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Divergent medial amygdala projections regulate approach–avoidance conflict behavior

Nature Neurosciencevolume 22pages565575 (2019) | Download Citation


Avoidance of innate threats is often in conflict with motivations to engage in exploratory approach behavior. The neural pathways that mediate this approach–avoidance conflict are not well resolved. Here we isolated a population of dopamine D1 receptor (D1R)-expressing neurons within the posteroventral region of the medial amygdala (MeApv) in mice that are activated either during approach or during avoidance of an innate threat stimulus. Distinct subpopulations of MeApv-D1R neurons differentially innervate the ventromedial hypothalamus and bed nucleus of the stria terminalis, and these projections have opposing effects on investigation or avoidance of threatening stimuli. These projections are potently modulated through opposite actions of D1R signaling that bias approach behavior. These data demonstrate divergent pathways in the MeApv that can be differentially weighted toward exploration or evasion of threats.

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Datasets supporting the findings in this study and custom codes used for imaging analysis are available from the corresponding author upon reasonable request.

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  1. 1.

    Elliot, A. J. The hierarchical model of approach-avoidance motivation. Motiv. Emot. 30, 111–116 (2006).

  2. 2.

    Choi, G. B. et al. Lhx6 delineates a pathway mediating innate reproductive behaviors from the amygdala to the hypothalamus. Neuron 46, 647–660 (2005).

  3. 3.

    Burnett, C. J. et al. Hunger-driven motivational state competition. Neuron 92, 187–201 (2016).

  4. 4.

    Padilla, S. L. et al. Agouti-related peptide neural circuits mediate adaptive behaviors in the starved state. Nat. Neurosci. 19, 734–741 (2016).

  5. 5.

    Blanchard, D. C. & Blanchard, R. J. Innate and conditioned reactions to threat in rats with amygdaloid lesions. J. Comp. Physiol. Psychol. 81, 281–290 (1972).

  6. 6.

    Martinez, R. C., Carvalho-Netto, E. F., Ribeiro-Barbosa, E. R., Baldo, M. V. & Canteras, N. S. Amygdalar roles during exposure to a live predator and to a predator-associated context. Neuroscience 172, 314–328 (2011).

  7. 7.

    Olds, M. E. & Olds, J. Approach-avoidance analysis of rat diencephalon. J. Comp. Neurol. 120, 259–295 (1963).

  8. 8.

    Dielenberg, R. A., Hunt, G. E. & McGregor, I. S. “When a rat smells a cat”: the distribution of Fos immunoreactivity in rat brain following exposure to a predatory odor. Neuroscience 104, 1085–1097 (2001).

  9. 9.

    McGregor, I. S., Hargreaves, G. A., Apfelbach, R. & Hunt, G. E. Neural correlates of cat odor-induced anxiety in rats: region-specific effects of the benzodiazepine midazolam. J. Neurosci. 24, 4134–4144 (2004).

  10. 10.

    Chung, A. S., Miller, S. M., Sun, Y., Xu, X. & Zweifel, L. S. Sexual congruency in the connectome and translatome of VTA dopamine neurons. Sci. Reports 7, 11120 (2017).

  11. 11.

    Bromberg-Martin, E. S., Matsumoto, M. & Hikosaka, O. Dopamine in motivational control: rewarding, aversive, and alerting. Neuron 68, 815–834 (2010).

  12. 12.

    Vincenz, D., Wernecke, K. E. A., Fendt, M. & Goldschmidt, J. Habenula and interpeduncular nucleus differentially modulate predator odor-induced innate fear behavior in rats. Behav. Brain Res. 332, 164–171 (2017).

  13. 13.

    Ipser, J. C., Kariuki, C. M. & Stein, D. J. Pharmacotherapy for social anxiety disorder: a systematic review. Expert. Rev. Neurother. 8, 235–257 (2008).

  14. 14.

    Garcia, R. Neurobiology of fear and specific phobias. Learn. Mem. 24, 462–471 (2017).

  15. 15.

    Dong, H. W., Petrovich, G. D. & Swanson, L. W. Topography of projections from amygdala to bed nuclei of the stria terminalis. Brain Res. Rev. 38, 192–246 (2001).

  16. 16.

    Swanson, L. W. The amygdala and its place in the cerebral hemisphere. Ann. NY Acad. Sci. 985, 174–184 (2003).

  17. 17.

    Gross, C. T. & Canteras, N. S. The many paths to fear. Nat. Rev. Neurosci. 13, 651–658 (2012).

  18. 18.

    Lein, E. S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007).

  19. 19.

    Heusner, C. L., Beutler, L. R., Houser, C. R. & Palmiter, R. D. Deletion of GAD67 in dopamine receptor-1 expressing cells causes specific motor deficits. Genesis 46, 357–367 (2008).

  20. 20.

    Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

  21. 21.

    Zhang, T. Y., Chrétien, P., Meaney, M. J. & Gratton, A. Influence of naturally occurring variations in maternal care on prepulse inhibition of acoustic startle and the medial prefrontal cortical dopamine response to stress in adult rats. J. Neurosci. 25, 1493–1502 (2005).

  22. 22.

    Unger, E. K. et al. Medial amygdalar aromatase neurons regulate aggression in both sexes. Cell Reports 10, 453–462 (2015).

  23. 23.

    Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22–29 (2012).

  24. 24.

    Kollack-Walker, S., Don, C., Watson, S. J. & Akil, H. Differential expression of c-fos mRNA within neurocircuits of male hamsters exposed to acute or chronic defeat. J. Neuroendocrinol. 11, 547–559 (1999).

  25. 25.

    Lin, D. et al. Functional identification of an aggression locus in the mouse hypothalamus. Nature 470, 221–226 (2011).

  26. 26.

    Liu, Y. X. et al. Psychological stress on female mice diminishes the developmental potential of oocytes: a study using the predatory stress model. PLoS One 7, e48083 (2012).

  27. 27.

    Hong, W., Kim, D. W. & Anderson, D. J. Antagonistic control of social versus repetitive self-grooming behaviors by separable amygdala neuronal subsets. Cell 158, 1348–1361 (2014).

  28. 28.

    Choi, J. S. & Kim, J. J. Amygdala regulates risk of predation in rats foraging in a dynamic fear environment. Proc. Natl Acad. Sci. USA 107, 21773–21777 (2010).

  29. 29.

    Ghosh, K. K. et al. Miniaturized integration of a fluorescence microscope. Nat. Methods 8, 871–878 (2011).

  30. 30.

    Chen, T. W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

  31. 31.

    Pardo-Bellver, C., Cádiz-Moretti, B., Novejarque, A., Martínez-García, F. & Lanuza, E. Differential efferent projections of the anterior, posteroventral, and posterodorsal subdivisions of the medial amygdala in mice. Front. Neuroanat. 6, 33 (2012).

  32. 32.

    Petrovich, G. D., Canteras, N. S. & Swanson, L. W. Combinatorial amygdalar inputs to hippocampal domains and hypothalamic behavior systems. Brain Res. Rev. 38, 247–289 (2001).

  33. 33.

    Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

  34. 34.

    Blanchard, R. J. & Blanchard, D. C. Attack and defense in rodents as ethoexperimental models for the study of emotion. Prog. Neuropsychopharmacol. Biol. Psychiatry 13, Suppl, S3–S14 (1989).

  35. 35.

    Chuong, A. S. et al. Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Nat. Neurosci. 17, 1123–1129 (2014).

  36. 36.

    Tervo, D. G. et al. A designer aav variant permits efficient retrograde access to projection neurons. Neuron 92, 372–382 (2016).

  37. 37.

    Flagel, S. B. et al. A selective role for dopamine in stimulus-reward learning. Nature 469, 53–57 (2011).

  38. 38.

    Berridge, K. C. & Robinson, T. E. What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Res. Rev. 28, 309–369 (1998).

  39. 39.

    Dong, H. W. & Swanson, L. W. Projections from bed nuclei of the stria terminalis, posterior division: implications for cerebral hemisphere regulation of defensive and reproductive behaviors. J. Comp. Neurol. 471, 396–433 (2004).

  40. 40.

    Marowsky, A., Yanagawa, Y., Obata, K. & Vogt, K. E. A specialized subclass of interneurons mediates dopaminergic facilitation of amygdala function. Neuron 48, 1025–1037 (2005).

  41. 41.

    Schultz, W. Multiple dopamine functions at different time courses. Annu. Rev. Neurosci. 30, 259–288 (2007).

  42. 42.

    Pezze, M. A. & Feldon, J. Mesolimbic dopaminergic pathways in fear conditioning. Prog. Neurobiol. 74, 301–320 (2004).

  43. 43.

    Yang, H. et al. Laterodorsal tegmentum interneuron subtypes oppositely regulate olfactory cue-induced innate fear. Nat. Neurosci. 19, 283–289 (2016).

  44. 44.

    Silva, B. A. et al. Independent hypothalamic circuits for social and predator fear. Nat. Neurosci. 16, 1731–1733 (2013).

  45. 45.

    Hari Dass, S. A. & Vyas, A. Copulation or sensory cues from the female augment Fos expression in arginine vasopressin neurons of the posterodorsal medial amygdala of male rats. Front. Zool. 11, 42 (2014).

  46. 46.

    Bergan, J. F., Ben-Shaul, Y. & Dulac, C. Sex-specific processing of social cues in the medial amygdala. eLife 3, e02743 (2014).

  47. 47.

    Wang, L., Chen, I. Z. & Lin, D. Collateral pathways from the ventromedial hypothalamus mediate defensive behaviors. Neuron 85, 1344–1358 (2015).

  48. 48.

    Golden, S. A. et al. Persistent conditioned place preference to aggression experience in adult male sexually-experienced CD-1 mice. Genes Brain. Behav. 16, 44–55 (2017).

  49. 49.

    Ferrari, P. F., van Erp, A. M., Tornatzky, W. & Miczek, K. A. Accumbal dopamine and serotonin in anticipation of the next aggressive episode in rats. Eur. J. Neurosci. 17, 371–378 (2003).

  50. 50.

    Couppis, M. H. & Kennedy, C. H. The rewarding effect of aggression is reduced by nucleus accumbens dopamine receptor antagonism in mice. Psychopharmacology (Berl.) 197, 449–456 (2008).

  51. 51.

    Zhuang, X., Masson, J., Gingrich, J. A., Rayport, S. & Hen, R. Targeted gene expression in dopamine and serotonin neurons of the mouse brain. J. Neurosci. Methods 143, 27–32 (2005).

  52. 52.

    Gore, B. B. & Zweifel, L. S. Genetic reconstruction of dopamine D1 receptor signaling in the nucleus accumbens facilitates natural and drug reward responses. J. Neurosci. 33, 8640–8649 (2013).

  53. 53.

    Sparta, D. R. et al. Construction of implantable optical fibers for long-term optogenetic manipulation of neural circuits. Nat. Protoc. 7, 12–23 (2011).

  54. 54.

    Resendez, S. L. et al. Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nat. Protoc. 11, 566–597 (2016).

  55. 55.

    Sanz, E. et al. Fertility-regulating Kiss1 neurons arise from hypothalamic POMC-expressing progenitors. J. Neurosci. 35, 5549–5556 (2015).

  56. 56.

    Weber, F. et al. Control of REM sleep by ventral medulla GABAergic neurons. Nature 526, 435–438 (2015).

  57. 57.

    Kim, J. G. et al. Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding. Nat. Neurosci. 17, 908–910 (2014).

  58. 58.

    Chand, A. N., Galliano, E., Chesters, R. A. & Grubb, M. S. A distinct subtype of dopaminergic interneuron displays inverted structural plasticity at the axon initial segment. J. Neurosci. 35, 1573–1590 (2015).

  59. 59.

    Ting, J. T., Daigle, T. L., Chen, Q. & Feng, G. Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics. Methods Mol. Biol. 1183, 221–242 (2014).

  60. 60.

    Carter, M. E., Soden, M. E., Zweifel, L. S. & Palmiter, R. D. Genetic identification of a neural circuit that suppresses appetite. Nature 503, 111–114 (2013).

  61. 61.

    Soden, M. E. et al. Genetic isolation of hypothalamic neurons that regulate context-specific male social behavior. Cell Rep. 16, 304–313 (2016).

  62. 62.

    Stamatakis, A. M. & Stuber, G. D. Activation of lateral habenula inputs to the ventral midbrain promotes behavioral avoidance. Nat. Neurosci. 15, 1105–1107 (2012).

  63. 63.

    Zhou, P. et al. Efficient and accurate extraction of in vivo calcium signals from microendoscopic video data. eLife 7, e28728 (2018).

  64. 64.

    Pnevmatikakis, E. A. et al. Simultaneous denoising, deconvolution, and demixing of calcium imaging data. Neuron 89, 285–299 (2016).

  65. 65.

    Jimenez, J. C. et al. Anxiety cells in a hippocampal-hypothalamic circuit. Neuron 97, 670–683.e6 (2018).

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We thank M. Soden for technical advice and assistance with slice electrophysiology and members of the Zweifel lab for scientific discussion. We thank C. Campos, A. Bowen and R. Palmiter for their assistance with calcium imaging studies. We also thank J. Allen for assistance in the production of AAV viral vectors. This work was funded by the US National Institutes of Health (P50MH10642 and R01MH094536 to L.S.Z).

Author information


  1. Department of Pharmacology, University of Washington, Seattle, WA, USA

    • Samara M. Miller
    • , Daniele Marcotulli
    •  & Larry S. Zweifel
  2. Department of Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona, Italy

    • Daniele Marcotulli
  3. Departments of Biology and Anthropology, University of Washington, Seattle, WA, USA

    • Angela Shen
  4. Department of Psychiatry, University of Washington, Seattle, WA, USA

    • Larry S. Zweifel


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S.M.M. and L.S.Z designed the experiments. S.M.M. and L.S.Z wrote the manuscript. S.M.M. performed all viral injection surgeries, behavior experiments, and slice electrophysiology. D.M. analyzed all calcium imaging data. Behavioral analysis and histology was performed by S.M.M. and A.S. L.S.Z purified all viral vectors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Larry S. Zweifel.

Integrated supplementary information

  1. Supplementary Figure 1 MeApv D1R neurons are activated by multisensory innate fear stimuli.

    (a) Behavioral schematic (b-c) quantification and histological images of Fos levels in MEApv D1R neurons during exposure to threat stimuli. Fos is significantly induced in MEAPV D1R neurons over controls when mice are exposed to predator odor (PO), robobug (RB), and resident-intruder threat stimuli (RI) (n = 3 mice, 1-way ANOVA F(3, 189) = 55.05, P < 0.0001, Bonferroni’s multiple comparisons test). Scale bar: 15 μm. (d) Total number of virally labeled MEApv-D1R neurons did not differ across assays (n = 3 mice, 1-way ANOVA P > 0.05 Bonferroni’s multiple comparisons test). (e) Total Fos was significantly higher following predator odor (PO), robobug (RB), and resident-intruder (RI) assays (n = 3 mice, 1-way ANOVA, F(3,14) = 12.31, P = 0.008). Center values represent mean and error bars represent s.e.m.

  2. Supplementary Figure 2 Histological and behavioral characterization of calcium imaging experiments.

    (a) Schematic confirming viral targeting of GCaMP6m and microendoscope placement (n = 4 mice). Out of 24 mice injected with GCaMP6m, 4 mice were successfully implanted with lenses and used for this study. (b) Top: schematic of viral constructs for GCamP6m and HM3-mCherry expression. Bottom: histology showing GCamP6m (green) and HM3 (red) targeting to MeApv of D1R-Cre mice with lens implanted above. Histological verification was replicated in all four experimental animals. Scale bar: 100 μm. (c-f) Distribution of behavioral epochs during approach and hide box behavior for robobug and predator odor assays. (g) Mean time spent during investigation or hide box for predator odor and robobug assays. (n = 4 mice). Center values represent mean, error bars represent s.e.m. (h-K) Comparison of average z-scored ∆F for avoidance cells vs. approach cells during periods of (h) avoidance or (i) investigation of the robobug, (j) avoidance and (k) investigation of predator odor (n = 4 mice; two-tailed Wilcoxon rank sum test; the box represents the 25th–75th percentiles with smallest and largest data points falling within a 1.5 inter quartile range below and above the box, respectively. Whiskers represent the minimum and maximum.). (i) Correlation between percentage of cells active during investigation of robobug and predator odor (n = 4 mice; Pearson correlation test). (j) Same as (i) for male and female odorant assays. (m-n) Overlap of hide box active cells (m) and approach cells (n) in predator odor and robobug assays.

  3. Supplementary Figure 3 MeApv D1R neurons are activated during investigation of conspecific odorant.

    (a) Top: probability of distribution of peak fluorescence for all cells active during investigation of conspecific odorant (dashed line represents start of active investigation of male odorant, n = 4 mice); Middle: heat plot of calcium responses to odorant for all the active cells aligned to an investigation (n = 4 mice); Bottom: average traces of all cells activated cells during an approach and investigation epoch ± SEM (n = 4 mice). (b) Selectivity of cells active in mice during investigation of conspecific odorants (n = 4 mice) relative to approach and avoidance investigation of robobug and predator odor. (c and d) Distribution of behavioral epochs during approach and hide box behavior for conspecific odorants. (e) Percent of activated cells was not correlated with investigation time. (n = 4 mice; Pearson correlation test). (f) Time spent in hide box or in active investigation in response to conspecific odorant (n = 4 mice). Center values represent mean and error bars represent s.e.m.

  4. Supplementary Figure 4 Spatial distribution of electrophysiological recordings in VMH and BNST.

    (a) Example image and schematic showing location of cells patched in BNST and corresponding responses for ChR2 functional connectivity experiments (n = 31 cells/5 mice). (b) Same as (a) but for VMH (n = 15 cells/ 4 mice). Scale bar: 100 μm. (c) Example trace showing delayed inhibitory response in BNST that was blocked by application of CNQX (delay ~9.1 ms).

  5. Supplementary Figure 5 MeApv D1R pathways are activated by multisensory innate fear stimuli.

    (a) Injection schematic, quantification and histological images of Fos levels in VMH and BNST MeApv-projecting neurons during exposure to threat stimuli. (b) More neurons in the MeApv were labeled by RetroBeads injected into the BNST than into the VMH (n = 12 mice, P = 0.0012, two-tailed unpaired t test). (c) Total Fos was significantly higher following predator odor (PO), robobug (RB), and resident-intruder (RI) assays following RetroBead injections into the BNST or VMH (n = 3 mice/group, 2-way ANOVA, effect of assay F(3,16) = 35.56, P < 0.0001). (d) There is significantly more Fos induction in VMH-projecting MeApv neurons compared with BNST-projecting MeApv neurons during predator odor and robobug exposure (n = 3 mice/group, **P = 0.002, *P = 0.0134, two-tailed unpaired t test). (e) Histology showing Fos expression in BNST and VMH projecting MeApv neurons during exposure to threat stimuli. Scale bar: 10 μm. For each behavioral assay, histology was verified in two other animals. Center values represent mean and error bars represent s.e.m.

  6. Supplementary Figure 6 Confirmation of ChR2 viral targeting and optical cannula placement.

    (a) Rang of spread of ChR2 viral coverage within MEApv for each of the three experimental groups (n = 51 mice total for all three groups) with example image. Scale bar: 100 μm. Location of optical fibers and ChR2 terminals within (b) VMH (c) BNST and (d) optical fiber placement in MeApv for cell body stimulation. Scale bar: 50 μm.

  7. Supplementary Figure 7 Activation of VMH-, but not BNST-, projecting MeApv D1R neurons elicits real-time place-avoidance behavior.

    (a) Representative real time place preference (RTPP) location plots. (b) Quantification of RTPP (right) shows that optogenetic activation of VMH-projecting MEApv D1R neurons reduces time spent in light paired chamber, but activation of BNST-projecting MEApv D1R neurons has no effect (2-way ANOVA F(2, 29) = 3.31, P = 0.05; Bonferroni’s multiple comparisons); alternatively shown as time spent in unpaired side subtracted from paired side (1-way ANOVA F(2, 35) = 4.295, P = 0.036, Tukey’s multiple comparisons) . (c) Total distance traveled was reduced in VMH stimulated versus BNST stimulated MEApv-D1R terminals in the RTPP assay (1-way ANOVA F(2, 29) = 5.766, P = 0.0082; Tukey’s multiple comparison test). Center values represent mean and error bars represent s.e.m.

  8. Supplementary Figure 8 Global activation of MEApv D1R neurons biases approach over avoidance.

    (a) Schematic of viral injection of AAV1-FLEX-ChR2-mCherry and optic fiber implant into MEApv of D1RCre/+ mice. (b) Optogenetic activation MeApv-D1R neurons increases approach to predator odor (n = 7 mice/group; Time spent in hide box: P = 0.0459, two-tailed unpaired t test; Latency to approach: *P = 0.0459, unpaired t test; Investigation time: P = 0.0804, unpaired t test; Frequency of investigations: *P = 0.0104, unpaired t test). (c) During exposure to robobug, optogenetic activation of MeAPV-D1R neurons results in significantly shorter latency to sniff predator odor (n = 7 mice/group; *P = 0.0186, two-tailed unpaired t test) and less time in hide box during exposure to predator odor (*P = 0.0186, unpaired t test); investigation of predator odor is not affected (P = 0.11, unpaired t test). (d) Optogenetic activation of MEAPV-D1R neurons does not significantly affect behavior during resident intruder assay. (n = 7, 11 mice/group; grooming: P = 0.4021; fighting: P = 0.1252; investigation: P = 0.9951; unpaired t test). (e) Optogenetic activation of MEAPV-D1R neurons does not significantly affect preference for side of chamber or locomotion during RTPP (n = 8, 10 mice; 2 way ANOVA F(1, 16) = 0.01, P = -0.9420, Bonferroni’s multiple comparison; P = 0.6869, two-tailed unpaired t test). Center values represent mean and error bars represent s.e.m.

  9. Supplementary Figure 9 Confirmation of JAWS viral targeting and optical cannula placement.

    (a) Injection site of AAV2-Retro-FLEX-JAWS-GFP into VMH and (b) bilateral cannula placement over MeApv with cell body transfection of JAWS in MeApv (n = 21 animals). Scale bar: 50 μm. (c) Injection site of AAV2.Retro.JAWS into BNST, and (d) bilateral cannula placement over MeApv with cell body transfection of JAWS in MeApv (n = 19 mice). Scale bar: 50 μm.

  10. Supplementary Figure 10 Infusion of SKF 81,297 into MeApv.

    (a) Confirmation of bilateral cannula placement over MeApv. (b) SKF 81,297 does not affect locomotion, as seen in both the predator odor and robobug assays (n = 6 mice). Scale bar: 100 μm. Center values represent mean and error bars represent s.e.m.

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