Abstract
To survive in a complex social group, one needs to know who to approach and, more importantly, who to avoid. In mice, a single defeat causes the losing mouse to stay away from the winner for weeks1. Here through a series of functional manipulation and recording experiments, we identify oxytocin neurons in the retrochiasmatic supraoptic nucleus (SOROXT) and oxytocin-receptor-expressing cells in the anterior subdivision of the ventromedial hypothalamus, ventrolateral part (aVMHvlOXTR) as a key circuit motif for defeat-induced social avoidance. Before defeat, aVMHvlOXTR cells minimally respond to aggressor cues. During defeat, aVMHvlOXTR cells are highly activated and, with the help of an exclusive oxytocin supply from the SOR, potentiate their responses to aggressor cues. After defeat, strong aggressor-induced aVMHvlOXTR cell activation drives the animal to avoid the aggressor and minimizes future defeat. Our study uncovers a neural process that supports rapid social learning caused by defeat and highlights the importance of the brain oxytocin system in social plasticity.
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Data availability
Raw values associated with each figure panel can be found in the source data files. Fibre photometry recording data, behaviour annotations, tracking and raw representative histology images can be downloaded from Zenodo (https://doi.org/10.5281/zenodo.8417540). Behaviour videos and additional histology images are available from the corresponding authors upon reasonable request. They are not deposited to a public database owing to their large size and size limitation of online depositories. Illustrations of the coronal brain sections in Figs. 1a, 2a,g,m, 3a,h, 4j, 5a and 6a,h,n and Extended Data Figs. 4a, 5a, 6a, 9a,k, 10a, 12a, 14a and 15a were adapted from the Allen Brain Reference Atlas (https://atlas.brain-map.org). Source data are provided with this paper.
Code availability
Matlab codes used in this study can be downloaded from Zenodo (https://doi.org/10.5281/zenodo.8417540).
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Acknowledgements
We thank R. C. Froemke, G. Buzsáki and all members from the Lin Laboratory for discussions and comments; R. Kenmochi for cartoon illustrations; and M. Karadas for helping set up the shock–odour pairing chamber. This research was supported by NIH grants R01MH101377 (D.L.), R01MH124927 (D.L.), 1R01HD092596 (D.L.), U19NS107616 (D.L., A.C.M. and R.W.T.) and 1R01NS125271 (R.W.T.), the Uehara Memorial Foundation (T.O.), a JSPS Overseas Research Fellowship (T.O.), an Osamu Hayaishi Memorial Scholarship (T.O.) and the Ichiro Kanehara Foundation (T.O.).
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D.L. conceived the project, designed experiments, analysed data, prepared figures, wrote the paper and supervised the project. T.O. co-designed experiments, performed all histology, functional manipulation and in vivo recording experiments in males, analysed data, prepared figures and co-wrote the paper. R.Y. performed all slice recording experiments, analysed data and prepared relevant figures and texts. Y.J. performed all functional manipulation and in vivo recording experiments in females and did most of the animal tracking. D.W. performed preliminary slice recordings. R.T. assisted with behaviour testing and analyses. B.D. and A.C.M. assisted with animal tracking. X.W. assisted with initial Oxtr KO characterization. G.Z., C.X.W. and J.-J.L. assisted with behaviour annotation. R.W.T. supervised X.W., G.Z., C.X.W. and J.-J.L. and provided critical feedback to the project.
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Extended data figures and tables
Extended Data Fig. 1 One-day 10-min social defeat is sufficient to induce social avoidance of winner-like conspecifics.
a. Experimental timeline. b. Cartoon illustration of the social interaction (SI) test (top) and a video frame overlaid with DLC tracking results (bottom). Red dot: body center; green dot: head center; yellow dot: nose point; blue dot: cup center. c. The resident-intruder (RI) test illustration and procedure. d. The latency to first defeat in male and female mice. e. The total defeat duration during the 10-min RI test in male and female mice. f. Video frames from SI tests overlaid with the movement trajectories of the SW mother aggressor (maroon) and C57 test female (pink) during the 1st (top) and 4th (bottom) minute of the test. g. The percentage of time the male (g1) and female (g2) test mice and the aggressors spent on staying stationary (velocity <1 pixel/frame) over the course of the RI tests. h. Heatmaps showing the body center location of a representative female mouse during pre-defeat and post-defeat SI tests. i-j. Representative traces showing the distance between the test animal body center to the cup center (i) and the movement velocity of the test animal (j) in pre- and post-defeat SI tests. Color shades indicate manually annotated behaviors. k. Distribution of the distance between the test animal’s body center and cup center during the pre-defeat (gray) and post-defeat (color) SI tests for males (k1) and females (k2). Shades shown in gray represent the distance range considered as “around cup”. l. The percentage of total time the male (l1) and female (l2) test mice spent around the aggressor cup (distance <250 pixels) during SI tests. m. The percentage of total time the male (m1) and female (m2) test mice spent investigating the aggressor cup during SI tests. n. The average duration of each investigation episode of the male (n1) and female (n2) test mice during SI tests. o. The cup approach frequency of the male (o1) and female (o2) test mice during SI tests. p. Accumulative plots showing the distribution of movement velocity when the male (p1) and female (p2) test mice are far away (distance > 300 pixels) from the constrained aggressor during the pre-defeat (gray) and post-defeat (color) SI tests. q. The median velocity when the male (q1) and female (q2) test mice are far away (distance > 300 pixels) from the constrained aggressor during SI tests. r. Experimental design to test whether social avoidance after defeat is specific to the same aggressor. The aggressor in the RI and SI tests is the same in the AAA paradigm. Different SW aggressors are used for RI and SI tests in the ABA paradigm. s. Distribution of the distance between the test animal’s body center and constrained aggressor during the pre- and post-defeat SI tests for AAA (s1) and ABA (s2) paradigms. t. Accumulative plots showing the distribution of movement velocity when the test mice are far away (distance > 300 pixels) from the constrained aggressor during the pre-defeat and post-defeat SI tests in AAA (t1) and ABA (t2) paradigms. u. The total defeat time during the RI tests in AAA and ABA paradigms is comparable. v. The change index of time spent around the constrained aggressor during the SI tests in AAA and ABA paradigms. The change index is defined as (Ppost-Ppre)/(Ppost+Ppre). Ppre and Ppost are the behavior parameter values during the pre-defeat and post-defeat SI tests, respectively. w. The change index of investigation time of the constrained aggressor during the SI tests in AAA and ABA paradigms. x. The change index of the median movement velocity when the test animal is far away from the aggressor in the SI tests in AAA and ABA paradigms. Plots with shades and error bars represent mean ± s.e.m. Circles and lines represent individual animals. Numbers on the plots indicate the number of animals. Mann-Whitney test (d, v, w), unpaired t-test (e, u, x), two-way repeated measure ANOVA with Sidak’s multiple comparisons test (g), Wilcoxon matched-pairs signed rank test (l1, m1, o1, l2, m2, and n2), and paired t-test (n1, q1, o2, and q2). All statistical tests are two-tailed. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 2 Defeated animals do not avoid conspecifics with genetic backgrounds different from the aggressor.
a. Experimental timeline. b. The left shows a cartoon illustration and a snapshot of the multi-animal social interaction (MSI) test. The right shows the stimulus animals used for male and female MSI tests. c-d. Heatmaps showing the body center location of a representative male (c) and a female (d) mouse in pre- and post-defeat MSI tests. e-f. Total time male test mice spent investigating (e) and around (f) each constrained animal during pre- and post-defeat MSI tests. g. The number of approaches towards each cup during pre- and post-defeat MSI tests. h-j. Data from female mice. Plots follow the convention of e-g. Plots with error bars represent mean±s.e.m. Lines represent individual animals. Numbers on the plots indicate the number of animals. (e-j) Two-way repeated measure ANOVA with Sidak’s multiple comparisons test. All statistical tests are two-tailed. **p < 0.01, and ****p < 0.0001. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 3 The relationship between OXTR and defeat-induced c-Fos and Esr1 in the VMHvl.
a. Representative histological images showing c-Fos (red) and OXTR (OXTRZsGreen, green) expression in the aVMHvl (Bregma: −1.50 mm) and pVMHvl (Bregma: −1.82 mm) in OXTRCre:Ai6 (OXTRZsGreen) male mice after attack or social defeat. Insets showing enlarged views of the boxed areas. Dashed lines mark the boundary of the aVMH. Scale bars: 50 µm. b. The number of c-Fos and OXTR double-positive cells after attack and defeat in the aVMHvl (Bregma: −1.34 to −1.50 mm) and pVMHvl (Bregma: −1.66 to −1.82 mm). c. Representative histological images showing OXTR (green) and Esr1 (red) expression in the aVMHvl (Bregma: −1.50 mm) and pVMHvl (Bregma: −1.82 mm) in OXTRCre:Ai6 (OXTRZsGreen) male mice. Insets showing enlarged views of the boxed areas. Dashed lines mark the boundary of the VMH. Scale bars: 100 µm. d. Number of OXTR and Esr1 positive cells in the aVMHvl and pVMHvl. e. The percentage of Esr1 and OXTR double-positive cells in OXTR positive cells in the aVMHvl and pVMHvl. f. The percentage of Esr1 and OXTR double-positive cells in Esr1 positive cells in the aVMHvl and pVMHvl. Plots with error bars represent mean±s.e.m. (b, d, e and f) n = three 40-µm sections were counted per region per animal, 3 animals per group. Circles in b and lines in d-f represent individual animals. (b and d) Two-way repeated measure ANOVA with Sidak’s multiple comparisons test. (e) Paired t-test. (f) Mann-Whitney test. All statistical tests are two-tailed. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 4 aVMHvlOXTR cells increase responses to the aggressor after defeat in male mice.
a. Schematics of virus injection and a representative histology image. Dashed line marks the aVMH. Scale bar: 200 µm. Brain illustration in is based on a reference atlas from https://atlas.brain-map.org/. b. Cartoon illustration of the RI test. c-e. Representative Z-scored GCaMP6f traces during RI tests with a BC male intruder (c), a SW male intruder (d), or resident SW (e). c2, d2 and e2 show the enlarged boxed areas. f. Post-event histograms (PETHs) of GCaMP6f signal aligned to initial opponent encounters, only including sessions without defeat or attack during the first 10 s. g. Peak GCaMP6f response within the first 10 s of RI tests. h-i. PETHs of GCaMP6f signals aligned to close investigation (h) and agonistic interactions (i). j. Averaged Z-scored responses during various social behaviors. k. Experimental timeline. l. Heatmaps showing the body center location of a representative test male during pre- and post-defeat SI tests. m. Distribution of the distance between the test animal’s body center and cup center during pre- and post-defeat SI tests. n. The percentage of total time test mice spent around the aggressor cup during pre- and post-defeat SI tests. o. The percentage of total time the test mice spent investigating the constrained aggressor during pre- and post-defeat SI tests. p. Representative Z-scored GCaMP6f traces from a recording male mouse during pre-defeat (p1) and post-defeat (p2) SI tests. Shades mark investigation events. q. PETHs of Z-scored GCaMP6f signals aligned to the onset of investigating aggressor during pre- and post-defeat SI tests. r. Average Z-scored ΔF/F of aVMHvlOXTR cells during investigating aggressor in pre- and post-defeat SI tests. s. Scatter plot showing the correlation between investigation time change index and change in Z-scored GCaMP response to the aggressor during post-defeat SI tests from the pre-defeat level. t. Representative Z-scored GCaMP trace (black) overlaid with the velocity trace (orange) during the post-defeat SI test. Blue indicates the period when the test animal quickly retreated from the aggressor. u. PETHs of Z-scored GCaMP signal (black) and velocity (orange) aligned to the retreat onset. v. The retreat onset GCaMP signal is significantly higher than the retreat offset signal. w. Representative Z-scored GCaMP trace (black) overlaid with the velocity trace (orange) during the post-defeat SI test. Gray indicates when the test animal stayed immobile and far from the aggressor. x. PETHs of Z-scored GCaMP signal (black) and velocity (orange) aligned to immobility onset. Immobility trials are defined as velocity <1 pixel/frame lasting for > 0.5 s. y. The mean GCaMP signal during immobility. Plots with shades and error bars represent mean±s.e.m. Lines and circles represent individual animals. Numbers on the plots indicate the number of animals. One-way ANOVA with Tukey’s multiple comparisons (g), Two-way repeated-measure ANOVA with Sidak’s multiple comparisons (j), Wilcoxon matched-pairs signed rank test (n and o), paired t-test (r, v), one-sample t-test (y), and Pearson cross-correlation (s). All statistical tests are two-tailed. *p < 0.05. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 5 Female aVMHvlOXTR cells increase responses to the aggressor after defeat.
a. Schematics of virus injection and a representative histology image. Dashed lines mark the boundary of the aVMH. Scale bar: 200 µm. Brain illustration is based on a reference atlas from https://atlas.brain-map.org/. b. Experimental timeline. c. Cartoon illustration of the SI test. d-e. Representative Z-scored GCaMP6f traces from a recording female mouse during RI tests with a non-aggressive naïve C57 (d) and an aggressive lactating SW (e) female mouse. d2 and e2 show the zoomed-in view of the boxed area in d1 and e1, respectively. f. PETHs of Z-scored GCaMP6f signals aligned to C57 intruder introduction and introduction of the test mouse to the SW lactating female’s cage. Only sessions with no defeat during the first 10 s are included. g. The peak GCaMP6f response within the first 10 s of intruder/resident encounter. Only sessions with no defeat or attack during the first 10 s are included. h-i. PETHs of Z-scored GCaMP6f signals aligned to the onset of investigating C57 intruders (h), and investigating and being defeated by SW residents (i). j. Average Z-scored ΔF/F of aVMHvlOXTR cells during various social behaviors in the RI tests. k. Heatmaps showing the body center location of a representative test female during pre-defeat and post-defeat SI tests. l. Distribution of the distance between the test animal’s body center and cup center during pre- and post-defeat SI tests. m. The percentage of the total time the test mice spent around the aggressor cup during pre- and post-defeat SI tests. n. The percentage of the total time the test mice spent investigating the aggressor cup during pre- and post-defeat SI tests. o. Representative Z-scored GCaMP traces from a recording female during pre-defeat (o1) and post-defeat (o2) SI tests. Shades represent investigation events. p. PETHs of Z-scored GCaMP signals aligned to the onset of investigating the constrained aggressor during pre- and post-defeat SI tests. q. Average Z-scored ΔF/F of aVMHvlOXTR cells during investigating aggressor in pre- and post-defeat SI tests. r. Representative Z-scored GCaMP trace (black) overlaid with the velocity trace (orange) during the post-defeat SI test. Gray indicates the periods when the test animal stayed immobile and far from the aggressor, and blue indicates a fast retreat event. s. PETHs of Z-scored GCaMP signal (black) and velocity (orange) aligned to the retreat onset. t. The GCaMP signal at the retreat onset is significantly higher than the signal at the retreat offset. u. PETHs of Z-scored GCaMP signal (black) and velocity (orange) aligned to immobility onset. Immobility trials are defined as velocity <1 pixel/frame lasting > 0.5 s. v. The mean GCaMP signal during immobility. Plots with shades and error bars represent mean±s.e.m. Circles and lines represent individual animals. Numbers on the plots indicate the number of animals. Kruskal-Wallis test with Dunn’s multiple comparisons test (j), Wilcoxon matched-pairs signed rank test (n), unpaired t-test (g), paired t-test (m, q, t), and one-sample t-test (v). All statistical tests are two-tailed. *p < 0.05, **p < 0.01, and ****p < 0.0001. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 6 aVMHvlOXTR cells increase response to the aggressor after defeat in female mice.
a. Virus injection site and a representative histology image. Scale bar: 200 µm. Brain illustration is based on a reference atlas from https://atlas.brain-map.org/. b. Experimental timeline and cartoon illustration of the behavior assay. c-e. Summary plots showing the investigation time (c), time around each cup (d), and number of approach (e) during pre- and post-defeat MSI tests. E: Empty; Cv: C57 virgin female; Sv: unfamiliar SW virgin female; Sm: SW lactating female aggressor. f. Representative raw traces showing the Z-scored GCaMP6 signal in the pre- (f1) and post-defeat (f2) MSI tests. Shades represent investigation episodes. Empty cup investigation episodes are not marked. g. PETHs aligned to the investigation onset of different stimuli in pre- and post-defeat MSI tests. h. The mean Z-scored GCaMP6 signal during investigation of different targets in pre- and post-defeat MSI tests. h1 and h2 are the same data shown in different arrangements. i. Scatter plots showing the correlation between change index in investigation time and change in Z-scored GCaMP responses to various social targets after defeat from the pre-defeat level. Plots with error bars and shades represent mean±s.e.m. Circles and lines represent individual animals. Numbers on the plots indicate the number of animals. (c, d, e, h) Two-way repeated measure ANOVA with Sidak’s multiple comparisons test. (i) Pearson cross-correlation. All statistical tests are two-tailed. *p < 0.05, **p < 0.01, and ***p < 0.001. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 7 Defeat experience enhances aggressor cue-induced c-Fos in aVMHvlOXTR cells during subsequent encounters.
a. Experimental design. CCC: SI (Constrained SW)-SI (Constrained SW)-SI (Constrained SW) (top); DDC: RI (Defeat)-RI (Defeat)-SI (Constrained SW) (bottom). b and c. Representative images showing the expression of OXTR (OXTRZsGreen, green) and c-Fos (red) in the aVMHvl (b) and pVMHvl (c) in animals experienced CCC or DDC. Insets showing enlarged views of the boxed areas in the aVMHvl and pVMHvl. Dashed lines mark the boundary of the VMH. Scale bars: 50 µm. d. The percentage of CCC and DDC-induced c-Fos cells that express OXTR in the aVMHvl and pVMHvl. Numbers indicate the number of animals. Plots with error bars represent mean±s.e.m. Circles indicate individual animals. Two-way repeated measure ANOVA with Sidak’s multiple comparisons test. All statistical tests are two-tailed. *p < 0.05. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 8 No change in excitability of aVMHvlOXTR cells one day after defeat.
a. Representative recording traces of aVMHvlOXTR cells under specific current steps, ranging from 50 pA to 250 pA, from single-housed (SH), defeated (D) and socially interacted (SI) male mice. b. Frequency-current (F-I) curve of aVMHvlOXTR cells in SH, D, and SI groups. Two-way repeated measure ANOVA with Sidak’s multiple comparison test. *p < 0.05 for D vs. SI comparisons. If not indicated, p > 0.05. c. Resting Membrane Potential (RMP) of aVMHvlOXTR cells in SH, D, and SI groups. d. Rheobase of aVMHvlOXTR cells in SH, D, and SI groups. e. Input resistance of aVMHvlOXTR cells in SH, D, and SI groups. Plots with error bars represent mean±s.e.m. Circles in c-e represent individual recording cells. Numbers on the plots indicate the number of cells. Cells are from 3-4 male mice per group. One-way ANOVA with Tukey’s multiple comparisons test (c) and Kruskal-Wallis test with Dunn’s multiple comparisons test (d-e). All statistical tests are two-tailed. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 9 aVMHvlOXTR cells do not respond to non-social aversive odors.
Virus injection schematics and a representative histology image. Scale bar: 200 µm. Brain illustration is based on a reference atlas from https://atlas.brain-map.org/. a. Experimental timeline for testing 2MT responses and cartoon illustration of the behavioral assay. b. Distribution of the distance between the test animal’s body center and constrained aggressor (gray) or 2MT (orange) during the test. The test animals were defeated one day before the recording. c. The percentage of the total time the test mice spent around the constrained aggressor or 2MT (distance <250 pixels) during the test. Only animals that showed clear avoidance of the aggressor (< 20% of total time investigating the constrained aggressor) were included in the analysis. d. The percentage of the total time the test mice spent investigating the constrained aggressor or 2MT. e. Accumulative plots showing the distribution of movement velocity when the test mice are far away (distance > 300 pixels) from the constrained aggressor or 2MT. f. The median velocity of the test mice when far from the constrained aggressor or 2MT. g. A representative trace showing continuous Z-scored GCaMP signal during aggressor (magenta) and 2MT (orange) encounters. Shade represents investigation episodes. Dashed lines indicate the constrained aggressor and 2MT introduction. h. PETHs of Z-scored GCaMP signals aligned to the onset of investigating the aggressor and 2MT. i. Average Z-scored ΔF/F of aVMHvlOXTR cells during aggressor and 2MT investigation. j. Virus injection schematics. Brain illustration is based on a reference atlas from https://atlas.brain-map.org/. k. Experimental timeline for the shock-odor conditioning and testing. US-paired odor is always delivered after unpaired odor presentation. l. A representative trace showing the movement velocity of an animal when exposed to shock-paired and unpaired odors one day after shock-odor conditioning. Bars indicate pre-odor and odor delivery periods for calculation in (n). m. A summary of median velocity before and during odor delivery in the post-conditioning test. n. A representative Z-scored GCaMP recording trace of an animal when exposed to shock-paired and unpaired odors one day after shock-odor conditioning. Bars indicate pre-odor and odor delivery periods for calculation in (p). o. A summary of mean GCaMP response (Z-scored ΔF/F) before and during odor delivery in the post-conditioning test. Plots with error bars and shades represent mean±s.e.m. Numbers on the plots indicate the number of animals. Lines represent individual animals. Wilcoxon matched-pairs signed rank test (d); Paired t-test (e, g and j); One-way ANOVA with repeated measures followed by Tukey’s multiple comparisons test (n and p). All statistical tests are two-tailed. *p < 0.05. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 10 aVMHvlOXTR cells do not respond to non-social aversive cues or noxious somatosensory stimuli in head-fixed animals.
a. (left) Virus injection location and the schematics of head-fixed fiber photometry recording of aVMHvlOXTR cells. Brain illustration is based on a reference atlas from https://atlas.brain-map.org/. (right) Experimental timelines. The responses to shock-paired and unpaired odors are recorded in one session one day after the shock-odor conditioning. The responses to saline, 2MT, and aggressor urine are recorded in a separate session one day after defeat. b. Representative raw GCaMP trace of aVMHvlOXTR cells during delivery of saline (b1), aggressor urine (b2), 2MT (b3), shock-unpaired odor (b4) and shocked-paired odor (b5). All stimuli are presented on Q-tips placed approximately 1 cm in front of the mouse nose for 10 s. c. PETHs of Z-scored GCaMP signals aligned to the onset of various odor presentations. Red dotted horizontal lines indicate Z = 0. Gray and black vertical dashed lines indicate the onset and average offset of stimulus presentation. d. Average Z-scored ΔF/F during various stimulus presentations. Circles represent individual animals. One-way ANOVA with repeated measures followed by Tukey’s multiple comparisons test. *p < 0.05, and **p < 0.01. e. Representative raw GCaMP trace of aVMHvlOXTR cells during gentle touch (e1), back pinch (e2), back poke (e3), and tail pinch (e4). All stimuli were manually delivered. f. PETHs of Z-scored GCaMP signals aligned to the onset of various somatosensory stimuli. Gray and black vertical dashed lines indicate the onset and average offset of stimulus presentation. g. Average Z-scored ΔF/F during various stimulus presentations. Lines represent individual animals. One-way ANOVA with repeated measures followed by Tukey’s multiple comparisons test. Plots with error bars and shades represent mean±s.e.m. Numbers on the plots indicate the number of animals. All statistical tests are two-tailed. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 11 Behavior changes induced by optogenetic activation of aVMHvlOXTR and SOROXT cells.
a. Experimental timeline, light delivery protocol, and cartoon illustration of the behavioral assay. b. Video frames from SI tests overlaid with movement trajectories of a GFP (gray) and a ChR2 animal (green) during interleaved light-on (ON) and light-off (OFF) trials. c-f. Average distance to the constrained male (c), percentage of time spent investigating the constrained BC male (d), frequency of approaching the cup (e), and the median movement velocity (f) during light-on (blue) and light-off periods in GFP (black) and ChR2 (green) groups. Statistical results were between ON and OFF periods in ChR2 animals. All p > 0.05 for GFP animals. g. Experimental timeline, stimulation protocol, and cartoon illustration of the behavioral test. h. Representative traces showing the movement velocity of an GFPOXTR animal (h1) and an ChR2OXTR animal (h2) during the light-on (blue shade) and light-off period. i. Plots showing the distribution of movement velocity during the light-on and light-off periods of GFP (i1) and ChR2 (i2) animals. j. The percentage of time the animals spent immobile (velocity <1 pixel/frame). k. The percentage of time the animals spent flight (velocity > 20 pixels/frame). l. Experimental timeline and schematic illustration of the real-time place preference test. m. Heatmaps showing the body center distribution of representative GFPOXTR and ChR2OXTR animals during the 10-min RTPP tests. n. The percentage of time the animals spent in the light-paired chamber. Circles represent individual animals. o. Heatmaps showing the body center distribution of representative GFPOXT and ChR2OXT animals during the 10-min RTPP tests. p. The percentage of time the animals spent in the light-paired chamber. Plots with shades and error bars represent mean±s.e.m. Lines and circles represent individual animals. Numbers indicate the number of animals. Two-way RM ANOVA with Sidak’s multiple comparisons test (c-f, j-k) and unpaired t-test (n, p). All statistical tests are two-tailed. *p < 0.05, **p < 0.01, and ***p < 0.001. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 12 SOROXT affects aVMHvl cell activity by activating OXTR, not glutamatergic synaptic transmission.
a. Strategy to evaluate OXTR knockout efficiency. b. Representative recording traces of GFP cells from knockout (KO) and control (Ctrl) sides under TGOT perfusion (red bar). c. Number of aVMHvl cells (from 4 animals) depolarized ( > 4 mV) by TGOT in KO and control sides. Chi-square’s test. **p < 0.01. d. Representative traces showing the membrane potential changes of two aVMHvl cells, one recorded in ACSF (d1) and the other in the presence of 1 µM L-368, 889, a highly specific OXTR antagonist (d2). e. The number of cells depolarized (ΔRMP > 4 mV) by the SOROXT optogenetic stimulation (20 Hz, 1 ms, 5 min) and not. n = 23 (without OXTRA) cells from 5 animals and 23 (with OXTRA) cells from 4 animals. Chi-square’s test. ****p < 0.0001. f-h. Histology images showing oxytocin (OXT, red) immunostaining and Vglut2 (green) expression in the PVN (f), SON (g), and SOR (h) from Vglut2Cre:Ai6 male mice. Scale bars: 50 µm. i. The percentage of OXT-positive cells that express Vglut2 in the PVN, SON, and SOR. Circles represent individual animals. Plotted as mean±s.e.m. n = 3 male mice. j. Slice recording schematics. k-l. Representative voltage clamp recording traces from aVMHvlOXTR cells when a 1 ms light pulse (blue vertical bar) was delivered to activated PVNOXT (k) or SOROXT (l) input. m. None of the aVMHvlOXTR cells showed light-evoked EPSC during PVNOXT (0/14 cells) or SOROXT optogenetic activation (0/12 cells). See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 13 Overlap between OXT and defeat-induced c-Fos.
a. Defeat-induced c-Fos (red) and oxytocin (OXT, green). Scale bars: 50 µm. b-c. Number of c-Fos+OXT+ cells (b) and percentage of c-Fos+ cells in OXT+ cells (c) in different regions. Every other brain section was counted. Plotted as mean±s.e.m. Lines represent individual animals. One-way RM ANOVA with Tukey’s multiple comparisons (b-c). All statistical tests are two-tailed. **p < 0.01. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 14 SOROXT cells do not increase responses to aggressors after defeat in male mice.
a. Schematics of virus injection and a representative histological image for fiber photometry recording of SOROXT cells in male mice. The dashed line marks SOR. Scale bar: 200 µm. Brain illustration is based on a reference atlas from https://atlas.brain-map.org/. b. Experimental timeline. c. Heatmaps showing the body center location of a recording mouse in MSI tests before and after defeat. E: empty; BC: familiar non-aggressive Balb/C male; C57: unfamiliar C57 male; and SW: SW aggressor. d. Time spent around each cup during MSI tests before and after defeat. e-f. Representative Z-scored GCaMP6f traces from a male recording mouse during pre-defeat (e) and post-defeat (f) MSI tests. Shades represent investigation periods of different constrained stimulus animals. Periods investigating the empty cup are not marked. g. PETHs of Z-scored GCaMP6f signals aligned to the onset of investigation of different constrained stimuli. Gray: pre-defeat; Color: post-defeat. h. Average Z-scored ΔF/F of SOROXT cells during the investigation of various constrained stimuli in the pre-defeat and post-defeat MSI tests. Plots with shades and error bars represent mean±s.e.m. Lines represent individual animals. Numbers on the plots indicate the number of animals. Two-way repeated measure ANOVA with Sidak’s multiple comparisons (d, h). All statistical tests were two-tailed. See Supplementary Table 1 for detailed statistics.
Extended Data Fig. 15 SOROXT cells in female mice are activated by noxious stimuli.
a. Schematics of virus injection and a representative histological image for fiber photometry recording of SOROXT cells in female mice. The dashed line marks SOR. Scale bar: 200 µm. Brain illustration is based on a reference atlas from https://atlas.brain-map.org/. b. Experimental timeline. c-d. Representative Z-scored GCaMP6f traces of SOROXT cells from an animal that encountered a naïve C57BL/6 female intruder (c) or a SW lactating female mouse in the SW cage (d). c2 and d2 show enlarged views of boxed areas in c1 and d1, respectively. e. PETH of Z-scored GCaMP6f signal aligned to C57 female intruder introduction. As all lactating mothers attacked the test mouse within 10 s, the introduction response cannot be isolated. f. The peak GCaMP6f response within the first 10 s after C57 intruder introduction. g-h. PETHs of Z-scored GCaMP6f signals aligned to close investigation (CI) of C57BL/6 female intruders (g), investigating and being defeated by SW mothers (h). i. Average Z-scored ΔF/F of SOROXT cells during various social behaviors. j. Heatmaps showing the body center location of a recording mouse in MSI tests before and after defeat. E: empty; Cv: familiar C57BL/6 virgin female; Sv: unfamiliar virgin SW female; and Sm: SW mother. k. Time spent around each cup during MSI tests before and after defeat. l. Representative Z-scored GCaMP6f traces from a female recording mouse during pre-defeat (l1) and post-defeat (l2) MSI tests. Shades represent investigation periods of different constrained stimulus animals. Periods investigating the empty cup are not marked. m. PETHs of Z-scored GCaMP6f signals aligned to the onset of investigation of different constrained stimuli. Gray: pre-defeat; Color: post-defeat. n. Average Z-scored ΔF/F of SOROXT cells during the investigation of various constrained stimuli in the pre-defeat and post-defeat MSI tests. o. Schematics of head-fixed fiber photometry recording of SOROXT cells and presented stimuli. p. Representative raw GCaMP6f trace of SOROXT cells during delivery of aggressor urine on a Q-tip, gentle touch, back pinch, back poke, and tail pinch. q. PETHs of Z-scored GCaMP6f signals aligned to the onset of aggressor urine presentation (q1), gentle touch (q2), back pinch (q3), back poke (q4), and tail pinch (q5). Gray and black dashed lines indicate the onset and average duration of stimulus delivery, respectively. r. Average Z-scored ΔF/F during various stimulus delivery. Plots with shades and error bars represent mean±s.e.m. Circles and lines represent individual animals. Numbers on the plots indicate the number of animals. Kruskal-Wallis test with Dunn’s multiple comparisons test (i); Two-way repeated measure ANOVA with Sidak’s multiple comparisons test (k, n); and One-way repeated measure ANOVA with Tukey’s multiple comparisons test (r). All statistical tests are two-tailed. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. See Supplementary Table 1 for detailed statistics.
Supplementary information
Supplementary Information
Supplementary notes, including additional discussion regarding the role of the SOROXT–aVMHvlOXTR circuit in defeat-induced social avoidance and social fear.
Supplementary Table 1
Detailed statistical results for all analyses shown in Figs. 1–6, Extended Data Figs. 1–15 and Supplementary Fig. 1.
Supplementary Video 1
Fibre photometry recording of aVMHvlOXTR cell GCaMP signals during MSI tests 1 day before and 1 day after defeat. Before defeat, the cells showed little response to all stimulus constrained animals. After defeat, the cells increased responses to the constrained SW aggressor but not other social stimuli.
Supplementary Video 2
After optogenetic activation of aVMHvlOXTR cells (light on), the undefeated naive animal showed strong avoidance of the constrained SW aggressor.
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Osakada, T., Yan, R., Jiang, Y. et al. A dedicated hypothalamic oxytocin circuit controls aversive social learning. Nature 626, 347–356 (2024). https://doi.org/10.1038/s41586-023-06958-w
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DOI: https://doi.org/10.1038/s41586-023-06958-w
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