction state in tdTomato control mice (ctrl) and mice shown in d with activation or suppression of VGAT⁺ MRN neurons. Bars indicate median values, error bars are bootstrapped standard error and circles represent individual experimental sessions. n = 20, 11 and 10 experiments from 10, 6 and 5 mice in tdTomato, VGAT⁺ neuron activation and VGAT⁺ suppression groups, respectively. f, Schematic of calcium fibre photometry recording from VGAT⁺ MRN neurons in mice exposed to multiple novel objects. g, Heat map of z-scored calcium activity traces of an example experiment (top) and average z-scored calcium activity trace (bottom; mean ± s.e.m., averaged over all events from 5 mice (n = 66 and 193 deep interaction and switch events)) of VGAT⁺ MRN neurons during object interactions aligned either to the onset of deep interactions (left) or the time of switching between objects (right; early example traces are from switching objects following a deep interaction, later example are traces during switching between objects without deep interactions). h, Median z-scored calcium activity (median ± bootstrap standard error) of VGAT⁺ MRN neurons during disengaged states (n = 190 events from 5 mice), exploratory states (n = 139 events from 5 mice) and perseverative states (n = 51 events from 5 mice). i, Schematic of experimental design to quantify levels of exploration during a T-maze test. During the test, in each trial photo-stimulation is applied throughout the central arm until mice turn into the left or right arm. j, Percentage of trials with entrance into the non-rewarded arm (median ± bootstrap standard error), indicating exploratory behaviour in tdTomato control mice (n = 5 mice) and mice with optogenetic activation of VGAT⁺ MRN neurons (green, 5 mice) trained in the same task. k, Schematic of experimental design to quantify levels of perseverative behaviour during a reversal test in the T-maze. l, Percentage of trials with entrance into the previously rewarded (but now non-rewarded) arm during the reversal T-maze test (median ± bootstrap standard error) in tdTomato control mice (n = 8 mice) compared with mice with optogenetic suppression of VGAT MRN neurons (blue, 5 mice). *P < 0.05; **P < 0.01; ***P < 0.001. See Extended Data Table 1 for statistics. Source data

Fig. 2. VGluT2-expressing MRN neurons drive exploratory behaviour.

a, Left, schematic of optogenetic activation or suppression of VGluT2⁺ MRN neurons. Right, example image of virus expression in VGluT2⁺ MRN neurons with optic fibre positions. Scale bar, 0.5 mm. b,c, Median fraction of time spent in the perseverative (blue), exploratory (green) and disengaged state (orange) during the MNOI test in mice with activation (b, left) or suppression (b, right) of VGluT2⁺ MRN neurons and tdTomato control mice (c). n = 20, 10 and 11 experiments from 10, 5 and 5 mice in tdTomato, VGluT2⁺ excitation and VGluT2⁺ inhibition groups, respectively. d, Schematic of calcium fibre photometry recording from VGluT2⁺ MRN neurons in mice exposed to multiple novel objects. e, Heat map of z-scored calcium activity traces in an example experiment (top) and average z-scored calcium activity trace (bottom; mean ± s.e.m., all events from 5 mice (n = 17 deep interaction and 67 switch events)) of VGluT2⁺ MRN neurons aligned either to the onset of deep object interactions (left) or the time of switching between objects (right). f, z-scored calcium activity of VGluT2⁺ MRN neurons (median ± bootstrap standard error) during disengaged, exploratory and perseverative states. n = 72 disengaged, 53 exploratory and 13 perseverative state events from 3 mice. g, Experimental design to quantify levels of exploration during a T-maze test. h, Percentage of trials with entrance into the non-rewarded arm during the T-maze test, in tdTomato control mice (n = 5 mice) and mice with activation of VGluT2⁺ MRN neurons (left; 5 mice). i, Experimental design to quantify perseverance during a reversal test in the T-maze. j, Percentage of trials with entrance into the previously rewarded arm in tdTomato control mice (n = 8 mice) and mice with suppression of VGluT2⁺ MRN neurons (left; 5 mice). *P < 0.05; ***P < 0.001. In c,h,j, bars indicate median, error bars are bootstrapped standard error and circles represent individual experimental sessions. See Extended Data Table 1 for statistics. Source data

Fig. 3. Effect of manipulation of VGAT- and VGluT2-expressing MRN neurons on valence.

a, Schematic of the experimental design for a self-stimulation test. b, Preference for returning to the opto-linked nose-poke port (100 × (number of entries into the opto-linked nose-poke port − number of entries into the non-stimulation nose-poke port)/total number of entries into both nose-poke ports) in tdTomato control mice (n = 9) and mice with activation or suppression of VGAT⁺ (n = 5 and 6 mice) and VGluT2⁺ (n = 5 and 9 mice) MRN neurons. c, Schematic of the TMT aversion test. d, Number of approaches of the aversive TMT-coated object in control mice compared to mice with activation or suppression of VGAT⁺ and VGluT2⁺ MRN neurons. e, Escape probability after approaching a TMT-coated object in control mice (n = 9) compared with mice with VGluT2⁺ suppression (n = 13 mice), VGluT2⁺ activation (n = 11), VGAT⁺ suppression (n = 11) and VGAT⁺ activation (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001. In b,d,e, bars indicate median, error bars are bootstrapped standard error and circles represent individual experimental sessions. See Extended Data Table 1 for statistics. Source data

Fig. 4. Activity of SERT-expressing MRN neurons is necessary for task engagement.

a, Left, experimental design of optogenetic activation or suppression of serotonergic (SERT⁺) MRN neurons. Right, image of virus expression in SERT⁺ MRN neurons with optic fibre position. Scale bar, 0.5 mm. b,c, Median fraction of time spent in each interaction state during the MNOI test in mice with activation (b, left) or suppression (b, right) of SERT⁺ MRN neurons and in tdTomato control mice (c). n = 20, 13 and 20 experiments from 10, 4 and 8 mice in tdTomato, SERT+ activation and SERT+ suppression groups, respectively. d, Experimental design of a self-stimulation test. e, Preference for returning to the opto-linked nose-poke port (100 × (number of entries into the opto-linked nose-poke port − number of entries into the non-stimulation nose-poke port)/total number of entries into both nose-poke ports) in control mice (n = 9) and mice with activation (n = 6) or suppression (n = 6) of SERT⁺ MRN neurons. f, Experimental design of the nose poke–reward association task. g, Examples of task performance (including timing of nose pokes, licks and completed (compl.) trials) of a tdTomato control mouse and a mouse with suppression of SERT⁺ MRN neurons. Blue boxes indicate photo-stimulation time periods. h, Number of completed trials during photo-stimulation in tdTomato control mice and mice with suppression or activation of SERT⁺ MRN neurons. n = 14, 7 and 5 mice, respectively. i, Schematic of calcium fibre photometry recording from SERT⁺ MRN neurons in mice exposed to multiple objects. j, Example traces of z-scored calcium activity of SERT⁺ MRN neurons, aligned to the onset of object interactions, for the first (blue), second, third and fourth (grey) deep interaction with the same object. k, Average z-scored calcium activity trace (mean ± s.e.m.) during object interactions for first (top; blue; 8 trials) and following deep interactions (top; grey; 25 trials), and during brief interactions (bottom; green; 52 trials). n = 4 mice. l, Median z-scored calcium response of SERT⁺ MRN neurons during the first deep interaction (n = 8 events) compared with following deep interactions with the same object (n = 25 events) and brief interactions (n = 52 events) from 4 mice. *P < 0.05; ***P < 0.001. In c,e,h,l, bars indicate median, error bars are bootstrapped standard error and circles represent individual experimental sessions. See Extended Data Table 1 for statistics. Source data

Fig. 5. LHb input to MRN drives disengagement.

a, Retrograde tracing from MRN using retroAAV (left), and image of retrogradely labelled neurons in LHb (right). Scale bar, 0.25 mm. b, Schematic of optogenetic activation or suppression of LHb input to MRN (left), and image of ChR2-expressing LHb axons in the MRN with optic fibre positions (right). Scale bar, 0.5 mm. c, Schematic of the self-stimulation test (left), and preference for returning to the opto-linked nose-poke port (calculated as in Fig. 4e) in control mice compared with mice with suppression or activation of LHb input to the MRN (right; n = 9, 5 and 9 mice, respectively). d,e, Median fraction of time spent in the three interaction states during the MNOI test in mice with activation (d, left) and suppression (d, right) of LHb input to the MRN and in control mice (e). n = 20, 20 and 15 experiments from 10, 9 and 5 mice in control, activation and suppression groups, respectively. f, Schematic of nose-poke reward association task with activation of LHb input to the MRN (left) and examples of task performance (including timing of nose pokes, licks and completed (compl.) trials) of a control mouse and a mouse with activation of LHb input to the MRN. Light blue boxes indicate laser stimulation periods. Right, average number of completed trials during laser stimulation in tdTomato mice and mice with activation of LHb input in the MRN (n = 14 and 10 mice, respectively). g, Experimental design for recording calcium signals in MRN cell types using fibre photometry, while optogenetically activating LHb input to the MRN. h, z-Scored calcium traces (mean ± s.e.m.) aligned to laser onset, showing the effect of optogenetic activation of LHb input on the activity of VGAT⁺ (left; n = 4 mice), VGluT2⁺ (middle; n = 5 mice) and SERT+ (right; n = 4 mice) neurons in MRN. i, Top, schematic of experimental design to obtain calcium recordings from SERT⁺ MRN neurons, while activating LHb input to the MRN during the first deep interaction with objects. Bottom, z-scored calcium activity (mean ± s.e.m.) aligned to onset of first deep object interaction with and without optogenetic activation of LHb input to the MRN. n = 10 (laser off) versus 7 (laser on) events from 3 mice. ***P < 0.001. In c,e,f, bars indicate median values, error bars are bootstrapped standard error and circles represent individual experimental sessions. See Extended Data Table 1 for statistics. Source data

Fig. 6. LHA input to MRN bidirectionally controls perseverance.

a, Retrograde tracing from MRN using retroAAV (top), and image of retrogradely labelled neurons in LHA (bottom). 3V, 3rd ventricle; DM, dorsomedial hypothalamus; EP, entropeduncular nucleus; MeA, medial amygdala; Subl, subincertal nucleus; VMH, ventromedial hypothalamus. Scale bar, 0.5 mm. b, Schematic of optogenetic activation or suppression of LHA VGAT⁺ input to MRN (top) and image of LHA axons in MRN with optic fibre positions (bottom). Scale bar, 0.5 mm. c, Schematic of the self-stimulation test (left), and preference for returning to the opto-linked nose-poke port (calculated as in Fig. 4e) in control mice (n = 9) and mice with suppression or activation of LHA VGAT⁺ input to the MRN (right; n = 5 and 6 mice, respectively). d,e, Median fraction of time spent in the interaction states during the MNOI test in mice with activation (d, left) and suppression (d, right) of the LHA VGAT⁺ input to the MRN and in control mice (e). n = 20, 23 and 15 experiments from 10, 9 and 8 mice in control mice, activation and suppression groups. f, Left, experimental design to quantify levels of perseverance during a reversal test in the T-maze. Right, percentage of trials with entrance into the previously rewarded (but now non-rewarded) arm in control mice (n = 8) and mice with activation of LHA VGAT⁺ input to the MRN (8 mice). g, Left, experimental design to quantify levels of exploration during a T-maze test. Right, percentage of trials with entrance into the non-rewarded arm in control mice (n = 5) and mice with suppression of LHA VGAT⁺ input to the MRN (5 mice). h, Experimental design for recording calcium signals in MRN VGAT⁺ neurons while optogenetically activating LHA VGAT⁺ input to the MRN. i, z-scored calcium traces (mean ± s.e.m.) aligned to laser onset, showing the effect of optogenetic activation of LHA VGAT⁺ input on the activity of the VGAT⁺ MRN neurons (n = 5 mice). *P < 0.05; **P < 0.01; ***P < 0.001. In c,e–g, bars indicate median values, error bars are bootstrapped standard error and circles represent individual experimental sessions. See Extended Data Table 1 for statistics. Source data

Extended Data Fig. 1. Expression and overlap of VGAT, VGluT2 and SERT in MRN, and interaction states extracted by hidden Markov model.

a, Example image of multi-colour single molecule in-situ mRNA hybridization in MRN. DAPI is shown in blue, and SERT+, VGAT+, and VGluT2+ cells are in red, magenta, and green, respectively. White arrows indicate the location of example cells. b, Overlap between the four colours of the example slice shown in a. c, Venn diagram of MRN cells expressing SERT, VGAT and VGluT2, shown in percentages of all counted MRN neurons (N = 620 neurons from 5 slices, 3 mice). d, Schematic of experimental design to express eGFP in Cre-expressing cells in the MRN of SERT-Cre mice. e, Example of multi-colour single molecule in-situ mRNA hybridization in MRN. DAPI is shown in blue, and SERT+ and eGFP+ cells in magenta and green, respectively. White arrows indicate the location of example cells. The bottom right panel shows the overlap between the three colours. f, Venn diagram of SERT+ and SERT- MRN cells expressing eGFP, shown in percentage of counted neurons (N = 51 neurons from 4 slices, 3 mice). g, Average duration of object interactions (left, P = 2.3 × 10⁻⁶, two-sided paired t-test; N = 20 experiments from 10 mice) and frequency of object interaction (right, P = 0.0001, two-sided Wilcoxon signed rank test) in exploratory (green) and perseverative (blue) states in control mice during the MNOI test. h, Median transition probabilities at the time of switching between the three interaction states in control mice. Bar graphs show the transition probability from each state to the other two states (median values and individual experiments). From exploratory state to disengaged vs. perseverative state: P = 0.0003, two-sided Wilcoxon signed rank test; from disengaged state to exploratory vs. perseverative state: P = 0.0302, two-sided paired t-test; from perseverative state to disengaged state vs. exploratory state: P = 0.8047, two-sided paired t-test, N = 20 experiments from 10 mice). *: p-value < 0.05, ***: p-value < 0.001. Bars depict median, error bars are bootstrapped standard error and circles indicate individual experiments.

Extended Data Fig. 2. Impact of tonic and phasic optogenetic stimulation of VGAT+ MRN neurons on object interaction.

a, Schematic of experimental design: optogenetic activation or suppression of VGAT+ MRN neurons, using ChR2 or stGtACR2 (left) and example image of virus expression in VGAT+ MRN neurons with optic fibre position (right). DRN: dorsal raphe nucleus, MRN: median raphe nucleus, PAG: periaqueductal gray. b, Number of switches between objects during the MNOI test in control (ctrl) mice and mice with activation (act) and suppression (supp) of VGAT + MRN neurons (ctrl vs. act: P = 0.00591, ctrl vs. supp: P = 2.3 × 10⁻⁷, two-sided t-test with Bonferroni multi-comparison correction). N = 20, 11 and 10 experiments from 10, 6 and 5 mice in control, VGAT+ activation and VGAT+ suppression groups. c, Duration of deep interactions (when mice grab, bite or carry the object) with each object during the MNOI test of mice in b (ctrl vs. act: P = 0.0102, ctrl vs. supp: P = 1.6 × 10⁻⁶, two-sided t-test with Bonferroni multi-comparison correction). d, Schematic of the experimental design to examine the effect of phasic 2-second suppression of VGAT+ MRN neurons in the MNOI test (with 5 objects) when the animal was not interacting with an object. e, Transition probability from no object interaction within the 2-second stimulation window (in experiment in d) to brief interaction (left, tdTomato ctrl (N = 5 experiments from 5 mice) vs VGAT+ supp. (N = 7 experiments from 7 mice), P = 0.0025; two-sided Mann-Whitney U test), to deep interaction (middle, ctrl vs VGAT+ supp.: P = 4.2 × 10⁻⁵; two-sided t-test), and probability to remain not interacting with the objects (right, ctrl vs VGAT+ supp.: P = 0.0054; two-sided t-test). f, Phasic 2-second suppression of VGAT+ MRN neurons in the MNOI test when the animal’s snout is close to an object. g, Probability within the 2-second stimulation window (in experiment in f) to switch to a different object (left, ctrl (N = 5 experiments from 5 mice) vs VGAT+ supp. (N = 16 experiments from 7 mice), P = 0.0012; two-sided Mann-Whitney U test), to transition to a deep interaction (middle, ctrl vs VGAT+ supp.: P = 0.0016; two-sided Mann-Whitney U test), and to transition to no-interaction (right, ctrl vs VGAT+ supp.: P = 0.4660; two-sided Mann-Whitney U test). h, Phasic 2-second suppression of VGAT+ MRN neurons in the MNOI test during deep interaction with an object. i, Probability (in experiment in h) to switch object (left, ctrl (N = 7 experiments from 5 mice) vs VGAT+ supp. (N = 15 experiments from 7 mice), P = 0.3733; two-sided Mann-Whitney U test), to persist in deep interaction (middle, ctrl vs VGAT+ supp.: P = 0.0857; two-sided Mann-Whitney U test), and to transition to no-interaction (right, ctrl vs VGAT+ supp.: P = 0.1025; two-sided Mann-Whitney U test). j, Median duration of deep interactions after 2-second laser stimulation during deep object interactions (in experiments in h, in ctrl vs VGAT+ supp.: P = 0.0015; two-sided Mann-Whitney U test). *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001. In b, c, e, g, i and j bars depict mean, error bars are standard error and circles indicate individual experiments.

Extended Data Fig. 3. Effect of MRN cell type manipulation on motor behaviour in an open field, and on arousal and valence.

a, Linear velocity over the course of a 2-minute open field test in laser-on and laser-off conditions, averaged over mice in a tdTomato control group (ctrl; N = 8 mice), and in groups of mice with suppression of SERT+ (SERT+ supp; N = 7 mice), suppression of VGAT+ (VGAT+ supp; N = 7 mice), and activation of VGluT2+ (VGluT2+ act; N = 7 mice) MRN neurons. b, Optogenetic stimulation effect on average linear velocity over time (100 × (linear velocity during laser-on period - linear velocity during laser-off period)/linear velocity during laser-off period) in mice in a. Control mice (N = 8) are compared to SERT+ supp (N = 7 mice, P > 0.9999), VGAT+ supp (N = 7 mice, P > 0.9999) and VGluT2+ act (N = 7 mice, P > 0.9999); two-sided t-test with Bonferroni multi-comparison correction. c, Optogenetic stimulation effect on averaged angular velocity over time (100 × (angular velocity during laser-on period - angular velocity during laser-off period)/angular velocity during laser-off period) in mice in a. Control mice are compared to SERT+ supp (P > 0.9999), VGAT+ supp (P = 0.5144) and VGluT2+ act (P = 0.1110); two-sided t-test with Bonferroni multi-comparison correction. d, Optogenetic stimulation effect on time spent rearing (100 × (time spent rearing in laser-on period – time spent rearing in laser-off period)/total test duration) in mice in a. Control mice are compared to mice with SERT+ supp (P = 0.6436), VGAT+ supp (P = 0.6800) and VGluT2+ act (P = 0.1739); two-sided t-test with Bonferroni multi-comparison correction. e, Optogenetic stimulation effect on time spent grooming (100 × (time spent grooming in laser-on period – time spent grooming in laser-off period)/total test duration) in mice in a. Control mice are compared to mice with SERT+ supp (P = 0.3647), VGAT+ supp (P = 0.3885) and VGluT2+ act (P > 0.9999); two-sided t-test with Bonferroni multi-comparison correction. In b-e, bars depict mean, error bars are standard error and circles indicate individual experiments. f, Schematic of the experimental design for quantifying fear responses to an innately aversive dark looming stimulus, with three different stimulus contrast levels (10%, 50%, and 90%), with and without optogenetic activation of VgluT2+ MRN neurons. g, Heatmaps show running speed (cm/s) of an example mouse in response to 90% contrast looming stimuli in laser off and laser on trials. h, Escape probability of mice in response to looming stimuli with different contrasts, during activation of VgluT2+ MRN neurons (laser on) and control trials (laser off). N = 3 mice, 10% contrast: P = 0.1181; 50% contrast: P > 0.9999; 90% contrast: P > 0.9999, two-sided paired t-test. i, Schematic of the experimental design to examine the effects of optogenetic manipulation of VGluT2+ and VGAT+ MRN neurons on running speed and arousal level, measured using pupil size and whisker activity. j, Z-scored running speed evoked by optogenetic stimulation in control mice and mice with activation or suppression of VGluT2+ or VGAT+ MRN neurons. N = 15, 11, 9, 8 and 11 mice, respectively; P > 0.9999, P = 0.0229, P = 0.3880 and P = 0.1210 for comparing control mice to activation or suppression of VGluT2+ neurons, and activation or suppression of VGAT+ neurons, respectively; two-sided t-test with Bonferroni multi-comparison correction. k, Z-scored pupil size over time (mean ± s.e.m.) in control mice (grey) and mice with activation or suppression of VGluT2+ and VGAT+ MRN neurons, averaged over trials and aligned to onset of optogenetic manipulation. Light blue box indicates the laser stimulation period. l, Median z-score of pupil size during laser stimulation from traces in k. N = 15, 11, 10, 10 and 11 mice; P = 8.0 × 10⁻⁶, P = 6.7 × 10⁻⁵, P = 0.0015 and P = 0.0660 for comparing control mice to activation or suppression of VGluT2+ neurons, and activation or suppression of VGAT+ neurons, respectively; two-sided t-test with Bonferroni multi-comparison correction. m,n, same as k,l but for z-scored whisker activity (summation of absolute frame-by-frame differences in pixel luminance). N = 15, 11, 10, 8 and 11 mice, respectively; P = 0.0165, P = 0.0195, P = 0.0153 and P = 0.2356 for comparing control mice to activation or suppression of VGluT2+ neurons, and activation or suppression of VGAT+ neurons, respectively; two-sided t-test with Bonferroni multi-comparison correction. *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001. Bars in h, k, l and n depict median, error bars are bootstrapped standard error and circles indicate individual experiments.

Extended Data Fig. 4. Choosing the rewarded arm suppresses VGAT+ neurons and choosing the non-rewarded arm activates VGluT2+ neurons in MRN.

a, Schematic of calcium fibre photometry recordings from VGAT+ and VGluT2+ MRN neurons in mice during a T-maze test, after training. b, Schematic of the experimental design to express GCaMP6f in VGAT+ MRN neurons and implant a fibre for photometry. c,d, Left, Heatmap shows z-scored calcium activity traces of VGAT+ MRN neurons of an example mouse (left) and average z-scored calcium activity trace across mice (right, mean ± s.e.m., N = 9 mice) aligned to turns towards the non-rewarded arm (c), and aligned to turns towards the rewarded arm (d). e, Median z-scored calcium activity of VGAT+ MRN neurons during 3-second trials when the rewarded or the non-rewarded arm was chosen (N = 9 mice, P = 5.7 × 10⁻⁵, two-sided paired t-test). Error bars indicate bootstrapped standard error and individual data points are averaged z-scored activity over trials in individual mice. f, Schematic of the experimental design to express GCaMP6f in VGluT2+ MRN neurons and implant a fibre for photometry. g,h, same as c,d, but for calcium activity of VGluT2+ MRN neurons. i, Median z-scored calcium activity of VGluT2+ MRN neurons during trials in which the rewarded or the non-rewarded arm was chosen (N = 6 mice, P = 0.0266, two-sided paired t-test). Data point and error bars indicate median ± bootstrapped standard error. *: p-value < 0.05, ***: p-value < 0.001.

Extended Data Fig. 5. VGAT+ and VGluT2+ MRN neuron manipulation causes exploitation and exploration in multi-choice tasks.

a, Left, Schematic of the 3-armed bandit task with lick-ports P1 to 3 to examine the effects of optogenetic suppression of VGAT + MRN neurons. Reward probability at P3 is constant at 50%, and reward probabilities at P1 and P2 fluctuate between 25% and 75% in blocks of changing lengths. b, Schematic of experimental design for optogenetic suppression of VGAT+ MRN neurons. c, Behaviour of an example VGAT-Cre mouse in a control session with no laser stimulation. X-axis shows trials and the orange and green colours indicate the blocks where P2 and P1 have the high-reward probability (0.75), respectively. Y-axis shows the chosen lick-ports and if reward was delivered (green circle) or not (red cross). d, Behaviour of an example mouse following suppression of VGAT+ MRN neurons during exploitation of a high-reward port (P1). Light blue bar indicates the laser stimulation period. e, Suppression of VGAT+ MRN neurons (blue shading indicates laser-on period) after the first occurrence of choosing the high-probability arm for 3 sequential trials in a block) decreased the animals’ exploration probability (probability of switching to one of the other two ports) during the laser-on period (blue circles), compared to control trials without laser under the same condition (black circles). N = 6 mice, P = 3.2 × 10⁻⁴, two-way repeated measures ANOVA (two-sided). Light blue box indicates the laser stimulation period. Data points and error bars show median ± bootstrapped standard error. f, Schematic of the reward probabilities in the 3-armed bandit task to examine the effects of optogenetic manipulation of VGluT2+ MRN neurons on exploration. reward probability at P3 is constant at 50%, and reward probabilities at P1 and P2 fluctuate between 10% and 90% in blocks of changing lengths. g, Schematic of experimental design for optogenetic activation of VGluT2+ MRN neurons. h, The same as c but for an example VGluT2-Cre mouse in a control session with reward probabilities as shown in f without optogenetic stimulation. i, Left, Examples of mouse behaviour following activation of VGluT2+ MRN neurons during exploitation of a high-reward port (P1). Light blue bar indicates the laser stimulation period. X-axis shows trials. Y-axis shows the chosen lick-ports and if reward was delivered (green circle) or not (red cross). j, Brief activation of VGluT2+ MRN neurons (laser on: blue circles) during a stable period of exploitation (choosing the high-probability arm for 5 sequential trials) increased the exploration probability (probability of switching to one of the other ports) over the next 4 trials, compared to control trials under the same condition (laser off: black circles). N = 11 mice, P = 6.5 × 10⁻⁸, two-way repeated measures ANOVA (two-sided). Light blue shading indicates the laser stimulation period. Data points and error bars show median ± bootstrapped standard error. k, Schematic of nose-poke reward association task with distractor nose poke ports, which have no association with reward. Mice with expression of ChR2 in VgluT2+ or VGAT+ MRN receive brief laser stimulation (1-2 s) upon entering a region of interest around the reward-associated (main) nose poke port in a subset of trials (see Methods). l, Median number of interactions with distractors per laser stimulation trial over all the completed laser stimulation trials in control mice and mice with optogenetic activation of VGluT2+ MRN neurons (P = 0.0027, two-sided chi-square test, N = 7 vs. 3 mice). m, The same as l, but in control mice and mice with optogenetic activation of VGAT+ MRN neurons (P > 0.9999, two-sided chi-square test, N = 7 vs. 3 mice). **: p-value < 0.01, ***: p-value < 0.001. In l and m bars depict mean, error bars are standard error and circles indicate individual experiments.

Extended Data Fig. 6. Impact of tonic and phasic optogenetic stimulation of VGluT2+ MRN neurons on object interaction.

a, Schematic of experimental design: optogenetic activation or suppression of VGluT2+ MRN neurons, using ChR2 or stGtACR2. b, Number of switches between objects during the MNOI test in control mice (ctrl) and mice with activation (act) and suppression (supp) of VGluT2+ MRN neurons (ctrl vs. act: P = 3.2 × 10⁻⁶, ctrl vs. supp: P > 0.9999, two-sided t-test with Bonferroni multi-comparison correction). N = 20, 10 and 11 experiments from 10, 5 and 5 mice in control, VGluT2+ activation and VGluT2+ suppression groups. c, Schematic of the experimental design to examine the effect of phasic 2-second activation of VGluT2+ MRN neurons in the MNOI test (with 5 objects) when the animal was not interacting with an object. d, Transition probability from no object interaction within the 2-second stimulation window (in experiment in c) to brief interaction (left, tdTomato ctrl (N = 5 experiments from 5 mice) vs VGluT2+ act. (N = 9 experiments from 7 mice), P = 4.7 × 10⁻⁵; two-sided t-test), to deep interaction (middle, ctrl vs VGluT2+ act.: P = 0.0100; two-sided Mann-Whitney U test), and probability to remain not interacting with the objects (right, ctrl vs VGluT2+ act.: P = 4.6 × 10⁻⁴; two-sided t-test). e, Phasic 2-second activation of VGluT2+ MRN neurons in the MNOI test when the animal’s snout was close to an object. f, Probability within the 2-second stimulation window (in experiment in e) to switch to a different object (left, ctrl (N = 5 experiments from 5 mice) vs VGluT2+ act. (N = 20 experiments from 7 mice), P = 0.0012; two-sided Mann-Whitney U test), to transition to a deep interaction (middle, ctrl vs VGluT2+ act.: P = 7.4 × 10⁻⁴; two-sided Mann-Whitney U test), and to transition to no-interaction (right, ctrl vs VGluT2+ act.: P = 0.0602; two-sided Mann-Whitney U test). g, Phasic 2-second activation of VGluT2+ MRN neurons in the MNOI test during deep interaction with an object. h, Probability (in experiment in g) to switch object (left, ctrl (N = 7 experiments from 5 mice) vs VGluT2+ act. (N = 14 experiments from 7 mice), P = P = 0.0076; two-sided Mann-Whitney U test), to persist in deep interaction (middle, ctrl vs VGluT2+ act.: P = 0.0090; two-sided t-test), and to transition to no-interaction (right, ctrl vs VGluT2+ act.: P = 0.2126; two-sided Mann-Whitney U test). *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001. In b, d, f and h bars depict mean, error bars are standard error and circles indicate individual experiments.

Extended Data Fig. 7. Effect of manipulation of MRN cell types on reinforcement.

a, Top: schematic of the self-stimulation test in control mice expressing tdTomato in MRN (N = 9). Second row: number of entries (irrespective of time spent in the port) into the opto-linked (black) and non-stimulation (grey) nose-poke ports over the course of the test (averaged over mice). Third row: cumulative number of entries into the opto-linked (black) and non-stimulation (grey) nose-poke ports over the course of the test. Each line shows behaviour in an individual experimental session. Bottom: number of entries into the opto-linked port plotted against number of entries into the non-stimulation nose-poke port for individual experiments. b, The same as a but for mice with suppression of VGluT2+ MRN neurons (N = 5). c, The same as a but for mice with activation of VGluT2+ MRN neurons (N = 9). d, The same as a but for mice with suppression of VGAT+ MRN neurons (N = 5). e, The same as a but for mice with activation of VGAT+ MRN neurons (N = 6). f, The same as a but for mice with suppression of SERT+ MRN neurons (N = 6). g, The same as a but for mice with activation of SERT+ MRN neurons (N = 6). h, Probability of interaction with objects over the course of an MNOI test (without laser stimulation), one minute after a 2-min MNOI test with laser stimulation, averaged over mice in tdTomato control group (ctrl, N = 6 experiments from 6 mice), and in groups of mice with suppression (supp) of SERT+ and VGAT+, and activation (act) of VGluT2+ MRN neurons (N = 7, 9 and 11 experiments from 7 mice in each group). i, Fraction of time spent in interaction with objects in mice shown in h. Ctrl vs. supp. SERT: P = 0.0420, ctrl vs. supp. VGAT: P = 0.0024, ctrl vs. act. VGluT2: P = 0.0019, two-sided t-test with Bonferroni multi-comparison correction. Bars indicate median values, error bars are bootstrapped standard error and circles individual experimental sessions. *: p-value < 0.05, **: p-value < 0.01.

Extended Data Fig. 8. Impact of SERT+ MRN neuron phasic stimulation on engagement and arousal.

a, Example image of virus expression (stGtACR2-fusionRed) in SERT+ neurons in the MRN with optic fibre position (left), with zoomed-in sections showing labelled neurons in MRN (middle) but not in the dorsal raphe nucleus (DRN, right). b, Schematic of the experimental design to examine the effect of phasic 2-second suppression of SERT+ MRN neurons in the MNOI test (with 5 objects) when the animal was not interacting with an object. c, Transition probability from no object interaction within the 2-second stimulation window (in the experiment in b) to brief interaction (left, tdTomato control mice (ctrl, N = 5 experiments from 5 mice) vs SERT+ supp. (N = 7 experiments from 7 mice), P = 0.0174; two-sided t-test), to deep interaction (middle, ctrl vs SERT+ supp.: P = 0.0202; two-sided Mann-Whitney U test), and probability to remain not interacting with objects (right, ctrl vs SERT+ supp.: P = 4.4 × 10⁻⁴; two-sided t-test). d, Phasic 2-second suppression of SERT+ MRN neurons in the MNOI test when the animal’s snout was close to an object. e, Transition probability within the 2-second stimulation window (in experiment in d) to switch to another object (left, ctrl (N = 5 experiments from 5 mice) vs SERT+ supp. (N = 8 experiments from 7 mice), P = 0.1330; two-sided t-test), to transition to a deep interaction (middle, ctrl vs SERT+ supp.: P = 0.0016; two-sided Mann-Whitney U test), and to transition to no-interaction (right, ctrl vs SERT+ supp.: P = 0.0326; two-sided Mann-Whitney U test). f, Phasic suppression of SERT+ MRN neurons in the MNOI test during deep interaction with an object. g, Probability (in experiment in f) to switch object within the 2-second time window (top, ctrl (N = 7 experiments from 5 mice) vs SERT+ supp. (N = 10 experiments from 7 mice), P = 0.3579; two-sided Mann-Whitney U test), to persist in deep interaction (middle, ctrl vs SERT+ supp.: P = 0.0024; two-sided t-test), and to transition to no-interaction (right, ctrl vs SERT+ supp.: P = 0.0062; two-sided Mann-Whitney U test). Bars in c, e and g depict mean, error bars are standard error and circles indicate individual experiments. h, Schematic of the experimental design to examine the effects of optogenetic manipulation of SERT+ MRN neurons on arousal level, measured using pupil size and whisker activity. i, Z-scored pupil size over time (mean ± s.e.m.) in control mice (grey) and mice with activation or suppression of SERT+ MRN neurons, averaged over trials and aligned to onset of optogenetic manipulation. Light blue box indicates the laser stimulation period. j, Median z-score of pupil size during laser stimulation from traces in i. N = 15, 8 and 7 mice; P > 0.9999 and P = 0.0004 for comparing control mice to activation or suppression of SERT+ neurons, respectively; two-sided t-test with Bonferroni multi-comparison correction. k,l, same as i,j but for z-scored whisker activity (summation of absolute frame-by-frame differences in pixel luminance). N = 15, 8 and 7 mice, respectively; P > 0.9999 and P = 0.0010 for comparing control mice to activation or suppression of SERT+ neurons, respectively; two-sided t-test with Bonferroni multi-comparison correction. Bars in j and l depict median, error bars are bootstrapped standard error and circles indicate individual experiments. m, Schematic of the fibre photometry recording during interactions with food or an aversive TMT-coated object. n, Average z-scored calcium trace (mean ± s.e.m., n = 4 mice) of SERT+ MRN neurons aligned to the onset of interactions with an aversive, TMT-covered object. o, Average z-scored calcium trace of SERT+ MRN neurons aligned to the first deep interaction (left) and further deep interactions with a food pellet (right). p, Average z-scored calcium trace of SERT+ MRN neurons aligned to the onset of disengaged state events (N = 75 events from 5 mice) during the MNOI test. *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Extended Data Fig. 9. Inputs to MRN and specific MRN cell types.

a, Schematic of experimental design to express eGFP in MRN-projecting neurons, using a retrograde AAV (left) and example image of the virus expression in MRN (right). b, Fraction of MRN-projecting cells out of all eGFP-expressing cells in the brain in the brain areas with highest density of labelled neurons (N = 5 mice). ACC: Anterior cingulate cortex, OFC: Orbitofrontal cortex, PrL: Prelimbic cortex, NAc: Nucleus accumbens, LHb: Lateral habenula, LHA: Lateral hypothalamic area, ZI: Zona incerta, VTA: ventrolateral tegmental area, PAG: Periaqueductal gray, IPN: Interpeduncular nucleus, DRN: Dorsal raphe nucleus, LDTg: Laterodorsal tegmental nucleus. c, Schematic of experimental design to label neurons presynaptic of VGAT+, VGluT2+, and SERT+ neurons in MRN, using a mono-transsynaptic rabies virus approach. d, Fraction of neurons innervating VGAT+ MRN neurons (blue), VGluT2+ MRN neurons (green), and SERT + MRN neurons (orange) out of the total number of presynaptic neurons from long-range projections (excluding areas close to the MRN, such as DRN and PAG; N = 7, 5 and 5 VGAT-Cre, VGluT2-Cre, and SERT-Cre mice). ACC: Anterior cingulate cortex, OFC: Orbitofrontal cortex, PrL: Prelimbic cortex, LHb: Lateral habenula, LHA: Lateral hypothalamic area, IPN: Interpeduncular nucleus, ZI: Zona incerta. e, Example image of LHA neurons innervating VGAT+ MRN neurons (left), VGluT2+ MRN neurons (middle), and SERT+ MRN neurons (right). f, Number of VGAT+ MRN-innervating, VGluT2+ MRN-innervating, and SERT+ MRN-innervating neurons in LHA, normalized by the total number of starter cells in MRN (N = 7, 5 and 5 VGAT-Cre, VGluT2-Cre, and SERT-Cre mice). VGAT vs VGluT2: P = 0.0379, VGAT vs SERT: P > 0.9999, VGluT2 vs SERT: P = 0.0476; two-sided Mann-Whitney U test with Bonferroni multi-comparison correction. Note that this approach cannot distinguish between GABAergic and glutamatergic presynaptic neurons in the LHA. g, Example image of LHb neurons innervating VGAT+ MRN neurons (left), VGluT2+ MRN neurons (middle), and SERT+ MRN neurons (right). Scale bar indicates 0.5 mm. h, Number of VGAT+ MRN-innervating, VGluT2+ MRN-innervating, and SERT + MRN-innervating neurons in LHb, normalized by the total number of the starter cells in MRN (N = 7, 5 and 5 VGAT-Cre, VGluT2-Cre, and SERT-Cre mice). VGAT vs VGluT2: P = 0.0152, VGAT vs SERT: P > 0.9999, VGluT2 vs SERT: P = 0.0476; two-sided Mann-Whitney U test with Bonferroni multi-comparison correction. *: p-value < 0.05. Bars in b, d, f and h depict median, error bars are bootstrapped standard error and circles indicate individual experiments.

Extended Data Fig. 10. Activation of MRN inputs from LHA and LHb has little direct impact on VTA.

a, Top, Schematic of experimental design to label MRN-projecting and ventral tegmental area (VTA)-projecting neurons, with GFP and tdTomato, respectively. Bottom, Example of MRN-projecting (green) and VTA-projecting (magenta) neurons in LHb, and their overlap. b, Schematic of experimental design to record the effect of activation of LHb input to MRN on the activity of MRN and VTA neurons, using high-density multi-channel Neuropixels probes. c, Top, Example raster plot (each row is one trial, each dot is one spike) of activity of one example MRN single-unit over the laser stimulation trials, aligned to the onset of laser stimulation. The firing rate averaged over trials is shown in green. The right green y-axis indicates the firing rate in Hz. The right-top corner shows the spike shape. Bottom, same as top but for a VTA single-unit. The firing rate averaged over trials is shown in magenta. d, Number of MRN and VTA neurons (median ± bootstrapped standard error) activated within 10 ms from the onset of laser stimulation of LHb input to MRN (N = 3 mice, P = 0.0102, two-sided paired t-test). e, Average firing rate of MRN (left) and VTA (right) single-units, while activating LHb axons in MRN, aligned to laser onset (mean ± s.e.m.). Firing rate comparison 50 ms before vs after laser onset in MRN: P = 7.1 × 10⁻¹²; and VTA: P = 0.98; two-sided Wilcoxon signed-rank test. f, Same as a, but for MRN-projecting (green) and VTA-projecting (magenta) neurons in LHA, and their overlap. g, Schematic of experimental design to record the effect of activation of LHA GABAergic input to MRN on the activity of MRN and VTA neurons, using high-density multi-channel Neuropixels probes. h, Same as c, but for optogenetic activation of GABAergic LHA axons in MRN. i, Number of MRN and VTA neurons (median ± bootstrapped standard error) suppressed within 100 ms from the onset of laser stimulation of LHA GABAergic input to MRN (N = 3 mice, P = 0.0123, two-sided paired t-test). j, Average firing rate of single units in MRN (left) and VTA (right), while activating LHA GABAergic axons in MRN, aligned to laser onset (mean ± s.e.m.). Firing rate comparison 50 ms before vs after laser onset in MRN: P = 2.5 × 10⁻⁸, and VTA: P = 0.15; two-sided Wilcoxon signed-rank test. *: p-value < 0.05, ***: p-value < 0.001.

Extended Data Fig. 11. Impact of manipulation of LHb input to MRN on valence and arousal and fibre photometry calcium recordings from these projections.

a, Schematic of experimental design for optogenetic suppression or activation of LHb input to MRN. b, Left: schematic of the real-time place preference test. Right: preference for the opto-linked chamber (100 × (duration of time spent in the opto-linked chamber - duration of time spent in the non-stimulation chamber) / total time) in control mice and mice with activation or suppression of LHb input to MRN (N = 7, 5 and 8 mice, respectively; P = 0.0053 and P = 0.0034 for comparing control mice to activation or suppression of LHb input to MRN, respectively; two-sided t-test with Bonferroni multi-comparison correction). c, Schematic of the experimental design: chronic laser activation (4 days, 24 h per day) followed by the sucrose preference test without laser stimulation on the 5th day. d, Sucrose preference in control mice and mice after repeated optogenetic activation of LHb input to MRN (N = 3 and 4 mice, respectively, P = 0.0027, two-sided t-test). e, Experimental design to examine the effects optogenetic manipulation of LHb input to MRN on arousal level, measured using pupil size and whisker activity. f, Z-scored pupil size over time (mean ± s.e.m.) in control mice and mice with suppression or activation of LHb input to MRN, averaged over laser stimulation trials and aligned to laser onset. The light blue box indicates the laser stimulation period. g, Median z-scored pupil size during the laser stimulation period from traces in f. N = 15, 10 and 12 mice; P > 0.9999 and P = 0.0037 for comparing control mice to suppression or activation of LHb input to MRN, respectively; two-sided t-test with Bonferroni multi-comparison correction. h,i, same as f,g but for z-scored whisker activity (summation of absolute frame-by-frame differences in pixel luminance). N = 15, 10 and 12 mice; P > 0.9999 and P = 2.4 × 10⁻⁵ for comparing control mice to suppression or activation of LHb input to MRN, respectively; two-sided t-test with Bonferroni multi-comparison correction. In b, d, g and i bars depict median, error bars are bootstrapped standard error and circles indicate individual experiments. j, Schematic of calcium fibre photometry recording from LHb input to MRN in mice exposed to either novel objects or aversive TMT-coated objects. k, Schematic of the experimental design to express GCaMP6f in LHb neurons and implant a fibre to image LHb input to MRN. l, Left, Heatmap shows z-scored calcium activity of LHb input to MRN (top) and their average z-scored calcium activity trace (mean ± s.e.m.) across mice (bottom, N = 4 mice, activity during baseline vs during escape: P = 2.9 × 10⁻⁵; two-sided Wilcoxon signed rank test) aligned to time of fast retreat (escape) after an approach of a TMT-coated object. m, Same as l, but calcium activity (mean ± s.e.m.) aligned to start of a deep interaction with a novel object (N = 3 mice, activity during baseline vs during deep interactions: P = 0.7084; two-sided Wilcoxon signed rank test). **: p-value < 0.01, ***: p-value < 0.001.

Extended Data Fig. 12. Impact of manipulation of LHA VGAT+ input to MRN on valence, arousal and deep object interactions, and fibre photometry calcium recordings from these projections.

a, Example image of virus expression in VGluT2+ neurons in lateral hypothalamic area (LHA,in green; left) of VgluT2-Cre mice, showing absence of axon terminals in MRN (right). 3 V: 3^rd ventricle, DM: dorsomedial hypothalamus, EP: entropeduncular nucleus, LHA: lateral hypothalamic area, MeA: medial amygdala, Subl: subincertal nucleus, VMH: ventromedial hypothalamus. DRN: dorsal raphe nucleus, MRN: median raphe nucleus, PAG: periaqueductal gray, PnO: pontine reticular formation. b, Schematic of experimental design for optogenetic suppression or activation of LHA VGAT+ input to MRN. c, Schematic of the real-time place preference test. d, Preference for the opto-linked chamber (100 × (duration of time spent in the opto-linked chamber - duration of time spent in the non-stimulation chamber) / total time) in control mice and mice with activation or suppression of LHA VGAT+ input to MRN (N = 7, 4 and 6 mice, respectively; P = 0.04865 and P = 0.0001 for comparing control mice to activation or suppression of LHA VGAT+ input to MRN, respectively; two-sided t-test with Bonferroni multi-comparison correction). e, Experimental design to examine the effects of optogenetic manipulation of LHA VGAT + MRN input on arousal level, measured using pupil size and whisker activity. f, Z-scored pupil size over time (mean ± s.e.m.) in control mice and mice with suppression or activation of LHA VGAT+ input to MRN, averaged over laser stimulation trials and aligned to laser onset. The light blue bar indicates the laser stimulation period. g, Median z-scored pupil size during the laser stimulation period from traces in f. N = 15, 11 and 20 mice; P > 0.9999 and P = 0.0044, for comparing control mice to suppression or activation of LHA input, respectively; two-sided t-test with Bonferroni multi-comparison correction. h,i, same as f,g but for z-scored whisker activity (summation of absolute frame-by-frame differences in pixel luminance). N = 15, 11 and 20 mice; P > 0.9999 and P = 0.1352 for comparing control mice to suppression or activation of LHA input, respectively; two-sided t-test with Bonferroni multi-comparison correction. j, Schematic of the TMT aversion test. k, Number of approaches of the aversive TMT-covered object in control mice and mice with suppression or activation of LHA VGAT+ input to MRN. N = 9, 10 and 11 mice, respectively; P = 0.0760 and P = 0.2107 for comparing control mice to suppression or activation of LHA input, respectively; two-sided t-test with Bonferroni multi-comparison correction. l, Escape probability after approaching the TMT-covered object for mice shown in k. P = 0.0258 and P = 0.0005 for comparing control mice to suppression or activation of LHA input, respectively; two-sided t-test with Bonferroni multi-comparison correction. m, Schematic of the MNOI test. n, Duration of deep interactions with each object during the MNOI test in control mice (ctrl), mice with suppression of VGAT + MRN neurons (supp. vgat) and mice with activation of LHA VGAT+ input to the MRN (act. lha). Ctrl vs. supp. vgat: P = 1.6 × 10⁻⁶, ctrl vs. act. lha: P = 7.6 × 10⁻⁶, two-sided t-test with Bonferroni multi-comparison correction. N = 20, 10 and 23 experiments from 10, 5 and 9 mice in ctrl, supp. vgat and act. lha groups. o, Duration of deep interactions with each object in the MNOI test in control mice (ctrl), mice with activation of VGAT + MRN neurons (act. VGAT) and mice with suppression of LHA VGAT+ input to the MRN (supp. LHA), Ctrl vs. act. VGAT: P = 0.0102, ctrl vs. supp. LHA: P = 0.0001, two-sided t-test with Bonferroni multi-comparison correction. N = 20, 11 and 15 experiments from 10, 6 and 8 mice in ctrl, act. VGAT and supp. LHA groups. p, Schematic of calcium fibre photometry recording from GABAergic LHA input to MRN in mice exposed to multiple novel objects. q, Schematic of the experimental design to express GCaMP6f in VGAT + LHA neurons and implant a fibre to image LHA input to MRN. r, Heatmap of individual z-scored calcium activity traces of VGAT + LHA input to MRN of an example mouse (left) and average z-scored calcium activity trace (mean ± s.e.m.) across mice (N = 5 mice, activity during baseline vs during deep interactions: P = 5.4 × 10⁻⁸; two-sided Wilcoxon signed rank test) (right) during object interactions aligned to the onset of deep interaction with an object. s, Heatmap of z-scored calcium activity traces of VGAT + LHA input to MRN of the same mouse as r (left) and average z-scored calcium activity trace (mean ± s.e.m.) across mice (before-interaction baseline vs activity after switching object: P = 0.0672; two-sided Mann-Whittney U test) (right) during object interactions aligned to the time of switching between objects. t, Median z-scored calcium activity of VGAT + LHA input to MRN during disengaged states (N = 261 events from 5 mice), exploratory states (N = 195 events, disengaged vs exploratory P > 0.9999) and perseverative states (N = 71 events, disengaged vs perseverative: P = 0.0002), two-sided nested ANOVA with Bonferroni multi-comparison correction. *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001. In panels d, g, i, k, l, n, o and t bars depict median, error bars are bootstrapped standard error and circles indicate individual experiments.

Extended Data Fig. 13. Summary of how MRN neurons affect interaction states.

The schematic summarizes the findings of how MRN cell-types and their inputs from LHA and LHb regulate perseverative, exploratory and disengaged states.