Functional identification of an aggression locus in the mouse hypothalamus

Journal name:
Nature
Volume:
470,
Pages:
221–226
Date published:
DOI:
doi:10.1038/nature09736
Received
Accepted
Published online

Abstract

Electrical stimulation of certain hypothalamic regions in cats and rodents can elicit attack behaviour, but the exact location of relevant cells within these regions, their requirement for naturally occurring aggression and their relationship to mating circuits have not been clear. Genetic methods for neural circuit manipulation in mice provide a potentially powerful approach to this problem, but brain-stimulation-evoked aggression has never been demonstrated in this species. Here we show that optogenetic, but not electrical, stimulation of neurons in the ventromedial hypothalamus, ventrolateral subdivision (VMHvl) causes male mice to attack both females and inanimate objects, as well as males. Pharmacogenetic silencing of VMHvl reversibly inhibits inter-male aggression. Immediate early gene analysis and single unit recordings from VMHvl during social interactions reveal overlapping but distinct neuronal subpopulations involved in fighting and mating. Neurons activated during attack are inhibited during mating, suggesting a potential neural substrate for competition between these opponent social behaviours.

At a glance

Figures

  1. Fos catFISH analysis of cell activation during fighting versus mating.
    Figure 1: Fos catFISH analysis of cell activation during fighting versus mating.

    af, c-fos expression patterns following single (a, b) or two sequential (cf) social interactions. Boxed areas are enlarged to right of each panel. Blue, Topro-3 nuclear counterstain. Red, c-fos cytoplasmic transcripts (cRNA probe); yellow dots, nuclear c-fos transcripts (red cRNA plus green intron probe signals). Scale bars, 10µm. g, Percentage of total cells expressing c-fos after the 2nd behaviour (nuclear signal) that also expressed c-fos after the 1st behaviour (nuclear + cytoplasmic signal) (one-way ANOVA with Bonferroni correction). *P<0.05, **P<0.01, ***P<0.001.

  2. Response patterns of a VMHvl neuron during social encounters.
    Figure 2: Response patterns of a VMHvl neuron during social encounters.

    a, Video frames taken from consecutive trials with intruder animals of the indicated sex and strain. bf, Average firing rate (over 0.5s bins; ±s.e.m.) during indicated behavioural episodes (manually annotated, frame-by-frame) from five exemplar cells. ‘Before,’ before introducing stimulus animal; ‘No contact,’ periods during encounter without physical contact between intruder and resident . g, Recordings from the cell in e, middle. Blue trace, superimposed individual spikes; red line, average spike shape. Scale bars, 200µV, 200µs. Raster plots illustrate 300s of continuous recording. Coloured shading and arrow mark manually annotated behavioural episodes. h, Schematics indicating cell response type at each recording site from Bregma level −1.35mm to −1.65mm. Anatomical structures based on Allen Brain Atlas (http://www.brain-map.org). fx, fornix; ARH, arcuate nucleus; v3, third ventricle; TU, tuberal nucleus.

  3. Summary of cell responses in VMHvl during mating and fighting.
    Figure 3: Summary of cell responses in VMHvl during mating and fighting.

    a, b, Percentage of cells excited (red) or inhibited (blue) during encounters with male (a) or female (b) mice. c, Numbers of cells exhibiting statistically significant changes in firing rate (see Methods) towards males or females. d, Firing rate changes for all 104 recorded cells, averaged over entire encounter with males or females. e, Firing rate changes averaged over all 104 recorded cells, during various behavioural episodes. Grey, behaviour not applicable (N/A) to the stimulus animal.

  4. Optogenetic activation of VMHvl elicits attack in mice.
    Figure 4: Optogenetic activation of VMHvl elicits attack in mice.

    a, Schematic illustrating optic fibre placement; VMHvl shaded in blue. be, Fos induction (red) in EF1α::ChR2–EYFP-expressing (green) cells at 1h post-illumination. Fos+ cells outside EYFP+ region may be synaptic targets of ChR2-activated cells. Blue, fluorescent Nissl stain. f, LacZ expression identifies infected cell bodies (red). Scale bar, 500µm. gj, LacZ expression (red) and native ChR2–EYFP fluorescence (green) largely overlaps. Boxed areas in c, h enlarged at lower right. Scale bars in be, gj, 50µm or 10µm (insets). k, Raster plots illustrating behavioural episodes (legend below) in a ChR2-expressing male paired with a female in two consecutive tests. l, Attack onset/offset latencies (relative to initiation versus termination of illumination) towards indicated intruders, **P<0.01). m, Efficacy of light-stimulated attack. ‘Optimized light intensity’, laser power yielding average maximal response in each animal (range: 1–3.3mWmm−2). ‘1mWmm−2’, average response obtained at this power (t-test, P = 0.06). n, Percentage of animals attacking moving versus non-moving anaesthetized animals or inflated glove (yellow shading). o, Percentage of trials inducing attacks towards female during successive stages of mating. *P<0.05, ** P<0.01 (one-way ANOVA with Bonferroni correction). ns, not significant. p, Distribution of infected cells in each animal, plotted as cells per section in VMHvl posterior portion versus that in (VMHdm+VMHc) region. Colour code indicates whether illumination induced freeze/flight (green), attack (red) or no change in behaviour (blue). See also Supplementary Footnote 4 for further statistical analysis.

  5. Reversible inhibition of natural aggression by genetic silencing of VMHvl
    Figure 5: Reversible inhibition of natural aggression by genetic silencing of VMHvl

    a, Anti-GFP antibody staining (green) in mice bilaterally infected with AAV2-GluClα + AAV2-GluClβ. Scale bar, 500µm. be, Overlap between GluCl-expressing (green) and Fos-expressing (red) cells, 1h after fighting. Blue, Topro-3 nuclear stain. Inset in c represents boxed area. Yellow cells are double-labelled. Scale bars, 50µm or 10µm (inset). f, g, Percent change in cumulative attack duration (f) and latency (g) during a 600s resident-intruder trial before versus 24h after IVM injection. Test, GluCl virus-injected animals (n = 33) (red bar). Control, no surgery (white bar, n = 12), saline (black bar, n = 6) or GluClβ virus-injected animals (n = 12, grey bar) (**P<0.01, *P<0.05, t-test). h, Cumulative attack duration during repeated cycles of IVM injection or washout (*P<0.05, Bonferroni after tests of two-way ANOVA with repeated measures). Test, GluCl virus-injected animals (n = 12); Control, saline (n = 6). i, j, Percent change in mount duration (i) or latency (j) in test (n = 12) versus control (GluClβ virus injected, n = 12) males paired with females. k, Percentage of infected cells in posterior portion of VMHvl (Bregma −1.4–1.8mm) plotted against extent of aggression suppression after IVM injection. The Pearson correlation coefficient is significantly higher than 0 (P<0.001). See Supplementary Fig. 19 for further analysis.

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Author information

Affiliations

  1. Division of Biology 216-76, California Institute of Technology, 1201 East California Boulevard, Pasadena, California 91125, USA

    • Dayu Lin,
    • Hyosang Lee &
    • David J. Anderson
  2. Howard Hughes Medical Institute, California Institute of Technology, 1201 East California Boulevard, Pasadena, California 91125, USA

    • Dayu Lin &
    • David J. Anderson
  3. Allen Institute for Brain Science, Seattle, Washington, 98103, USA

    • Maureen P. Boyle &
    • E. S. Lein
  4. Computation and Neural Systems 136-93, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA

    • Piotr Dollar &
    • Pietro Perona

Contributions

D.L. designed, carried out and analyzed preliminary fos catFISH experiments and all other experiments, and co-wrote the manuscript; M.B. and E.L. performed additional fos catFISH experiments; P.D. and P.P. developed custom behaviour annotation software; H.L. performed some of the optogenetic experiments; D.J.A. conceived the project, suggested experiments, analysed data and co-wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Information (2.8M)

    The file contains Supplementary Figures 1-19 with legends, Supplementary Methods, Supplementary Footnotes 1-4 and additional references.

Movies

  1. Supplementary Movie 1 (10.7M)

    The sound in this movie corresponds to the response of a neuron recorded from a C57BL/6 male during investigation and attack of a BALB/c male. Viewers are kindly requested not to upload this movie to other publicly accessible sites.

  2. Supplementary Movie 2 (12.8M)

    The sound in this movie corresponds to the response of the same neuron shown in Supplementary Movie 1 during investigation and mounting of a C57BL/6 female. Viewers are kindly requested not to upload this movie to other publicly accessible sites.

  3. Supplementary Movie 3 (9.5M)

    The movie shows an attack towards a BALB/c female is induced by light stimulation of a C57BL/6 male mouse in the VMHvl region. Viewers are kindly requested not to upload this movie to other publicly accessible sites.

  4. Supplementary Movie 4 (9.2M)

    The movie shows an attack towards a glove, which is induced by stimulating VMHvl in a C57BL/6 male mouse. Viewers are kindly requested not to upload this movie to other publicly accessible sites.

Additional data