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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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cpp tocris cat no 0247  (Tocris)
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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(-)-bicuculline methochloride  (Bio-Techne corporation)
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with <t>Anisomycin</t> (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.
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Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with Anisomycin (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.

Journal: Neuron

Article Title: Engram Cell Excitability State Determines the Efficacy of Memory Retrieval.

doi: 10.1016/j.neuron.2018.11.029

Figure Lengend Snippet: Figure 1. Engram Cell-Specific High-Excitability State in Response to Memory Recall (A) Schematic of the experimental strategy (see text and STAR Methods). (B) Behavioral protocol: on day 0, mice are taken off doxycycline diet (Dox Off). On day 1, mice are subjected to contextual fear conditioning (CFC) and the expression window is closed (Dox On). On day 2, mice are re-exposed to the conditioned context A and sacrificed 5 min (5m) or 3 hr (3h) after recall to perform ex vivo patch-clamp recording of dentate granule (DG) cells. An additional control, a no recall group (NR), was not subjected to context re-exposure. (C) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (D–F) Intrinsic electrophysiological properties:membrane resistance (Rm) vsrheobase of DG granule cells belonging to NR(D), 5m (E), and 3h (F) groups (top, example traces). ChR2-EYFP+ engramcells are ingreen; non engramcells are inblack. Red box: increased Rmand rheobase inthe ChR2-EYFP+ engramcells ofthe 5m group. (G) Temporal dynamic of the excitability state. Top: behavioral protocol; bottom: membrane resistance of ChR2-EYFP+ (green) and ChR2-EYFP (black) cells. (H and I) The high-excitability state is NMDA dependent (H) and its decay to baseline is protein synthesis dependent (I). Top: behavioral protocol. Mice were injected with CPP (5 mg/Kg i.p.) before recall in context A (H) or with Anisomycin (150 mg/Kg i.p.) after recall in context A (I). Mice were sacrificed for patch-clamp recordings 5 min (H) or 24 hr (I) after recall in context A. Data are represented as mean ± SEM, and statistical significance is assessed by two-tailed unpaired t test for D–F, H, and I (t test *p < 0.05, **p < 0.01, ***p < 0.001) and two-way ANOVA with Sidak correction for multiple comparison for Figure 1G: df = 283, Frow(6, 171) = 9.000, Ftime(1, 114) = 21.81 (*p < 0.05, **p < 0.01). Number of mice used: NR: 6, 5m: 10, 10m: 5, 30m: 3, 1h: 8, 2h:3, 3h: 7, 24h: 6, CPP: 3, and ANI: 4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-cFos Synaptic System Cat# 226-003 Anti-GFP AbCam Cat# ab13970 Anti-RFP Rockland Cat# 600-901-379 CF555 Streptavidin Biotium Cat# 29038 Anti-GFP Invitrogen Cat# A10262 Anti-Kir2.1 Neuromab Cat# 75-210 Anti-PAN Sigma Cat# S-8809 fAB fragment Jackson Immunoresearch Cat# 115-007-003 Bacterial and Virus Strains AAV9-TRE-ChR2-EYFP Liu et al. 2012 N/A AAV9-TRE-RFP Ramirez et al. 2013 N/A AAV9-TRE-Kir2.1-RFP This article N/A Biological Samples Chemicals, Peptides, and Recombinant Proteins Biocytin Sigma Cat# B4261 CPP Tocris Cat# 0247 Anisomycin Tocris Cat# 1290/10 ML133 Sigma Cat# SML0190 CsCl Sigma Cat# C3011 ZD7288 Tocris Cat# 1000/10 Tertiapin LQ Tocris Cat# 4339/1 Quinine Tocris Cat# 4114/50 Glibenclamide Tocris Cat# 0911/100 Experimental Models: Organisms/Strains Mouse: cfos-tTA/cfos-shEGFP Jackson https://www.jax.org/strain/018306 Software and Algorithms ImageJ-Fiji NIH https://fiji.sc/ Excel Microsoft https://products.office.com/en-us/excel GraphPad Prism https://www.graphpad.com IgorPro7 Wavemetrics https://www.wavemetrics.com

Techniques: Expressing, Ex Vivo, Patch Clamp, Control, Membrane, Injection, Two Tailed Test, Comparison

Figure 2. Kir2.1 Regulates the State of The DG Granule Cell Membrane Excitability (A) DG granule cell (inset) response to current steps. (B) Somatic voltage clamp analysis of current-voltage (I-V) relationship revealed an inwardly rectifying current. (C) Leaky current in response to a negative voltage step is sensitive to ML133 (50 mM), a Kir2.1 blocker. Right: paired analysis, control in red and ML133 in black (n = 15, paired t test ***p < 0.001) and percentage of leaky current sensitive to ML133 and residual one. (D) Correlation between membrane resistance (Rm) and the pharmacologically isolated Kir2.1 current. (E) Top: schematic of the experimental strategy (see text and STAR Methods). Bottom: behavioral protocol (same as Figure 1B). DG cells ChR2-EYFP+ and ChR2-EYFP were recorded 5 minutes after recall. (F) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (G) Inward current in response to a negative voltage step before and after ML133 application in ChR2-EYFP and ChR2-EYFP+ cells. (H) Quantification of total leaky current, Kir2.1 current and residual (non Kir2.1) leaky current in ChR2-EYFP and ChR2-EYFP+ cells. (I) Schematic of the experimental strategy (same as Figure 1B). Anisomycin (ANI) was injected immedialtely after training and mice were perfused 24 hr later. (J) DG granule cell ChR2-EYFP+ (green) stained with a Kir2.1 antibody (red). (K) Confocal images of DG granule cells ChR2-EYFP (double-arrowhead) and ChR2-EYFP+ (single-arrowhead) stained for Kir2.1 (red). (L) Paired analysis of Kir2.1 expression, ChR2-EYFP vs ChR2-EYFP+ (mean in red, two-tailed paired t test, **p < 0.01).

Journal: Neuron

Article Title: Engram Cell Excitability State Determines the Efficacy of Memory Retrieval.

doi: 10.1016/j.neuron.2018.11.029

Figure Lengend Snippet: Figure 2. Kir2.1 Regulates the State of The DG Granule Cell Membrane Excitability (A) DG granule cell (inset) response to current steps. (B) Somatic voltage clamp analysis of current-voltage (I-V) relationship revealed an inwardly rectifying current. (C) Leaky current in response to a negative voltage step is sensitive to ML133 (50 mM), a Kir2.1 blocker. Right: paired analysis, control in red and ML133 in black (n = 15, paired t test ***p < 0.001) and percentage of leaky current sensitive to ML133 and residual one. (D) Correlation between membrane resistance (Rm) and the pharmacologically isolated Kir2.1 current. (E) Top: schematic of the experimental strategy (see text and STAR Methods). Bottom: behavioral protocol (same as Figure 1B). DG cells ChR2-EYFP+ and ChR2-EYFP were recorded 5 minutes after recall. (F) Confocal image of two biocytin-filled DG cells, ChR2-EYFP+ and ChR2-EYFP. (G) Inward current in response to a negative voltage step before and after ML133 application in ChR2-EYFP and ChR2-EYFP+ cells. (H) Quantification of total leaky current, Kir2.1 current and residual (non Kir2.1) leaky current in ChR2-EYFP and ChR2-EYFP+ cells. (I) Schematic of the experimental strategy (same as Figure 1B). Anisomycin (ANI) was injected immedialtely after training and mice were perfused 24 hr later. (J) DG granule cell ChR2-EYFP+ (green) stained with a Kir2.1 antibody (red). (K) Confocal images of DG granule cells ChR2-EYFP (double-arrowhead) and ChR2-EYFP+ (single-arrowhead) stained for Kir2.1 (red). (L) Paired analysis of Kir2.1 expression, ChR2-EYFP vs ChR2-EYFP+ (mean in red, two-tailed paired t test, **p < 0.01).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-cFos Synaptic System Cat# 226-003 Anti-GFP AbCam Cat# ab13970 Anti-RFP Rockland Cat# 600-901-379 CF555 Streptavidin Biotium Cat# 29038 Anti-GFP Invitrogen Cat# A10262 Anti-Kir2.1 Neuromab Cat# 75-210 Anti-PAN Sigma Cat# S-8809 fAB fragment Jackson Immunoresearch Cat# 115-007-003 Bacterial and Virus Strains AAV9-TRE-ChR2-EYFP Liu et al. 2012 N/A AAV9-TRE-RFP Ramirez et al. 2013 N/A AAV9-TRE-Kir2.1-RFP This article N/A Biological Samples Chemicals, Peptides, and Recombinant Proteins Biocytin Sigma Cat# B4261 CPP Tocris Cat# 0247 Anisomycin Tocris Cat# 1290/10 ML133 Sigma Cat# SML0190 CsCl Sigma Cat# C3011 ZD7288 Tocris Cat# 1000/10 Tertiapin LQ Tocris Cat# 4339/1 Quinine Tocris Cat# 4114/50 Glibenclamide Tocris Cat# 0911/100 Experimental Models: Organisms/Strains Mouse: cfos-tTA/cfos-shEGFP Jackson https://www.jax.org/strain/018306 Software and Algorithms ImageJ-Fiji NIH https://fiji.sc/ Excel Microsoft https://products.office.com/en-us/excel GraphPad Prism https://www.graphpad.com IgorPro7 Wavemetrics https://www.wavemetrics.com

Techniques: Membrane, Control, Isolation, Injection, Staining, Expressing, Two Tailed Test