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-g and 1-g

group, p< 0.0001 for 2-g and 10-g group. (d) Freezing responses for mice bilaterally infused with APV (2 g) 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.

Nature Neuroscience: doi:10.1038/nn.4370

Supplementary Figure 2

Drug spread in ACx.

Coronal slices of mouse brain showing the spread of 1 l of FITC (Molecular Weight: 389), at 30 min after infusion. Scale bar: 500m.

Nature Neuroscience: doi:10.1038/nn.4370

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 m. (b) Zoom-in images showing co-

expression of ChR2 and hM4D. Scale bar: 10 m. (c) Proportion of ChR2/hM4D expressing neurons.

Nature Neuroscience: doi:10.1038/nn.4370

Supplementary Figure 4

Virus expression of hM4D and eArch3.0 in LA.

(a) Left: Image showing expression of AAV-hM4D in LA. Scale bar: 500m. Right: Image showing hM4D-expressing LA axons in ACx.

Scale bar: 100 m. (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 m. Right: Image

showing eArch3.0-expressing LA axons in ACx. Scale bar: 100 m.

Nature Neuroscience: doi:10.1038/nn.4370

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).

Nature Neuroscience: doi:10.1038/nn.4370

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 m. (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 m.

Nature Neuroscience: doi:10.1038/nn.4370

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.

Nature Neuroscience: doi:10.1038/nn.4370

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 m. (b) Zoom-in view of the

labeled L2/3 neurons. Scale: 100 m. (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 m. (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.

Nature Neuroscience: doi:10.1038/nn.4370

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 m.

Nature Neuroscience: doi:10.1038/nn.4370

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.).

Nature Neuroscience: doi:10.1038/nn.4370

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 m.

Nature Neuroscience: doi:10.1038/nn.4370