1. Direct Medial Entorhinal Cortex Input to Hippocampal CA1 Is Crucial for Extended Quiet Awake Replay 
Jun Yamamoto, Susumu Tonegawa 
Neuron 
Volume 96, Issue 1, Pages e4 (September 2017)
DOI: /j.neuron
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2. Figure 1 Ripple Bursts under Long-Track Exposure
(A) Long-track setup of the 6.3 m linear track in a visual cue-surrounded room. The folded track was set up on a table and elevated by 10 cm from the surface of the table. Three explicitly large visual cues were on the white surrounding walls. The experimenter and recording setups were isolated with a thick black curtain. Bottom panel shows an example of an animal trajectory during RUN. Two reward sites were in the left bottom corner in the panel. Animals were able to freely run and turn corners of the track.
(B) Recording configuration in dorsal CA1 with a silicone linear probe. A solid line on the top panel and a dotted line on the bottom panel show electrode track in CA1.
(C) Examples of identified single and ripple bursts in CA1. Top panel: wideband LFP ranging from striatum oriens to striatum radiatum of the dorsal CA1. Middle panel: same recording position as top panel; color-coded LFP ripple band amplitude. Bottom panel: multi-unit activities and sum of MUAs (STAR Methods).
(D) Proportion of different types of ripple bursts during quiet awake (QAW) and slow-wave sleep (SWS). QAW, singlet, 54.1% ± 8.2%; doublet, 28.6% ± 6.2%; triplet, 14.3% ± 4.2%; other, 3.1% ± 1.8%; SWS, singlet, 47.6% ± 10.3%; doublet, 32.1% ± 6.2%; triplet, 18.2% ± 4.2%; other, 2.7% ± 1.4%; paired t test, tsinglet(1,235) = 1.462, psinglet = 0.106; tdoublet(638) = 1.176, pdoublet = 0.228; ttriplet(331) = 0.562, ptriplet = 0.559; tother(63) = 1.091, pother = 0.279, Bonferroni corrected.
(E) Ripple occurrence per behavioral session. QAW, 2.4 ± 0.37 (10 s−1); SWS, 6.5 ± 0.81 (10 s−1); paired t test, t(2,267) = 3.291, ∗∗∗p = 2.6 × 10−8.
(F) Distribution of ripple occurrence per ripple type. QAW, singlet, 1.22 ± 0.39 (10 s−1); doublet, 0.75 ± 0.29 (10 s−1); triplet, 0.32 ± 0.16 (10 s−1); other, 0.11 ± 0.04 (10 s−1); SWS, singlet, 3.21 ± 0.37 (10 s−1); doublet, 1.62 ± 0.31 (10 s−1); triplet, 0.88 ± 0.19 (10 s−1); other, 0.32 ± 0.12 (10 s−1); paired t test, tsinglet(1,235) = 3.293, ∗∗∗psinglet = 1.3 × 10−7; tdoublet(638) = 2.498, ∗∗pdoublet = 0.006; ttriplet(331) = 2.502, ∗∗ptriplet = 0.008; tother(63) = 2.263, ∗pother = 0.032, Bonferroni corrected.
(G) Histogram of inter-ripple interval. Red arrowheads denote local peaks (around 110, 220, 330, and 440 ms) in the monotonous Poisson distribution.
(H) Examples of detected singlet-ripple location on the long track (blue dots, singlet start position).
(I) Examples of detected doublet-ripple location on the long track (green dots, doublet start position).
(J) Examples of detected triplet-ripple location on the long track (red dots, triplet start position).
Data are represented as mean ± SD.
Neuron  , e4DOI: ( /j.neuron )
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3. Figure 2
Quiet Awake-Specific Ripple Burst-Associated Alternating Burst Activities in MEC-CA1 Network
(A) An example of singlet sharp-wave ripple. Top panel: color-coded ripple band LFPs of superficial MEC and dorsal CA1 cell layer. Middle panel: associated superficial MEC MUA with elevated burst activities (red downward arrowheads). Green dotted trace is smoothed sum of MEC MUA (STAR Methods). Bottom panel: detected CA1 MUA and its smoothed sum (blue dotted traces) with elevated ripple activities (red upward arrowhead).
(B) Same as in (A): examples of doublets and a triplet, interregional burst interaction during quiet awake.
(C) Same as in (A): examples of non-interregional ripple bursts during slow-wave sleep.
(D) Cross-correlation between MEC and CA1 using ripple band LFP peak power times (top) and LFP and spikes (bottom) during QAW. Blue trace, 10 ms bin; red trace, smoothed trend line (Gaussian kernel, σ = 20 ms).
(E) Cross-correlation between MEC and CA1 using MEC ripple band LFP peak power times and CA1 MUA spike times (top) and LFP and spikes (bottom) during SWS. Blue trace, 10 ms bin; red trace, smoothed trend line (Gaussian kernel, σ = 20 ms).
(F) Granger causal directional effect of interregional bursts between MEC and CA1 during quiet awake. MEC to CA1, 0.57 ± 0.09; MEC to CA1, 0.32 ± 0.08; z = 2.674, ∗∗p = 0.004, Wilcoxon rank-sum test. Dotted line denotes 95% significance level. Red dotted line, ripple power peak times between MEC and CA1 shuffled; blue dotted line, spike theta phase preserved while temporal association between MEC and CA1 randomized.
(G) No directional effect between MEC and CA1 during slow-wave sleep. MEC to CA1, 0.12 ± 0.05; MEC to CA1, 0.17 ± 0.06; z = 0.552, p = 0.681, Wilcoxon rank-sum test. Dotted lines denote 95% significance level. Red dotted line, ripple power peak times between MEC and CA1 shuffled; blue dotted line, spike theta phase preserved while temporal association between MEC and CA1 randomized.
Data are represented as mean ± SD.
Neuron  , e4DOI: ( /j.neuron )
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4. Figure 3
Effects of Optogenetic Intervention of MECIII Input on Ripple Bursts
(A) Sagittal sections of MEC and CA1 area. MECIII-Cre mice were bilaterally infected with the Cre-specific AAVrh8-hSyn1-DIO-eArchT-eYFP virus and silicone linear probes with an optical fiber were implanted in both areas.
(B) Examples of light stimulation during quiet awake when animals were in an open arena. Top panel: light OFF condition. Bottom panel: light ON condition. For each panel, traces from top are as follows: light timing, detected MUA ripples, ripple band LFP, wide-band LFP traces, and classified ripple bursts.
(C) Fundamental sharp-wave ripple parameters during quiet awake, including ripple frequency (light OFF, 149.3 ± 4.4 Hz; light ON, 148.7 ± 4.9 Hz; paired t test, t(302) = 1.003, p = 0.302) and single ripple duration (light OFF, 91.2 ± 6.3 ms; light ON, 90.8 ± 5.8 Hz; paired t test, t(302) = 0.575, p = 0.553) during optical stimulation.
(D) Distribution of ripple occurrence during quiet awake by ripple burst type. Singlet, light OFF, 1.32 ± 0.23 (10 s−1); light ON, 2.29 ± 0.21 (10 s−1), tsingle(638) = 3.093, ∗∗psingle = 0.002; doublet, light OFF, 0.72 ± 0.12 (10 s−1); light ON, 0.42 ± 0.11 (10 s−1), tdouble(304) = 2.681, ∗∗pdouble = 0.007; triplet, light OFF, 0.30 ± 0.06 (10 s−1); light ON, 0.12 ± 0.05 (10 s−1), ttriple(138) = 2.235, ∗∗ptriple = 0.009; other, light OFF, 0.10 ± 0.03 (10 s−1); light ON, 0.06 ± 0.02 (10 s−1), paired t test, tother(34) = 1.018, pother = 0.342; paired t test, Bonferroni corrected.
(E) Distribution of ripple bursts with optogenetic stimulation during quiet awake. Singlet, light OFF, 57.5% ± 9.3%; light ON, 74.2% ± 10.2%, tsingle(638) = 1.841, psingle = 0.061; doublet, light OFF, 27.2% ± 4.2%; light ON, 17.4% ± 2.7%, tdouble(304) = 2.301, ∗pdouble = 0.032; triplet, light OFF, 13.2% ± 3.5%; light ON, 7.3% ± 2.1%, ttriple(138) = 2.129, ∗ptriple = 0.025; other, light OFF, 2.1% ± 0.6%; light ON, 1.1% ± 0.8%, tother(34) = 1.903, pother = 0.061; paired t test, Bonferroni corrected.
(F) Same as in (C) but during slow-wave sleep. Ripple frequency (light OFF, 150.2 ± 4.7 Hz; light ON, 151.4 ± 6.3 Hz, paired t test, t(428) = 0.755, p = 0.462) and single ripple duration (light OFF, 95.3 ± 7.2 ms; light ON, 92.5 ± 5.8 Hz, paired t test, t(428) = 0.225, p = 0.824) during optical stimulation.
(G) Same as in (D) but during slow-wave sleep. Singlet, light OFF, 3.13 ± 0.39 (10 s−1); light ON, 3.19 ± 0.37 (10 s−1), tsingle(948) = 0.552, psingle = 0.693; doublet, light OFF, 1.49 ± 0.29 (10 s−1); light ON, 1.54 ± 0.31 (10 s−1), tdouble(428) = 0.248, pdouble = 0.835; triplet, light OFF, 0.77 ± 0.16 (10 s−1); light ON, 0.79 ± 0.19 (10 s−1), ttriple(231) = 0.325, ptriple = 0.769; other, light OFF, 0.25 ± 0.04 (10 s−1); light ON, 0.21 ± 0.12 (10 s−1), paired t test, tother(63) = 0.221, pother = 0.895; paired t test (mean ± SD), Bonferroni corrected.
(H) Distribution of ripple bursts with optogenetic manipulation during slow-wave sleep. Singlet, light OFF, 54.6% ± 9.3%; light ON, 55.7% ± 11.5%, tsingle(948) = 0.635, psingle = 0.524; doublet, light OFF, 32.2% ± 6.1%; light ON, 31.5% ± 5.2%, tdouble(428) = 0.921, pdouble = 0.382; triplet, light OFF, 13.1% ± 4.0%; light ON, 12.5% ± 3.1%, ttriple(231) = 1.748, ptriple = 0.081; other, light OFF, 0.5% ± 0.2%; light ON, 0.7% ± 0.2%, tother(63) = 1.349, pother = 0.226; paired t test, Bonferroni corrected.
Data are represented as mean ± SD.
Neuron  , e4DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions

5. Figure 4
MECIII Input Is Indispensable for Long-Range CA1 Replay in Quiet Awake
(A) Comparison of the fundamental parameters of individual ripples. Peak frequency, CT, 152.5 ± 4.1 Hz; MT, 151.4 ± 5.0 Hz, paired t test, t(336) = 0.738, p = 0.486; duration, CT, 89.3 ± 6.9 ms; MT, 92.4 ± 5.2 ms, t(336) = 0.633, p =
(B) Examples of ripple burst-associated replays in CT and MT. Top panel: decoded replay trajectory. Current location is shown as a solid green line. Bottom panel: detected replay candidates (red) using MUA (blue).
(C) Examples of fragmented replay trajectories in MECIII input-blocked mice (MT). Blue solid lines are detected fragmented replays. Fragmentation index, CT,1.3 ± 0.4; MT, 3.3 ± 0.6, paired t test, t(142) = 3.022, ∗∗p =
(D) Examples of positive and negative correlated replays. Current locations at the time of replay indicated as green solid lines.
(E) Examples of replay trajectories of MECIII-blocked MT and CT. Each line represents single replay events and is color-coded with single ripple (blue) or doublets (green) or triplets (red). Replay start and end locations were used to draw each line.
(F) Group data of the spatial coverage. CT, 57.3% ± 9.3%; MT, 25.7% ± 8.3%, z = 3.298, ∗∗∗p = 1.5 × 10−5, Wilcoxon rank-sum test.
(G) Distribution of ripple occurrence by ripple burst type. Singlet, CT, 1.29 ± 0.18 (10 s−1); MT, 2.30 ± 0.16 (10 s−1), tsingle(328) = 3.295, ∗∗∗psingle = 2.4 × 10−4; doublet, CT, 0.72 ± 0.12 (10 s−1); MT, 0.42 ± 0.11 (10 s−1), tdouble(147) = 2.817, ∗∗pdouble = 0.006; triplet, CT, 0.30 ± 0.06 (10 s−1); MT, 0.12 ± 0.05 (10 s−1), ttriple(63) = 2.856, ∗∗ptriple = 0.008; other, CT, 0.10 ± 0.03 (10 s−1); MT, 0.06 ± 0.02 (10 s−1), paired t test, tother(21) = 1.829, pother = 0.072; paired t test, Bonferroni corrected.
(H) Distribution of ripple bursts during quiet awake. Singlet, CT, 54.0% ± 8.3%; MT, 79.3% ± 10.4%, tsingle(328) = 2.327, ∗psingle = 0.021; doublet, CT, 28.6% ± 6.2%; MT, 13.9% ± 4.6%, tdouble(147) = 2.832, ∗∗pdouble = 0.006; triplet, CT, 13.2% ± 4.2%; MT, 7.3% ± 2.5%, ttriple(63) = 3.243, ∗∗ptriple = 0.002; other, CT, 3.1% ± 2.1%; MT, 2.7% ± 1.2%, tother(21) = 2.063, pother = 0.053; paired t test, Bonferroni corrected.
Data are represented as mean ± SD.
Neuron  , e4DOI: ( /j.neuron )
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6. Figure 5
MECIII Input Is Dispensable for Both Ripple Bursts and for Long-Range CA1 Replay during Slow-Wave Sleep
(A) An example of sleep period after RUN session expressing elevated sharp-wave ripples during prolonged immobility.
(B) Replay fidelity measurement by replay score. Quiet awake, 0.63 ± 0.12; slow-wave sleep, 0.41 ± 0.10, z = 2.844, ∗∗p = 0.004, Wilcoxon rank-sum test.
(C) Same as in Figure 4D. Examples of statistically significant slow-wave sleep replay episodes.
(D) Same as in Figure 4E. Spatial coverage plot of slow-wave sleep replays.
(E) Group data of spatial coverage of sleep replays. CT, 53.2% ± 25.3%; MT, 49.7% ± 22.9%, p = 0.092, Wilcoxon rank-sum test.
(F) Distribution of ripple occurrence by ripple burst type. Singlet, CT, 3.24 ± 0.39 (10 s−1); MT, 3.19 ± 0.37 (10 s−1), tsingle(544) = 0.539, psingle = 0.731; doublet, CT, 1.53 ± 0.29 (10 s−1); MT, 1.54 ± 0.19 (10 s−1), tdouble(221) = 1.088, pdouble = 0.268; triplet, CT, 0.84 ± 0.16 (10 s−1); MT, 0.79 ± 0.19 (10 s−1), ttriple(102) = 1.295, ptriple = 0.291; other, CT, 0.24 ± 0.04 (10 s−1); MT, 0.21 ± 0.12 (10 s−1), paired t test, tother(42) = 0.717, pother = 0.482; paired t test, Bonferroni corrected.
(G) Distribution of ripple bursts during slow-wave sleep. Singlet, CT, 51.0 %± 10.3%; MT, 54.3% ± 12.1%, tsingle(544) = 1.206, psingle = 0.203; doublet, CT, 24.3% ± 6.7%; MT, 27.2% ± 4.8%, tdouble(221) = 0.602, pdouble = 0.552; triplet, CT, 14.3% ± 4.4%; MT, 10.5% ± 2.2%, ttriple(102) = 1.611, ptriple = 0.128; other, CT, 10.4% ± 2.4%; MT, 8.2% ± 1.1%, tother(42) = 1.310, pother = 0.098; paired t test, Bonferroni corrected.
Data are represented as mean ± SD.
Neuron  , e4DOI: ( /j.neuron )
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7. Figure 6
CA3 Input Is Crucial for CA1 Ripples and Replays Regardless of Behavioral State
(A) Schematic of CA3-Cre-eArchT mouse, Nmice = 4. CA3 input is optogenetically blocked by axon terminal stimulation in CA1 area. Bilaterally injected and stimulated.
(B) Viral expression pattern in CA3-Cre (KA1-Cre) mouse. Unilaterally recorded spiking activities and LFPs.
(C) Group data of CA3 input blockade in CA1 area. Ripple peak frequency (light OFF, 155.4 ± 4.4 Hz; light ON, 143.2 ± 4.9 Hz, paired t test, t(739) = 0.387, p = 0.681), single ripple duration (light OFF, 97.3 ± 6.2 ms; light ON, 75.2 ± 10.3 ms, paired t test, t(739) = 1.726, p = 0.063), and occurrence (light OFF, 0.81 ± 0.051 Hz; light ON, 0.051 ± 0.05 Hz, paired t test, t(395) = 3.294, ∗∗∗p = 1.2 × 10−8).
(D) An example of optogenetic blockade of ripples during quiet awake. Top panel: green line indicates animal’s position. Bottom panel: MUA as blue lines. Detected ripples as red lines.
(E) An example of optogenetic blockade of ripples during slow-wave sleep. Amber arrowheads highlight residual ripple events during blockade.
(F) An example of position reconstruction in a CA3-Cre-eArchT mouse.
(G) Examples of residual replay (three left top panels) and bootstrap results (three left bottom panels) that correspond to arrowheads in (D). No significant replays during CA3 blockade, light OFF, 9.8% ± 1.4%; light ON, 1.2% ± 0.6%, z = 3.291, ∗∗∗p = 6.2 × 10−6, Wilcoxon rank-sum test (right panel).
Data are represented as mean ± SD.
Neuron  , e4DOI: ( /j.neuron )
Copyright © 2017 Elsevier Inc. Terms and Conditions