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Posterior amygdala regulates sexual and aggressive behaviors in male mice

Nature Neuroscience volume 23, pages 1111–1124 (2020)Cite this article

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Abstract

Sexual and aggressive behaviors are fundamental to animal survival and reproduction. The medial preoptic nucleus (MPN) and ventrolateral part of the ventromedial hypothalamus (VMHvl) are essential regions for male sexual and aggressive behaviors, respectively. While key inhibitory inputs to the VMHvl and MPN have been identified, the extrahypothalamic excitatory inputs essential for social behaviors remain elusive. Here we identify estrogen receptor alpha (Esr1)-expressing cells in the posterior amygdala (PA) as a main source of excitatory inputs to the hypothalamus and key mediators for mating and fighting in male mice. We find two largely distinct PA subpopulations that differ in connectivity, gene expression, in vivo responses and social behavior relevance. MPN-projecting PAEsr1+ cells are activated during mating and are necessary and sufficient for male sexual behaviors, while VMHvl-projecting PAEsr1+ cells are excited during intermale aggression and promote attacks. These findings place the PA as a key node in both male aggression and reproduction circuits.

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Fig. 1: Largely distinct subpopulations of PAEsr1+ neurons project to the MPN and VMHvl.
Fig. 2: PAEsr1+ cells provide monosynaptic excitatory inputs to MPNEsr1+ and VMHvlEsr1+ cells.
Fig. 3: Largely distinct transcriptional profiles for PA subregions projecting to MPN and VMHvl.
Fig. 4: Different response patterns of PAEsr1+MPN and PAEsr1+VMHvl cells during social behaviors.
Fig. 5: Pharmacogenetic inhibition of PAEsr1+MPN and PAEsr1+VMHvl cells impairs sexual and aggressive behaviors.
Fig. 6: Pharmacogenetic activation of PAEsr1+MPN and PAEsr1+VMHvl cells differentially promote sexual and aggressive behaviors in naive males.
Fig. 7: Inputs and outputs of PAEsr1+MPN and PAEsr1+VMHvl cells.

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Data availability

We have uploaded the RNA-seq data from this manuscript to the Gene Expression Omnibus (GEO) under accession number GSE151798. All other data that support the findings of this study are available from the corresponding authors upon request.

Code availability

MATLAB code ScanImage is available at https://github.com/bernardosabatini/SabalabAcq/. MATLAB codes for behavioral annotation and tracking are available from https://github.com/pdollar/toolbox/. Custom TDT OpenEx programs and MATLAB codes for fiber photometry data analysis will be provided upon request to the corresponding authors.

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Acknowledgements

We thank C. Loomis at the NYULMC Experimental Pathology Research Laboratory for help on laser capture microdissection, A. Heguy and Y. Zhang at the NYULMC Genome Technology Center for help on RNA-seq and V. Varshini at the NYULMC Bioinformatics Laboratory for help with sequence alignment. This research was supported by a JSPS overseas fellowship (T.Y.); an Uehara postdoctoral fellowship (T.Y.); NIH grants R01MH107742 and R01MH108594 (B.L.) and R00NS087098 and DP2NS105553 (N.X.T.); the Leon Levy Foundation (N.X.T.); the Dana Foundation (N.X.T.); the Alfred P. Sloan Foundation (N.X.T. and D.L.); the Whitehall Foundation (N.X.T. and D.L.); NIH grants R01MH101377, R21MH105774, 1R01HD092596 and U19NS107616 (D.L.); the Mathers Foundation (D.L.); an Irma T. Hirschl Career Scientist Award (D.L.); and a McKnight Scholar Award (D.L.).

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Authors and Affiliations

  1. Neuroscience Institute, New York University Langone Medical Center, New York, NY, USA

    Takashi Yamaguchi, Dongyu Wei, Soomin C. Song, Nicolas X. Tritsch & Dayu Lin

  2. Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA

    Byungkook Lim

  3. Department of Psychiatry, New York University Langone Medical Center, New York, NY, USA

    Dayu Lin

  4. Center for Neural Science, New York University, New York, NY, USA

    Dayu Lin

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  1. Takashi Yamaguchi
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Contributions

D.L. conceived the project, designed experiments, analyzed data and wrote the paper. T.Y. co-designed and performed most experiments, analyzed data and co-wrote the paper. D.W. and S.C.S. performed slice recording and analyzed data. N.X.T. supervised the slice recording experiment, analyzed data and edited the paper. B.L. generated AAV-Ef1α-fDIO-hM4Di-mCherry and AAV-Ef1α-fDIO-hM3Dq-mCherry.

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Correspondence to Takashi Yamaguchi or Dayu Lin.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Lisa Stowers, Larry Zweifel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 PAEsr1+ cells provide the largest inputs to the medial hypothalamus among all the Esr1+ cells in the amygdala, related to Fig. 1.

a, The retrogradely labeled Esr1+ cells in the amygdala after HSV injection into VMHvl (green) and MPN (red). Scale bar: 200 μm. Images are representative of n = 3 mice. b, c, The distributions of mCherry+ (MPN projecting, b) and EYFP+ cells (VMHvl projecting, c) after injecting HSV-hEf1α-LS1L-mCherry into the MPN and HSV-hEf1α-LS1L-EYFP into the VMHvl of Esr1-2A-Cre male mice. Data in b and c are presented as mean ± s.e.m. PA: posterior amygdala; MeApd: medial amygdala posterior dorsal subdivision; MeApv: medial amygdala posterior ventral subdivision; MeAad: medial amygdala anterior dorsal subdivision; MeAav: medial amygdala anterior ventral subdivision; CoApl: cortical amygdala posterolateral part; CoApm: cortical amygdala posteromedial part; CoAa: cortical amygdala anterior part; BLAa: basolateral amygdala anterior part; BLAp: basolateral amygdala posterior part; BMAp: basomedial amygdala posterior part; PAA: piriform-amygdalar area.

Extended Data Fig. 2 Characterization of the neurotransmitter type of PAEsr1+ neurons, related to Fig. 2.

a and b, Overlay between Vglut1 (a) or Vgat (b) (green) and Esr1 (red) in the PA from bregma level −2.00 to −2.90 mm. Vglut1 and Vgat are visualized using Vgat-ires-Cre × Ai6 and Vglut1-ires-Cre × Ai6 lines, respectively. Right showing the enlarged view of the boxed area. Scale bars: 200 μm (bottom) and 20 μm (upper right). c, The percentage of Vglut1+ and Esr1+ cells in the total neuronal populations in PA, the percentage of PAEsr1+ cells that are glutamatergic or GABAergic, and the percentage of glutamatergic cells expressing Esr1 and the percentage of GABAergic cells expressing Esr1. n = 2 animals for each group. Data in c are presented as mean.

Extended Data Fig. 3 Topographical Fos expression patterns in the PA after mating and fighting, related to Fig. 4.

a-c, Representative images showing the expression of c-Fos (green) and Esr1 (red) in the PA at bregma level −2.30 mm (left) and −2.75 mm (right) after handling (a) (n = 3 animals), mating (b) (n = 3 animals) or fighting (c) (n = 4 animals). Right showing the enlarged views of the boxed areas. Scale bars: 200 μm (left) and 20 μm (right). d, The number of Fos+ neurons in the PA increased significantly after mating and fighting. One-way ANOVA with Tukey’s multiple comparison test. e, Majority of Fos+ cells induced by mating or fighting express Esr1 in the PA. Red and blue dashed lines mark the percentage of Esr1+ cells in the total PA population. Two-tailed unpaired t-test. f, The number of Fos+ neurons expressing Esr1 in the PA along the anterior-posterior axis after handling control (gray), mating (red) or fighting (blue). All data in d, e, and f are presented as mean ± s.e.m. *p < 0.05, ***p < 0.001. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 4 Virus injection and expression sites and optic fiber placements for recording and functional manipulation experiments, related to Figs. 4, 5 and 6.

a–c, Coronal brain sections at the bregma level of MPN, VMHvl and PA showing the virus injection or expression sites (dots) and optic fiber placements (lines) in fiber photometry and pharmacogenetics experiments. Each dot or line represents one animal. Injection and recording sites in each animal are unilateral in (a) and bilateral in (b, c). Brain atlas images are modified from ref. 50.

Extended Data Fig. 5 PAEsr1+MPN cells but not PAEsr1+VMHvl cells increase activity during initiation of sexual behaviors, related to Fig. 4.

a, Representative Ca2+ trace (black) of PAEsr1+ cells and the test animal’s movement velocity (red) during interactions with a female mouse. Vertical lines mark the computer detected velocity changing points that are either followed by mounting attempts within 3 s (magenta) or not (blue). Gray shades mark the manually annotated attempted mount (dark gray), shallow-thrust (median gray) and deep-thrust (light gray). b, Ca2+ signal aligned to the onset of movement initiation, either followed by sexual behaviors (left) or not (right). c, Bar graphs showing that the slope of the Ca2+ signal is significantly positive between 0 and 1 s (shades in b) after movement initiation only if the animal later initiated mounting. The average latency from movement initiation to manually annotated mounting initiation is 1.3 s. d, Velocity of the animal aligned to the automatically detected velocity changing points, either followed by sexual behaviors (left) or not (right). e, Bar graphs showing the velocity change from −1–0 s to 0–1 s in (d) does not differ between trials followed by sexual behaviors and those not. n = 5 animals in b–e. f–j, PAEsr1+VMHvl cells showed no increase in Ca2+ activity during the movement initiation regardless whether it is followed by attempted mounting or not. Plots are organized in parallel to those shown in a–e. n = 6 animals in g–j. All data in c, e, h and j are presented as mean ± s.e.m. One sample two-tailed t-test in c and h. Two-tailed paired t-test in e and j. *p < 0.05. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 6 No change in PAEsr1+MPN and PAEsr1+VMHvl Ca2+ signal during non-social interaction and no change in fluorescence signal during social behaviors in GFP control animals, related to Fig. 4.

a and c, Representative traces showing the GCaMP6 signal of PAEsr1+MPN (a) and PAEsr1+VMHvl (c) cells during object interaction. Gray shades mark sniffing object episodes. (b and d) PETHs of Ca2+ signal (ΔF/F) of PAEsr1+MPN (b) and PAEsr1+VMHvl cells (d) aligned to sniffing object. n = 7 (PAEsr1+MPN) and 5 (PAEsr1+VMHvl) animals. e, A representative image showing GFP (green) expression in the PAEsr1+ cells. Blue: DAPI. Scale bar: 200 µm. Yellow dashed lines indicate the optic fiber location. f, g, Representative Ca2+ traces during interaction with a female (f) and a male intruder (g) introduced into the home cage of the recording mouse. Colored shades mark the behavioral episodes. h, The peak ΔF/F during various social behaviors of all animals. n = 3 animals. All data in h are presented as mean ± s.e.m. One-way ANOVA with post-hoc Tukey’s test. p > 0.05. i–q, PETHs of fluorescence signals aligned to intruder introduction (i, j), sniffing female (k), sniffing male (l), attempted mounting (m), shallow-thrust (n), deep-thrust (o), ejaculation (p), and attack (q). Gray and bold color lines indicate results from individual animals and the population average, respectively. Vertical dashed blue lines indicate time 0. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 7 hM4Di mediated PAEsr1+ cell inactivation impaired male mouse sexual behaviors. Related to Fig. 5.

a, c, d, CNO injection prolonged the latencies to attempt mount (a), shallow thrust (c) and deep thrust (e) in PAEsr1+ hM4Di but not mCherry expressing animals. b, d, f, The durations of shallow thrust (d) and deep thrust (f) significantly decreased after CNO injection in test but not control groups. Note that each test lasts 10 min. For animals that did not show the relevant behaviors within the testing period, the latency will be set at 600 s. n = 5 animals in a–f. All data in a–f are presented as mean ± s.e.m. Two-way ANOVA with repeated measures followed by Bonferroni multiple comparison test. *p < 0.05; **p < 0.01. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 8 hM3Dq mediated activation of PAEsr1+MPN and PAEsr1+VMHvl promotes sexual and aggressive behaviors in naïve male mice in a CNO dose dependent manner, related to Fig. 6.

a, Representative raster plots showing the behaviors towards male and female intruders after saline, 0.1 mg/kg CNO and 0.5 mg/kg CNO i.p. injection in PAEsr1+MPN hM3Dq expressing animals. Scale bar: 60 s. b-g, Both 0.1 mg/kg and 0.5 mg/kg CNO shortened the latency to attempted mount, shallow thrust and increased the duration of attempted mount and shallow thrust while only 0.5 mg/kg promoted female-directed aggression. h–m, CNO injection into PAEsr1+MPN hM3Dq expressing animals did not promote male-directed aggression regardless of the CNO concentration. n, Representative raster plots showing the behaviors towards male and female intruders after saline, 0.1 mg/kg CNO and 0.5 mg/kg CNO i.p. injection in PAEsr1+VMHvl hM3Dq expressing animals. Scale bar: 60 s. o-z, 0.5 mg/kg CNO i.p. injection in PAEsr1+VMHvl hM3Dq expressing animals caused a significant decrease in attack latency (s and y) and an increase in attack duration (t and z) towards both male and female intruders. Animals with 0.1 mg/kg CNO showed an increased trend of attack. Each test lasts 10 min. For animals that did not show the relevant behaviors within the testing period, the latency will be set at 600 s. n = 8 animals in b–m and o–z. All data in b–m and o–z are presented as mean ± s.e.m. Repeated measure One-way ANOVA with Geisser-Greenhouse’s correction followed by Tukey’s multiple comparison test in p; Friedman test followed by Dann’s multiple comparisons test in b–g, l–o and q–z. *p < 0.05; **p < 0.01. Brain atlas images in a are modified from ref. 50. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 9 Optogenetic activation of PAEsr1+VMHvl neurons promotes aggression while optogenetic activating PAEsr1+MPN cells fails to cause behavioral change in naïve male mice, related to Fig. 6.

a, Experimental schematics. b, Representative images showing the expressions of ChR2-EYFP (green) and DAPI (blue) in PAEsr1+MPN (top) and PAEsr1+VMHvl (bottom) cells. Scale bar: 200 μm. Dashed lines outline the PA. Solid white lines indicate the placement of optic fibers. c, Test schedule. d, e, Representative raster plots illustrating behaviors against various intruders during and between light stimulation. Scale bar: 60 s. Top showing light-on period in cyan. Bottom showing behaviors. f–j, Optogenetic activation of PAEsr1+MPN cells did not cause any measurable change in sexual and aggressive behaviors towards any intruder. k–o, The percentage of trials that animals attacked (k), average latency to attack during each trial (l), and the average duration of attack per trial (m) towards male intruder, but not castrated male and female intruder, increased with light stimulation. n, o, Light stimulation did not change duration of sniffing (n) or mounting (o) towards any intruders. p–t, Animals expressing EYFP in PAEsr1+VMHvl neurons showed no behavioral changes during light stimulation. n = 5 animals for each group. All data in f-t shown as mean ± s.e.m. Two-tailed paired t-test (i, k, l, m, n, s and t) and Wilcoxon matched-pairs signed rank test (g, h, j, l, o-t). *p < 0.05. For detailed statistics information, see Supplementary Table 1.

Extended Data Fig. 10 A basic wiring diagram showing triple descending projections from the cerebral hemisphere to the behavior control column that drives social behaviors and general exploration, related to Figs. 1, 7.

In parallel to the classical cortico-striato-nigral circuit that controls general exploration, PA/vSub (cortex equivalent), MeA (striatum equivalent) and BNST (pallidum equivalent) modulate the rostral part of the behavior control column (including MPN and VMHvl) through triple descending projections – a cortical excitatory projection, an striatal inhibitory projection and a pallidal disinhibitory projection. SNr: substantia nigra pars reticulate.

Supplementary information

Reporting Summary

Supplementary Table 1

Statistical results related to Figs.1–7 and Extended Data Figs. 3 and 5–9.

Supplementary Table 2

Normalized counts of genes expressed in PA-VMHvl, PA-MPN and BMAp regions based on RNA-seq.

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Yamaguchi, T., Wei, D., Song, S.C. et al. Posterior amygdala regulates sexual and aggressive behaviors in male mice. Nat Neurosci 23, 1111–1124 (2020). https://doi.org/10.1038/s41593-020-0675-x

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