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Divergent medial amygdala projections regulate approach–avoidance conflict behavior

Nature Neurosciencevolume 22pages565575 (2019) | Download Citation

Abstract

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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Acknowledgements

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

Affiliations

  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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Contributions

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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https://doi.org/10.1038/s41593-019-0337-z