b bla - nature · of rats injected (black arrows) bilaterally into the bla with arc antisense (as)...

Supplementary Figure 1

Ye et al.

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The behavioral outcomes of blocking Arc expression in dHC and BLA during retrieval.

(a) Mean latency ± s.e.m. of rats injected (black arrow) with Arc AS or SC into dHC 2 d after training (Tr) and memory retention tested (T) 1 h later (unpaired two-tailed Student t-test; t14 = 0.2768, P = 0.786, n = 8, 8; 2 independent experiments). (b) Mean latency ± s.e.m. of rats injected (black arrows) bilaterally into the BLA with Arc antisense (AS) or control scrambled (SC) oligodeoxynucleotides (ODNs) 1 h before each 10 s retrieval in the 3 retrievals (3Rs) group or at the matched time points after training (Tr) in the non-retrieval (NoR) group (two-way ANOVA followed by Bonferroni post hoc test; treatment F3, 65 = 50.82, P < 0.0001; testing F2, 65 = 2.01, P = 0.1418; interaction F6, 65 = 0.98, P = 0.4470; n = 8, 8, 9, 8; 3 independent experiments). T1-T3: tests as shown in the schema. RS: reminder footshock. * P < 0.05, ** P < 0.01, *** P < 0.001. Histological images showing the injection sites are presented in Supplementary Figure 10.

Nature Neuroscience: doi:10.1038/nn.4443


Context retrieval does not change the levels of total CREB, cofilin or TrkB in dHC and mPFC.

Examples and relative quantitative western blots analyses of dHC and mPFC extracts obtained from rats trained (Tr) and euthanized (eut, red arrows) 1 h after 1 or 3 memory retrievals (1R or 3Rs), or 3 exposures to a novel context (3Cs), or at the matched time point in the non-retrieval (NoR) group. Naïve rats (N) served as reference control. Data are presented as mean percentage ± s.e.m. of the mean values of the N group (one-way ANOVA followed by Newman-Keuls post hoc test; dHC: CREB F4, 30 = 0.3876, P = 0.8158, n = 9, 7, 5, 8, 6; cofilin F4, 30 = 0.2894, P = 0.8825, n = 9, 7, 5, 8, 6; mPFC: CREB F4, 32 = 0.6654, P = 0.6206, n = 9, 8, 5, 8, 7; cofilin F4, 32 = 1.545, P = 0.2128, n = 9, 8, 5, 8, 7; TrkB F4, 32 = 0.5108, P = 0.7282, n = 9, 8, 5, 8, 7; 3 independent experiments).


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Ye et al.


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Both memory enhancement and extinction increase the levels of Arc, pCREB and p-cofilin in dHC.

Cropped examples and relative quantitative western blots analyses of dHC extracts obtained from rats trained (Tr) and euthanized (eut, red arrows) 1 h after 3 memory retrievals (3Rs) or extinction (T + Ext), or at the matched time point in the non-retrieval (NoR) group. Naïve rats (N) served as reference control. Data are presented as mean percentage ± s.e.m. of the mean values of the N group (one-way ANOVA followed by Newman-Keuls post hoc test; Arc F3, 25 = 10.03, P = 0.0002, n = 8, 7, 7, 7; pCREB F3, 24 = 9.310, P = 0.0003, n = 7, 7, 7, 7; pcofilin F3, 25 = 7.325, P = 0.0011, n = 8, 7, 7, 7; 3 independent experiments). * P < 0.05, ** P < 0.01, *** P < 0.001. Full-length blots are presented in Supplementary Figure 11.


Ye et al.


amCitrine HA-hM4Di DAPI

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Immunofluorescent staining of brain sections following dHC injection of AAV8/hSyn-HA-hM4Di-IRES-mCitrine.

Staining for HA-hM4Di is shown in red, mCitrine in green, the nuclear marker DAPI in blue, and merged images are shown on the right. Images of corresponding brain atlas are shown on the right of the merged immunofluorescent staining images, with red box indicating where the immunofluorescent staining images are taken from. (a) Images taken with Olympus VS120 wide-field epi-fluorescent microscope showing immunofluorescent staining of mCitrine and HA-hM4Di in dHC. (b) Images taken with Leica SP5 confocal microscope showing immunofluorescent staining of mCitrine and HA-hM4Di in the PL and IL. The images on the right show enlarged views of dHC projection terminals in the PL and IL cortex that contain hM4Di, corresponding to the white boxed - area in the merged images.



Retrograde labeling of dHC neurons projecting to PL; and Arc expression in mPFC after blocking dHC-to-PL cortex projections.

(a) Distribution of the retrograde tracer CTB-Alexa Fluor 555 at the injection site in PL, as well as the spread and retrograde labeling of neurons in adjacent mPFC sections. (b) Retrograde labeling of hippocampal neurons in the same brain injected with CTB-Alexa Fluor 555 in PL. Note that there is sparse labeling of dorsal hippocampal neurons in CA1 and CA2 subregions, indicating direct projections from this part of hippocampus to PL. The images were taken with Olympus VS120 wide-field epi-fluorescent microscope: CTB-Alexa Fluor 555 (red), DAPI (blue). Enlarged images on the bottom are taken from the white boxed - area in the upper images. (c) Examples and quantification of immunofluorescent staining of Arc in the PL and IL obtained from rats euthanized (eut, red arrows) 1 h after 3 memory retrievals (3Rs) or at the matched time points for the non-retrieval (NoR) groups from the rats injected with AAV8/hSyn-HA-hM4Di-IRES-mCitrine or control AAV8/hSyn-GFP into dHC, and CNO or control vehicle (veh) into PL (black arrows). Data are presented as mean percentage ± s.e.m. of the mean values of the NoR + veh group (one-way ANOVA followed by Newman-Keuls post hoc test; PL F4, 21 = 25.36, P < 0.0001, n = 5, 5, 6, 5, 5; IL F4, 21 = 8.694, P = 0.0003, n = 5, 5, 6, 5, 5; 2 independent experiments). ** P < 0.01, *** P < 0.001.


Ye et al.

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Chicago Sky Blue dye diffusion in PL; and memory retention after blocking Arc expression in PL.

(a) Representative sections of one rat brain at +3.24mm, +2.76mm and +2.52mm from bregma, analyzed under light microscopy for Chicago Sky Blue (1%) diffusion 1 h after PL injection (6 independent experiments). (b) Mean latency ± s.e.m. of rats bilaterally injected (black arrows) into the PL cortex with Arc antisense (AS) or control scrambled (SC) oligodeoxynucleotides (ODNs) 1 h before each 10 s retrieval in the 3 retrievals (3Rs) group or at the matched time points after training (Tr) in the non-retrieval (NoR) group. Memory retention was tested (T1-T3) as shown in the schema. RS: reminder footshock (two-way ANOVA followed by Bonferroni post hoc test; treatment F2, 88 = 60.88, P < 0.0001; testing F2, 88 = 0.57, P = 0.5688; interaction F4, 88 = 0.73, P = 0.5714; n = 12, 13, 13; 3 independent experiments). ** P < 0.01, *** P < 0.001. Histological images showing the injection sites are presented in Supplementary Figure 10.



Training and context retrieval-mediated induction of GluA1 and GluA2.

Cropped examples and relative quantitative western blots analyses of mPFC extracts obtained from rats trained (Tr) and euthanized (eut, red arrows) 1 h after 1 or 3 memory retrievals (1R or 3Rs), or 3 exposures to a novel context (3Cs), or at the matched time point in the non-retrieval (NoR) group. Naïve rats (N) served as reference control. Data are presented as mean percentage ± s.e.m. of the mean values of the N group (one-way ANOVA followed by Newman-Keuls post hoc test; GluA1 F4, 26 = 6.146, P = 0.0013, n = 10, 8, 4, 5, 4; GluA2 F4, 26 = 4.557, P = 0.0064, n = 10, 8, 4, 5, 4; 3 independent experiments). * P < 0.05, ** P < 0.01, *** P < 0.001. Full-length blots are presented in Supplementary Figure 12.



Molecular changes in the PL cortex after training, memory enhancement and extinction.

Cropped examples and relative quantitative western blot analyses of PL extracts obtained from rats trained (Tr) and euthanized (eut, red arrows) 1 h after 3 memory retrievals (3Rs), extinction (T + Ext) or at the matched time point in the non-retrieval (NoR) group. Naïve rats (N) served as reference control. Data are presented as mean percentage ± s.e.m. of the mean values of the N group (one-way ANOVA followed by Newman-Keuls post hoc test; Arc F3, 33 = 15.82, P < 0.0001, n = 10, 9, 9, 9; pCREB F3, 21 = 8.344, P = 0.0008, n = 6, 6, 6, 7; pcofilin F3, 22 = 7.033, P = 0.0017, n = 6, 6, 7, 7; BDNF F3, 22 = 3.421, P = 0.0350, n = 6, 6, 7, 7; pTrkB F3, 21 = 5.461, P = 0.0062, n = 6, 6, 7, 7; NLGN1 F3, 33 = 4.449, P = 0.0099, n = 10, 9, 9, 9; NLGN2 F3, 34 = 3.620, P = 0.0227, n = 10, 9, 10, 9; 3 independent experiments). Full-length blots are presented in Supplementary Figure 12.


Ye et al.

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Training, retrieval and extinction do not change actin level.

Cropped examples and relative quantitative western blots analyses of PL extracts obtained from rats trained (Tr) and euthanized (eut, red arrows) 1 h after 3 memory retrievals (3Rs) or extinction (T + Ext), or at the matched time point in the non-retrieval (NoR) group. Naïve rats (N) served as reference control. Data are presented as mean percentage ± s.e.m. of the mean values of the N group (one-way ANOVA followed by Newman-Keuls post hoc test; actin per lane F3, 28 = 0.2485, P = 0.8617, n = 8, 8, 8, 8; actin / GAPDH F3, 28 = 0.3735, P = 0.7727, n = 8, 8, 8, 8; 3 independent experiments).


Ye et al.


Fig.3

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Histological images showing the injection sites for all the behavioral experiments presented in the main figures.

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Full-length pictures of the blots presented in the main figures.

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b bla - nature · of rats injected (black arrows) bilaterally into the bla with arc antisense (as)...

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