It is hypothesized that a memory is encoded in a subset ofneurons, 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 stillpoorly understood. Here we show that the engram cells exhibit a remarkable synchronousactivity 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-learningsleep 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 engramcells; through a photoconvertible fluorescent protein Kikume Green Red (KikGR). The neuronalactivity of hippocampal CA1 neurons was observed, through Ca2+ influx with GCaMP7 in freely-moving animals by miniature head-mount fluorescent microscopy. Our advanced imagingsystem of engram cells and non-engram cells provides deeper insights into the dynamics of theneural activity during contextual memory processing. Engram cells exhibit highly repetitiveactivity corresponding to remarkable synchrony during novel context exposure. Populationvector 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 duringlearning. Furthermore, Non-negative Matrix Factorization (NMF) analysis extracted characteristicensembles 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 non-engram 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.
Ø Activity pattern extraction (Non-negative Matrix Factorization)
Ø Dot product between patterns (Pattern matching)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.
Ø Matching score (MS)To compare the activity of engram cells and non-engram cells across different sessions, we calculated a matching score across
sessions, 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 Population vector distance (PVD)The population vector distance is defined by the Mahalanobis distance, which implies the distance between 2 groups of
neuronal activity vector normalized by their variance.
Optogenetic activation of c-fos-Tet-tagged cells in CA1 induces recall of contextual memory②
P < 0.01, one-way ANOVA; *P < 0.05, **P < 0.01, Scheffe’s post-hoc test
Repetitive activity is a characteristic feature of Engram cells during
contextual learning③
Ensemble activity patterns are preferentially reactivated in Engram cells during both resting and retrieval sessions④
5. Ratio of sum of all correlations within the first 60s
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, Yasunori Hayashi4,9,10, Takeshi Sakurai6, Makoto Osanai11,12, Tomoki Fukai2,4, Kaoru Inokuchi1,2
①
②
③
②
ROIs=Engram
c-fos promoter tTA TRE KikGR
Lentivirus
OFF Dox
tTA
DoubleTg mice
Thy1 gene G-CaMP7 2A RFP Thy1 gene
c-fos::tTA system with lentivirus to label engram cellsOhkawa et al., Cell Reports (2015) Vol. 11 p261-
・ Labeling of engram
Nat. Neurosci., 2013 Vol. 16 p264-
5 mm 5 mm
KiKGR (G)CA1 KiKGR (R) Nucleus MergeKiKGR (G)
control
365 nm light
Before After 365 nmUnder nVista
・ For Ca2+ imagingThy1::G-CaMP7 Tg established byDrs Masaaki Sato and
Yasunori Hayashi
CaMKII KikGRLentivirus
ContextApre-exposure
Context AImmediate shock
1 day
ON-Dox
2 day
s
~1 hr
c-fos::tTA Tg
Memory test inNew Cage
Optical stimulation ofCA1 cell ensemble
Freezing↑
LVinjection
FreezingFreezingFreezingFreezing(FreezingFreezing(FreezingFreezing(Freezing
(FreezingFreezing
(FreezingFreezing(FreezingFreezing(FreezingFreezing
(
Freezing
(
FreezingFreezing
(
FreezingFreezing
(
Laser OFF Laser ONLaserON/ OFF
2 days
Habituation inNew Cage with bedding
Memory test inContext A
3-5 hr
ContextBpre-exposure
Context AImmediate shock
1 day
ON-Dox
2 day
s
~1 hr
c-fos::tTA Tg
Memory test inNew Cage
Optical stimulation ofCA1 cell ensemble
Freezing↑
LVinjection
FreezingFreezingFreezingFreezing(FreezingFreezing(FreezingFreezing(Freezing
(FreezingFreezing
(FreezingFreezing(FreezingFreezing(FreezingFreezing
(
Freezing
(
FreezingFreezing
(
FreezingFreezing
(
Laser OFF Laser ONLaserON/ OFF
2 days
Habituation inNew Cage with bedding
Memory test inContextB
3-5 hr
Contextpre-exposure IS Test
Context B
Context A
Context A
Context A
Context B
Context A
Unpaired
Paired
0
10
20
30
40
50
Free
zing
(%)
**
UnpairedPaired
1. Context pre-exposure facilitation effect on immediate shock deficit procedure (CPFE procedure)
3. Labeling efficacy of c-fos-Tet Tag system
Ohkawa et al., Cell Reports (2015) Vol. 11 p261-
** P < 0.002, Welch’s t test, two-tailed
1-2 min, P > 0.82, one-way ANOVA
3-4 min, P < 0.01, one-way ANOVA; * P < 0.05, ** P < 0.01, Scheffe’s post-hoc test
## P < 0.01, Paired t test, two-tailed P > 0.52, one-way ANOVA
100 μm
Test 1
Test 2
Test 1 Test 2
4. Sum of all correlations within the first 60s (NV#69)
N =10, * P < 0.05, Wilcoxon signed-ranks test (two-tailed)
3. Analysis of Correlation Matrices Repetition (NV#69)
1. Ca2+ signal of Engram and Non-Engram during learning
2. Wave form of Ca2+ event
④
③
KiKGR=Engram
①
KiKGR
④
3. Pattern #5 and 9 of Engram in Session A of NV#196 5. Matching score of Engram and Non-engramcells relative to session A
1. Schedule Ca2+ imaging duringcontext memory processing
2. Dot product between patterns of different sessions extracted by NMF analysis (NV#196)
4. Engram patterns appeared in learning session are significantly highly reactive in both of either NREM or REM sleep and retrieval session compared with Non-engram patterns
Engram Non-engram
Paired
Unpaired
2. Engram of a context information is actually formed in CA1
●We declare no Conflict of Interest (COI).
EEG
EMG
Non-REM
REM
N=6, * P < 0.05; ** P < 0.01, Paired t test (two-tailed)
EYFP / Paired ChR2 / UnpairedChR2 / Paired
50 μm
Engram cellsNon-engram cells
Sum
of O
verla
ps o
f Cor
rela
tions
for a
Giv
en R
efer
ence
tim
e
0
1
2
3
Reference Time [s]0 10 20 30 40 50 60
4
5
Time Pattern # Time
Synchronyǁ
Synchronous activity
(C)Patternsǁ
Cell ensemble
(B)
・ ・ ・Pattern #1Pattern #2Pattern #3
Neu
ron
#
Neuron #1 2 3 4
・ ・ ・・ ・ ・
1 2 34
Data (D)Non-negativeMatrixFactorization(NMF)
N =6, * P < 0.05; ** P<0.01; # P<0.05 between Engram and Non-engram, Paired t test (one-tailed)
Engr
amN
on-e
ngra
m
1
21
41
401
1
101
201
301
Cel
l num
ber
Cel
l num
ber
0
2
4
6
8
10
0 60 120 180 240 300 360Time [s]
(NV#69)
(NV#69)
Engram cells Non-engram cells
Ca2+
sig
nal
0 10 20 30 40 50 60Time [s]
0 10 20 30 40 50 60Time [s]
0
8
The number of patterns are determined by AICc.
(= AICc)The data matrix (D) is approximatedby two matrices of Patterns (B) andSynchrony (C).
0
10
20
30
40
1-2 min 3-4 min4Hz Light
EYFP / PairedChR2 / PairedChR2 / Unpaired
Free
zing
(%)
***##
50
0
5
10
15
EYFPPaired
ChR2Paired
ChR2Unpaired%
of E
YFP
or C
hR2+
cel
ls /
DA
PI
n.s.20
Engram cells
0
10
20
30
40
50
Com
pare
d Ti
me
[s]
Reference Time [s]0 10 20 30 40 50 60
60
0
0.02
0.04
0.06
-0.04
-0.02
0
3
1
Engramcells
Non-engramcells
*
Ratio
of s
um o
f all
corr
elat
ions
with
inth
e fir
st 6
0s 2
iB iCiC
iDiD
iD
iEiE
iEiE
iBiB iB
iA iA iA iA
iC iC
iD
11
6
1
1161
1581
0
0.2
0.4
0.6
0.8
1
11
6
1
16
11
6
1
16
11
6
1
13
7
1
19
11
6
1
16
11
6
1
16
13
7
1
19
13
7
1
19
13
7
1
19
13
7
1
19
13
7
1
19
13
7
1
19
13
7
1
19
iFiF
iFiF
iD
13
7
1
19
1581 1581 1581 1581
1161 16 1161 16 1161 16 1161 16
1161 1161
iB
iA
iC
1161 16 1161 16
iE1371 19
iF
Com
pare
d Se
ssio
n
Reference SessionCBA D E
C
B
A
D
E
F
F0
0.2
0.4
0.6
0.8
1
iB iCiC
iDiD
iD
iEiE
iE
iBiB iB
iA iA iA iA
iC iC
iD
65331 97
39
20
1
58
iE
39201 58
37191 55
i Fi F
iF
iB
iA
iC
iD
43
22
1
iF
37191 55
i F
iE39201 58
39
20
1
58
39
20
1
58
37
19
1
55
65331 97 65331 97 65331 97 65331 97
39201 58 39201 58 39201 58
43
22
1
43
22
1
43
22
1
43
22
1
39
20
1
58
39
20
1
58
39
20
1
58
37
19
1
55
37
19
1
55
39
20
1
58
39201 58 39201 58 39201 58
Pattern #5 in Session A of Animal ID#196 Session A
Pattern #9 in Session A of Animal ID#19615 30 45 60
Neuron ID#In
tens
ityIn
tens
ityIn
tens
ityTime [s]
Overlay of Pattern #5 and #9
0
1
2
0
1
2
0
1
2
0 70 140 210 350280
0 70 140 210 350280
0 70 140 210 350280
15 30 45 60
15 30 45 60
Rela
tive
Mag
nitu
de
01234
Rela
tive
Mag
nitu
de
01234
Rela
tive
Mag
nitu
de
01234
Mat
chin
g Sc
ore
(MS)
Rel
ativ
e to
Sess
ion
A
EngramNon-engram
SessionsCBA D E
00.20.40.60.8
1
F
#
n.s.
Learning(A)
REM(D)
Retrieval(E)
20 40 60 80
1000
Patterns of Learning (Session A) [%]
*
**
n.s.n.s.
Time
Learning
Replay Retrieval
Ø Contextual memory in the hippocampus is represented as distinct subsets of synchronous activity (defined by Ca2+ transients) that comprise several ensembles of engram cells.
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.
Non-engram cells
Reference Time [s]0 10 20 30 40 50 60
0
0.02
0.04
0.06
-0.04
-0.02
Engram cells
0
10
20
30
40
50
Com
pare
d Ti
me
[s]
Reference Time [s]0 10 20 30 40 50 60
60
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
Com
pare
d Se
ssio
n
Reference SessionCBA D E
C
B
A
D
E
F
F
Com
pare
d Se
ssio
n
Reference SessionCBA D E
C
B
A
D
E
F
F0
0.2
0.4
0.6
0.8
1
iB iCiC
iDiD
iD
iEiE
iEiE
iBiB iB
iA iA iA iA
iC iC
iD
11
6
1
1161
1581
0
0.2
0.4
0.6
0.8
1
11
6
1
16
11
6
1
16
11
6
1
13
7
1
19
11
6
1
16
11
6
1
16
13
7
1
19
13
7
1
19
13
7
1
19
13
7
1
19
13
7
1
19
13
7
1
19
13
7
1
19
iFiF
iFiF
iD
13
7
1
19
1581 1581 1581 1581
1161 16 1161 16 1161 16 1161 16
1161 1161
iB
iA
iC
1161 16 1161 16
iE1371 19
iF
N =6, ** P < 0.01; *** P<0.001; # P<0.05 between Engram and Non-engram, Paired t test (one-tailed)
Poul
atio
n Ve
ctor
Dis
tanc
e (P
VD)
Rela
tive
to S
essi
on A
0
1
2
3
4
Diffe
renc
e in
PVD
bet
wee
nEn
gram
and
Non
-eng
ram
cel
ls
0
0.5
1
1.5
2
SessionsCBA D E F
SessionsCBA D E F
*** *** *** ** **
#EngramNon-engram
Engram cells original data maintain higher MS across sessions compared to shuffled data
Mat
chin
g Sc
ore
(MS)
Rel
ativ
e to
Sess
ion
A
SessionsCBA D E
0
0.2
0.4
0.6
0.8
1
F
Original data of engramShuffled data of engram
Diff
eren
ce in
MS
betw
een
Orig
inal
and
Shu
ffled
dat
a
SessionsCBA D E
0
0.2
0.4
0.6
0.8
1
F
* * *n.s. n.s.
-0.2
-0.4
N =6, * P < 0.05 between Engram original and shuffled data, Paired t test (one-tailed)
⑤
PVD is more consistent in Engram cells than Non-engram cells across sessions⑥
Free
zing
(%)
***
0
10
20
30
40
50
EYFPPaired
ChR2Paired
ChR2Unpaired
60