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Neural circuits underlying a psychotherapeutic regimen for fear disorders

Naturevolume 566pages339343 (2019) | Download Citation

Abstract

A psychotherapeutic regimen that uses alternating bilateral sensory stimulation (ABS) has been used to treat post-traumatic stress disorder. However, the neural basis that underlies the long-lasting effect of this treatment—described as eye movement desensitization and reprocessing—has not been identified. Here we describe a neuronal pathway driven by the superior colliculus (SC) that mediates persistent attenuation of fear. We successfully induced a lasting reduction in fear in mice by pairing visual ABS with conditioned stimuli during fear extinction. Among the types of visual stimulation tested, ABS provided the strongest fear-reducing effect and yielded sustained increases in the activities of the SC and mediodorsal thalamus (MD). Optogenetic manipulation revealed that the SC–MD circuit was necessary and sufficient to prevent the return of fear. ABS suppressed the activity of fear-encoding cells and stabilized inhibitory neurotransmission in the basolateral amygdala through a feedforward inhibitory circuit from the MD. Together, these results reveal the neural circuit that underlies an effective strategy for sustainably attenuating traumatic memories.

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All data used in this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Y.-S. Kim for providing the PLCß4 knockdown virus, G. Buzsáki for advising us on silicon probe recording in freely moving mice, and J. J. Shin for discussions on slice recordings. This work was supported by IBS grant IBS-R001-D1.

Reviewer information

Nature thanks J. Johansen, G. Quirk and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Jinhee Baek, Sukchan Lee

Affiliations

  1. Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, South Korea

    • Jinhee Baek
    • , Sukchan Lee
    • , Taesup Cho
    • , Seong-Wook Kim
    • , Minsoo Kim
    • , Yongwoo Yoon
    • , Ko Keun Kim
    • , Junweon Byun
    •  & Hee-Sup Shin
  2. Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea

    • Jinhee Baek
    •  & Jaeseung Jeong
  3. Department of Physiology, Seoul National University College of Medicine, Seoul, South Korea

    • Sukchan Lee
    •  & Sang Jeong Kim
  4. Department of Basic Science, University of Science and Technology, Daejeon, South Korea

    • Junweon Byun
    •  & Hee-Sup Shin

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Contributions

J. Baek, S.L. and H.-S.S. designed the experiments and wrote the manuscript. J. Baek performed in vitro and in vivo electrophysiology and optogenetic experiments. S.L. performed behavioural experiments. S.-W.K. contributed to genetic studies. M.K. and Y.Y. contributed to histological work. T.C. performed in vitro electrophysiology. K.K.K. and J. Byun contributed to in vitro electrophysiology analysis. J. Byun performed blinded counting. S.J.K. aided in the interpretation of data and contributed to editing the manuscript. J.J. and H.-S.S. supervised the project and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Jaeseung Jeong or Hee-Sup Shin.

Extended data figures and tables

  1. Extended Data Fig. 1 Effect of ABS pairing on fear extinction of strong fear memory and effect on memory reactivation and reconsolidation.

    a, One day after fear conditioning (0.7 mA foot shock), visual stimulation was presented during fear extinction (n = 7 mice for each group). Mixed-design ANOVA for extinction: F4,30 = 78.62, P = 1.85 × 10−15 for group effect. One-way ANOVA for recall test: F4,30 = 53.95, P = 2.81 × 10−13. b, Effects of ABS pairing on fear relapse (n = 7 mice for each group). Two-way ANOVA: F1,36 = 138.521, P = 6.73 × 10−14 for group effect. Post hoc multiple comparison with Bonferroni correction; ***P < 0.001. Asterisks above bars indicate significant difference in comparison to recall. c, Effects of ABS pairing during memory reactivation (CS, n = 8; ABS + CS, n = 8 mice). Student’s t-test, two-sided: t(14) = −3.9058, P = 0.001584 for memory reactivation; t(14) = 0.2411, P = 0.813 for PR-LTM; **P < 0.01. Data shown as mean ± s.e.m. See Supplementary Table 1 for statistical details.

  2. Extended Data Fig. 2 Single-unit recording of SC.

    a, Coronal sections showing the positions of the silicon probes (left) and tetrodes (right). SGS, stratum griseum superficiale; SGI, stratum griseum intermediale; SGP, stratum griseum profundum. b, Schematic of 64-channel silicon probes used for SC recordings. c, Example waveforms of recorded neurons from a single shank. d, Probe tracks (left) and tetrode tip locations (right). e, Example single-unit responses of the SC to sensory stimulation (500-ms bins; pie charts, n = 109 cells). Sensory stimulation blocks were pseudo-randomly presented. f, Averaged SC responses during 5 s after stimulus onset (n = 109 cells). Mixed-design ANOVA: F3,324 = 15.4, P = 2.17 × 10−19 for stimulation effect. g, h, Positive responses of SC neurons from CS group (g; n = 33 cells) and ABS + CS group (h; n = 62 cells) during fear extinction. i, Averaged positive responses across extinction trials (early, second-to-fifth trials; mid, sixth-to-tenth trials; late, eleventh-to-fifteenth trials; samples from g, h). Mixed-design ANOVA: F1,93 = 7.621, P = 0.00695 for group effect. j, k, Negative responses of SC neurons from CS group (j; n = 10 cells) and ABS + CS group (k; n = 8 cells) during fear extinction. l, Averaged negative responses across extinction trials (samples from j, k). Mixed-design ANOVA: F1,16 = 0.71, P = 0.412 for group effect. Mean ± s.e.m.; post hoc multiple comparison with Bonferroni correction; *P < 0.05. See Supplementary Table 1 for statistical details.

  3. Extended Data Fig. 3 Freezing behaviour and correlation with SC activity during fear extinction.

    a, b, Fear extinction (a) and subsequent retention tests (b) with SC single-unit recordings (CS, n = 10; ABS + CS, n = 8 mice). Mixed-design ANOVA for extinction: F1,16 = 29.73, P = 5.32 × 10−5 for group effect. Mixed-design ANOVA for retention tests: F1,16 = 32.65, P = 3.2 × 10−5 for group effect. Mean ± s.e.m.; post hoc multiple comparison with Bonferroni correction; *P < 0.05, **P < 0.01, ***P < 0.001. Asterisks above bars indicate significant difference in comparison to recall. cf, Pearson’s correlation analyses of SC positive responses (CS, n = 9; ABS + CS, n = 8 mice) during fear extinction with freezing during late extinction trials (c; a block of the last three extinction trials), recall test (d), spontaneous recovery test (e) or renewal test (f). gj, Pearson’s correlation analyses of SC negative responses (CS, n = 5; ABS + CS, n = 5 mice) during fear extinction with freezing during late extinction trials (g), recall test (h), spontaneous recovery test (i) or renewal test (j). See Supplementary Table 1 for statistical details.

  4. Extended Data Fig. 4 Single-unit recording of MD.

    a, Coronal section showing the position of the recording sites (red arrow). HB, habenular nucleus; PVT, paraventricular thalamic nucleus. b, c, An example spike sorting result from a single tetrode. b, Example feature plot showing clusters of candidate spikes; c, average waveforms of isolated units from the tetrode. d, Tetrode tip locations in MD. e, f, Positive responses of MD neurons in CS group (e; n = 49 cells) and ABS + CS group (f; n = 63 cells) g, Averaged positive responses across extinction trials (early, second-to-fifth trials; mid, sixth-to-tenth trials; late, eleventh-to-fifteenth trials; samples from e, f). Mixed-design ANOVA: F1,110 = 17.83, P = 4.99 × 10−5 for group effect. h, i, Negative responses of MD neurons in CS group (h; n = 31 cells) and ABS + CS group (i; n = 44 cells) during fear extinction. j, Averaged negative responses of the MD across extinction trials (samples from h, i). Mixed-design ANOVA: F1,73 = 1.762, P = 0.188 for group effect. Mean ± s.e.m.; post hoc multiple comparison with Bonferroni correction; ***P < 0.001. See Supplementary Table 1 for statistical details.

  5. Extended Data Fig. 5 Freezing behaviour and correlation with MD activity during fear extinction.

    a, b, Fear extinction (a) and subsequent retention tests (b) with MD single-unit recordings (CS, n = 6; ABS + CS, n = 8 mice). Mixed-design ANOVA for extinction: F1,12 = 13.85, P = 0.000292 for group effect. Mixed-design ANOVA for retention tests: F1,12 = 33.1, P = 9.11 × 10−5 for group effect. Mean ± s.e.m.; post hoc multiple comparison with Bonferroni correction; **P < 0.01, ***P < 0.001. cf, Pearson’s correlation analyses of MD positive responses (CS, n = 6; ABS + CS, n = 8 mice) during fear extinction with freezing during late extinction trials (c, a block of the last three extinction trials), recall test (d), spontaneous recovery test (e) or renewal test (f). gj, Pearson’s correlation analyses of MD negative responses (CS, n = 4; ABS + CS, n = 8 mice) during fear extinction with freezing during late extinction trials (g), recall test (h), spontaneous recovery test (i) or renewal test (j). See Supplementary Table 1 for statistical details.

  6. Extended Data Fig. 6 Plcb4 deletion disturbing MD activity blocks the effects of ABS paired extinction.

    a, Effects of the Plcb4 knockout (KO) on ABS paired extinction (wild-type (WT) CS, n = 5; WT ABS + CS, n = 5; KO CS n = 5; KO ABS + CS n = 7 mice). Mixed-design ANOVA for fear extinction: F3,18 = 57.56, P = 2.01 × 10−9 for group effect. One-way ANOVA for recall test: F3,18 = 35.24, P = 9.6 × 10−8. b, Effects of Plcb4 knockdown in MD on ABS paired extinction (shControl CS, n = 4; shControl ABS + CS, n = 7; shPlcb4 CS, n = 4; shPlcb4 ABS + CS, n = 5 mice). Mixed-design ANOVA for fear extinction: F3,16 = 19.25, P = 1.47 × 10−5 for group effect. One-way ANOVA for recall test: F3,16 = 26.18, P = 2.07 × 10−6. Mean ± s.e.m; ***P < 0.001. See Supplementary Table 1 for statistical details. cj, Knockdown of Plcb4 in the MD by injection of shRNA lentiviral vector. Double fluorescence labelling of PLCβ4 expression with DAPI counterstain in the MD of shControl-injected mice (cf) and shPlcb4-injected mice (gj). Histology was confirmed for all mice in b after behavioural experiments. df, hj, Higher magnification images corresponding to the rectangles in c, g, respectively. Scale bars, 1,000 μm (c, g); 100 μm (df, hj).

  7. Extended Data Fig. 7 Verification of viral expression and functional connectivity of the SC–MD pathway.

    a, Retrograde tracer CTB (green) was injected into the MD. Only 6.12% (37/600) of CTB-positive neurons were GABA-positive and only 4.38% (37/844) of GABA-positive neurons were CTB-positive. Experiments were repeated with three mice (two slices per mouse) with similar results, and combined cell numbers are presented. White arrow indicates a CTB-positive GABAergic neuron in the SC. Scale bar, 20 μm. b, Illustration of viral injections in SC and fibre placement in MD. c, Coronal section showing a neuron expressing eNpHR3.0–eYFP in SC. Viral expression was confirmed in 20 mice after behavioural experiments (Fig. 2g–i). d, Coronal section showing fibres expressing eNpHR3.0–eYFP in MD. Viral expression was confirmed in 20 mice after behavioural experiments (Fig. 2g–i). e, Optical fibre placements for SC–MD silencing experiments. f, ChR2–YFP virus injection in SC and slicing position for whole-cell recording of MD neurons (blue dashed line). g, A sample trace of action potentials recorded from MD neurons in slice culture in response to ChR2 stimulation of the SC–MD pathway. h, Optical fibre placements for SC–MD photostimulation experiments.

  8. Extended Data Fig. 8 Single-unit recording of BLA neurons and their classification.

    a, Coronal section (left) and illustration (right) showing the position of the recording site. LA, lateral nucleus of the amygdala; BA, basal nucleus of the amygdala. b, An example spike sorting showing clusters of candidate spikes (left) and average waveforms of four isolated units (right) from a single tetrode. c, Heat map and classified BLA responses during extinction trials (1-s bins; χ2(2) = 16.204, P = 0.0003029 (CS, n = 190; ABS + CS n = 227 cells). d, e, Average positive responses (d; CS, n = 67; ABS + CS, n = 63 cells) and negative responses (e; CS, n = 36; ABS + CS, n = 84 cells) in the BLA during fear extinction (1-s bins). Mann–Whitney U-test, two-sided: P = 0.3736 for positive responses; P = 0.296 for negative responses. f, g, Pearson’s correlation analysis of BLA positive responses (f; CS, n = 8, ABS + CS, n = 6 mice) or negative responses (g; CS, n = 8, ABS + CS, n = 9 mice) during fear extinction with average freezing level during spontaneous recovery and renewal. h, Proportions of the classified BLA responses (χ2(3) = 2.0536, P = 0.5613). ik, Averaged pip responses (20-ms bins) of classified fear cells (i; CS, n = 34; ABS + CS, n = 42 cells), resistant cells (j; CS, n = 21; ABS + CS, n = 16 cells) and extinction cells (k; CS, n = 24; ABS + CS, n = 30 cells) during the first extinction trial (left) and the last extinction trial (right). ln, Time course of averaged pip responses (left) and trial responses (right) of fear cells (l; samples from i), resistant cells (m; samples from j) and extinction cells (n; samples from k) during fear extinction (early, second-to-fifth trials; mid, sixth-to-tenth trials; late, eleventh-to-fifteenth trials). Mixed-design ANOVA for pip responses: F1,74 = 0.513, P = 0.476 for group effect of fear cells; F1,35 = 2.859, P = 0.0998 for group effect of resistant cells; F1,52 = 0.345, P = 0.559 for group effect of extinction cells. Mixed-design ANOVA for trial responses: F1,74 = 4.775, P = 0.032 for group effect of fear cells; F1,35 = 4.846, P = 0.0344 for group effect of resistant cells; F1,52 = 0.638, P = 0.428 for group effect of extinction cells. Mean ± s.e.m.; post hoc multiple comparison with Bonferroni correction. See Supplementary Table 1 for statistical details.

  9. Extended Data Fig. 9 Freezing behaviour and correlation with BLA activity during fear extinction.

    a, b, Fear extinction (a) and subsequent retention tests (b) with BLA single-unit recordings (CS, n = 8; ABS + CS, n = 9 mice). Mixed-design ANOVA for extinction: F1,15 = 19.46, P = 0.000505 for group effect. Mixed-design ANOVA for retention tests: F1,15 = 27.29, P = 0.000103 for group effect. Mean ± s.e.m.; post hoc multiple comparison with Bonferroni correction; **P < 0.01, ***P < 0.001. cf, Pearson’s correlation analyses of fear-cell trial responses (CS, n = 8; ABS + CS, n = 9 mice) with freezing during late extinction trials (c; a block of the last three extinction trials), recall test (d), spontaneous recovery test (e) or renewal test (f). gj, Pearson’s correlation analyses of resistant-cell trial responses (CS, n = 7; ABS + CS, n = 7 mice) with freezing during late extinction trials (g), recall test (h), spontaneous recovery test (i) or renewal test (j). kn, Pearson’s correlation analyses of extinction-cell trial responses (CS, n = 8; ABS + CS, n = 7 mice) with freezing during late extinction trials (k), recall test (l), spontaneous recovery test (m) or renewal test (n). See Supplementary Table 1 for statistical details.

  10. Extended Data Fig. 10 The MD drives feedforward inhibition in the BLA.

    a, Fear extinction training for ex vivo mIPSC recordings in the BLA (conditioned (cond), n = 3; 1 d CS, n = 2; 1 d ABS + CS, n = 3; 7 d CS, n = 3; 7 d ABS + CS, n = 3 mice). Statistical analysis was not performed because of the small sample size. b, Optical fibre placements for MD–BLA silencing experiments. c, Viral injections used to visualize the MD–BLA projection. The results (d, e) were replicated with seven mice including five mice obtained after whole-cell recording (h). d, Coronal section under excitation with low laser power optimized for visualizing fluorescence in MD area. e, Coronal section under excitation with high laser power optimized for visualizing fluorescence in the BLA complex. CeA, central amygdala. f, Viral injection (top) and whole-cell recording (bottom) for the feedforward inhibition test. g, Sample traces evoked by photostimulation of MD fibres. h, Averaged latencies of EPSCs (B6/J, n = 7; Grik4-cre, n = 8 cells) and IPSCs (B6/J, n = 11; Grik4-cre, n = 6 cells) from the laser onset to 10% rise time. i, j, Light-evoked outward currents recorded at +10 mV were blocked by bicuculline (i) or CNQX and d-AP5 (j), indicating that recorded currents represent feedforward inhibition. k, Fear extinction training for ex vivo recording of MD–BLA synaptic transmission (CS, n = 3; ABS + CS, n = 3 mice). Mixed-design ANOVA: F1,4 = 7.305, P = 0.0539 for group effect. Data shown as mean ± s.e.m. See Supplementary Table 1 for statistical details.

Supplementary information

  1. Reporting Summary

  2. Supplementary Table

    This table contains detailed statistical results.

  3. Video 1: Fear extinction with ABS-paired CS reduces freezing behaviour.

    The CS group (conventional extinction group) was presented only with the auditory CS during the whole extinction trials (a,b). The first extinction trial of the ABS-paired group (c) was presented only with the auditory CS. Then, the alternating bilateral sensory stimulation (ABS) was paired with the CS beginning at the second extinction trials (d).

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https://doi.org/10.1038/s41586-019-0931-y

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