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. 2023 Dec;26(12):2131-2146.
doi: 10.1038/s41593-023-01475-5. Epub 2023 Nov 9.

Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors

Julieta E Lischinsky  1 , Luping Yin  2 , Chenxi Shi  2   3 , Nandkishore Prakash  4 , Jared Burke  2   5 , Govind Shekaran  2   5 , Maria Grba  2   5 , Joshua G Corbin  4 , Dayu Lin  6   7   8
Affiliations

Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors

Julieta E Lischinsky et al. Nat Neurosci. 2023 Dec.

Abstract

Social behaviors are innate and supported by dedicated neural circuits, but the molecular identities of these circuits and how they are established developmentally and shaped by experience remain unclear. Here we show that medial amygdala (MeA) cells originating from two embryonically parcellated developmental lineages have distinct response patterns and functions in social behavior in male mice. MeA cells expressing the transcription factor Foxp2 (MeAFoxp2) are specialized for processing male conspecific cues and are essential for adult inter-male aggression. By contrast, MeA cells derived from the Dbx1 lineage (MeADbx1) respond broadly to social cues, respond strongly during ejaculation and are not essential for male aggression. Furthermore, MeAFoxp2 and MeADbx1 cells show differential anatomical and functional connectivity. Altogether, our results suggest a developmentally hardwired aggression circuit at the MeA level and a lineage-based circuit organization by which a cell's embryonic transcription factor profile determines its social information representation and behavioral relevance during adulthood.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. MeAFoxp2 and MeADbx1 cells are essentially non-overlapping transcriptionally defined subpopulations.
a, Immunostaining of Foxp2 and GFP (Dbx1-derived cells) in the MeAp of Dbx1cre;Ai6 male mice. Left bottom shows the enlarged view of boxed areas. b, Percentage of MeAFoxp2 and MeADbx1 cells in the total MeAp population. c, The number of Foxp2-only, Dbx1-derived-only and double-positive cells in each side of the MeAp from bregma −1.4 mm to −2.1 mm. d, The total number of counted Foxp2-only, Dbx1-derived-only and double-positive cells in each side of the posterodorsal and posteroventral MeA (MeApd and MeApv). e, The total number of Foxp2-only, Dbx1-derived-only and double-positive cells in each side of the MeAp. f, Immunostaining of Foxp2 (magenta) and GFP (marking Vgat or Vglut2, cyan) in the MeA of Vgatcre;Ai6 or Vglut2cre;Ai6 male mice. The left bottom shows the enlarged view of boxed areas. g, Percentage of MeAFoxp2 cells overlapping with Vgat+ or Vglut2+ cells in the MeAp. h, Percentage of Foxp2+Vgat+ cells over the total Foxp2+ cells in the MeApd and MeApv. i, Percentage of Foxp2+Vglut2+ cells over the total Foxp2+ cells in the MeApd and MeApv. j, Triple in situ hybridization of Vgat (left, orange), Vglut2 (right, orange) and GFP (marking Dbx1-derived cells, green) in the MeAp of Dbx1cre;Ai6 male mice. Left bottom shows the enlarged view of boxed areas. k, Percentage of MeADbx1 cells overlapping with Vgat+ or Vglut2+ cells in the MeAp. l, Percentage of Dbx1-derived+Vgat+ cells over the total Dbx1-derived cells in the MeApd and MeApv. m, Percentage of Dbx1-derived+Vglut2+ cells over the total Dbx1-derived cells in the MeApd and MeApv. For b–e and g–i, every third of 50-µm brain sections was counted. For k–m, every sixth of 20-µm brain sections was counted. The Allen Brain Reference Atlas was used to determine the MeAp subdivisions. n, number of animals. n = 3 mice for all groups. b, Unpaired t-test, P = 0.0018. g–i,k–m, Paired t-tests; P = 0.0004 (g), P = 0.0132 (h), P = 0.0075 (k). All statistical tests are two-tailed. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, otherwise P > 0.05. See Source Data Fig. 1 for more detailed statistics. Source data
Fig. 2
Fig. 2. Distinct responses to social cues of MeAFoxp2 and MeADbx1 cells in head-fixed naive mice.
a, Schematics showing the timeline of stimulus presentation. b, Schematics of viral injection strategy for targeting MeAFoxp2 and MeADbx1 cells. c, Representative histology images of viral injection, denoting GCaMP6f expression (green), Foxp2 antibody (red) and DAPI (blue) staining in Foxp2cre and Dbx1cre;LSL-FlpO mice. White dotted lines mark the optic fiber tracks. d, Percentage of cells co-expressing Foxp2 and GCaMP6f over the total number of GCaMP6f cells in the MeA of Foxp2cre and Dbx1cre;LSL-FlpO mice. e1–e4, Top, representative Ca2+ traces of MeAFoxp2 cells during the presentation of an adult male (e1), an adult female (e2), a pup (e3) and an object (e4). Colored shades represent the duration of the stimulus presentation. Bottom, corresponding heat maps of the z-scored Ca2+ responses (Fz) per animal before and after the onset of each stimulus in MeAFoxp2 cells. f1–f4, Responses of MeADbx1 cells to various stimuli in head-fixed naive male mice. g,h, Average peri-stimulus histograms (PSTHs) of Ca2+ signals from MeAFoxp2 (g) and MeADbx1 (h) cells aligned to the onset (left) and offset (right) of various stimulus presentations. Open circles indicate significantly increased responses (q < 0.05) from the baseline (Fz = 0). Colored lines and shades represent mean responses ± s.e.m. across animals. Dashed lines mark time 0. i,j, Peak Fz signal of MeAFoxp2 (i) and MeADbx1 (j) cells during the presentation of social and non-social stimuli. k, PI of MeAFoxp2 and MeADbx1 cells to different social stimuli. For example, PImale is calculated as (Fzmale − 0.5 × (Fzfemale + Fzpup)) / (|Fzmale| + 0.5 × |Fzfemale + Fzpup|). d, Unpaired t-test. g–h, One-sample t-test for each stimulus with a null hypothesis Fz = 0, corrected for repeated testing with FDR of 0.05. i,j, One-way repeated-measures ANOVA followed by Tukey’s multiple comparisons tests; P < 0.0001 (i) and P = 0.0004 (j) (interaction term). k, Two-way repeated-measures ANOVA followed by Sidak’s multiple comparison tests; P = 0.0006 (interaction term). All statistical tests are two-tailed. n, number of animals. Data are mean ± s.e.m; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, otherwise P > 0.05. See Source Data Fig. 2 for more detailed statistics. Source data
Fig. 3
Fig. 3. Differential response patterns of MeAFoxp2 and MeADbx1 cells during fighting and mating in socially experienced male mice.
a, Schematics of viral strategies and the fiber photometry setup. b, Experimental timeline for Ca2+ recordings in freely moving experienced male mice. c,f,i–l, Representative Ca2+ traces and PETHs of MeAFoxp2 (c–f) and MeADbx1 (i–l) cells during interactions with an adult male, an adult female, a pup and an object. Dashed black lines in PETHs represent the behavior onset at time 0; blue lines in Ca2+ traces indicate time 0 when the intruder is introduced. g,m, Introduction responses of MeAFoxp2 (g) and MeADbx1 (m) cells, calculated as the peak Ca2+ signal within the first 100 s after stimulus introduction. h,n, Average Ca2+ responses of MeAFoxp2 (h) and MeADbx1 (n) cells during behaviors toward various conspecific intruders and a novel object. o, PIs of MeAFoxp2 and MeADbx1 cells showing the relative introduction response magnitudes across different social stimuli. p, PIs of MeAFoxp2 and MeADbx1 cells showing the relative investigation response magnitudes across different social stimuli. g,h,n–p, Mixed-effects analysis followed by Sidak’s multiple comparisons tests; P < 0.0001 (interaction term). m, Friedman test followed by FDR correction; P = 0.0023. h,n, One-sample t-test with null hypothesis Fz = 0, corrected for repeated testing with FDR of 0.05. Parentheses indicate number of animals. All statistical tests are two-tailed. Data are mean ± s.e.m; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #q < 0.05, otherwise P > 0.05 and q > 0.05. See Source Data Fig. 3 for more detailed statistics. Source data
Fig. 4
Fig. 4. Comparison of MeAFoxp2 cell responses in naive versus non-aggressive and aggressive experienced male mice.
a–d, Representative Ca2+ traces of MeAFoxp2 cells during the presentation of an adult male (a), an adult female (b), a pup (c) and an object (d) in naive male mice. e, Average PETHs of MeAFoxp2 cell responses aligned to investigation onset in naive male mice. The dashed black line represents the behavior onset at time 0. f, Average Fz score of MeAFoxp2 cells during investigation of different stimuli in naive male mice. g–i, Representative heat maps showing trial-by-trial Ca2+ signal in normalized Fz (by subtracting the signal at time 0) of MeAFoxp2 cells while investigating a male intruder in a naive (g), experienced non-aggressive (experienced NA) (h) and experienced aggressive (experienced A) (i) male mouse. Black short lines denote the timepoints when Fz ≥ 1. Black dots denote the investigation offsets. j, Average PETHs of MeAFoxp2 cell responses aligned to investigation onset in naive (purple), experienced NA (orange) and experienced A (pink) male mice. k, Percent of male investigation trials in which MeAFoxp2 cells reach Fz ≥ 1. l, Latency of MeAFoxp2 cells to respond (Fz > 1) in responsive trials. m, Average Fz score of MeAFoxp2 cells during male investigation. n, Male PIs of MeAFoxp2 cell responses during investigation across experience. o, Average male investigation duration per trial. f, Friedman test followed by multiple comparison tests with FDR correction; P = 0.0006. One-sample t-test for each stimulus with null hypothesis Fz = 0, corrected for repeated testing with FDR of 0.05. k–m,o, One-way ANOVA followed by Tukey’s multiple comparison tests; P = 0.0013 (interaction term) (k), P = 0.0278 (interaction term) (l), P = 0.0037 (interaction term) (m). m, One-sample t-test with null hypothesis Fz = 0, corrected for repeated testing with FDR of 0.05. n, Kruskal–Wallis test followed by the multiple comparison tests with FDR correction. Parentheses and n indicate the number of animals per group. k–o, Naive group n = 14; Experienced NA group n = 7; Experienced A group n = 14. All statistical tests are two-tailed. Data are mean ± s.e.m. #q < 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, otherwise P > 0.05 and q > 0.05. See Source Data Fig. 4 for more detailed statistics. Source data
Fig. 5
Fig. 5. MeAFoxp2 cell responses before, during and after puberty in developing male mice.
a, Schematics of virus injection and a representative histology image indicating GCaMP6f expression (green), Foxp2 antibody (red) and DAPI (blue) staining in Foxp2cre male mice. White dotted lines mark the fiber end. b, Timeline of virus injection, fiber placement and recordings. c, Pup at P11 before viral surgery and juvenile at P25 before experimental recording. d, Timeline of behavioral test during the recording day. Stimuli were presented in a pseudo-random order. e–h, Representative Fz-scored Ca2+ traces of MeAFoxp2 cells during interactions with an anesthetized (e1–g1) or freely moving (h1) male, an anesthetized (e2–g2) or freely moving (h2) female or a pup (e3–h3) in a male mouse at different ages. Average Fz score during social investigation (e4–h4) of animals at different ages. Average Fz score of the entire intruder session (e5–h5) of animals at different ages. i, Average Fz score of MeAFoxp2 cell responses during male (purple), female (red) and pup (blue) investigation in male mice of different ages. j, Average Fz score of MeAFoxp2 cells per intruder session at different ages. k, Male investigation PIs at different ages. e4–h4,e5–h5, One-way repeated-measures ANOVA followed by Tukey’s multiple comparison tests; interaction terms: P = 0.0003 (e4), P = 0.0071 (e5), P = 0.0084 (f4), P = 0.0295 (g4), P = 0.0009 (h4), P = 0.0026 (h5). One-sample t-test for each behavior with null hypothesis Fz = 0, corrected for repeated testing with FDR of 0.05. i,j, Two-way repeated-measures ANOVA followed by Sidak’s multiple comparison tests; P = 0.1007 (interaction term) (i) and P = 0.0137 (interaction term) (j). The color of the line specifies the social stimulus that evokes significantly different responses over development. k, Kruskal–Wallis test followed by multiple comparison tests with FDR correction. n = 9 (P25), 6 (P30–P32), 6 (P40–P44) and 7 (>P56) mice. n, number of animals. All statistical tests are two-tailed. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #q < 0.05, otherwise P > 0.05 and q > 0.05. See Source Data Fig. 5 for more detailed statistics. Source data
Fig. 6
Fig. 6. Differences in the anatomical and functional inputs of MeAFoxp2 and MeADbx1 cells for sensory processing.
a,c, The timeline of monosynaptic retrograde rabies tracing of MeAFoxp2 (a) and MeADbx1 (c) cells and distribution of starter cells (mCherry+GFP+). b,d, Representative images showing the location of starter MeAFoxp2 cells in a Foxp2cre mouse (b) or MeADbx1 cells in a Dbx1cre;LSL-FlpO mouse (d). TVA-mCherry (red), Rabies-GFP (green) and DAPI (blue) staining. Scale bars, 100 µm (top right). e, Distribution of retrogradely labeled cells. f,h, Representative histological images with cells retrogradely labeled from MeAFoxp2 (f) and MeADbx1 (h) cells. g,i, Overview of inputs into MeAFoxp2 (g) and MeADbx1 (i) cells. j,l, Recording strategy examining synaptic inputs from the AOB to MeAFoxp2 (j) and MeADbx1 (l) cells. k,m, Representative images showing ChrimsonR (red) expression in the olfactory bulb (OB) and ChrimsonR fibers in the MeA. Green: GFP expressed in Foxp2 (k) and Dbx1 (m) cells. Blue: DAPI staining. n,t, The distribution of synaptic responses of MeAFoxp2 (n) and MeADbx1 (t) cells to OB terminal activation. o,u, Representative traces showing optogenetically (1 ms, 605 nm) evoked IPSCs (oIPCSs) and EPSCs (oEPSCs) before and after bath application of TTX and TTX + 4-AP. p–s, Amplitude (p,r) and latency (q,s) of oIPSCs and oEPSCs in MeAFoxp2 and MeADbx1 cells. v–w, oIPSCs in MeAFoxp2 (v) and MeADbx1 (w) cells were abolished by TTX and failed to recover after applying TTX + 4-AP. x, oEPSCs in MeADbx1 cells were abolished by TTX but recovered after TTX + 4-AP application. a,c, n, number of animals for a–i. e, Two-way ANOVA followed by Sidak’s multiple comparison tests; n = 4 mice per group; P < 0.0001. r,s, Mann–Whitney test; P = 0.0002 (r) and P < 0.0001 (s). v–x, Friedman test followed by multiple comparison tests with FDR correction; P = 0.0014, n = 7 cells from three male mice (v); P = 0.0041 and P = 0.0017, respectively (w,x), n = 6 cells from three male mice. n–s, n = 23 cells from three male mice for MeAFoxp2 group and n = 33 cells from three male mice for MeADbx1group, where n is the number used for statistical analysis. All statistical tests are two-tailed. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, otherwise P > 0.05. See Extended Data Table 1 for brain region abbreviations. See Source Data Fig. 6 for more detailed statistics. Source data
Fig. 7
Fig. 7. MeAFoxp2 cells bi-directionally modulate territorial aggression, whereas MeADbx1 cells do not.
a, Strategies for chemogenetic activation of MeAFoxp2 and MeADbx1 cells. b, Representative histological images of hM3Dq (red) expression in the MeA of Foxp2cre and Dbx1cre;LSL-FlpO mice. Top right shows an enlarged view of the MeA infection. Blue: DAPI. c, Experimental timeline of chemogenetic activation experiments. d, Representative raster plots showing behaviors toward male intruders of five Foxp2hM3Dq and five Foxp2mCherry male mice after i.p. injection of saline or CNO. e, Percentage of Foxp2hM3Dq and Foxp2mCherry male mice that attacked a male intruder after saline or CNO injection. f,g, Percent of time Foxp2hM3Dq and Foxp2mCherry mice spent attacking (f) and investigating (g) a male intruder. h–k, Follows the conventions in d–g. CNO injection into Dbx1hM3Dq mice caused a reduction in social investigation but did not change aggressive behaviors toward a male intruder. l, Strategies for chemogenetic inactivation of MeAFoxp2 and MeADbx1 cells. m, Representative histological images showing hM4Di (red) expression in the MeA of Foxp2cre and Dbx1cre;LSL-FlpO mice. Top right shows an enlarged view of the MeA infection. Blue: DAPI. n, Experimental timeline of chemogenetic inactivation experiments. o, Representative raster plots showing behaviors toward male intruders of five Foxp2hM4Di and five Foxp2mCherry mice after i.p. injection of saline or CNO. p–r, Percent of time Foxp2hM4Di and Foxp2mCherry male mice spent investigating (p) and attacking (q) a male intruder and the latency to first attack (r). s–v, Follows the conventions in o–r. CNO injection into Dbx1hM4Di or Dbx1mCherry mice did not change any male-directed behaviors in comparison to those after saline injection. e,i, McNemar’s test; P = 0.0412 (e). f,g,j,k,p–r,t–v, Two-way repeated-measures ANOVA followed by Sidak’s multiple comparison tests; interaction terms: P = 0.0004 (f), P = 0.004 (g), P = 0.2653 (k), P = 0.0550 (p), P = 0.0091 (q) and P = 0.1836 (r). n, number of animals. All statistical tests are two-tailed. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, otherwise P > 0.05. See Source Data Fig. 7 for more detailed statistics. Source data
Fig. 8
Fig. 8. Outputs of MeAFoxp2 and MeADbx1 cells.
a,c, Strategies for anterograde viral tracing of MeAFoxp2 (a) and MeADbx1 (c) cells. Pie charts showing the distribution of infected cells. b,d, Representative histological images showing the infected cells in Foxp2cre (b) and Dbx1cre;LSL-FlpO (d) mice. Green: GCaMP6f expression. Blue: DAPI staining. e,g, Representative histological images showing MeAFoxp2 (e) and MeADbx1 (g) projections at various downstream regions. The gain of PA and BNST images in g was reduced to avoid complete saturation. f,h, Overviews of MeAFoxp2 (f) and MeADbx1 (h) cell outputs. i, The intensity of MeAFoxp2 and MeADbx1 projection fields in various regions, calculated as the average pixel intensity in a given region divided by the maximum mean intensity value among all regions with ≥0.2 normalized intensity and the VMHvl. j, The intensity of fibers, originating from MeAFoxp2 and MeADbx1 cells, at AVPV and MPOA (anterior medial hypothalamic regions) over the VMHvl and PMv (posterior medial hypothalamic regions). The dotted line denotes y = 1. k, The intensity of fibers, originating from MeAFoxp2 and MeADbx1 cells, at BNSTpr over that in BNSTif. The dotted line denotes y = 1. i, Two-way repeated-measures ANOVA followed by Sidak’s multiple comparison tests. j,k, Two-tailed unpaired t-test; P = 0.0081 (j) and P = 0.0095 (k). Parentheses and n indicate number of animals. All statistical tests are two-tailed. Data are mean ± s.e.m. **P < 0.01, otherwise P > 0.05. One-sample t-test for each behavior with null hypothesis ratio = 1, corrected for repeated testing with FDR of 0.05. #q < 0.05, otherwise q > 0.05. See Extended Data Table 1 for brain region abbreviations. See Source Data Fig. 8 for more detailed statistics. Source data
Extended Data Fig. 1
Extended Data Fig. 1. Characterization of MeAFoxp2 and MeADbx1 cell distribution in the MeApd, related to Fig. 1.
(a) Representation of a histological MeAp image. The open circles and the dashed line indicate the optic tract used to determine the medial/lateral location of MeApdFoxp2 and MeApdDbx1 cells. (b-f) Distribution of MeApdFoxp2 (magenta) and MeApdDbx1 (green) cell distance to the optic tract at Bregma −1.5 mm (b), −1.6 mm (c), −1.7 mm (d), −1.8 mm (e) and −1.9 mm (f). (g) Representation of a histological MeAp image. The open circles and the dashed line indicate the MeA dorsal edge used to determine the dorsal/ventral location of MeApdFoxp2 and MeApdDbx1 cells. (h-l) Distribution of MeAFoxp2 (magenta) and MeADbx1 (green) cell distance from the dorsal edge at Bregma −1.5 mm (h), −1.6 mm (i), −1.7 mm (j), −1.8 mm (k) and −1.9 mm (l). Magenta and green dashed lines denote the median medial-lateral (b-f) and ventral-lateral (h-l) locations of the MeApdFoxp2 and MeApdDbx1 cells, respectively. White arrows indicate how the distance to the optic tract (a) or the dorsal edge of the MeApd (g) is calculated. n = 133-568 from 3 animals for each group (where n is the number used for statistical analysis). (b-f, h-l) Two-tailed Mann Whitney tests; (b) p = 0.0018, (c) p = 0.0002, (e, i-l) p < 0.0001. **p < 0.01; ***p < 0.001; ***p < 0.0001. Otherwise, p > 0.05. See Source Data Extended Data Fig. 1 for more detailed statistics. Source data
Extended Data Fig. 2
Extended Data Fig. 2. Additional characterization of MeAFoxp2 and MeADbx1 cell responses in experienced male mice, related to Fig. 3.
(a) Average PETHs of MeAFoxp2 Ca2+ signal aligned to the onset (left) and offset (right) of investigation only (blue) and investigation-followed-by-attack (purple) trials. Open circles denote the period with significantly different responses (p < 0.05). (b) Average PETHs of MeADbx1 cell responses aligned to the onset (left) and offset (right) of investigation only (blue) and investigation-followed-by-attack (purple) trials. The two traces do not differ significantly at any time point. (a and b) Two-tailed one-sample t-test at each time point corrected for repeated testing with FDR 0.05. Parentheses denote the number of animals. Data are mean ± SEM. See Source Data Extended Data Fig. 2 for more detailed statistics. Source data
Extended Data Fig. 3
Extended Data Fig. 3. MeAFoxp2 cell responses to male and female mice from multiple strains, related to Fig. 3.
(a-d) Representative Ca2+ traces of MeAFoxp2 cells during the presentation of a BALB/c male (a), a C57BL/6 male (b), a C57BL/6 female (c), and a 129S4/SvJae female (d) mouse. (e) Average PETHs of MeAFoxp2 cell responses aligned to investigation onset of mice from different strains. The dashed black line represents the behavior onset at time zero. (f) Average Fz scores of MeAFoxp2 cells during investigation of male and female mice from different strains; p = 0.0052. Friedman test followed by multiple comparison tests with correction for FDR of 0.05. n = number of animals. Data are mean ± SEM; *p < 0.05, **p < 0.01. Otherwise, p > 0.05. See Source Data Extended Data Fig. 3 for more detailed statistics. Source data
Extended Data Fig. 4
Extended Data Fig. 4. Quantification of behaviors towards different intruders in Foxp2GCaMP and Dbx1GCaMP male mice, related to Fig. 3.
(a) Percent of time naïve Foxp2GCaMP, socially experienced Foxp2GCaMP and socially experienced Dbx1GCaMP male mice spent investigating a male intruder. (b) The fraction of naïve Foxp2GCaMP, socially experienced Foxp2GCaMP and socially experienced Dbx1GCaMP male mice that attacked a male intruder. (c-e) Percent of time naïve Foxp2GCaMP, socially experienced Foxp2GCaMP and socially experienced Dbx1GCaMP male mice spent attacking a male intruder (c), as well as the latency to first attack (d) and total number of attack events (e). (f-l) Percent of time Foxp2GCaMP and Dbx1GCaMP male mice spent investigating (f), mounting (g), and intromitting (j) a receptive female intruder, as well as the total number of mounting events per recording session (h), latency to first mount (i), total number of intromission events (k) and latency to ejaculation (l). (m) Percent of time Foxp2GCaMP and Dbx1GCaMP male mice spent investigating a pup intruder. (a, c-m) Two-tailed unpaired t-tests (if pass normality test) or two-tailed Mann Whitney tests (if not pass normality test) between naïve Foxp2GCaMP and socially experienced Foxp2GCaMP animals, and between socially experienced Foxp2GCaMP and Dbx1GCaMP animals; (d) p = 0.0148, (f) p < 0.0001, (h) p = 0.0039 between naïve Foxp2GCaMP and socially experienced Foxp2GCaMP animals, (j) p = 0.0045 between naïve Foxp2GCaMP and socially experienced Foxp2GCaMP animals, (k) p = 0.0016 between naïve Foxp2GCaMP and socially experienced Foxp2GCaMP animals, (m) p = 0.0025 between socially experienced Foxp2GCaMP and socially experienced Dbx1GCaMP male mice. (b) Two-tailed Chi-square tests; p = 0.0023 between naïve Foxp2GCaMP and socially experienced Foxp2GCaMP animals. n = number of animals. Data are mean ± S.E.M.; *p < 0.05, **p < 0.01, ***p < 0.001. Otherwise, p > 0.05. See Source Data Extended Data Fig. 4 for more detailed statistics. Source data
Extended Data Fig. 5
Extended Data Fig. 5. GCaMP viral expression and investigation time in Foxp2cre mice at P25, related to Fig. 5.
(a) Percent of time P25 Foxp2cre mice spent investigating an anaesthetized male, anaesthetized female or pup intruder after fiber implantation surgery at P24 or without surgery. Two-way ANOVA followed by Sidak’s multiple comparison tests. All p values > 0.05. Parentheses denote the number of animals. Data are mean ± SEM. (b) Schematic of virus injection and a representative histology image indicating GCaMP6f expression (green), Foxp2 antibody (red), and DAPI (blue) staining in Foxp2cre male mice at P25. White dotted lines mark the fiber end. See Source Data Extended Data Fig. 5 for more detailed statistics. Source data
Extended Data Fig. 6
Extended Data Fig. 6. Additional behavioral assays after chemogenetic activation and inactivation of MeAFoxp2 cells, related to Fig. 7.
(a) In Foxp2hM3Dq and Foxp2mCherry male mice, the latency to attack a male intruder after CNO injection did not differ from that after saline injection. Only animals that showed attack after both saline and CNO injections were included in this analysis. (b) Percent of time Foxp2hM3Dq male mice spent attacking a male intruder during three consecutive days of testing after 1st day i.p. saline injection (SAL), 2nd day CNO injection (CNO), and 3rd day saline injection (SAL2). (c) No changes in velocity (pixels/frame) in Foxp2hM3Dq or Foxp2mCherry male mice were observed in a 5 min period 30 min after CNO or saline injection when the test animal was alone in its cage. (d) Number of Foxp2hM3Dq or Foxp2mCherry male mice that attacked pups vs. those that did not after saline or CNO injection. Each circle represents one mouse. (e) Percentage of time Foxp2hM3Dq or Foxp2mCherry male mice spent investigating the pup after saline or CNO injection. (f) Representative raster plots showing the behaviors towards an adult female intruder of 5 Foxp2hM3Dq and 5 Foxp2mCherry mice after i.p. injection of saline or CNO. (g-l) Between CNO-injected and saline-injected days, there is no difference in any parameters related to male sexual behaviors in Foxp2mCherry and Foxp2hM3Dq male mice. (m) Representative raster plots showing the behaviors towards an adult female intruder of 5 Foxp2hM4Di and 5 Foxp2mCherry mice after i.p. injection of saline or CNO. (n-s) Between CNO-injected and saline-injected days, there is no difference in any parameters related to male sexual behaviors in Foxp2mCherry and Foxp2hM4Di male mice. (b) Friedman tests followed by multiple comparison tests with FDR correction; p < 0.0001. (c, e, g-l, n-s) Two-way repeated measures ANOVA followed by Sidak’s multiple comparison tests; (d) Two-tailed McNemar’s test. n = number of animals. Data are mean ± SEM. **p < 0.01, ***p < 0.001. Otherwise, p > 0.05. See Source Data Extended Data Fig. 6 for more detailed statistics. Source data
Extended Data Fig. 7
Extended Data Fig. 7. The aggression level of Foxp2cre and Dbx1cre;LSL-FlpO naïve male mice, related to Fig. 7.
(a) Percent of time Foxp2cre and Dbx1cre;LSL-FlpO male mice spent investigating a male intruder. (b) Percent of Foxp2cre and Dbx1cre;LSL-FlpO male mice that attacked a male intruder. (c) Percent of time Foxp2cre and Dbx1cre;LSL-FlpO male mice spent attacking a male intruder. (d) Attack latency of Foxp2cre and Dbx1cre;LSL-FlpO male mice that attacked the male intruder. (e) Number of attacks of Foxp2cre and Dbx1cre;LSL-FlpO male mice towards a male intruder. Data are the same as those in Fig. 7e–k after day 1 saline injection. (a,d) Two-tailed unpaired t-test. (b) Two-tailed Chi-Square test; p = 0.0455 (c,e) Two-tailed Mann-Whitney test; (c) p = 0.0465. n = number of animals. Data are mean ± SEM; *p < 0.05. Otherwise, p > 0.05. See Source Data Extended Data Fig. 7 for more detailed statistics. Source data
Extended Data Fig. 8
Extended Data Fig. 8. Additional behavioral assays during chemogenetic activation and inactivation of MeADbx1 cells, related to Fig. 7.
(a) In Dbx1hM3Dq and Dbx1mCherry male mice, the latency to attack a male intruder after CNO injection did not differ from that after saline injection. Only animals that showed attack after both saline and CNO injections were included in this analysis. (b) Velocity (pixels/frame) of Dbx1hM3Dq or Dbx1mCherry male mice in a 5 min period after 30 min CNO or saline injection when the test animal was alone in its home cage. (c) Number of Dbx1hM3Dq and Dbx1mCherry male mice that attacked pups vs. those that did not after saline or CNO injection. Each circle represents one mouse. (d) Percentage of time Dbx1hM3Dq and Dbx1mCherry male mice spent investigating the pup after saline or CNO injection. (e) Representative raster plots showing the behaviors towards an adult female intruder of 5 Dbx1hM3Dq and 5 Dbx1mCherry mice after i.p. injection of saline or CNO. (f-k) No difference in male sexual behaviors after CNO injection in comparison to saline injection in Dbx1mCherry or Dbx1hM3Dq male mice. (l) Representative raster plots showing the behaviors towards an adult female intruder of 5 Dbx1hM4Di and 5 Dbx1mCherry mice after i.p. injection of saline or CNO. (m-r) No difference in male sexual behaviors after CNO injection in comparison to saline injection in Dbx1mCherry or Dbx1hM4Di male mice. (a, b, d, f-k, m-r) Two-way repeated measures ANOVA followed by Sidak’s multiple comparison tests. (c) Two-tailed McNemar’s test. n = number of animals. Data are mean ± SEM. All p > 0.05. See Source Data Extended Data Fig. 8 for more detailed statistics. Source data
Extended Data Fig. 9
Extended Data Fig. 9. Brain regions downstream of MeAFoxp2 and MeADbx1 cells, related to Fig. 8.
(a-b) Representative images of 6 brain regions showing the GFP fibers originating from MeAFoxp2 (a) and MeADbx1 (b) cells.
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