Abstract
Innate defensive behaviors triggered by environmental threats are important for animal survival. Among these behaviors, defensive attack toward threatening stimuli (for example, predators) is often the last line of defense. How the brain regulates defensive attack remains poorly understood. Here we show that noxious mechanical force in an inescapable context is a key stimulus for triggering defensive attack in laboratory mice. Mechanically evoked defensive attacks were abrogated by photoinhibition of vGAT+ neurons in the anterior hypothalamic nucleus (AHN). The vGAT+ AHN neurons encoded the intensity of mechanical force and were innervated by brain areas relevant to pain and attack. Activation of these neurons triggered biting attacks toward a predator while suppressing ongoing behaviors. The projection from vGAT+ AHN neurons to the periaqueductal gray might be one AHN pathway participating in mechanically evoked defensive attack. Together, these data reveal that vGAT+ AHN neurons encode noxious mechanical stimuli and regulate defensive attack in mice.
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Data availability
All data supporting the findings of this study are provided within the paper and its Supplementary Information. All additional information will be made available upon reasonable request to the authors. Source data are provided with this paper.
Code availability
The MATLAB code for data analyses is available from the corresponding author upon reasonable request.
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Acknowledgements
We thank the members of the Neuroscience Pioneer Club for valuable discussions. This work was supported by the National Natural Science Foundation of China (31925019 to P.C. and 32171018 to F.Z.), the National Key R&D Program of China (2021ZD0202701 to P.C.), the Natural Science Foundation of Hebei Province (C2020206027 to F.Z.) and institutional grants from the Chinese Ministry of Science and Technology to the National Institute of Biological Sciences (NIBS). All data are archived in the NIBS.
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Contributions
P.C., C.Z., Z.Z. and F.Z. conceived the study. Z.X. performed injections and fiber implantation. H.G., X.C., T.T. and Y.X. performed behavioral tests. C.S. and H.G. performed EMG recording. H.G. performed fiber photometry recording. Z.X., X.C. and M.H. conducted histological analyses. M.H. maintained the snake. C.S. and H.G. performed slice physiology. J.Z., W.L., Z.Z., F.Z. and Z.T. provided reagents. D.L., C.S., H.G., Z.X. and P.C. analyzed data. P.C. wrote the manuscript.
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Peer review information Nature Neuroscience thanks Richard Palmiter, Philip Tovote 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 Analyses of sensory-triggered defensive attack.
(a) Behavioral ethograms of two example mice exhibiting mechanically evoked defensive attack in an enclosed arena with (top) and without (bottom) ambient light. (b) Quantitative analyses of time spent for biting-like attack of mice in response to mechanical stimuli on the tail in an arena with and without ambient light. (c) Quantitative analyses of time for biting-like attack of mice in response to a dummy snake with or without providing mechanical stimuli on four limbs. (d, e) An example picture (left) and quantitative analyses of time spent for biting-like attack (right) showing defensive attack to a plastic lid (d) and a wood block (e) that were linked to mechanical stimuli in an enclosed arena. (f) Quantitative analyses of time spent in biting-like attack of male and female mice in response to mechanical stimuli on the tail (clip + ). (g) Schematic diagrams showing the test mice were subject to an alligator clip (a context with high escapability) (left) or an alligator clip connected to a heavy dummy snake (a context with low escapability) (right). (h) Quantitative analyses of biting-like attack in mice, showing the time spent for defensive attack against an alligator clip with a dummy snake (low escapability) was significantly higher than that against an alligator clip without a dummy snake (high escapability). (i) Schematic diagrams showing the test mice were subject to an alligator clip in a large arena (a context with high escapability) (left) or a small arena (a context with low escapability) (right). (j) Quantitative analyses of biting-like attack in mice, showing the time spent for defensive attack against an alligator clip in small arena (low escapability) was significantly higher than that against an alligator clip in large arena (high escapability). Numbers of mice were indicated in the graphs (b-f, h, j). Data in (b-f, h, j) are means ± SEM. Statistical analyses in (b-f, h, j) were performed by two-sided Student t-tests (n.s. P > 0.1; *** P < 0.001). For the P values, see Supplementary Table 4.
Extended Data Fig. 2 Analyses of cell-type specificity of vGlut2-IRES-Cre and vGAT-IRES-Cre lines in the AHN.
(a, b) Example micrographs showing the distributions of vGat mRNA (a) and vGlut2 mRNA (b) in the AHN of WT mice. (c) Quantitative analyses of number of AHN cells expressing vGat mRNA (green) and vGlut2 mRNA (red) in coronal sections, as indicated by the distance to bregma. (d-f) An example coronal section showing the distribution of EGFP (green) and vGat mRNA (red) in the AHN of vGAT-IRES-Cre mice, which were injected with AAV-DIO-EGFP in the AHN (d). Example micrographs (e) and statistical analyses (f) showing EGFP and vGat mRNA were mostly colocalized in the same AHN neurons. (g-i) An example coronal section showing the distribution of EGFP (green) and vGlut2 mRNA (red) in the AHN of vGAT-IRES-Cre mice, which were injected with AAV-DIO-EGFP in the AHN (g). Example micrographs (h) and statistical analyses (i) showing EGFP and vGlut2 mRNA were largely segregated in different AHN cells. (j-l) An example coronal section showing the distribution of EGFP (green) and vGat mRNA (red) in the AHN of vGlut2-IRES-Cre mice, which were injected with AAV-DIO-EGFP in the AHN (j). Example micrographs (k) and statistical analyses (l) showing EGFP and vGat mRNA were mostly segregated in different AHN cells. (m-o) An example coronal section showing the distribution of EGFP (green) and vGlut2 mRNA (red) in the AHN of vGlut2-IRES-Cre mice, which were injected with AAV-DIO-EGFP in the AHN (m). Example micrographs (n) and statistical analyses (o) showing EGFP and vGlut2 mRNA were mostly colocalized in the same AHN neurons. Arrows indicate the dually labeled cells (e, n). Numbers of mice are indicated in the graphs (c, f, i, l, o). Data in (c, f, i, l, o) are means ± SEM. Statistical analyses in (c) were performed by One-Way ANOVA (*** P < 0.001). The experiment (d, e, g, h, j, k, m, n) was repeated three times independently with similar results.
Extended Data Fig. 3 GCaMP signals of AHN vGAT + neurons recorded with fiber photometry in non-social contexts.
(a) Schematic diagram showing a mouse receiving noxious mechanical stimuli applied with a clip. (b) An example trace of normalized EGFP fluorescence changes (ΔF/F) in a mouse that received noxious mechanical stimuli applied with a clip which were indicated by red vertical lines in the ethogram. (c) Averaged EGFP fluorescence changes of an example mouse before and after the initiation of noxious mechanical stimuli. (d) Schematic diagram showing a mouse exhibiting risk assessment to snake. (e) An example trace of normalized GCaMP fluorescence changes (ΔF/F) in a mouse that exhibited risk assessment to snake, which was indicated by red vertical lines. (f) Averaged GCaMP fluorescence changes (ΔF/F) of an example mouse before and after the initiation of risk assessment. (g) Schematic diagram showing a mouse exhibiting exploration to a wood block. (h) An example trace of normalized GCaMP fluorescence changes (ΔF/F) in a mouse that exhibit wood-block exploration, which was indicated with red vertical lines. (i) Averaged GCaMP fluorescence changes (ΔF/F) of an example mouse before and after the initiation of object exploration. (j) Schematic diagram showing a mouse on top of a hot-plate (25 °C or 55 °C). (k) Two example traces of normalized GCaMP fluorescence changes (ΔF/F) in a mouse that was placed onto a hot-plate at either 55 °C or 25 °C. (l) Averaged GCaMP fluorescence changes (ΔF/F) of an example mouse before and after the limbs touched the hot plate. (m) Quantitative analyses of EGFP fluorescence changes of vGAT+ AHN neurons before and after the initiation of mechanical stimuli. (n-p) Quantitative analyses of GCaMP fluorescence changes (ΔF/F) before and after the initiation of risk assessment (n), object exploration (o), and touching the hot plate (p). Data in (c, f, i, l-p) were means ± SEM (error bars). Numbers of mice were indicated in the graphs (m-p). Statistical analyses (m-p) were performed by two-sided Student t-tests (*** P < 0.001; * P < 0.05; n.s. P > 0.1). For the P values, see Supplementary Table 4. Scale bars are labeled in the graphs.
Extended Data Fig. 4 GCaMP signals of vGAT + AHN neurons recorded with fiber photometry in social contexts.
(a) Schematic diagram showing a male C57BL/6 mouse was attacked by a male CD1 mouse. (b) An example trace of normalized GCaMP fluorescence changes (ΔF/F) in vGAT+ AHN neurons of a C57BL/6 mouse that was attacked by a male CD1 mouse. Attacks were indicated by red vertical lines. (c) Averaged GCaMP fluorescence changes (ΔF/F) of an example C57BL/6 mouse before and after the initiation of social attack by CD1 mouse. (d) Schematic diagram showing a male C57BL/6 mouse exhibiting social investigation to a male C57BL/6 mouse. (e) An example trace of normalized GCaMP fluorescence changes (ΔF/F) in a C57BL/6 mouse that exhibited social investigation to a male C57BL/6 mouse. Social investigation was indicated by red vertical lines. (f) Averaged GCaMP fluorescence changes (ΔF/F) of an example C57BL/6 mouse before and after the initiation of social investigation. (g) Schematic diagram showing a male C57BL/6 mouse exhibiting social investigation to a female C57BL/6 mouse. (h) An example trace of normalized GCaMP fluorescence changes (ΔF/F) in a C57BL/6 mouse that exhibited social investigation to a female C57BL/6 mouse. Social investigation was indicated by red vertical lines. (i) Averaged GCaMP fluorescence changes (ΔF/F) of an example C57BL/6 mouse before and after the initiation of social investigation. (j-l) Quantitative analyses of GCaMP fluorescence changes (ΔF/F) of 7 test mice before and after the initiation of social attack by a CD1 mouse (j), social investigation to a male C57BL/6 mouse (k) or female C57BL/6 mouse (l). Data in (c, f, i-l) were means ± SEM (error bars). Numbers of mice are indicated in the graphs (j-l). Statistical analyses were performed by two-sided Student t-tests (*** P < 0.001). For the P values, see Supplementary Table 4. Scale bars are labeled in the graphs.
Extended Data Fig. 5 Single unit recording from AHN vGAT + neurons with an optrode.
(a) An example micrograph showing the optrode track above the AHN, in which ChR2-mCherry was expressed in vGAT+ AHN neurons. Note the recording site was marked by electrolytic lesion (arrow) in the AHN. For the analyses of specific expression of ChR2-mCherry in vGAT+ AHN neurons, see Fig. 6b and Supplementary Fig. 9a. (b) Principal component analyses of light-evoked spikes (blue) and spontaneous spikes (black) of an example putative AHN vGAT+ neuron. Gray dots represent noise. Inset shows example waveforms of light-evoked spike (Light) and spontaneous spike (Spon). (c) Raster plots of example units of putative vGAT+ AHN neurons showing their responses to mechanical stimuli (Mech) and to cotton swab with snake feces (Feces). (d) Schematic diagrams showing the recording sites marked by electrolytic lesions were within the anterior part (AHA), central part (AHC) and posterior part (AHP) of the AHN. (e) Quantitative analyses of peak Z-Score of 15 single units that were localized in the AHA (n = 4), AHC (n = 6), and AHP (n = 5). Data in (e) were means ± SEM (error bars). Numbers of mice were indicated in the graphs (e). Scale bars were labeled in the graphs. The experiment (a) was repeated three times independently with similar results.
Extended Data Fig. 6 Contribution of LPB and PVT neurons to mechanically evoked defensive attack.
(a, b) Example coronal sections showing the expression of hM4Di-mCherry in the LPB (a) and PVT (b) of WT mice. (c) Quantitative analyses of time spent for biting-like attack toward the dummy snake in mice with and without chemogenetic inactivation of LPB neurons. (d) Quantitative analyses of time spent for biting-like attack toward the dummy snake in mice with and without chemogenetic inactivation of PVT neurons. (c) Quantitative analyses of time spent for biting-like attack toward the dummy snake in mice with and without chemogenetic inactivation of both PVT and LPB neurons. Data in (c-e) are means ± SEM (error bars). Numbers of mice are indicated in the graphs (c-e). Statistical analyses (c-e) were performed by two-sided Student t-tests (*** P < 0.001; n.s. P > 0.1). For the P values, see Supplementary Table 4. Scale bars are labeled in the graphs. The experiment (a, b) was repeated seven times independently with similar results.
Extended Data Fig. 7 Divergent projections of vGAT + AHN neurons.
(a) Schematic diagram showing the strategy to map the divergent projections of vGAT AHN neurons. (b) Example coronal brain section showing the injection center with fluorescence signals of EGFP-Syb2 in the AHN of vGAT-IRES-Cre mice. (c-g) Example coronal brain sections showing EGFP + synaptic terminals of vGAT+ AHN neurons in the target brain regions, including MPOA/MPA (c), LS (d), VMH (e), PMD (f), and PAG (g). Scale bars are labeled in the graphs. The experiment (b-g) was repeated five times independently with similar results.
Extended Data Fig. 8 Functional validation of GABAergic inhibition from vGAT + AHN neurons to the downstream target brain regions.
(a) Schematic diagrams showing injection of AAV-DIO-ChR2-mCherry into the AHN of vGAT-IRES-Cre mice (left) and whole-cell recording of light-evoked postsynaptic currents (PSCs) from the target neurons in the downstream areas of the AHN in acute brain slices (right). (b-f) Example traces and quantitative analyses showing the effect of antagonists of glutamate receptors (APV/CNQX) and GABAa receptor (PTX) on the amplitude of light-evoked PSCs recorded from neurons in the LS (b), VMHdm (c), PMD (d), VMHvl (e) and vlPAG (f). Data in (b-f) are means ± SEM (error bars). Numbers of cells are indicated in the graphs (b-f). Statistical analyses were performed by two-sided Student t-tests ( ***, P < 0.001). For the P values, see Supplementary Table 4.
Extended Data Fig. 9 Activation of vGAT + AHN-PAG pathway and AHN-LS pathway.
(a) Example coronal sections showing ChR2-mCherry expression is largely restricted within the AHN of vGAT-IRES-Cre mice (left) and the optical-fiber tracks above the ChR2-mCherry+ axon terminals in the vlPAG (right). (b-d) Quantitative analyses of time spent for freezing (b), risk assessment toward snake (c), and avoidance from snake (d) of mice evoked by activation of vGAT+ AHN-vlPAG pathway. (e) Example coronal sections showing ChR2-mCherry expression is largely restricted within the AHN of vGAT-IRES-Cre mice (left) and the optical-fiber tracks above the ChR2-mCherry+ axon terminals in the LS (right). (f-h) Quantitative analyses of time spent for freezing (f), risk assessment to snake (g), and avoidance from snake (h) of mice evoked by activation of vGAT+ AHN-LS pathway. Scale bars are labeled in the graphs. Data in (b-d, f-h) are means ± SEM (error bars). Numbers of mice are indicated in the graphs (b-d, f-h). Statistical analyses were performed by two-sided Student t-tests (***, P < 0.001). For the P values, see Supplementary Table 4. The experiment (a, e) was repeated seven times independently with similar results.
Extended Data Fig. 10 Experiments to rule out the involvement of collateral activation.
(a) Left, an example coronal section showing ChR2-mCherry expression largely restricted in the AHN and the optical-fiber track above the AHN of vGAT-IRES-Cre mice. Right, an example coronal section showing the ChR2-mCherry+ axon terminals in the vlPAG and the cannula track above these axon terminals. (b) Schematic diagram showing the procedure for infusing saline and PTX (100 μM) into the vlPAG combined with measuring biting-like attack to the live snake evoked by activation of vGAT+ AHN neurons. (c) Quantitative analyses of light-evoked biting-like attack to live snake in mice with vlPAG treated with different doses of Saline. (d) Left, example coronal section showing expression of ChR2-mCherry expression largely restricted in the AHN and the track of cannulae above the AHN. Right, example coronal section showing the ChR2-mCherry+ axon terminals in the vlPAG and the optical fiber track above the axon terminals. Scale bars are labeled in the graphs. Data in (c) are means ± SEM (error bars). Numbers of mice are indicated in the graphs (c). Statistical analyses (c) were performed by One-Way ANOVA (n.s., P > 0.1). For the P values, see Supplementary Table 4. The experiment (a, d) was repeated six times independently with similar results.
Supplementary information
Supplementary Information
Supplementary Figs. 1–8, Supplementary Tables 1–4 and approval form from the NIBS Administrative Panel on Laboratory Animal Care
Supplementary Video 1
Behavioral responses of a male mouse to a dummy snake coated with and without snake feces
Supplementary Video 2
Behavioral responses of a male mouse to a dummy snake equipped with an alligator clip to apply mechanical stimuli on the tail
Supplementary Video 3
Behavioral responses of a male mouse to neutral object equipped with an alligator clip to apply mechanical stimuli on the tail
Supplementary Video 4
Photoinhibition of vGAT+ AHN neurons of a male mouse reversibly abrogated mechanically evoked defensive attack to the dummy snake
Supplementary Video 5
Photostimulation of vGAT+ AHN neurons of a male mouse promoted mechanically evoked defensive attack
Supplementary Video 6
Photostimulation of vGAT+ AHN neurons of a male mouse evoked biting attack to a live snake in the arena
Supplementary Video 7
Photostimulation of vGAT+ AHN neurons of a male mouse evoked biting attack to a wood block in the arena
Supplementary Video 8
Photostimulation of vGAT+ AHN neurons of a male mouse abrogated its ongoing social aggression against a male intruder
Supplementary Video 9
Photostimulation of vGAT+ AHN neurons of a male mouse did not evoke biting attack or mounting to another male mouse in the arena
Supplementary Video 10
Photostimulation of vGAT+ AHN neurons of a male mouse did not evoke biting attack or mounting to a female mouse in the arena
Supplementary Video 11
In the presence of both a live snake and a male mouse, photostimulation of vGAT+ AHN neurons evoked biting attack selectively to the live snake rather than the male C57BL/6 mouse in the arena
Supplementary Video 12
In the presence of both a live snake and a female mouse, photostimulation of vGAT+ AHN neurons evoked biting attack selectively to the live snake rather than the female mouse in the arena
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Xie, Z., Gu, H., Huang, M. et al. Mechanically evoked defensive attack is controlled by GABAergic neurons in the anterior hypothalamic nucleus. Nat Neurosci 25, 72–85 (2022). https://doi.org/10.1038/s41593-021-00985-4
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DOI: https://doi.org/10.1038/s41593-021-00985-4
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