Abstract
Neural circuits underlying auditory fear conditioning have been extensively studied. Here we identified a previously unexplored pathway from the lateral amygdala (LA) to the auditory cortex (ACx) and found that selective silencing of this pathway using chemo- and optogenetic approaches impaired fear memory retrieval. Dual-color in vivo two-photon imaging of mouse ACx showed pathway-specific increases in the formation of LA axon boutons, dendritic spines of ACx layer 5 pyramidal cells, and putative LA–ACx synaptic pairs after auditory fear conditioning. Furthermore, joint imaging of pre- and postsynaptic structures showed that essentially all new synaptic contacts were made by adding new partners to existing synaptic elements. Together, these findings identify an amygdalocortical projection that is important to fear memory expression and is selectively modified by associative fear learning, and unravel a distinct architectural rule for synapse formation in the adult brain.
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Change history
02 July 2018
In the version of this article initially published, Fig. 7f purported to show an example of a multi-synapse spine. However, the structure in question included a mitochondrion and microtubules, meaning that it was actually a segment of dendritic shaft. A new image showing an example of a bona fide spine has been substituted. The legend has been changed to state that the image shows two boutons rather than three. The error has been corrected in the HTML and PDF versions of the article. The original and corrected figures are shown in the accompanying Author Correction.
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Acknowledgements
We thank N. Xu for help with chronic window surgery, Q. Hu for confocal imaging, Y. Kong and B. Zhang for electron microscopy, L. Han for behavioral analysis, L. Zhou for slice recording, and Y. Dan for critical comments and suggestions. This work was supported by grants from Ministry of Science and Technology (973 Program, 2011CBA00400, M.-m.P.) and Chinese Academy of Sciences (Strategic Priority Research Program, XDB02020001, M.-m.P.), and a SIBS-SA scholarship to Y.Y.
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Y.Y., D.-q.L. and M.-m.P. designed the experiments. Y.Y. and D.-q.L. performed the experiments and analyzed the data. W.H. performed in utero electroporation experiments. Y.S. and J.D. performed the electrophysiology experiments. Y.Z. guided spine data analysis. Y.Y., D.-q.L. Y.Z. and M.-m.P. wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 ACx plasticity is required for fear conditioning.
(a) Freezing time of conditioned and control mice before CS presentation and during CS presentation in a new context. Student’s t-test, p> 0.5 for baseline, p< 0.0001 during CS. (b) Diagram showing bilateral infusion of drugs in ACx. (c) Freezing responses in recall test for mice bilaterally infused with different doses of APV at 30 min before fear conditioning. Student’s t-test, p> 0.05 for 0.5-μg and 1-μg group, p< 0.0001 for 2-μg and 10-μg group. (d) Freezing responses for mice bilaterally infused with APV (2 μg) and muscimol immediately after fear conditioning. Student’s t-test, p = 0.028 for APV, p = 0.009 for muscimol. Error bar, s.e.m.
Supplementary Figure 2 Drug spread in ACx.
Coronal slices of mouse brain showing the spread of 1 μl of FITC (Molecular Weight: 389), at 30 min after infusion. Scale bar: 500μm.
Supplementary Figure 3 Coexpression of ChR2 and hM4D in LA for slice recording.
(a) Image showing ChR2 (red) and hM4D (green) expressing neurons in LA. Scale bar: 100 μm. (b) Zoom-in images showing co-expression of ChR2 and hM4D. Scale bar: 10 μm. (c) Proportion of ChR2/hM4D expressing neurons.
Supplementary Figure 4 Virus expression of hM4D and eArch3.0 in LA.
(a) Left: Image showing expression of AAV-hM4D in LA. Scale bar: 500μm. Right: Image showing hM4D-expressing LA axons in ACx. Scale bar: 100 μm. (b) Freezing responses for mice expressing hM4D in LA neurons in two recall tests. Mice were first tested when CNO was infused into ACx. The same mice were tested again 24 h later when saline was infused into ACx. Each line represents data from one mouse. Paired t-test, p= 0.032. (c) Left: Image showing expression of AAV-eArch3.0 in LA. Scale bar: 500 μm. Right: Image showing eArch3.0-expressing LA axons in ACx. Scale bar: 100 μm.
Supplementary Figure 5 Criteria for bouton identification.
(a, b) Fluorescent intensity was measured along identified axons. Bright swellings were identified as boutons when the peak intensity was over 3 fold that of the axon shaft (blue dotted line). (c, d) When changing the threshold to 2 fold (green solid line in B), results of bouton counting were not significantly different using the two different criteria. Each data point represents results from one image stack (>100 boutons each).
Supplementary Figure 6 Retrograde tracing using cholera toxin subunit B (CTB) showed that neurons in LA, MG and ACC project to ACx.
(a) CTB was injected into the superficial layers of ACx. Scale: 100 μm. (b-e) Retrograde labeled neurons were found in the contralateral ACx (b), MG (c), LA (d) and frontal cortex including Cg1, Cg2 and M2 (e). Scale: 100 μm.
Supplementary Figure 7 Correlation of bouton/spine formation with freezing responses.
Correlation analyses showed weak correlation between LA axon bouton formation and freezing responses, and between ACx spine formation and freezing responses.
Supplementary Figure 8 No changes in the spine dynamics in the apical dendrites of ACx L2/3 neurons.
(a) Image showing that L2/3 neurons were labeled with tdTomato using in-utero electroporation. Scale: 500 μm. (b) Zoom-in view of the labeled L2/3 neurons. Scale: 100 μm. (c) Example images obtained by repeated imaging of the same apical dendrites of ACx L2/3 neurons in control and conditioned mice. Green and red arrows, newly formed and eliminated spines, respectively, as compared to -1d. Scale bar, 2 μm. (d) Percentages of spine formation and elimination at apical dendrites of L2/3 neurons in control and conditioned mice at 2h (control: n = 7; conditioned: n = 6) and 3d (control: n = 8; conditioned: n = 6). Mann-Whitney U-test, p> 0.4 for all comparison. Error bar, s.e.m.
Supplementary Figure 9 Example images demonstrating separation of GFP and YFP signals.
(a) Signals obtained with 535/50 bandpass filter, both GFP and YFP signals were collected. (b) Signals obtained with 495/40 bandpass filter. Only GFP signals were collected. (c) YFP-only signals were obtained by subtracting GFP signals from GFP+YFP signals. (d) Merged image of GFP (green) and post-processing YFP (red) signals. Scale bar: 2 μm.
Supplementary Figure 10 Formation rate of boutons/spines in labeled synaptic pairs 2 h after conditioning.
(a) Percentages of newly formed boutons/spines in labeled LA-ACx synaptic pairs (red circles), as compared to those randomly selected (black circles) boutons/spines in the same animal, in conditioned (n = 6) and control (n = 4) mice at 2h after conditioning. Paired t-test, p = 0.03 for bouton formation in conditioned group. p> 0.3 for other comparison.(d, e) Similar to (c), except that labeled synaptic pairs were in MG-ACx (conditioned, n = 4; control, n = 5; p> 0.2) or ACC-ACx connections (conditioned, n = 4; control, n = 4; p> 0.2 for bouton, p = 0.02 for spine in conditioned, p = 0.0021 for spine in control group.).
Supplementary Figure 11 Examples showing that new synapses were mostly made by adding new boutons to existing spines or new spines to existing boutons.
(a) Example showing a new spine (red) growing onto an existing bouton (green). (b) Example showing a new bouton growing onto an existing spine. (c) Example showing de novo formation of a pairs of bouton and spine. (d) Example showing a new bouton growing onto an existing spine with a presynaptic partner labeled. (e) Example showing a new spine growing onto an existing bouton with a postsynaptic partner labeled. (f) Example showing a new spine replacing an existing postsynaptic partner of an existing bouton. Scale bar: 1 μm.
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Yang, Y., Liu, Dq., Huang, W. et al. Selective synaptic remodeling of amygdalocortical connections associated with fear memory. Nat Neurosci 19, 1348–1355 (2016). https://doi.org/10.1038/nn.4370
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DOI: https://doi.org/10.1038/nn.4370
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