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A thalamo-amygdalar circuit underlying the extinction of remote fear memories

Nature Neuroscience volume 24, pages 964–974 (2021)Cite this article

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Abstract

Fear and trauma generate some of the longest-lived memories. Despite the corresponding need to understand how such memories can be attenuated, the underlying brain circuits remain unknown. Here, combining viral tracing, neuronal activity mapping, fiber photometry, chemogenetic and closed-loop optogenetic manipulations in mice, we show that the extinction of remote (30-day-old) fear memories depends on thalamic nucleus reuniens (NRe) inputs to the basolateral amygdala (BLA). We found that remote, but not recent (1-day-old), fear extinction activates NRe-to-BLA inputs, which become potentiated upon fear reduction. Furthermore, both monosynaptic NRe-to-BLA and total NRe activity increase shortly before freezing cessation, suggesting that the NRe registers and transmits safety signals to the BLA. Accordingly, pan-NRe and pathway-specific NRe-to-BLA inhibition impairs, whereas their activation facilitates, remote fear extinction. These findings identify the NRe as a crucial BLA regulator for extinction and provide the first functional description of the circuits underlying the attenuation of consolidated fear memories.

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Fig. 1: Remote fear extinction recruits an IL→NRe→BLA pathway.
Fig. 2: The NRe bidirectionally modulates remote fear memory extinction.
Fig. 3: Freezing cessation during remote fear memory recall and extinction is preceded by increased NRe activity.
Fig. 4: Behavioral closed-loop optogenetic manipulation of the NRe during remote fear memory extinction bidirectionally modulates freezing cessation.
Fig. 5: NRe projections to the BLA are recruited upon and regulate freezing cessation during remote extinction.
Fig. 6: NRe projections to the BLA are potentiated upon remote fear memory extinction.
Fig. 7: NRe inputs to the BLA bidirectionally modulate remote fear memory extinction.

Data availability

All data are available from the authors upon reasonable request.

Code availability

All code used in this manuscript is available at https://zenodo.org/record/4588295.

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Acknowledgements

We thank M. Curdy, F. Wyler, M. Santoboni, M. Kintscher, F. Habibollahi, C. Yildirim, R. Gros, A. Goossens, C. Benquet, D. Joly, T. Hübscher, A.-R. Warter, L. Duvaud, L. Ho Dac and N. Sato for their contributions in histological procedures, the EPFL BIOP core facility for their technical assistance with image analysis, the Bertarelli Foundation Gene Therapy Core Facility at EPFL for AAV production and L. de Lecea, S. Tyree (Stanford) and N. Banterle (EPFL) for their assistance with fiber photometry. Funding: B.A.S. is supported by an EMBO long-term fellowship (ALT1605-2014, Marie Curie Actions, LTFCOFUND2013, GA-2013-609409). M.F.M.-R. is supported by an UNAM-DGECI International Scholarship from the National Autonomous University of Mexico (DGECI/DG/DFI/SME/1731/2016). J.G. is an MQ fellow (MQ15FIP100012) and a NARSAD Independent Investigator (24497). H.H. is supported by a NENS Exchange Grant. The laboratory of J.G. is supported by the European Research Council (ERC-2015-StG 678832), the Swiss National Science Foundation (31003 A_155898), the National Competence Center for Research (NCCR) SYNAPSY (51NF40-185897) and the Vallee Foundation (VS-2019-27). Work from the C.S. laboratory is supported by grants from the SNSF (31003A-176206) and the NCCR SYNAPSY (158776 and 185897).

Author information

Author notes

  1. Bianca A. Silva

    Present address: National Research Council of Italy, Institute of Neuroscience, Humanitas Clinical and Research Center – IRCCS, Rozzano, Milan, Italy

  2. Hendrik Heiser

    Present address: Laboratory of Neural Circuit Dynamics, Brain Research Institute, University of Zürich, Zürich, Switzerland

  3. Lukas van den Heuvel

    Present address: Laboratory of Quantitative Neurosciences, Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands

  4. Maria Fernanda Martinez-Reza

    Present address: Physiological Genomics, Biomedical Center (BMC), Ludwig-Maximilians Universität München, Planegg, Germany

Authors and Affiliations

  1. Laboratory of Neuroepigenetics, Brain Mind Institute, School of Life Sciences, École Polytechnique Fédérale Lausanne, Lausanne, Switzerland

    Bianca A. Silva, Allison M. Burns, Hendrik Heiser, Lukas van den Heuvel, Giulia Santoni, Maria Fernanda Martinez-Reza & Johannes Gräff

  2. Laboratory of Behavioral Genetics, Brain Mind Institute, School of Life Sciences, École Polytechnique Fédérale Lausanne, Lausanne, Switzerland

    Simone Astori & Carmen Sandi

  3. International Max Planck Research School “Neurosciences”, Georg-August-Universität Göttingen, Göttingen, Germany

    Hendrik Heiser

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Contributions

This study was planned and conceptualized by B.A.S. and J.G. B.A.S. carried out the experiments and analyzed data. S.A. performed and analyzed electrophysiological experiments under the guidance of C.S. A.M.B. contributed to fiber photometry data analysis. G.S. contributed to viral injections and behavioral experiments. H.H., L.v.d.H. and M.F.M.-R. contributed to histology and image analysis. The paper was written by B.A.S. and J.G. and commented on by all authors.

Corresponding author

Correspondence to Johannes Gräff.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Larry Zweifel 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 cFos and AAV2r quantifications in the IL, NRe and BLA upon recent and remote fear memory extinction.

a, Quantification of cFos+ cells in the IL (left) and NRe (right) of brain sections used for functional connectivity analysis following recent fear memory extinction (Fig. 1). Unpaired t-test. IL: P = 0.6748, N = 10 mice/group; NRe: P = 0.381, N = 8 (Ext), 10 (No shock) animals/group. b, Quantification of AAV2r+ cells in the IL and NRe of brain sections used for functional connectivity analysis following recent fear memory extinction (Fig. 1). Unpaired t-test. IL→BLA: P = 0.876, N = 8 (Ext), 8 (No shock) animals/group; NRe→BLA: P = 0.3599, N = 8 (Ext), 10 (No shock) animals/group; IL→NRe: P = 0.5688, N = 10 (Ext), 10 (No shock) animals/group. c, Quantification of cFos+ cells in the IL (left) and NRe (right) of brain sections used for functional connectivity analysis following remote fear memory extinction (Fig. 1). Unpaired t-test. IL: P = 0.1374, N = 9 (Ext), 8 (No shock) animals/group; NRe: P = 0.1349, N = 11 (Ext), 6 (No shock) animals/group. d, Quantification of AAV2r+ cells in the IL and NRe of brain sections used for functional connectivity analysis following remote fear memory extinction. Unpaired t-test. IL→BLA: P = 0.006, N = 9 (Ext), 8 (No shock) animals/group; NRe→BLA: P = 0.638, N = 11 (Ext), 6 (No shock) animals/group; IL→NRe: P = 0.0132, N = 9 (Ext), 9 (No shock) animals/group. e, Matched comparisons (within the same animals/group) of IL→BLA vs IL→NRe activation upon recent (left) or remote (right) fear memory extinction. Recent: Unpaired t-test: P = 0.1304, N = 7 animals/behavioral group. Two-tailed one-sample t-test (theoretical mean=1), IL→BLA, #P = 0.04; IL→NRe, P = 0.4476; Remote: Unpaired t-test: P = 0.0001, N = 9 animals/behavioral group. Two-tailed one-sample t-test (theoretical mean=1), IL→BLA, P = 0.6634; IL→NRe, ###P < 0.0001. BLA, basolateral amygdala; CFC, contextual fear conditioning; Ext, extinction; IL, infralimbic cortex; NRe, nucleus reuniens of the thalamus. Data are represented as mean ± SEM. Statistical analysis details for each figure panel are reported in Supplementary Table 1.

Extended Data Fig. 2 cFos mapping in BLA inputs.

Schematic representation of a, AAV2r injections into the BLA and b, behavioral groups used for cFos IHC in AAV2r-injected animals. Colocalization analysis of retrogradely traced cells from the BLA (AAV2r+) and cells activated upon remote fear memory recall and extinction (cFos+) in c, cortex: Two-way ANOVA, Control vs Extinction, F(1,39)=0.245, P = 0.62. Multiple comparison, Sidak. PL, N = 7 (No shock), 9 (Ext) mice/group; ACC, N = 8 (No shock), 9 (Ext) mice/group; IC, N = 6 (No shock), 6 (Ext) mice/group; d, midbrain: Two-way ANOVA, Control vs Extinction, F(1,46)=6.411, P = 0.0148. Multiple comparison, Sidak * P < 0.05. SUM, N = 6 (No shock), 19 (Ext) mice/group; VTA, N = 7 (No shock), 18 (Ext) mice/group; e, midline thalamus: Two-way ANOVA, Control vs Extinction, F(1,50)=3.989, P = 0.051. Multiple comparison, Sidak. Rh, N = 6 (No shock), 9 (Ext) mice/group; PVT, N = 8 (No shock), 7 (Ext) mice/group; IMD N = 7 (No shock), 6 (Ext) mice/group; CM, N = 8 (No shock), 7 (Ext) mice/group. f, hippocampus: unpaired t test P = 0.979. N = 7 (No shock), 8 (Ext) mice/group. ACC, anterior cingulate cortex; BLA, basolateral amygdala; CM, centromedial thalamus; IL, infralimbic cortex; IC, insular cortex (posterior, granular cortex); IMD, intermediodorsal nucleus of the thalamus; NRe, nucleus reuniens of the thalamus; PL, prelimbic cortex; PVT, periventricular thalamus; Rh, rhomboid nucleus; SUM, supramammilary nucleus; vCA1, ventral CA1; VTA, ventral tegmental area. Data are represented as mean ± SEM. Statistical analysis details for each figure panel are reported in Supplementary Table 1.

Extended Data Fig. 3 TRIO of a direct IL-NRe-BLA di-synaptic circuit.

(a) Schematic representation of the strategy for tracing the relationship between input and output (TRIO). An AAV2r-Cre virus was injected bilaterally into the BLA and Cre-dependent helper viruses (AAV8-DIO-TVA, AAV8-DIO-RG-GFP) were injected in the NRe. Retrograde transport of the AAV2r allows for selective expression of the Cre recombinase in NRe neurons projecting to the BLA. After Cre recombination, the helper AAVs express TVA, an avian receptor protein that confers infection capability to EnvA-pseudotyped rabies virus, and rabies glycoprotein B19G-GFP, which mediates the monosynaptic retrograde spread of rabies particles. Three weeks later, an EnvA-pseudotyped rabies virus carrying an mCherry coding sequence was injected into the NRe, which selectively infects TVA-expressing neurons and spreads retrogradely and trans-synaptically. (b) (Left) Example picture of starter cells in the NRe co-labeled with RG-GFP deriving from the Cre-dependent AAV and mCherry from the rabies virus. Scale bar = 250 μm. Zoom-in inset: scale bar = 100 μm. (Right) Retrogradely traced neurons in the IL identified by mCherry expression of transynaptically transported rabies. Scale bar = 250 μm. Zoom-in inset: scale bar = 100 μm. Arrowheads indicate mCherry+ neurons in the IL. (c) Schematic representation of mCherry+ neurons in the IL of one animal subjected to TRIO. (d) Quantification of the total number of mCherry+ neurons in the IL following TRIO tracing in experimental and control animals. N = 2 (Ctrl), 5 (TRIO) mice/group. (e) Schematic representation (top) and example pictures (bottom) of TRIO controls that did not receive AAV8-DIO-RG-GFP injections in the NRe. Data are represented as mean ± SEM. BLA, basolateral amygdala; IL, infralimbic cortex; NRe, nucleus reuniens of the thalamus; RG, rabies protein G.

Extended Data Fig. 4 Chemogenetic inhibition of the NRe induces persistent extinction impairment and is not due to unspecific CNO effects.

(a) Freezing responses (bottom) during the remote fear memory extinction paradigm (top) upon hM4Di/CNO inhibition of the NRe. Yellow bars indicate CNO administration during extinction but not at recall or SR. Control animals were injected with a homologous virus lacking the hM4Di sequence (AAV8-hSyn::mCherry) and received CNO administration identical to experimental animals. Two-way RM ANOVA, VEH vs CNO: F(1, 19)=7.047, P = 0.0156, multiple comparison, Sidak, * P < 0.05; N = 8(hM4Di), 13 (mCherry) animals/group. (b) Freezing responses (bottom) during the remote fear memory extinction paradigm (top) upon CNO administration in hM4Di or mCherry-injected animals in the NRe. Yellow bar indicates CNO inhibition at SR only. Two-way RM ANOVA, VEH vs CNO: F(1, 12)=0.14, P = 0.72, N = 6 (CNO), 8 (VEH) mice/group. (c) Representative picture of hM4Di-mCherry expression (virus: AAV8-hM4Di-mCherry) in a mis-injected animal. Scale bar = 800 μm. Zoom-in 200 μm. (d) Experimental timeline for CNO-mediated inhibition during remote fear memory recall, extinction and SR (top). Freezing responses (bottom) during the remote fear memory extinction paradigm and SR upon hM4Di-mediated inhibition of the NRe or its vehicle control. Yellow bars indicate days of CNO administration. Two-way RM ANOVA, VEH vs CNO: F(1, 41)=0.05, P = 0.82, N = 11 (CNO), 28 (VEH) mice/group. BL, baseline freezing; CFC, contextual fear conditioning; CNO, clozapine-N-oxide; NRe, nucleus reuniens of the thalamus; Rec, recall; SR, spontaneous recovery; VEH, vehicle. Data are represented as mean ± SEM. Statistical analysis details for each figure panel are reported in Supplementary Table 1.

Extended Data Fig. 5 NRe activation during remote fear memory extinction facilitates freezing reduction.

(a) Representative pictures of hM3Dq-mCherry expression (red) and cFos (green) in the NRe upon vehicle or CNO injection. Scale bar = 200 μm. (b) Quantification of cFos-positive cell density in the NRe upon vehicle or CNO injection. Unpaired t-test, P = 0.013. N = 5 (CNO), 6 (VEH) mice/group. (c) (Top) Experimental timeline: All animals underwent contextual fear conditioning (CFC) and received AAV8-CamKII::hM3Dq-mCherry injection one week later. Thirty days after CFC they underwent memory recall and subsequently the spaced extinction paradigm under CNO or vehicle treatment and were tested for spontaneous recovery (SR) two weeks later. (Bottom) Freezing responses during the remote fear memory extinction paradigm and SR upon hM3Dq/CNO activation of the NRe. Blue bars indicate days of CNO exposure. Two-way RM ANOVA, VEH vs CNO: F(1, 10)=4.227, P = 0.0668, multiple comparison, Sidak, * p < 0.05, N = 6 mice/behavioral group. (d) Freezing responses (Bottom) during the extinction memory test (ET) and SR (top) upon repeated hM3Dq-mediated home cage activation of the NRe in the absence of both the recall and the spaced extinction behavioral paradigm. Two-way RM ANOVA, VEH vs CNO: F(1, 14)=1.18, P = 0.296, N = 8 mice/group. BL, baseline freezing; CFC, contextual fear conditioning; CNO, clozapine-N-oxide; ET, extinction test; NRe, nucleus reuniens of the thalamus; Rec, recall; SR, spontaneous recovery; VEH, vehicle. Data are represented as mean ± SEM. Statistical analysis details for each figure panel are reported in Supplementary Table 1.

Extended Data Fig. 6 Activity rise in the NRe starts shortly before freezing cessation throughout remote fear memory extinction and is not time-locked to freezing initiation.

a, (Left) Example traces of photometry signals (reported as dF/F, see Methods) generated by 465 nm (black, Ca2+-dependent) and 405 nm (blue, Ca2+-independent) LED excitation during the first three days of extinction. Blue boxes indicate freezing bouts (0.5 s ≥ light blue < 1.5 s; dark blue ≥ 1.5 s). (Right) Mean signal ±2 s around cessation of freezing (indicated by the dashed lines) for >1.5 s freezing bouts from the corresponding behavioral session in the left panel. b,c, Heat maps represent photometry signals (reported as dF/F) for each freezing epoch and the following mobility epoch of one representative recall (b), and extinction (c), session. All heat maps are aligned to freezing cessation (0 s) and ordered by freezing epoch duration. Freezing epochs followed by mobility epochs shorter that 0.1 s were excluded from the analysis.

Extended Data Fig. 7 Characterization of NRe outputs in behaviorally naïve animals.

a, (Left) Schematic representation and representative picture (scale bar = 250 μm) of AAV8-CamKII::hM3Dq-mCherry injection in the NRe. (Right) Representative picture of hM3Dq-mCherry axonal expression in the medial prefrontal cortex (mPFC; left, scale bar = 250 μm), hippocampus (top-right, scale bar = 250 μm) and amygdala (bottom-right, scale bar = 100 μm); red=mCherry, blue=Hoechst. b, Representative pictures of cFos immunoreactivity in the mPFC (left), hippocampus (top right) and amygdala (bottom right) upon chemogenetic activation of the NRe. Animals were sacrificed 90 min after systemic CNO or vehicle administration. Scale bar 200 μm. c, Quantification of cFos immunoreactivity following NRe chemogenetic activation in the mPFC, two-way ANOVA, VEH vs CNO: F(1, 18)=2.82, P = 0.11, N = 5 (CNO), 6 (VEH) mice/group; hippocampus, two-way ANOVA, VEH vs CNO: F(1, 27)=3.17, P = 0.09, N = 5 (CNO), 6 (VEH) mice/group, and the amygdala, two-way ANOVA, VEH vs CNO: F(1, 35)=22.32, P < 0.0001, multiple comparison, Sidak * P < 0.05 N = 5 (CNO), 6 (VEH) mice/group. d, Schematic representation of AAV1-hSyn::Chronos-Tom injections in the NRe and subsequent ex vivo patch-clamp recordings in neurons of the central amygdala (CeA), and pyramidal cells from the basolateral amygdala (BLA) and the lateral amygdala (LA). e, (Left) Representative traces of evoked postsynaptic currents (EPSCs) elicited at −70 mV by brief consecutive LED pulses and (right) values of EPSC peak. One-way ANOVA: F(2, 32)=17.31, P < 0.0001; multiple comparison, Holm-Sidak, *** P < 0.001, n = 10 (CeA), 8 (LA), 15 (BLA) cells from N = 3-6 mice/group. f, Paired-pulse ratio (PPR) in the different amygdalar nuclei (color-coded). One-way ANOVA F(2, 28)=7.39, P = 0.0026; multiple comparison, Holm-Sidak, * P < 0.05, ** P < 0.01, n = 9 (CeA), 7 (LA), 15 (BLA) cells from N = 3-6 mice/group. BLA, basolateral amygdala, CeAl, lateral portion of the central amygdala, CeAm, medial portion of the central amygdala; DG, dentate gyrus; IL, infralimbic cortex; LA, lateral amygdala; mPFC, medial prefrontal cortex; PL, prelimbic cortex. Data are represented as mean ± SEM. Statistical analysis details for each figure panel are reported in Supplementary Table 1.

Extended Data Fig. 8 cFos activation analysis of NRe→BLA projections during remote fear memory extinction.

a, Representative pictures of cFos IHC in retrogradely traced cells from the BLA in the NRe following the remote recall session, the last extinction session and the no shock control group (for the experimental setup see Fig. 1). Scale bar = 100 μm. Arrowheads indicate double positive cells in the NRe (AAV2r+, cFos+). b, Colocalization analysis of retrogradely traced cells from the BLA (AAV2r+) and cells activated in the NRe (cFos+) in the remote recall, extinction and the no shock control group. Kruskal–Wallis test, P = 0.02, multiple comparison, Dunn * P < 0.05, ** P < 0.01, N = 6 (No shock), 14 (Recall), 11 (Ext) mice/group; Data are represented as mean ± SEM. Statistical analysis details for each figure panel are reported in Supplementary Table 1.

Extended Data Fig. 9 cFos activation and functional analysis of IL→BLA projections during remote fear memory extinction.

a, Schematic representation of the retrograde virus injection strategy. b, Representative pictures of cFos IHC in retrogradely traced cells from the BLA in the IL following the remote recall session, the last extinction session and the no shock control group (for experimental setup see Fig. 1). Scale bar = 100 μm c, Colocalization analysis of retrogradely traced cells from the BLA (AAV2r+) and cells activated in the IL (cFos+) in the remote recall, remote extinction and the no shock control group. Kruskal-Wallis test, multiple comparison, Dunn; P = 0.89, N = 8 (No shock), 7 (recall), 9 (Ext) mice/group. d, (Left) Schematic representation of the experimental strategy for DREADD-mediated manipulation of IL-deriving terminals in the BLA. (Right) Representative picture of hM4Di-mCherry expression at the level of the injection site in the IL (top) and cannula placement in the BLA (bottom). Notably, hM4Di-mCherry+ fibers can be observed in the NRe and BLA (scale bar = 500 μm). e, Selective hM4Di-mediated inhibition of IL-deriving terminals in the BLA does not affect remote fear memory extinction. Yellow bars indicate local BLA infusions of CNO/VEH at remote recall and throughout the remote fear memory extinction paradigm. Two-way RM ANOVA, VEH vs CNO: F(1, 7)=0.234, P = 0.643, multiple comparison, Sidak, N = 4 (VEH), 5 (CNO) mice/group. Data are represented as mean ± SEM. Statistical analysis details for each figure panel are reported in Supplementary Table 1.

Extended Data Fig. 10 Functional characterization of IL→NRe projections during remote fear memory extinction.

a, Schematic representation of the retrograde virus injection strategy. b, Representative pictures of cFos IHC in retrogradely traced cells from the NRe in the IL following the remote recall session, the last extinction session and the no shock control group (for experimental setup see Fig. 1). c, Colocalization analysis of retrogradely traced cells from the NRe (AAV2r+) and cells activated in the IL (cFos+) in the remote recall, remote extinction and the no shock control group. Kruskal-Wallis test, P = 0.031, N = 7 (No shock), 7 (recall), 9 (Ext) mice/group; multiple comparison, Dunn, * P < 0.05. Arrowheads indicate double positive cells in the IL (AAV2r+, cFos+). d, Schematic representation (left) and representative pictures (right) of the viral strategy for optogenetic stimulation of IL→NRe projections. AAV8 vectors carrying the activatory opsin Chronos-mCherry or mCherry only were injected bilaterally in the IL and an optic fiber was implanted above the NRe. Scale bar= 500 μm. e, Freezing behavior quantification upon optogenetic stimulation (20 Hz, 5 s light ON, 5 s light OFF, total duration=3 min) of IL→NRe projections during one remote fear memory extinction session. Two-way ANOVA for freezing time during light ON and OFF periods during one remote extinction session, interaction: F(1, 7)=17.42, P = 0.0042; ON vs OFF: F(1, 7)=21.49, P = 0.0024, multiple comparison, Sidak, * P < 0.05, ** P < 0.01, *** P < 0.001. N = 4 (Chronos), 5 (mCherry) mice/group. f, Schematic representation of the experimental approach and fiber photometry recording implant for IL→NRe fiber photometry recordings (top); representative picture of GCaMP6f expression and localization of the optical fiber implant in the IL (bottom). Scale bar = 500 μm. g, (Left) Example traces of photometry signals (reported as dF/F, see Methods) generated by 465 nm (black, Ca2+-dependent) and 405 nm (blue, Ca2+-independent) LED excitation during habituation, recall and the last extinction session. Blue boxes indicate freezing bouts (0.5 s ≥ light blue < 1.5 s; dark blue ≥1.5 s). (Right) Mean dF/F signal ±2 s around cessation of freezing (indicated by the dashed line, 0 s) for ≥1.5 s freezing bouts from the corresponding behavioral session in the left panel. h, (Left) Quantification of dF/F difference before and after freezing end. RM One-way ANOVA, F(2, 8)=0.989, P = 0.41, multiple comparison, Holm-Sidak, N = 5 animals. (Right) Signal power analysis of dF/F during remote fear memory extinction. Signal power was calculated as Σ(dF/F–av.dF/F)2. RM One-way ANOVA, F(2, 8)=6.72, P = 0.019, multiple comparison, Sidak, * P < 0.05. N = 5 animals. Data are represented as mean ± SEM. Statistical analysis details for each figure panel are reported in Supplementary Table 1.

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Silva, B.A., Astori, S., Burns, A.M. et al. A thalamo-amygdalar circuit underlying the extinction of remote fear memories. Nat Neurosci 24, 964–974 (2021). https://doi.org/10.1038/s41593-021-00856-y

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