1. Volume 23, Issue 8, Pages 2379-2391 (May 2018)
Activation of Entorhinal Cortical Projections to the Dentate Gyrus Underlies Social Memory Retrieval 
Celeste Leung, Feng Cao, Robin Nguyen, Krutika Joshi, Afif J. Aqrabawi, Shuting Xia, Miguel A. Cortez, O. Carter Snead, Jun Chul Kim, Zhengping Jia 
Cell Reports 
Volume 23, Issue 8, Pages (May 2018)
DOI: /j.celrep
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2. Cell Reports 2018 23, 2379-2391DOI: (10.1016/j.celrep.2018.04.073)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Inhibition of the EC-DG Circuit Blocks Social Recognition Memory Retrieval
(A) Positioning of the optical fiber in the dorsal DG and viral injections in the ventral EC.
(B) Inhibition and activation of neurotransmitter release at the EC-DG synapse.
(C) Images of dorsal and ventral brain sections showing the expression of ArchT-EYFP in the EC and the PP pathway, but not in the granule cells (GCLs) of the DG. Scale bars, 1,000 μm (coronal); 500 μm (DG); 100 μm (EC).
(D) The three-chamber social interaction (SI) test with a continuous 532-nm green light presented during sociability or discrimination.
(E) Inhibition during stage 2 or 3 had no effect on preference for stranger 1 (S1) over empty cage (E) in EYFP mice (EYFP: n = 8, p < ; EYFP stage 2: n = 8, p < 0.0001; EYFP stage 3: n = 9, p = ; two-tailed paired t test and two-tailed Wilcoxon matched-pairs signed-rank test).
(F) Inhibition during stage 2 or 3 had no effect on preference for stranger 2 (S2) over S1 in EYFP mice (EYFP: p = ; EYFP stage 2: p = ; EYFP stage 3: p = ; two-tailed paired t test and two-tailed Wilcoxon matched-pairs signed-rank test).
(G) Inhibition during stage 2 or 3 had no effect on discrimination scores of stage 3 in EYFP mice (F(2, 22) = , p = ; one-way ANOVA).
(H) Representative track plots and heatmaps showing the effect of ArchT on social discrimination.
(I) Inhibition of the EC-DG circuit by ArchT during stage 2 or 3 had no effect on sociability (ArchT: n = 11, p < ; ArchT stage 2: n = 12, p < ; ArchT stage 3: n = 11, p < ; two-tailed paired t test).
(J) Inhibition of the EC-DG circuit by ArchT during stage 2 or 3 abolished preference for S2 over S1 (ArchT: p = ; ArchT stage 2: p = ; ArchT stage 3: p = ; two-tailed paired t test).
(K) Decreased discrimination scores in ArchT stage 3 mice (F(2, 31) = 4.235, p = , one-way ANOVA; post hoc Holm-Sidak’s multiple comparisons with ArchT, ArchT stage 2 p = , ArchT stage 3 p = ).
(L) A modified multiple-trial social memory assay with a continuous 532-nm light presented during trials 5 and 7.
(M) ArchT and EYFP mice showed memory acquisition during trials 1–4. ArchT mice with inhibition on trial 5 showed an increased interaction time compared to EYFP mice (EYFP: n = 8, ArchT: n = 10; p = ; two-tailed unpaired t test). In trial 6 (light off), both ArchT and EYFP mice showed memory for the first stranger mouse. ArchT and EYFP mice with light showed similar preference for a novel mouse on trial 7.
(N) Normalized interaction time obtained from the multiple-trial memory test.
Error bars indicate SEM. See also Figures S1, S2, and S3.
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4. Figure 2
Activation of the EC-DG Circuit Facilitates Social Recognition Memory Retrieval
(A) The SI test with a 473-nm blue light during stage 3.
(B) Activation of the EC-DG circuit by ChR2 during stage 3 had no effect on preference for S1 over E (ChR2: n = 8, p < ; ChR2 stage 3: n = 10, p < 0.0001; two-tailed paired t test).
(C) Activation of the EC-DG circuit by ChR2 during stage 3 had no effect on preference for S2 over S1 (ChR2: p = ; ChR2 stage 3: p = ; two-tailed paired t test).
(D) No differences in discrimination scores between ChR2 stage 3 and control mice (p = ; two-tailed paired t test).
(E) A modified SI test with stage 2 being reduced to 2 min. The 473-nm light was presented during stage 2 only when the test mouse interacted with S1.
(F) Activation of the EC-DG circuit by ChR2 during stage 2 had no effect on preference for S1 (ChR2: n = 7, p < ; ChR2 stage 2: n = 7, p = ; two-tailed paired t test and Wilcoxon matched-pairs signed-rank test).
(G) Activation of the EC-DG circuit by ChR2 during stage 2 had no effect on the lack of preference for S2 over S1 (ChR2: p = ; ChR2 stage 2: p > ; two-tailed paired t test).
(H) No differences in discrimination scores between ChR2 stage 2 and control mice (p = ; two-tailed paired t test).
(I) A modified SI test with a 473-nm light presented during stage 3.
(J) Activation of the EC-DG circuit by ChR2 during stage 3 had no effect on preference for S1 over the empty cage (ChR2: n = 7, p < ; ChR2 stage 3: n = 8, p = ; two-tailed paired t test and Wilcoxon matched-pairs signed-rank test).
(K) Activation of the EC-DG circuit by ChR2 during stage 3 facilitated preference for S2 over S1 (ChR2: p = ; ChR2 stage 3: p = ; two-tailed paired t test).
(L) Discrimination scores showing significantly increased discrimination in ChR2 stage 3 compared to control group (p = ; two-tailed paired t test).
Error bars indicate SEM. See also Figures S2 and S4.
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5. Figure 3
Inducible Disruption of PAK Signaling in the Adult Brain Impairs Social Recognition Memory
(A) The generation of mPAK3-GFP dTg mice using the tTA/tetO-inducible system.
(B) Expression of mPAK3-GFP in dTg, but not in Tg-1 or in Tg-2, mice. Expression of mPAK3-GFP in dTg mice was turned off with the administration of DOX.
(C) Restricted expression of mPAK3-GFP in cortex (CX) and in hippocampus (HP), but not in cerebellum (CR) or in midbrain (MD).
(D) Coronal dorsal to ventral brain sections of dTg mice stained with anti-GFP and DAPI. Scale bar, 2,000 μm.
(E) HP section of dTg mice stained with anti-GFP and DAPI showing mPAK3-GFP expression in the DG-ML, but not in the DG-GCLs or CA1/CA3 areas. Scale bar, 500 μm.
(F) Horizontal brain section of dTg mice stained with anti-GFP. Scale bar, 1,000 μm.
(G) EC cortical sections of dTg mice stained with DAPI, anti-GFP, anti-CDP, anti-Reelin, or anti-Calbindin. Scale bar, 100 μm.
(H) Summary graph showing colocalization of mPAK3-GFP with CDP/Reelin-positive pyramidal neurons, but not with Calbindin-positive excitatory neurons, GABAergic neurons, or glial cells.
(I) Schematic of the three-chamber SI test.
(J) Normal preference for S1 over empty cage during stage 2 in dTg mice (WT: n = 13, p < ; dTg: n = 12, p < ; two-tailed paired t test).
(K) Impaired preference for S2 over S1 during stage 3 in dTg mice (WT: p = ; dTg: p = ; two-tailed paired t test).
(L) Lack of discrimination during stage 3 in dTg mice (p = ; two-tailed paired t test).
(M) Schematic of the five-trial social memory assay.
(N) WT, but not dTg, mice showed memory acquisition during trials 1–5 (WT: n = 15, p < 0.001; dTg: n = 15, p = ; two-way ANOVA; post hoc Holm-Sidak’s multiple comparisons for trial 1 versus trial 5). In trial 6, dTg mice showed significantly decreased interaction time compared to WT mice during the presentation of a novel mouse (p = ; two-tailed unpaired t test).
(O) Normalized interaction time (WT: n = 15, p = ; dTg: n = 15, p = ; two-way ANOVA; post hoc Holm-Sidak’s multiple comparisons for trial 1 versus trial 5).
Error bars indicate SEM. See also Figure S5.
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6. Figure 4
Intact Novel Recognition and Spatial Memory, but Impaired Social Memory in dTg Mice
(A) Preference for novel object 2 versus familiar object 1 (WT: n = 13, p = ; dTg: n = 11, p = 0.0058; two-tailed paired t test).
(B) Preference for novel object 3 versus object 2 (WT: p = ; dTg: p = ; two-tailed paired t test).
(C) Discrimination scores reflecting preferences for object 3 versus 2 (p = ; two-tailed unpaired t test).
(D) Performance in the visible platform (WT: n = 13; dTg: n = 11; genotype F(1, 60) =  p = ; two-way RM ANOVA).
(E) Learning acquisition in the hidden training (genotype F(1, 84) = 1.418 p = ; two-way RM ANOVA).
(F) Probe test (2 hr) (target p = ; other p = 0.5883; two-tailed unpaired Mann-Whitney test).
(G) Probe test (24 hr) (target p = ; other p = 0.6239; two-tailed unpaired t test).
(H) Latency to uncover buried pellets (WT: n = 10; dTg: n = 9; p = ; two-tailed Mann Whitney test).
(I) Habituation to the vanilla cue (WT: n = 9; dTg: n = 10; genotype F(1, 17) = 2.744, p = ; trial F(2, 34) = 57.51, p < ; two-way RM ANOVA).
(J) Habituation to the complex non-social cue (WT: n = 9; dTg: n = 10; genotype F(1, 17) = , p = ; trial F(2, 24) = 110.6, p < ; two-way RM ANOVA).
(K) Impaired habituation to the social cue in dTg mice (WT: n = 14; dTg: n = 13; genotype F(1, 25) = 4.121, p = ; trial F(2, 50) = 36.26, p < ; two-way RM ANOVA; post hoc Holm-Sidak’s multiple comparisons for trial 3, p = ).
Error bars indicate SEM.
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7. Figure 5
Acute PAK Inhibition within the EC-DG Circuit Impairs Social Recognition Memory
(A) Coronal sections of WT and dTg mice with systemic administration of DOX (50 mg/mL). Scale bar, 1,000 μm.
(B) Normal preference for S1 in dTg mice with systemic administration of DOX (WT with DOX: n = 14, p < ; WT without DOX: n = 8, p < 0.0001; dTg with DOX: n = 14, p < ; dTg without DOX: n = 7, p < ; two-tailed paired t test).
(C) Improved preference for S2 in dTg mice with systemic administration of DOX (WT with DOX: p = 0.0001; WT without DOX: p < ; dTg with DOX: p < ; dTg without DOX: p = ; two-tailed paired t test).
(D) Increased discrimination score in dTg mice with systemic administration of DOX (F(3, 39) = 5.192, p = ; one-way ANOVA; post hoc Holm-Sidak’s multiple comparisons, dTg with DOX versus dTg without DOX, p = ).
(E) Horizontal and coronal brain sections with local DOX infusion into the EC or BLA (50 mg/mL; 0.1 μL bilaterally for 5 days), respectively, showing mPAK3-GFP expression turned off in dTg. Scale bars, 1,000 μm.
(F) Normal preference for S1 in dTg mice with local DOX infusion into the EC or BLA (WT with DOX in EC: n = 9, p = ; WT with DOX in BLA: n = 6, p = ; dTg with DOX in EC: n = 7, p = ; dTg with DOX in BLA: n = 7, p = ; two-tailed Wilcoxon matched-pairs signed-rank test and two-tailed paired t test).
(G) Improved preference for S2 in dTg mice with local DOX infusion in the EC, but not in the BLA (WT with DOX in EC: p = ; WT with DOX in BLA: p = ; dTg with DOX in EC: p = ; dTg with DOX in BLA: p = ; two-tailed Wilcoxon matched-pairs signed-rank test and two-tailed paired t test).
(H) Increased discrimination score in dTg mice with local DOX infusion into the EC, but not in to the BLA (WT versus dTg for EC infusion, p = 0.7446; WT versus dTg for BLA infusion, p = ; two-tailed Mann Whitney test and two-tailed paired t test).
(I) Experimental procedure for infusion of the PAK inhibitor IPA3 or control PIR3.5 (5 nM, 1 μL/side over 2 min) followed by the SI test.
(J) Preference for S1 in WT and dTg mice with IPA3 or PIR3.5 infusion in the EC (WT PIR3.5: n = 11, p = ; WT IPA3: n = 10, p < ; dTg IPA3: n = 11, p = 0.0010; two-tailed Wilcoxon matched-pair signed-rank test and two-tailed paired t test).
(K) Impaired preference for S2 over S1 in WT and dTg mice with IPA3 infusion in the EC (WT PIR3.5: p = ; WT IPA3: p = ; dTg IPA3: p = ; two-tailed Wilcoxon matched-pair signed-rank test and two-tailed paired t test).
(L) Impaired discrimination scores during stage 3 in WT and dTg mice with IPA3 infusion in the EC (F(2, 29) = 8.583, p = , one-way ANOVA; post hoc Holm-Sidak’s multiple comparisons, p = ).
(M) Experimental procedure for drug infusion in the BLA followed by the SI test.
(N) Preference for S1 in WT and dTg mice with IPA3 or PIR3.5 infusion in the BLA (WT PIR3.5: n = 11, p < ; WT IPA3: n = 6, p < ; dTg PIR3.5: n = 12, p = ; dTg IPA3: n = 7, p < ; two-tailed Wilcoxon matched-pair signed-rank test and two-tailed paired t test).
(O) No preference for S2 over S1 in dTg mice with IPA3 or PIR3.5 infusion in the BLA (WT PIR3.5: p = ; WT IPA3: p < ; dTg PIR3.5: p = ; dTg IPA3: p = ; two-tailed Wilcoxon matched-pair signed-rank test and two-tailed paired t test).
(P) Impaired discrimination in dTg mice with IPA3 or PIR3.5 infusion in the BLA (F(3, 30) = 3.074; p = ; one-way ANOVA).
Error bars indicate SEM. See also Figure S6.
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8. Figure 6
Impaired Presynaptic Function at the EC-DG Synapse in dTg Mice
(A) Input/output curves of fEPSP versus stimulation intensity (WT: n = 10; dTg: n = 8; group F(1, 208) = 49.95, p < ; intensity F(12, 208) = 34.73, p < , two-way RM ANOVA). Scale bar, 0.35 mV/4 ms.
(B) Input/output curves of fEPSP versus prefiber volley (WT: n = 10; dTg: n = 8; group F(1, 208) = 49.95, p < , two-way RM ANOVA).
(C) Prefiber volley versus stimulation intensity. (WT: n = 10; dTg: n = 8; group F(1, 208) = , p = 0.5361, two-way RM ANOVA).
(D) Paired-pulse ratio (WT: n = 6; dTg: n = 8; group F(1, 108) = 39.06, p < ; ISI F(8, 108) = 2.340, p = 0.0233; 2-way RM ANOVA; post hoc Holm-Sidak’s multiple comparisons: p = for ISI 25, p = for ISI 40, p = for ISI 100). Scale bar, 0.2 mV/5 ms.
(E) mEPSC (WT: n = 11; dTg: n = 12; amplitude p = 0.7737, frequency p = ; two-tailed unpaired t test). Scale bar, 10 pA/1 s.
(F) EPSCAMPAR/EPSCNMDAR ratios (WT: n = 9; dTg: n = 8; p = ; two-tailed unpaired t test). Scale bar, 100 pA/50 ms.
(G) Current/voltage relationship of AMPAR-mediated synaptic currents (WT: n = 9; dTg: n = 8; group F(1, 120) = , p = ; potential F(7, 120) = 625.0, p < ; two-way RM ANOVA). Scale bar, 100 pA/50 ms.
(H) Representative electron micrographs of the DG molecular layer. Scale bars, 500 μm (15,000×), 250 μm (30,000×).
(I) Synapse density (WT: n = 25; dTg: n = 23; p = 0.5035; two-tailed unpaired t test).
(J) PSD length distribution (WT: n = 25; dTg: n = 24; group F(1, 237) = , p = ; length F(2, 237) = 130.3, p < ; two-way ANOVA).
(K) Number of synaptic vesicles (WT: n = 99; dTg: n = 94; p = ; two-tailed unpaired t test).
(L) Distribution of synaptic vesicles (WT: n = 98; dTg: n = 84; Group F(1, 1920) = 3.651, p = ; Distance from terminal F(9, 1920) = 140.0, p < 0.0001; two-way ANOVA, post hoc Holm-Sidak’s multiple comparisons between genotypes, distance 0–49 nm: p = ; 50–99 nm: p = 0.0012).
Error bars indicate SEM. See also Figure S7.
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9. Figure 7
Optical Activation of the EC-DG Terminals Reverses Social Memory Deficits in dTg Mice
(A) The positioning of optical fiber in the dorsal DG and injections of ChR2 viruses in the ventral EC in the dTg mice.
(B) Optical stimulation of the EC-DG synapse by ChR2 in dTg mice.
(C) Images showing expression of ChR2 that colocalizes with mPAK3-GFP in dTg mice. Scale bars: 1,000 μm (coronal), 500 μm (DG).
(D) The SI test with 473-nm of blue light during stage 3.
(E) Activation of the EC-DG circuit by ChR2 during stage 3 had no effect on normal sociability in dTg mice (ChR2: n = 13, p = ; ChR2-Stage 3: n = 16, p < 0.0001; two-tailed Wilcoxon matched-pairs signed rank test).
(F) Activation of the EC-DG circuit by ChR2 during stage 3 was sufficient to rescue the impaired preference for S2 in dTg (ChR2: p = ; ChR2-Stage 3: p < 0.0001; two-tailed paired t test).
(G) Improved discrimination in dTg mice with ChR2 activation during stage 3 (p = ; two-tailed paired t test).
(H) The modified SI test with the duration of stage 2 being reduced to 2 min and 473-nm light being presented during stage 2 only when interacting with S1.
(I) Activation of the EC-DG circuit by ChR2 during stage 2 had no effect on sociability in dTg mice (ChR2: n = 3, p < ; ChR2-Stage 2: n = 13, p = 0.0002; two-tailed paired t test and two-tailed Wilcoxon matched-pairs signed rank test).
(J) Activation of the EC-DG circuit by ChR2 during stage 2 had no effect on the impaired preference for S2 over S1 in dTg mice (ChR2: p = ; ChR2-Stage 2: p = ; two-tailed paired t test).
(K) Activation of the EC-DG circuit by ChR2 during stage 2 had no effect on the discrimination scores of stage 3 in dTg mice (p = ; two-tailed paired t test).
(L) The modified SI test with a 473-nm blue light presented during stage 3.
(M) Activation of the EC-DG circuit by ChR2 during stage 3 had no effect on sociability in dTg mice (ChR2: n = 12, p = ; ChR2-Stage 3: n = 12, p = 0.0005; two-tailed Wilcoxon matched-pairs signed rank test).
(N) Activation of the EC-DG by ChR2 during stage 3 rescued the impaired preference for S2 over S1 in dTg mice (ChR2: p = ; ChR2-Stage 3: p = ; two-tailed paired t test).
(O) Activation of the EC-DG circuit by ChR2 during stage 3 significantly increased discrimination scores of stage 3 in dTg mice (p = ; two-tailed paired t test).
Error bars indicate SEM.
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