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.