Integrating Neuronal Ensembles Constitute and Replay Hippocampal Engram○Khaled Ghandour1,2,3, ○Noriaki Ohkawa1,2,3, ○Chi Chung Alan Fung2,4, Hirotaka Asai1,2, Yoshito Saitoh1,2,3, Takashi Takekawa2,5, Reiko Okubo-Suzuki1,2,Shingo Soya6, Hirofumi Nishizono7, Mina Matsuo7, Masaaki Sato4,8,9, Masamichi Ohkura8,9, Junichi Nakai8,9,4,9,10
6
11,122,4
1,2
Yasunori Hayashi, Takeshi Sakurai , Makoto Osanai, Tomoki Fukai , Kaoru Inokuchi
1Dept Biochem, Grad Sch Med Pharm, Univ Toyama, Japan, 2CREST, JST, 3PRESTO, JST, 4RIKEN BSI, 5Fac Informatics, Kogakuin Univ, 6 WPI-IIIS, Univ Tsukuba,7 Life Sci Res Cent, Univ Toyama, 8Grad Sch Sci Eng, Saitama Univ, 9Brain Body Sys Sci Inst, Saitama Univ, 10Grad Sch Med, Kyoto Univ, 11Tohoku Univ Grad Sch Med, 12Grad Sch Biomed Eng, Tohoku Univ

Context B

30

EYFP / Paired③Ca2+ signal②405060

0102030Time [s]405060Freezing (%)

~1hr
~1116

1611166iB

1116*B**iEiEiDiDiFiF

65 15 9720 63911

20 6

39 11 58

19 63711

5516

20 7

39 135819

iAiA

4313

iF
iF

20 7

19 6

33 8

19

22 73911

iBiB

1 1
1 15816

20 63911

iBiB

5816

1 1
1 1

20 63911

iBiB5816

11
1 1391343
137iC1119 6116iC1116iC

1 1
1 1

1119

13

13

77

1
16iD

1116

16iD

1116

190E
D

0.6

C0.4

B

1iE

1930

2
1060

070

140

4
32
1030

45210

280

350

2
1060070

140210

280

350

2432
1030
Neuron ID#4560

10070

140

111 1

20 6 39 11 58iCiC

1943
1339130.2

22 720 7
1 1
1 1iCiC

19 637 11iDiD

5516

111 1iDiD

1943
13B
CDEFReference Session

022 7

111 1iEiE

5. Matching score of Engram and Non-engram
cells relative to session ASession A

45

22 75819

A
13

1 iCiC
0.8

A

7

1 1
1 1

39 11 58

F

7

120 7

20 6210280

350

EngramNon-engram

1#0.8
0.6
0.4

n.s.

0.20A

B

C D ESessions

5816

iFiF

16iEiE7iBiB

iF
iF

3711

432101520 61313

B C D
EF
Reference Session

1 11 113

Pattern #5 in Session A of Animal ID#196

RelativeMagnitude

iAiA

11⑤FTime [s]N =6, * P < 0.05; ** P<0.01; # P<0.05 between Engram and Non-engram, Paired t test (one-tailed)TimeØ Activity pattern extraction (Non-negative Matrix Factorization)

Ø Dot product between patterns (Pattern matching)

20 6

111 1

65 15 97190.2

N =10, * P < 0.05,Wilcoxon signed-ranks test (two-tailed)(= AICc)

iAiA

33 85819

Overlay of Pattern #5 and #9The data matrix (D) is approximatedby two matrices of Patterns (B) andSynchrony (C).

65 15 975516

0.415Non-engram
cells

3913

016

Pattern #9 in Session A of Animal ID#196

Engram
cells

3711

iB

1C11

3. Pattern #5 and 9 of Engram in Session A of NV#196

139 11iAiA

33 8

190.6

1525819

65 15 97

19iD

605516

33 8

22 7

160.8

D6iB

19E

1050

580.4

11FA

10.6

71 1
1 1

1 1
1 1iAiA131 11 165 15 97

Non-engram

4313

20 7iDiD

11114033 8iF

6A30iA

1 1
1 1150.81116220819 620 6

197iB

31039130.2

0Non-engram cells

03711

iB
iB
113639 110.2

Engram cells

4iA

3911

19iF
iF

5151911685819

20 6115516

iE
iE

11iF

iE
iA

1516

1030
Time [s]

858iE
iE

0.4

111020iA

155816iF-0.04

81087iE

0.87114. Sum of all correlations
within the first 60s (NV#69)

Ratio of sum of all
correlations within
the first 60s②Retrieval(E)

Pattern #Time

Engram cells original data maintain higher MS
across sessions compared to shuffled data
Original data of engram
Shuffled data of engram

10.8
0.6
0.4

Difference in MS between
Original and Shuffled data

Non-engram cells

615. Ratio of sum of all correlations
within the first 60s

0iA

1513iF

10 20
20 3030 4040 5050 60
6000 10
Reference TimeTime [s][s]Reference

1318-0.02

1011

Engram

19iE

2011④

KiKGR=Engramhrhr

019663①35iB30

Reference Time [s]③111116Matching Score (MS) Relative toSession A

Engram cells

tTA④KiKGR

hr

0.02

ROIｓ=Engram①35

16Compared Session(NV#69)OFF Dox

40

10.8
0.6
0.4

*0.2

n.s.

*

*
n.s.

0-0.2

0.2

-0.4

0A

B

CDESessions

FA

B

C
DESessions

F

N =6, * P < 0.05 between Engram original and shuffled data, Paired t test (one-tailed)

Learning

12
34Data (D)Non-negativeMatrix
Factorization(NMF)

To compare the patterns extracted across different sessions, cosine similarity between
2 vectors is used to quantify the similarity of 2 vectors. Pattern pair with > 0.6 isdefined as a significant pair in this work.

Patterns (B)ǁSynchrony (C)ǁCell ensemble

Synchronous
activity

Neuron #1 2 3 4Pattern #1

Pattern #2

Pattern #3

・・ ・・・ ・・・ ・

2. Wave form of Ca2+ event

REM(D)

N=6, * P < 0.05; ** P < 0.01, Paired t test (two-tailed)

iF

Time [s]

KikGR

0.04

Intensity

360

Patterns of Learning (Session A) [%]2. Dot product between patterns of different sessions extracted by NMF analysis (NV#196)

Intensity300

4. Engram patterns appeared in learning session are significantly highly reactive in both of either NREM orREM sleep and retrieval session compared with Non-engram patterns

0.06

Intensity240

N =6, ** P < 0.01; *** P<0.001; # P<0.05 between Engram and Non-engram, Paired t test (one-tailed)

Learning(A)

iE

180

Lentivirus
TRE

10 20 30 40 50 60
Reference[s]EngramTimecells
Non-engram
cells

Compared Session

120

P > 0.52, one-way ANOVA## P < 0.01, Paired t test, two-tailed

REM

Relative
Magnitude

c-fos::tTA system withlentivirus to label engram cells
Ohkawa et al., Cell Reports (2015) Vol. 11 p261-

60

3-4 min
4Hz LightEEG

Relative
Magnitude

0tTA

050

Sum of Overlaps of Correlations
for a Given Reference time01

c-fos promoter

Compared Time [s]Cell number

Non-engram

Thy1::G-CaMP7 Tg established byDrs Masaaki Sato andYasunori Hayashi

1-2 min, P > 0.82, one-way ANOVA

Ensemble activity patterns are preferentially reactivated inEngram cells during both resting and retrieval sessions

iD

Compared Time [s]Cell number

Engram-0.04

F1000ESessions

EMG-0.02

D802C

Non-REM

0100B*4・ For Ca2+ imaging

・ Labeling of engram

0.02

20

A

context memory processing

30

6

Thy1 gene

DoubleTg mice

0.04

40

8

F

1. Schedule Ca2+ imaging during

0.6

201

E

0.06

50

60

10④

D

3-4 min, P < 0.01, one-way ANOVA;* P < 0.05, ** P < 0.01,
Scheffe’s post-hoc test

1-2 min

ChR2
UnpairedC0n.s.

21200
ChR2PairedB

0.5

Sessions

5EYFPPaired

A##10001n.s.

4130

1*** *** *** ** **1.56060

EYFP / Paired
ChR2 / Paired
ChR2 / Unpaired

2240

2A RFP

Engram cells

40

10

3. Analysis of Correlation
Matrices Repetition (NV#69)

Repetitive activity is a characteristicfeature of Engram cells duringcontextual learning

101

G-CaMP7y50 μm

301

Thy1 geneda** P < 0.002, Welch’s t test,
two-tailed

15

Neuron #Ohkawa et al.,Cell Reports (2015) Vol. 11 p261-

Unpaired

20iC

Paired365 nmlightNat. Neurosci., 2013 Vol. 16 p264-10401

UndernVista

ChR2 / Unpaired

105 mmAfter 365 nm

ChR2 / Pairedn.s.

iD

20100 μm

Beforeda

3. Labeling efficacy of c-fos-Tet Tag system

15 mm

Memory test in Laser OFFContext B
Context A
Laser ONpre-exposure Immediate shock Context B
Memory test in Optical stimulation ofCA1 cell ensemble
New Cage

Laser ON/ OFF
Habituation inNew Cage with bedding

3#20control

LVinjection

Test 2Freezing (%)

c-fos::tTATg(ChR2Unpaired
P < 0.01, one-way ANOVA;*P < 0.05, **P < 0.01, Scheffe’s post-hoc test

50iC**ON-Dox% of EYFP or ChR2+ cells / DAPIFreezing (%)

Unpaired

25040Freezing↑((2 daysysContext A(((Context BChR2
PairedEngramNon-engram

0MergeEYFP
PairedUnpaired(NV#69)

Nucleus04

Difference in PVD between
Engram and Non-engram cells

Context A

iDiDContext A

2010Memory test in Laser OFF
Context A
Context A
Laser ONpre-exposure Immediate shock Context A
Memory test in Optical stimulation ofCA1 cell ensemble
New Cage

Laser ON/ OFF
Habituation inNew Cage with bedding

30

Matching Score (MS) Relative toSession AContext A

LVinjection((40Poulation Vector Distance (PVD)
Relative to Session Ac-fos::tTATg

PairedKikGR

KiKGR (R)*****CA1 KiKGR (G)ON-Doxday

Paired1s

TestdayIS

Freezing↑
Freezing(2 days2Context
pre-exposure

Test 2

Test 1

PVD is more consistent in Engram cells thanNon-engram cells across sessions

50(((2. Engram of a context information isactually formed in CA1

1. Context pre-exposure facilitation effect onimmediate shock deficit procedure(CPFE procedure)

1. Ca2+ signal of Engram and Non-Engram during learning
CaMKII*60③Lentivirus⑥

Test 1iCiC

neurons, which is activated by physiological input derived from a corresponding event, calledengram. The c-fos-TetTag system has been used to prove the engram theory, by manipulation ofcells that showed activity-dependent gene expression driven by learning. However, the physicaland basic activities that occur during learning and post learning in that group of cells, is still
poorly understood. Here we show that the engram cells exhibit a remarkable synchronous
activity representing the contextual memory in the form of several ensembles in engram cells.
These ensembles carry on their activity not only during learning but also during post-learning
sleep and retrieval sessions, in contrast to non-engram cells. A compatible imaging system wasestablished to observe the neuronal activity of ~1000 CA1 neurons and the labelled engram
cells; through a photoconvertible fluorescent protein Kikume Green Red (KikGR). The neuronal
activity of hippocampal CA1 neurons was observed, through Ca2+ influx with GCaMP7 in freelymoving animals by miniature head-mount fluorescent microscopy. Our advanced imaging
system of engram cells and non-engram cells provides deeper insights into the dynamics of theneural activity during contextual memory processing. Engram cells exhibit highly repetitive
activity corresponding to remarkable synchrony during novel context exposure. Population
vector distance (PVD) analysis indicates that total activity patterns of engram cells are stableand consistent across other sessions; sleep (NREM and REM) and retrieval, not only during
learning. Furthermore, Non-negative Matrix Factorization (NMF) analysis extracted characteristic
ensembles activity that were constructed by subgroup of engram cells, representing thepersistent synchronous activity even during post-learning sleep (NREM and REM) sessions andretrieval session, but not in a distinct context. In contrast, these features were not seen in nonengram cells. These results suggest that there are several fundamental characteristics of theengram cells that give them superiority in encoding the ongoing event and consolidating thepast ones.

Optogenetic activation of c-fos-Tet-tagged cells in CA1 induces
recall of contextual memory

iCiC

②

It is hypothesized that a memory is encoded in a subset ofTime

ReplayRetrieval

The number of patterns are determined by AICc.

Ø Matching score (MS)To compare the activity of engram cells and non-engram cells across different sessions, we calculated a matching score acrosssessions, which implies to what extent a pattern in the first session to be overlapped withany of the patterns extracted in the second session and vice versa.

Ø Contextual memory in the hippocampus is represented as distinct subsets of synchronous activity (defined by Ca2+ transients) thatcomprise several ensembles of engram cells.

Ø Population vector distance (PVD)

Ø In contrast to non-engram cells, these ensembles maintain their activity not only during learning but also during post-learning
sleep and retrieval sessions.

The population vector distance is defined by the Mahalanobis distance, which implies the distance between 2 groups ofneuronal activity vector normalized by their variance.

● We declare no Conflict of Interest (COI).