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Dorsolateral septum somatostatin interneurons gate mobility to calibrate context-specific behavioral fear responses

Nature Neuroscience (2019) | Download Citation

Abstract

Adaptive fear responses to external threats rely upon efficient relay of computations underlying contextual encoding to subcortical circuits. Brain-wide analysis of highly coactivated ensembles following contextual fear discrimination identified the dorsolateral septum (DLS) as a relay of the dentate gyrus–CA3 circuit. Retrograde monosynaptic tracing and electrophysiological whole-cell recordings demonstrated that DLS somatostatin-expressing interneurons (SST-INs) receive direct CA3 inputs. Longitudinal in vivo calcium imaging of DLS SST-INs in awake, behaving mice identified a stable population of footshock-responsive SST-INs during contextual conditioning whose activity tracked and predicted non-freezing epochs during subsequent recall in the training context but not in a similar, neutral context or open field. Optogenetic attenuation or stimulation of DLS SST-INs bidirectionally modulated conditioned fear responses and recruited proximal and distal subcortical targets. Together, these observations suggest a role for a potentially hard-wired DLS SST-IN subpopulation as arbiters of mobility that calibrate context-appropriate behavioral fear responses.

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The data that support the findings of this study and custom MATLAB codes are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Y. Ziv, C. Harvey, M. Kheirbek and members of the Sahay lab for their comments on the manuscript. A.B. acknowledges support from 2014 NARSAD Young Investigator Award, Bettencourt-Schueller Foundation, Philippe Foundation and 2016 MGH ECOR Fund for Medical Discovery (FMD) Postdoctoral Fellowship Awards. W.F. was supported by 2013 HSCI Harvard Internship Program Award. A.R. was supported by 2017 HSCI Harvard Internship Program Award. A.S. acknowledges support from the NIH Biobehavioral Research Awards for Innovative New Scientists (BRAINS; grant R01MH104175), NIH–R01AG048908, NIH-1R01MH111729, the Ellison Medical Foundation New Scholar in Aging, the Whitehall Foundation, an Inscopix Decode award, a NARSAD Independent Investigator Award, Ellison Family Philanthropic support, the Blue Guitar Fund, a Harvard Neurodiscovery Center–MADRC Center Pilot Grant award, Alzheimer’s Association Research Grant, a Harvard Stem Cell Institute Development grant and HSCI seed grant.

Author information

Author notes

  1. These authors contributed equally: Yuan Gao, Michael TaeWoo Kim.

Affiliations

  1. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

    • Antoine Besnard
    • , Michael TaeWoo Kim
    • , Hannah Twarkowski
    • , Alexander Keith Reed
    • , Tomer Langberg
    • , Wendy Feng
    •  & Amar Sahay
  2. Harvard Stem Cell Institute, Cambridge, MA, USA

    • Antoine Besnard
    • , Michael TaeWoo Kim
    • , Hannah Twarkowski
    • , Alexander Keith Reed
    • , Tomer Langberg
    • , Wendy Feng
    •  & Amar Sahay
  3. Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    • Antoine Besnard
    • , Michael TaeWoo Kim
    • , Hannah Twarkowski
    • , Alexander Keith Reed
    • , Tomer Langberg
    • , Wendy Feng
    •  & Amar Sahay
  4. BROAD Institute of Harvard and MIT, Cambridge, MA, USA

    • Antoine Besnard
    • , Michael TaeWoo Kim
    • , Hannah Twarkowski
    • , Alexander Keith Reed
    • , Tomer Langberg
    • , Wendy Feng
    •  & Amar Sahay
  5. Department of Biology, Boston University, Boston, MA, USA

    • Yuan Gao
    •  & Ian Davison
  6. Departments of Anatomy and Neurobiology and Biomedical Engineering, School of Medicine, University of California, Irvine, CA, USA

    • Xiangmin Xu
  7. Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, Heidelberg, Germany

    • Dieter Saur
  8. Institute of Translational Cancer Research and Experimental Cancer Therapy, and Department of Medicine II, Technische Universität Munchen, Munich, Germany

    • Dieter Saur
  9. Department of Psychiatry and Behavioral Science, University of Washington, Seattle, WA, USA

    • Larry S. Zweifel

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Contributions

A.B., Y.G., M.T.K., H.T., A.K.R., T.L., W.F., and I.D. performed experiments. X.X., D.S. and L.S.Z. contributed reagents. A.S. and A.B. codeveloped the concept, analyzed data and wrote the manuscript. A.S. conceived the project and supervised all aspects of the project.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Amar Sahay.

Integrated supplementary information

  1. Supplementary Figure 1 Brain-wide c-Fos expression associated with contextual fear discrimination (corresponds to Fig. 1).

    a) Immunohistochemistry for c-Fos in mice previously exposed to context B or A’ (30 min prior) as well as a naive control group (behavioral analysis shown in Supplementary Fig. 2c-e). Note the increase in number of c-Fos positive cells in dorsal DG following exposure to context B. Representative images for 3–6 independent animals per group (single experiment). Yellow arrowheads indicate representative c-Fos immunopositive cells. Scale bar: 100 μm. b-i) Immunohistochemistry for c-Fos in mice previously exposed to context B or A’ (60 min prior) as well as a naive control group. Representative images for 5 independent animals per group (single experiment) in dorsal DG (b), dorsal CA3 (c), dorsal CA1 (d), anterior DLS medial and lateral (e), anterior CPU medial (f), BNST dorsal and ventral (g), amygdala lateral and basolateral (h) and PAG lateral, dorsolateral and dorsomedial (i). Yellow arrowheads point to representative c-Fos immunopositive cells. Scale bar: 100 μm.

  2. Supplementary Figure 2 Immunohistochemical analysis of brain-wide c-Fos expression associated with contextual fear discrimination (corresponds to Fig. 1).

    a) Brain-wide c-Fos analysis in Context B and Context A’ groups as compared to naive controls (see Fig. 1f). Detailed quantifications in brain regions categorized according to changes in levels of c-Fos immunoreactivity: no change (blue), B and A’ significantly different from naive controls (grey) and B and A’ significantly different from each other (red). Data (means ± SEM; n = 5, 5, 5 mice per group) were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). *p < 0.05, context B or A’ versus controls, #p < 0.05, context B versus context A’. b) Examples of robust inter-regional correlations between c-Fos immunopositive cells (expressed as % controls) using within and between subject design in context A’ and B. c) Schematic representation of CFCDL timeline consisting of 5 block trainings (10 days) at the end of which mice were sacrificed 30 min following exposure to the safe context for c-Fos analysis (day 11). d) Freezing behavior upon final exposure to the safe context. Data (means ± SEM; n = 6, 4 mice per group) were analyzed with unpaired Student two-tailed T-test, ***p < 0.001, context B versus A’ (Statistics detailed in Supplementary Table 1). e) c-Fos analysis in dorsal, medial and ventral DG of Context B and A’ groups as compared to naive controls. Data (means ± SEM; n = 3, 6, 4 mice per group) were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (Immunohistochemistry shown in Supplementary Fig. 1a; Statistics detailed in supplementary Table 1). *p < 0.05, context B or A’ versus controls, #p < 0.05, context B versus context A’. f) Schematic representation of CFCDL timeline consisting of 5 block trainings with or without footshocks (10 days) at the end of which mice were sacrificed 60 min following exposure to context A (no footshock) or B for c-Fos analysis (day 11). g) Freezing behavior upon final exposure to context A or B. Data (means ± SEM; n = 4, 4, 4, 4 mice per group) were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). **p < 0.01, context A shocked throughout versus all groups. h) Immunohistochemistry for c-Fos in mice previously exposed to context B or A’ as well as a naive control group. Note the marked increase in the number of c-Fos positive cells in the DLS in context B mice. Representative images for 4 independent animals per group. Scale bar: 50 μm. i-j) Detailed quantifications in DLS medial and DLS lateral according to changes in levels of c-Fos immunoreactivity: no change (blue), B and A’ significantly different from naive controls (grey) and B and A’ significantly different from each other (red). Data (means ± SEM; n = 4, 4, 4, 4, 4 mice per group) were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). *p < 0.05, context B or A versus controls, #p < 0.05, context B shocked versus context B no-shock.

  3. Supplementary Figure 3 DLS c-Fos expression following contextual fear recall (corresponds to Fig. 1).

    a-c) c-Fos Immunohistochemistry in the anterior DLS of naïve mice (a) or mice previously exposed to context B (b) or A’(c) 60 min prior to sacrifice. Note the marked increase in number of c-Fos positive cells in context B mice. Representative images for 5 independent mice (M1–5) per group (single experiment). Scale bar: 50 μm.

  4. Supplementary Figure 4 Cav2 mapping of DLS afferents (corresponds to Fig. 2).

    a) Ai14 mice were injected with a Canine associated virus-2 encoding Cre recombinase (Cav2-Cre) virus in the DLS (red arrow). Cre-expressing cells (presynaptic partners) were retrogradely labeled with tdTomato at the site of injection (DLS) and in different brain regions such as the nucleus of the diagonal band, prefrontal cortex, anteromedial thalamus, nucleus reuniens and supramammillary nucleus. Representative images for 3 independent mice (single experiment). Scale bar: 100 μm. b) Presynaptic partners were identified in both CA3 and CA2 (as defined by RGS14 immunopositive cells). Scale bar: 100 μm. c) Quantifications of tdTomato expressing cells revealed presynaptically labeled cells in the Sub (subiculum) MS/DBN (medial septum/diagonal band nucleus), CA3/CA2, PFCx (prefrontal cortex), AM (anteromedial thalamic nucleus), SUM (supramammillary nucleus), Re (nucleus reuniens), LH (lateral hypothalamus) and CA1. Sparse tdTomato cells were found in the LM (lateral mammillary nucleus), Raphe, VTA (ventral tegmental area). No tdTomato cells were found in DG, AD (anterodorsal thalamic nucleus), PAG (periaqueductal gray), AV (anteroventral thalamic nucleus), CeA (central amygdala), CPu (caudate putamen), LA (lateral amygdala), NAcc (nucleus accumbens) and BNST (bed nucleus of stria terminalis). Data (means ± SEM; n = 3 mice per group) d) No presynaptic partners were detected in DG of Ai14 mice injected with Canine virus. Scale bar: 100 μm. e) No presynaptic partners were detected in CA3 of Ai14 mice not injected with Canine virus (Cav2-Cre). Nuclei are counterstained with DAPI. Representative images for 3 independent animals (single experiment). Scale bar: 100 μm.

  5. Supplementary Figure 5 Characterization of distribution of SST-INs in DLS (corresponds to Fig. 2).

    a) Immunohistochemistry for calbindin (CB), somatostatin (SST), calretinin (CR), parvalbumin (PV), neuropeptide Y (NPY), and choline acetyltransferase (ChAT) in the anterior DLS. Outlines denote medial and lateral parts of DLS and correspond to dorsal and ventral CA3 inputs to DLS (Besnard and Sahay, unpublished observations). Note the distribution of different neuronal populations throughout DLS except for ChAT, which is only expressed in the medial septum. Representative images for 3 independent animals per group (single experiment). Scale bar: 100 μm. b) Genetic labeling of Gad2 positive cells (Gad2-Cre::Ai14) and SST positive cells (SST-Cre::Ai14) with tdTomato reveals that SST-INs comprise a large proportion of DLS-INs. Representative images for 3 independent animals per group (single experiment). Scale bar: 100 μm. c-d) Detailed quantifications of tdTomato positive cells in DLS medial and DLS lateral along the rostro-caudal axis in Gad2-Cre::Ai14 and SST-Cre::Ai14 mice. Data (means ± SEM; n = 3, 3 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). e) Immunohistochemistry for somatostatin (SST), endogenous expression of tdTomato in the DLS of Gad2-Cre::Ai14, cell nuclei are counterstained with DAPI. Note the overlap between SST and tdTomato (white arrowheads) confirming that SST-INs are inhibitory in the DLS. Representative images for 3 independent animals (single experiment). Scale bar: 25 μm. f) Immunohistochemistry for EYFP in the DLS, NAcc, SUM, LH, PVNH, MS/DBN and BNST of Gad2-Cre mice and SST-Cre mice injected with AAV5-DIO-ChR2-EYFP. Representative images for 3 independent animals (single experiment). Scale bar: 100 μm.

  6. Supplementary Figure 6 Calcium imaging of SST-INs in DLS (corresponds to Fig. 3).

    a) Immunohistochemistry for GFP (GCaMP6m), tdTomato and c-Fos in the DLS of SST-Cre::Ai14 mice injected with AAV-DJ-DIO-GCaPM6m and perfused 30 min following CFC2. Cell nuclei are counterstained with DAPI. Note the expression of cytoplasmic GCaMP6m restricted to SST-INs expressing tdTomato (white arrowheads) and nuclear expression of c-Fos in a subset of these cells (yellow arrowheads). Representative images for 4 independent animals. Scale bar: 25 μm. b) Example of calcium transients detected with CNMF-E from 7 different cells during contextual fear conditioning in context A. Note the transients observed in response to some (not all) footshocks (vertical red lines). Scale bar: x axis:10 sec, y axis:5 sd. c-g) Percent time spent in the different compartments in the open-field (c), elevated plus maze (d), time spent freezing in context A and B, freezing bout duration (f) and number of freezing bouts (g) across two consecutive recording sessions (e). Data (means ± SEM; n = 8 mice) were analyzed using mixed factor two-way ANOVA (repeated measure over time)(Statistics detailed in supplementary Table 1). h) Average calcium transient frequency (Hz) for the overall population (n = 118 cells) identified with PCA-ICA in the OF, EPM, context A and context B (average of two consecutive sessions). Data (means ± SEM; n = 118 cells per group, and data distribution represented as violin plots) were analyzed using one-way ANOVA (repeated measures over time) followed by Tukey’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). *p < 0.05 for specific comparisons. i) Sorting of the 118 cells detected with PCA-ICA based on calcium transients observed in response to footshocks delivered during CFC1 and CFC2. Cells that never responded to foothsocks are shown in grey, cells that responded to 1, 2 or 3 footshocks during CFC1 in blue and CFC2 in green. Cells that responded to footshocks during CFC1 and CFC2 are shown in red. Linear regression was used to test correlations between responses to footshocks during CFC1 and CFC2 across foothsock responsive cells)(Statistics detailed in supplementary Table 1). r2 = 0.00; NS. j-k) Statistical comparison for the overlap of footshock responsive (SSTFSH) cells (63/118) and footshock unresponsive (SSTnon-FSH) cells (25/118) detected with PCA-ICA across CFC1 and CFC2 with a truncated null distribution (Statistics detailed in supplementary Table 1). l) Maximum calcium transient amplitude (df/f) for the population including cells that were found active across all behavioral states as identified with CNMF-E in the OF, EPM, context A and context B. Data (means ± SEM and data distribution represented as violin plots; n = 11–87 cells) were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). *p < 0.05 for specific comparisons. Area under the curve (df/f) for the overall population (n=121 cells) as identified with CNMF-E in the OF, EPM, context A and context B. Data (means ± SEM and data distribution represented as violin plots; n = 121 cells) were analyzed using one-way ANOVA (repeated measures over time) followed by Tukey’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). *p < 0.05 for specific comparisons. m) Sorting of the 121 cells detected with CNMF-E based on calcium transients frequency (Hz) during movement epochs in the OF on two consecutive sessions. Inactive cells are shown in grey, cells that were active exclusively during OF1 in blue and OF2 in green. Cells that were active during OF1 and OF2 are shown in red. Linear regression was used to test correlations between calcium transient frequency during OF1 and OF2 across cells active during both sessions (Statistics detailed in supplementary Table 1). r2 = 0.182; p < 0.01. n) Statistical comparison for the overlap of cells active during movement epochs (SSTMOB) cells detected with CNMF-E across OF1 and OF2 with a truncated null distribution (Statistics detailed in supplementary Table 1). o) Sorting of the 121 cells detected with CNMF-E based on calcium transients frequency (Hz) during exploration of the open arms in the EPM on two consecutive sessions. Inactive cells are shown in grey, cells that were active exclusively during EPM1 in blue and EPM2 in green. Cells that were active during EPM1 and EPM2 are shown in red. Linear regression was used to test correlations between calcium transient frequency during EPM1 and EPM2 across cells active during both sessions (Statistics detailed in supplementary Table 1). r2 = 0.126; NS. p) Statistical comparison for the overlap of cells active during exploration of the open arms (SSTOA) cells detected with CNMF-E across EPM1 and EPM2 with a truncated null distribution (Statistics detailed in supplementary Table 1). q) Direct comparisons of decoder analysis based on calcium transient frequency local minima (Onset) and maxima (Offset) predicting behavioral state transitions (freezing) in context A using 1, 2 or 3 SSTFSH cells (red) and SSTnon-FSH (orange) cells per mouse. Data (means ± SEM; n = 6,8 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time)(Statistics detailed in Supplementary Table 1).

  7. Supplementary Figure 7 Ca2+ imaging across multiple sessions (corresponds to Fig. 3).

    a) Spatial maps of segmented cells from CNMF-E were tracked across multiple imaging sessions (top to bottom) for each mouse (left to right). Individual cell maps show the maximum projection for Ca2+ transients observed during each session. Red and white arrowheads point to active and inactive cells, respectively. Note that each arrowhead (4 per mouse) indicates the same cell across 10 sessions for each mouse. b) Maximum projection of cells across sessions. Scale bar: 100 μm.

  8. Supplementary Figure 8 Cell registration across multiple sessions (corresponds to Fig. 3).

    a) Spatial maps of segmented cells from CNMF-E were uploaded to CellReg, which then probabilistically tracked cells across multiple sessions. b) Cell registration performed across 10 sessions allowed to generate a reliable cell map for each mouse. Two parameters were used to verify that a cell-pair represents indeed the same cell or different cells, namely, spatial correlation and the center of mass distance.

  9. Supplementary Figure 9 Optogenetic interrogation of SST-INs in DLS (corresponds to Figs. 4 and 5).

    a) Immunohistochemistry for somatostatin (SST) and enhanced yellow fluorescent protein (EYFP) in the DLS of SST-Cre mice injected with AAV5-DIO-EYFP. Note the overlap between SST and EYFP (white arrowheads). Representative images for 7 independent animals. Scale bar: 25 μm. b) Post-mortem histological control (related to Fig. 4) of AAV5-DIO-EYFP (virus: blue crosses; fiber optic: blue circles) as well as DIO-eNpHR3.0 (virus: red crosses; fiber optic: red circles) along the rostro-caudal axis of DLS. c) Post-mortem histological control (related to Fig. 5) of AAV5-DIO-EYFP (virus: blue crosses; fiber optic: blue circles) as well as DIO-ChR2 (virus: red crosses; fiber optic: red circles) along the rostro-caudal axis of DLS. d-e) Freezing analysis on block 7 for the effect of light silencing SST-INs in DLS across contexts for freezing bouts number (d) and duration (e). Data (means ± SEM; n = 7,7 mice per group) were analyzed using mixed factor two-way ANOVA (Statistics detailed in supplementary Table 1). f-g) In-depth analysis of the effect of SST cell body silencing on freezing bout duration revealed a main effect on short bouts (<10 sec) in context B but not A. Data (means ± SEM; n = 7, 7 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post-hoc test (detailed in Supplementary Table 1). *p < 0.05, DIO-EYFP versus DIO-eNpHR3.0. h-i) Freezing analysis on block 7 for the effect of light stimulating SST-INs (15 Hz) in DLS across contexts for freezing bouts number (h) and duration (i). Data (means ± SEM; n = 7,6 mice per group) were analyzed using mixed factor two-way ANOVA (Statistics detailed in supplementary Table 1). j-k) In-depth analysis of the effect of SST cell body stimulation (15 Hz) on freezing bout duration revealed a main effect on long bouts (<20 sec) in context A but not B. Data (means ± SEM; n = 7, 6 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post-hoc test (Statistics detailed in Supplementary Table 1). *p < 0.05, DIO-EYFP versus DIO-ChR2. l) Effect of 15 Hz light stimulation of SST cell bodies in DLS on SST-INs c-Fos expression 60 min following exposure to context B (day 21). Immunohistochemistry for SST and c-Fos (white arrowheads) in home cage controls (2 EYFP and 2 ChR2), EYFP light stimulated and ChR2 light stimulated animals. Representative images for 4,5,5 independent animals per group. Scale bar: 25 μm. m) ChR2 (Ch) light-stimulated animals showed greater overall expression of c-Fos in SST-INs as compared to controls (C) or EYFP (E) animals. Data (means ± SEM; n = 4, 5, 5 mice per group) were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). *p < 0.05, DIO-EYFP or DIO-ChR2 versus controls, #p < 0.05, DIO-ChR2 versus DIO-EYFP. n) In-depth analysis of c-Fos levels in SST-INs revealed distributions of cells with varying c-Fos levels in both groups and uncovered a significant increase in c-Fos expression in ChR2 expressing mice compared to controls and EYFP injected animals. Data (means ± SEM; n = 4, 5, 5 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time): c-Fos intensity F (10, 110) = 417.6, p < 0.001; treatment F (2, 11) = 31.7, p < 0.001; interaction F (20,110) = 11.07, p < 0.001 followed by Bonferroni’s multiple comparisons post-hoc test (Statistics detailed in supplementary Table 1). *p < 0.05, DIO-EYFP or DIO-ChR2 versus controls, #p < 0.05, DIO-EYFP versus DIO-ChR2.

Supplementary information

  1. Supplementary Figures 1–9

  2. Reporting Summary

  3. Supplementary Table 1

  4. Supplementary Table 2

  5. Supplementary Video 1

    Imaging during recall in Context A (raw calcium dynamics and df/f synced to behavior. Accelerated 4×).

  6. Supplementary Video 2

    Imaging during recall in Context B (raw calcium dynamics and df/f synced to behavior. Accelerated 4×).

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https://doi.org/10.1038/s41593-018-0330-y