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Interacting neural ensembles in orbitofrontal cortex for social and feeding behaviour

Nature volume 565, pages 645–649 (2019)Cite this article

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Abstract

Categorically distinct basic drives (for example, for social versus feeding behaviour1,2,3) can exert potent influences on each other; such interactions are likely to have important adaptive consequences (such as appropriate regulation of feeding in the context of social hierarchies) and can become maladaptive (such as in clinical settings involving anorexia). It is known that neural systems regulating natural and adaptive caloric intake, and those regulating social behaviours, involve related circuitry4,5,6,7, but the causal circuit mechanisms of these drive adjudications are not clear. Here we investigate the causal role in behaviour of cellular-resolution experience-specific neuronal populations in the orbitofrontal cortex, a major reward-processing hub that contains diverse activity-specific neuronal populations that respond differentially to various aspects of caloric intake8,9,10,11,12,13 and social stimuli14,15. We coupled genetically encoded activity imaging with the development and application of methods for optogenetic control of multiple individually defined cells, to both optically monitor and manipulate the activity of many orbitofrontal cortex neurons at the single-cell level in real time during rewarding experiences (caloric consumption and social interaction). We identified distinct populations within the orbitofrontal cortex that selectively responded to either caloric rewards or social stimuli, and found that activity of individually specified naturally feeding-responsive neurons was causally linked to increased feeding behaviour; this effect was selective as, by contrast, single-cell resolution activation of naturally social-responsive neurons inhibited feeding, and activation of neurons responsive to neither feeding nor social stimuli did not alter feeding behaviour. These results reveal the presence of potent cellular-level subnetworks within the orbitofrontal cortex that can be precisely engaged to bidirectionally control feeding behaviours subject to, for example, social influences.

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Fig. 1: Single-cell two-photon readout and control of OFC neuronal activity in vivo.
Fig. 2: Identifying and targeting activity-specific OFC neurons for cellular-resolution optogenetic perturbations.
Fig. 3: Activity-guided optogenetic stimulation of individual feeding-specific OFC neurons promotes caloric-reward licking.
Fig. 4: Selective optogenetic activation of juvenile-conspecific (social) responsive OFC neurons disrupts licking.

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Data availability

The data that support the finding of this study are available upon request from the corresponding author.

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Acknowledgements

We thank members of the Deisseroth laboratory for input and discussions, and particularly A. Crow for assistance with microscopy-related tasks. K.D. is supported by the Defense Advanced Research Projects Agency Neuro-FAST program, National Institute of Mental Health, National Institute on Drug Abuse, National Science Foundation, the Simons Foundation, the Tarlton Foundation, the Wiegers Family Fund, the Nancy and James Grosfeld Foundation, the H.L. Snyder Medical Foundation, and the Samuel and Betsy Reeves Fund. This work was also supported by HHWF (J.H.J.), NIDA (C.K.K.), Simons LSRF fellowship (J.H.M.), NSF GRFP (M.R.) and NIDDK (L.Y.).

Reviewer information

Nature thanks M. Krashes and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Joshua H. Jennings, Christina K. Kim, James H. Marshel

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Joshua H. Jennings, James H. Marshel, Misha Raffiee, Sean Quirin, Sally Pak, Charu Ramakrishnan & Karl Deisseroth

  2. Neurosciences Program, Stanford University, Stanford, CA, USA

    Christina K. Kim & Karl Deisseroth

  3. Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA

    Li Ye & Karl Deisseroth

  4. Howard Hughes Medical Institute, Stanford, CA, USA

    Li Ye & Karl Deisseroth

  5. CNC Program, Stanford University, Stanford, CA, USA

    Karl Deisseroth

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Contributions

J.H.J., C.K.K. and K.D. designed the experiments and wrote the paper. J.H.J. and C.K.K. performed all experiments and surgeries with contributions from M.R. J.H.J. and C.K.K. collected and analysed all datasets. J.H.M. and S.Q. assisted with the optical design of the two-photon microscope for simultaneous imaging and stimulation experiments. S.Q. developed the stimulation artifact removal scripts. M.R. assisted with behavioural experiments, constructed the supplementary videos, and performed histology and the freely moving behavioural experiments. M.R. designed the social contact, whisker and pupil-tracking scripts, and analysed related datasets. L.Y. processed and imaged all of the CLARITY samples. S.P. performed histology and Airyscan imaging of fixed brain slices. C.R. designed all constructs for viral packaging and provided crucial input on viral targeting strategies. K.D. supervised all aspects of the work.

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Correspondence to Karl Deisseroth.

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Extended data figures and tables

Extended Data Fig. 1 Targeting OFC for two-photon cellular-resolution Ca2+ imaging and optogenetic stimulation.

a, Confocal 10× tiled image of a 1-mm-thick coronal CLARITY section displaying the location of the GRIN lens and GCaMP6m expression within OFC. A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bar, 500 μm. b, Confocal 10× image from a 60-μm coronal slice depicting GRIN lens implantation and viral targeting site within OFC. Scale bar, 500 μm. c–e, Additional representative in vivo two-photon images of OFC cells co-expressing GCaMP6m and bReaChES-mCherry from a different focal plane. Images from a representative mouse; the experiment was repeated in n = 6 mice with similar results. Scale bars, 100 μm. f–h, Confocal 20× images of the OFC showing GABA immunolabelling (f) and CaMKIIα-GCaMP6m expression (g) with minimal overlap (h). Images from a representative mouse; the experiment was repeated in n = 4 mice with similar results. Scale bars, 50 μm.

Extended Data Fig. 2 Characterization of spiral stimulation through a GRIN lens.

a, Heat map representing the relative probability for multi-photon absorption across the FOV of a typical GRIN lens as measured by the ratio of the normalized images from a bulk fluorescence slide acquired by multi-photon (920 nm) and single-photon (488 nm) excitation, which accounts for photon collection losses. Scale bar, 50 μm. b, The power output of the 1,060-nm stimulation beam at the distal tip of a GRIN lens is constant across the entire FOV, indicating that the most probable loss mechanism for two-photon excitation is optical aberration. Stimulation laser power readouts were obtained while moving the spiral point at 50-μm increments across the FOV (500 μm). c, Cell masks of stimulation-targeted cells near the edge and centre of the GRIN lens from an example animal. Image from a representative mouse; the experiment was repeated in n = 6 mice with similar results. Scale bar, 100 μm. d, Mean Ca2+ activity of stimulation-targeted cells near the edge of the GRIN lens is comparable to the average activity responses of stimulation-targeted neurons located near the centre of the GRIN lens (10 stim trials, n = 4 stim-targeted cells from 1 mouse). The shaded region indicates stimulation time points. e, Average Ca2+ activity in response to the spiral-stimulation targets positioned 0 μm (red), 10 μm (blue), 20 μm (brown) and 40 μm (black) away (in the x–y plane) from the centre point of each stimulation-targeted neuron (10 stimulation trials per position, 30-s interval, 5-s spiral stimulation, 10 spiral-stimulation targets, n = 10 stimulation-targeted cells from one mouse). Shaded region indicates stimulation time points. f–i, Mean Ca2+ activity of stim-targeted cells in response to the spiral point distance (in the x–y plane) of 0 μm (f), 10 μm (g), 20 μm (h) and 40 μm (i; n = 10 stimulation-targeted cells from one mouse; this experiment was repeated in n = 3 mice with similar results). j, Representative zoomed-in Ca2+ activity traces during 5-s spiral-stimulation trials. k, Example Ca2+ traces from individual neurons showing responses before (purple) and following (orange) the removal of the spiral-stimulation artefact, present in the GCaMP acquisition channel owing to non-negligible GCaMP excitation with the stimulation light source (λ = 1,060 nm) during spiral stimulation. At 30-Hz image acquisition, for every 1 ms of the spiral photostimulation duration, the photostimulation artefact would contaminate ~17 consecutive image lines with an approximately uniform background. This artefact was estimated on a per-image-line basis as the average increase in signal during the stimulation frames from the image frames which occurred without photostimulation and then removed across each image line using custom MATLAB scripts (Methods).

Extended Data Fig. 3 Single-cell optogenetic stimulation of feeding-excited neurons.

a, Schematic outlining the experiment sequence for identification and stimulation of feeding- or social-excited cells. b, Cell masks of identified feeding-excited neurons (cyan) and spiral-stimulation targets (red) from a GCaMP6m (control) mouse. Image from a representative mouse; the experiment was repeated in n = 6 mice with similar results. Scale bar, 100 μm. c, Spiral stimulation of feeding-excited neurons in a GCaMP6m mouse did not produce time-locked optogenetic-evoked responses. d, During a caloric-reward baseline session (no stimulation), GCaMP6m + bReaChES and GCaMP6m mice did not exhibit a significant difference in licking. n = 6 mice per group, interaction F2,30 = 0.05, P = 0.95, two-way ANOVA followed by Bonferroni post hoc comparisons. e, Spiral stimulation of feeding-excited neurons did not significantly alter the latency to first lick following each caloric-reward delivery in GCaMP6m + bReaChES and GCaMP6m mice. n = 6 mice per group, interaction F1,20 = 0.15, P = 0.69, two-way ANOVA followed by Bonferroni post hoc comparisons. f, g, Average cumulative lick rate (30-s bins) across 10-min baseline and feeding-cell stimulation sessions in GCaMP6m (f) and GCaMP6m + bReaChES mice (g; n = 6 mice per group). h, GCaMP6m + bReaChES mice display increased cumulative licking across the entire feeding-cell stimulation session when compared to the baseline session. n = 20 feeding-stimulated cells per mouse, n = 6 mice, t5 = 8.250, P = 0.0004, two-sided paired t-test. i, Average lick rate from an example GCaMP6m + bReaChES animal during a feeding-cell stimulation session when the lick spout was empty (no caloric rewards; 10-min session; n = 20 feeding-stimulated cells). j, Stimulation of feeding-responsive cells in GCaMP6m + bReaChES mice did not alter licking for an empty lick spout (no caloric rewards; n = 4 mice, F2,6 = 1.19, P = 0.37, one-way ANOVA with repeated measures). **P < 0.001; n.s., non-significant (P > 0.05).

Extended Data Fig. 4 Single-cell stimulation of feeding-excited OFC neurons increases licking for caloric rewards, but not for saccharin.

a–c, Cell masks of feeding caloric-excited (a), saccharin-excited (b) and feeding (caloric + saccharin)-excited neurons (c) from an example animal. Images from one representative mouse; the experiment was repeated in n = 6 mice with similar results. Scale bars, 100 μm. d, The proportion of identified feeding-excited neurons is significantly greater than that for saccharin and feeding + saccharin cell types. n = 1,070 total cells, feeding-excited mean = 38 ± 8.1 cells, saccharin-excited mean = 20 ± 3.8 cells, feeding+saccharin mean = 8 ± 1.5 cells, n = 6 mice; F2,15 = 18.6, P < 0.0001, one-way ANOVA. e, Cell masks of identified feeding-excited neurons (cyan) and spiral-stimulation targets (red) from a GCaMP6m + bReaChES mouse. Image from one representative mouse; the experiment was repeated in six mice with similar results. Scale bar, 100 μm. f, Normalized mean Ca2+ activity of feeding-excited neurons across 20 trials of 5-s stimulation (n = 20 cells). g, Single-cell stimulation of feeding-excited neurons significantly increased caloric-reward licking in GCaMP6m + bReaChES mice compared to baseline sessions. n = 20 feeding-stimulated cells per mouse, n = 6 mice; interaction F2,10 = 18.0, P < 0.0001. h, Spiral stimulation of feeding-responsive cells did not significantly alter licking for a 0.1% saccharin solution. n = 20 feeding-stimulated cells per mouse, n = 6 mice; interaction F2,10 = 0.72, P = 0.99. i, Baseline licking for caloric and saccharin rewards did not significantly differ in GCaMP6m + bReaChES mice. n = 6 mice; interaction F2,10 = 3.21, P = 0.99. j, Single-cell stimulation of feeding-excited neurons significantly increased caloric licking in GCaMP6m + bReaChES mice when compared to saccharin licking (n = 6 mice, interaction F2,10 = 7.976, P = 0.002). Two-way ANOVA with repeated measures followed by Bonferroni post hoc comparisons. **P < 0.001, ***P < 0.0001; ***#P significant across all comparisons; n.s., non-significant (P > 0.05).

Extended Data Fig. 5 Activity signals in feeding-excited neurons in response to caloric rewards were enhanced and prolonged when caloric-reward delivery was paired with single-cell two-photon stimulation.

a, Mean Ca2+ signals in feeding-excited cells in response to caloric-reward delivery or 5-s spiral stimulation paired with caloric-reward delivery. n = 20 caloric-stimulated cells from 1 mouse. b, Average Ca2+ signals in feeding-excited cells during the first 5 s following stimulation + caloric reward were significantly greater than the activity responses from the first 5 s following caloric-reward delivery (n = 20 feeding-stimulated cells from 1 mouse; t19 = 2.950, P = 0.008, two-sided paired t-test). c, Mean peak Ca2+ signals in feeding-excited cells during stimulation + caloric-reward delivery were significantly greater than during caloric-reward delivery alone. n = 20 feeding-stimulated cells from 1 mouse; t19 = 2.122, P = 0.047, two-sided paired t-test. d, Example dF/F responses to caloric-reward deliveries on 4 longest lick-bout trials (orange; 0.67 s) and 4 shortest lick-bout trials (purple; 1.77 s) from an example animal during the baseline caloric-reward session. e, Corresponding lick rasters for long (orange) and short (purple) trials. Feeding-cell activity responses displayed significant changes during short versus long lick-bout trials. n = 81 feeding-excited cells, mean dF/F = 0.13 ± 0.019 versus 0.15 ± 0.020; Z = 3.4, P = 0.0007, two-sided Wilcoxon signed-rank test. f, g, Example dF/F responses to caloric rewards from another representative mouse (f) and corresponding lick rasters (g; n = 76 feeding-excited cells, mean dF/F = 0.073 ± 0.0083 versus 0.10 ± 0.012; Z = 2.6, P = 0.0098, two-sided Wilcoxon signed-rank test). *P < 0.05.

Extended Data Fig. 6 Head-fixed two-photon investigation of social behaviours.

a, The social-behavioural arena (left) and the opening of the social zone, where physical contact occurs between freely moving juvenile and head-fixed mice (right). b, The setup for monitoring social behaviour during in vivo head-fixed two-photon Ca2+ imaging. c, Identification of OFC cells that respond to physical contact with a juvenile social stimulus. d–f, Cell masks of social-zone (d; juvenile social-stimulus zone entry), social-contact (e; juvenile social-stimulus contact) and zone + contact-excited neurons (f) from an example animal. Images from one representative mouse; this experiment was repeated in six mice with similar results. Scale bars, 100 μm. g, The proportion of identified social-zone, social-contact, and zone + contact-excited neurons exhibited no significant differences. Mean number of juvenile zone entries = 18 ± 3, mean number of social contacts = 34 ± 6; n = 943 total cells from 5 mice, social-zone-excited mean = 54 cells ± 13, social-contact-excited mean = 48 cells ± 6.5, zone + contact-excited mean = 31 ± 4.9 cells; F2,12 = 2.1, P = 0.2, one-way ANOVA. h, i, The average percentage of social-zone (h) and social-contact (i) neurons that significantly responded during both social readouts. Neurons that exhibited a significant response to the first 5 s of social-zone entry or social contact compared to the previous 2 s of baseline activity were classified as social-zone or social-contact neurons using a two-sided Wilcoxon signed-rank test with a P < 0.05 threshold. All data are plotted as mean ± s.e.m; n.s., non-significant (P > 0.05).

Extended Data Fig. 7 Distinct juvenile-conspecific (social) excited neurons demonstrate minimal overlap with cells excited by the presentation of caloric rewards, novel objects or ultrasonic vocalization recordings.

a, Mean Ca2+ responses from feeding-excited cells in response to caloric-reward delivery (left; CV of responses to caloric-reward delivery = 1.74 ± 0.07; CV = (s.d. of response size)/(mean of response size), calculated per mouse) and to juvenile social-zone entries (right; CV of responses to social-zone entries = 13 ± 1.19, n = 822 caloric-excited cells from 11 mice; cells are sorted in the same order across the two behavioural stimuli.). Neurons were classified as feeding-responsive if a significant difference existed between the mean dF/F activity 2 s before and 2 s after the caloric-reward delivery (20 total caloric-reward deliveries) using two-sided Wilcoxon signed-rank test with a P < 0.05 threshold. b, Mean Ca2+ responses of social-excited neurons to caloric-reward deliveries (left; CV = 23 ± 6.80) and social-zone entries (right; CV = 1.69 ± 0.08, n = 331 social-excited cells from 11 mice). Social-responsive cells were identified by comparing the mean Ca2+ responses 2 s pre- and 5 s post-social-zone entry. Number of juvenile zone entries = 17 ± 1; two-sided Wilcoxon signed-rank test with a P < 0.05 threshold. c–e, Cell masks of feeding-excited (c), social-excited (d) and feeding + social-excited neurons (e) from an example animal. Images from a representative mouse; the experiment was repeated in six mice with similar results. Scale bars, 100 μm. f, Contingency table showing the total number of cells detected in the FOV, and the number of feeding-excited, social-excited, or feeding + social-excited (both) cells per mouse. g–j, Cell masks of juvenile-conspecific (g), adult-conspecific (h), novel-object (i; NO), and USV (j) excited neurons from an example animal. Images from a representative mouse; the experiment was repeated in six mice with similar results. Scale bars, 100 μm. For novel-object experiments, a custom 3D-printed mouse was presented to each real mouse for 3 s every 30 s during a 10-min recording. For USV experiments, a 50-kHz USV recording was played through a speaker for 3 s every 30 s during a 10-min imaging session. Number of juvenile zone entries = 17 ± 2; mean number of adult zone entries = 15 ± 2; 20 novel-object presentations; 20 total USV presentations). Novel-object and USV cells were identified by comparing the mean Ca2+ activity 2 s before and 3 s after novel-object or USV delivery using a two-sided Wilcoxon signed-rank test with a P < 0.05 threshold; juvenile- and adult-conspecific cells were identified by comparing the mean Ca2+ 2 s before and 5 s after social-zone entry. k, The proportion of cells that are excited by juvenile conspecific (26 cells ± 3.7), USV (7 cells ± 2.0), novel object (mean = 29 cells ± 7.8), adult conspecific (mean = 9 cells ± 2.0), juvenile conspecific + USV (0.7 cells ± 0.47), juvenile conspecific + novel object (5 cells ± 2.4), juvenile conspecific + adult conspecific (1.6 cells ± 0.84), novel object + adult conspecific (2.4 cells ± 0.90), novel object + USV (1.6 cells ± 0.43), adult conspecific + USV (1.1 cells ± 0.40), juvenile conspecific + USV + NO (0.3 cells ± 0.18), juvenile conspecific + USV + adult conspecific (0.3 cells ± 0.18), juvenile conspecific + NO + adult conspecific (0.7 cells ± 0.42), adult conspecific + NO + USV (0.3 cells ± 0.29), and juvenile conspecific + NO + USV + adult conspecific (0.1 cells ± 0.14). n = 1,268 total cells from 7 mice. l–o, Mean Ca2+ responses of cells excited by juvenile social-zone entries (l; n = 184 juvenile-conspecific excited cells; CV = 1.7 ± 0.12, CV = (s.d. of response size)/(mean of response size), calculated per mouse), adult social-zone entries (m; n = 66 adult-conspecific excited cells, CV = 4 ± 2.6), novel-object presentations (n; n = 201 NO-excited cells, CV = 1.67 ± 0.050), or USV deliveries (o; n = 50 USV-excited cells from 7 mice, CV = 1.85 ± 0.070). p–r, Mean Ca2+ activity of juvenile-conspecific excited neurons (n = 184 juvenile-conspecific excited cells from 7 mice) in response to adult social-zone entries (p; CV = 11 ± 2.4), novel-object presentations (q; CV = 11 ± 2.0), or USV deliveries (r; CV = 16 ± 4.0).

Extended Data Fig. 8 Social interaction disrupts caloric intake.

a–d, Setup for separate, counterbalanced 10-min sessions of free-access caloric licking during baseline (a), novel object (b), juvenile social interaction (c) and adult social interaction (d). e, Average cumulative lick rate (30-s bins) across 10-min baseline, novel object (3D-printed mouse), juvenile and adult social-interaction sessions (n = 6 mice). f, GCaMP6m + bReaChES mice display decreased cumulative licking within the first 2 min of the juvenile social-interaction session when compared to the first 2 min of the baseline, novel-object and adult social-stimulus sessions. n = 6 mice; interaction F5,15 = 3.398, P = 0.029, one-way ANOVA with repeated measures. g, Cumulative licking did not significantly differ within the last 2 min of the baseline, novel-object, juvenile and adult social-interaction sessions. n = 6 mice; interaction F5,15 = 1.714, P = 0.192, one-way ANOVA with repeated measures. h, Time course for interactions with juvenile, adult and novel object during each 2-min bin across each 10-min free-access licking session. n = 6 mice. i, Time spent interacting with juvenile conspecifics was significantly greater during the first 2 min than the last 2 min of the 10-min session and when compared to interactions with adult conspecifics and novel object. n = 6 mice; interaction F2,10 = 15.53, P = 0.0009, two-way ANOVA with repeated measures. *#P and **#P significant across all comparisons; n.s., non-significant (P > 0.05).

Extended Data Fig. 9 Activation of feeding- or social-excited cells does not alter whisker activity or pupil diameter and activation of NSNF neurons does not affect licking.

a, Representative whisker (orange) tracking from an example GCaMP6m + bReaChES mouse. b, c, Two-photon spiral stimulation of 20 feeding- or social-excited cells per animal does not significantly alter whisker activity. Animals are able to detect noise produced from the spiral-stimulation galvanometer mirrors during both no stimulation (laser shutter closed) and stimulation (laser shutter open) sessions, as demonstrated by a significant increase in classified whisking events during the spiral scanning period (b; unstimulated, 0 to 5 s versus −5 to 0 s: n = 5 mice, interaction F2,8 = 68.34, P = 0.02; and stimulated 0 to 5 s versus −5 to 0 s: n = 5 mice, interaction F2,8 = 68.34, P = 0.002, two-way ANOVA with repeated measures); this sensory response was not disrupted by spiral stimulation of feeding (b; n = 5 mice, interaction F2,8 = 2.31, P > 0.99, two-way ANOVA with repeated measures) or social (c; n = 6 mice, interaction F2,10 = 0.61, P > 0.99, two-way ANOVA with repeated measures) cells. d, Representative pupil-diameter tracking (purple) from an example GCaMP6m + bReaChES mouse. e, f, Pupil diameter is not significantly affected by stimulation of 20 feeding (e; n = 4 mice, F2,6 = 0.17, P = 0.85, one-way ANOVA with repeated measures) or social (f; n = 4 mice, F2,6 = 0.05, P = 0.95, one-way ANOVA with repeated measures) cells. g, Cell masks of neurons that do not display significant responses to either feeding or social stimuli (NSNF cells; orange) and spiral-stimulation targets (red) from a GCaMP6m + bReaChES mouse. Image from n = 1 representative mouse; this experiment was repeated in six mice with similar results. Scale bar, 100 μm. h, Normalized mean Ca2+ activity of NSNF neurons across 20 trials of 5-s stimulation. n = 20 stimulation-targeted NSNF cells. i, Single-cell stimulation of NSNF neurons did not significantly alter licking for caloric rewards in GCaMP6m + bReaChES mice when compared to baseline sessions. n = 20 NSNF-stimulated cells per mouse, n = 6 mice; interaction F2,10 = 1.76, P = 0.51, two-way ANOVA with repeated measures. j, Average cumulative lick rate (30-s bins) across 10-min baseline and NSNF cell stimulation sessions in GCaMP6m + bReaChES mice. n = 6 mice. n.s., non-significant (P > 0.05).

Extended Data Fig. 10 Alterations in local network activity from in vivo single-cell optogenetic stimulation of activity-specific OFC neurons.

a, Example field of view displaying stimulation-targeted feeding-excited (blue), non-targeted indirectly excited (green) and non-targeted indirectly inhibited (red) neurons from a GCaMP6m + bReaChES mouse. Image from a representative mouse; the experiment was repeated in six mice with similar results. Scale bar, 100 μm. b, Mean Ca2+ responses in feeding-excited neurons activated by direct stimulation (blue), and in non-targeted cells indirectly excited (green) or inhibited (red), in an example mouse. n = 10 successfully targeted feeding-excited cells, n = 13 non-targeted excited cells, n = 11 non-targeted inhibited cells. c, Mean Ca2+ responses of indirectly modulated cells in response to stimulation of 10 feeding-excited neurons from a GCaMP6m + bReaChES mouse (5-s stimulation delivered every 30 s for 10 trials). Neurons were considered responsive to the optogenetic stimulus if both the mean dF/F during the 5-s stimulation and the mean dF/F during the 1 s after the 5-s stimulation were significantly different from the baseline mean dF/F during the 5 s before the stimulation. d, Mean Ca2+ responses (in response to caloric-reward delivery during baseline imaging sessions) from NSNF neurons (that had been classified as such by virtue of individual-cell statistics during feeding and social experience) that were found at the population level to demonstrate significant excitation in response to optogenetic activation of feeding cells. n = 97 NSNF cells from 9 mice; mean response (dF/F during 0.5-s grey-shaded bar − dF/F 0 5 s before caloric delivery) = 0.012 ± 0.004; Z = 2.4, P = 0.016, two-sided Wilcoxon signed-rank test. e, Mean Ca2+ responses (in response to caloric-reward delivery during baseline imaging sessions) from NSNF cells (classified as such by virtue of individual-cell statistics during feeding and social experience) that were significantly inhibited by feeding-cell stimulation. n = 95 NSNF cells from 9 mice, mean response (dF/F during 0.5-s grey-shaded bar − dF/F 0.5 s before caloric delivery) = −0.014 ± 0.006; Z = −2.3, P = 0.023, two-sided Wilcoxon signed-rank test. f, The mean positive response of the excited cells was significantly greater than the mean negative response of the inhibited cells. n = 97 excited NSNF cells and 95 inhibited NSNF cells from 9 mice; Z = 3.2, P = 0.0012, two-sided Wilcoxon rank-sum test. g, Cell masks of stimulation-targeted juvenile-conspecific (social; yellow), non-targeted indirectly excited (green), and non-targeted indirectly inhibited (red) neurons from a GCaMP6m + bReaChES mouse. Image from a representative mouse; this experiment was repeated in six mice with similar results. Scale bar, 100 μm. h, Mean Ca2+ responses of social-excited neurons activated by direct stimulation (yellow) and non-targeted cells indirectly excited (green) or inhibited (red) from an example mouse Ten spiral-stimulation targets, n = 7 successfully targeted social cells, n = 20 non-targeted excited cells, n = 16 non-targeted inhibited cells. i, Mean Ca2+ responses of indirectly modulated cells in response to direct activation of social-excited neurons (5-s stimulation delivered every 30 s for 10 trials). j, The percentage of indirectly inhibited cells that were feeding-responsive was significantly greater for social-cell stimulation than for feeding-cell stimulation. Social-cell stimulation: 3 ± 1 indirectly inhibited feeding cells, 11 ± 4 total indirectly inhibited cells; n = 6 mice; feeding-cell stimulation: 0.8 ± 0.5 indirectly inhibited feeding cells, 12 ± 2 total indirectly inhibited cells; n = 9 mice; two-sided Mann–Whitney test, U = 4.50, P = 0.01. k, Average inhibitory responses of non-targeted feeding-excited cells across multiple trials of social-cell stimulation. Ten social-cell stimulation trials, 5-s stimulation, 30-s interval. l, The average magnitude of inhibition of non-targeted feeding cells from social-cell stimulation was significantly greater during the first stimulation trial compared to subsequent stimulation trials. Ten stimulation-targeted social cells per animal; n = 16 indirectly inhibited feeding cells from 6 mice; Z = −2.1, P = 0.04, two-sided Wilcoxon signed-rank test. m, Average inhibitory responses of non-targeted feeding cells during stimulation of non-social-responsive (NS) cells (10 non-social-responsive cell stimulation trials, 5-s stimulation, 30-s interval). n, The average relative inhibition of indirectly inhibited feeding-responsive neurons during the first non-social-cell stimulation trial was significantly less than the relative inhibition during subsequent stimulation trials. Ten stimulation-targeted non-social cells per mouse, n = 24 indirectly inhibited feeding cells from 9 mice; Z = 2.1, P = 0.04, two-sided Wilcoxon signed-rank test. *P < 0.05; n.s., non-significant (P > 0.05).

Supplementary information

Supplementary Information

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Reporting Summary

Supplementary Video 1

: Single-cell two-photon imaging and stimulation of OFC neurons in vivo A two-photon image sequence (right, 100 μm scale bar) synchronized with example Ca2+ traces (left) during sequential spiral stimulation of individual GCaMP6m+bReaChES-expressing OFC cells. Example cells (T1 – T10) targeted with spiral stimulation display reliable optogenetically-evoked time-locked activity responses during stimulation periods (5 s stim, 20 μm spirals, 1 ms spiral duration, 4 revolutions per site, 0.12 ms intersite interval) across multiple stimulation events, while example adjacent non-targeted cells (NT1 – NT3, grey) do not display direct responses to spiral stimulation (n = 10 representative targeted cells, n = 3 representative non-targeted cells, 10 stim trials shown, video playback speed x6).

Supplementary Video 2

: In vivo two-photon Ca2+ imaging of OFC neurons during licking response to caloric reward delivery A two-photon image sequence (left, 100 μm scale bar) and Ca2+ transients (right) from representative neurons classified as feeding-excited (F1 – F3, n = 3 cells, cyan) and non-feeding (NF1 – NF3, n = 3 cells, green) depicting an example animal’s feeding responses (top right) during liquid reward delivery. Recordings from example feeding-excited neurons reveal reliably increased Ca2+ responses during reward delivery in this activity-defined population.

Supplementary Video 3

: Two-photon optogenetic activation of individual OFC feeding-excited neurons in vivo Representative video (speed x5) showing Ca2+ responses (ΔF/F) from feeding-excited OFC neurons expressing GCaMP6m+bReaChES targeted with spiral stimulation (n = 20 cells, spiral target cell masks outlined in cyan, 100 μm scale bar). Feeding-excited cells targeted with sequential spiral stimulation (5 s stim, 20 μm spirals, 1 ms spiral duration, 4 revolutions per site, 0.12 ms intersite interval) at 30-second intervals display reliable time-locked activity responses across multiple stimulation trials (n = 5 total stimulation trials).

Supplementary Video 4

: In vivo two-photon Ca2+ imaging of OFC neurons during social interaction Videos (speed x2.5) of a same-sex juvenile-conspecific (social) stimulus freely-navigating through the social arena and zone (bottom right), while recording Ca2+ activity from the head-fixed adult (top right). Synchronized two-photon image sequence (left, 100 μm scale bar), and corresponding Ca2+ transients (center), illustrate responses of example social-excited (S1-S3, n = 3 cells, yellow) and non-social neurons (NS1-NS3, n = 3 cells, green) during multiple social interaction bouts (n = 3 bouts, highlighted in grey). Social-excited cells reliably display increased activity when the juvenile is in the social zone (i.e. while the juvenile mouse approaches and interacts with the head-fixed adult).

Supplementary Video 5

: In vivo two-photon Ca2+ imaging of OFC neurons during novel object interaction A synchronized two-photon image sequence (left, speed x4) during interaction between a representative head-fixed animal and a 3-D printed mouse (novel object; NO) depicts example cell responses (right) to novel object presentation. Cell responses are represented by Ca2+ transients from example NO-excited neurons (NO1 – NO2 cell masks in purple, n = 2 cells) and social-excited neurons (S1 – S2 cell masks in yellow, n = 2 cells). Activity during example novel object interaction periods (n = 2 interaction periods, highlighted in grey on right) is increased in NO-excited cells, but not in social-excited cells.

Supplementary Video 6

: Two-photon optogenetic activation of individual OFC juvenile-conspecific (social) excited neurons in vivo Representative video (speed x5) showing Ca2+ responses (ΔF/F) from stim-targeted social-excited OFC neurons expressing GCaMP6m+bReaChES (n = 20 cells, spiral target cell masks outlined in yellow, 100 μm scale bar). Social-excited cells targeted with sequential spiral stimulation (5 s stim, 20 μm spirals, 1 ms spiral duration, 4 revolutions per site, 0.12 ms intersite interval) at 30-second intervals display reliable time-locked activity responses across multiple stimulation trials (n=5 total stimulation trials).

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Jennings, J.H., Kim, C.K., Marshel, J.H. et al. Interacting neural ensembles in orbitofrontal cortex for social and feeding behaviour. Nature 565, 645–649 (2019). https://doi.org/10.1038/s41586-018-0866-8

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