Integrating Neuronal Ensembles Constitute and …Itishypothesizedthatamemoryisencodedinasubsetof...
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It is hypothesized that a memory is encoded in a subset of neurons, which is activated by physiological input derived from a corresponding event, called engram. The c-fos-TetTag system has been used to prove the engram theory, by manipulation of cells that showed activity-dependent gene expression driven by learning. However, the physical and 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 was established 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 freely- moving 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 the neural 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 stable and 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 the persistent synchronous activity even during post-learning sleep (NREM and REM) sessions and retrieval 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 the engram cells that give them superiority in encoding the ongoing event and consolidating the past 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 is defined 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 with any 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 memory ② 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 learning ③ Ensemble activity patterns are preferentially reactivated in Engram cells during both resting and retrieval sessions ④ 5. Ratio of sum of all correlations within the first 60s Integrating Neuronal Ensembles Constitute and Replay Hippocampal Engram ○ Khaled Ghandour 1,2,3 , ○ Noriaki Ohkawa 1,2,3 , ○ Chi Chung Alan Fung 2,4 , Hirotaka Asai 1,2 , Yoshito Saitoh 1,2,3 , Takashi Takekawa 2,5 , Reiko Okubo-Suzuki 1,2 , Shingo Soya 6 , Hirofumi Nishizono 7 , Mina Matsuo 7 , Masaaki Sato 4,8,9 , Masamichi Ohkura 8,9 , Junichi Nakai 8,9 , Yasunori Hayashi 4,9,10 , Takeshi Sakurai 6 , Makoto Osanai 11,12 , Tomoki Fukai 2,4 , Kaoru Inokuchi 1,2 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 cells Ohkawa 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 Merge KiKGR (G) control 365 nm light Before After 365 nm Under nVista ・ For Ca 2+ imaging Thy1::G - CaMP7 Tg established by Drs Masaaki Sato and Yasunori Hayashi CaMKII KikGR Lentivirus Context A pre-exposure Context A Immediate shock 1 day ON-Dox 2 days ~1 hr c-fos::tTA Tg Memory test in New Cage Optical stimul ation of CA1 cell ensemble Freezing↑ LV injection reezing reezing ez ( ee ee ( e e ( ( ( ( i i ( n n ( ( Laser OFF Laser ON Laser ON / OFF 2 days Habituation in New Cage with bedding Memory test in Context A 3-5 hr Context B pre-exposure Context A Immediate shock 1 day ON-Dox 2 days ~1 hr c-fos::tTA Tg Memory test in New Cage Optical stimul ation of CA1 cell ensemble Freezing↑ LV injection reezing reezing ez ( ee ee ( e e ( ( ( ( i i ( n n ( ( Laser OFF Laser ON Laser ON / OFF 2 days Habituation in New Cage with bedding Memory test in Context B 3-5 hr Context pre-exposure IS Test Context B Context A Context A Context A Context B Context A Unpaired Paired 0 10 20 30 40 50 Freezing (%) ** Unpaired Paired 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. Ca 2+ signal of Engram and Non-Engram during learning 2. Wave form of Ca 2+ 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 - engram cells relative to session A 1. Schedule Ca 2+ imaging during context 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 / Unpaired ChR2 / Paired 50 μm Engram cells Non-engram cells Sum of Overlaps of Correlations for a Given Reference time 0 1 2 3 Reference Time [s] 0 10 20 30 40 50 60 4 5 T i m e Pattern # T i m e Synchrony ǁ Synchronous activity ( C ) P atte r n s ǁ Cell ensemble (B) ・ ・ ・ P atte r n #1 P atte r n #2 P atte r n #3 Neuron # Neuron # 1 2 3 4 ・ ・ ・ ・ ・ ・ 1 2 3 4 D at a ( D ) Non - negative Matrix Factorization (NMF) N =6, * P < 0.05; ** P<0.01; # P<0.05 between Engram and Non-engram, Paired t test (one-tailed) Engram Non-engram 1 21 41 401 1 101 201 301 Cell number Cell number 0 2 4 6 8 10 0 60 120 180 240 300 360 Time [s] (NV#69) (NV#69) Engram cells Non-engram cells Ca 2+ signal 0 10 20 30 40 50 60 Time [s] 0 10 20 30 40 50 60 Time [s] 0 8 The number of patterns are determined by AICc. (= AICc) T he da t a m a t r i x (D) is approximated b y t w o matrices of Patterns (B) and Synchrony (C) . 0 10 20 30 40 1-2 min 3-4 min 4Hz Light EYFP / Paired ChR2 / Paired ChR2 / Unpaired Freezing (%) ** * ## 50 0 5 10 15 EYFP Paired ChR2 Paired ChR2 Unpaired % of EYFP or ChR2+ cells / DAPI n.s. 20 Engram cells 0 10 20 30 40 50 Compared Time [s] 0 10 20 30 40 50 60 60 0 0.02 0.04 0.06 -0.04 -0.02 0 3 1 Engram cells Non-engram cells * Ratio of sum of all correlations within the first 60s 2 iB iC iC iD iD iD iE iE iE iE iB iB iB iA iA iA iA iC iC iD 11 6 1 11 6 1 15 8 1 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 iF iF iF iF iD 13 7 1 19 15 8 1 15 8 1 15 8 1 15 8 1 11 6 1 16 11 6 1 16 11 6 1 16 11 6 1 16 11 6 1 11 6 1 iB iA iC 11 6 1 16 11 6 1 16 iE 13 7 1 19 iF Compared Session Reference Session C B A D E C B A D E F F 0 0.2 0.4 0.6 0.8 1 iB iC iC iD iD iD iE iE iE iB iB iB iA iA iA iA iC iC iD 65 33 1 97 39 20 1 58 iE 39 20 1 58 37 19 1 55 iF iF iF iB iA iC iD 43 22 1 iF 37 19 1 55 iF iE 39 20 1 58 39 20 1 58 39 20 1 58 37 19 1 55 65 33 1 97 65 33 1 97 65 33 1 97 65 33 1 97 39 20 1 58 39 20 1 58 39 20 1 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 39 20 1 58 39 20 1 58 39 20 1 58 Pattern #5 in Session A of Animal ID#196 Session A Pattern #9 in Session A of Animal ID#196 15 30 45 60 Neuron ID# Intensity Intensity Intensity Time [s] Overlay of Pattern #5 and #9 0 1 2 0 1 2 0 1 2 0 70 140 210 350 280 0 70 140 210 350 280 0 70 140 210 350 280 15 30 45 60 15 30 45 60 Relative Magnitude 0 1 2 3 4 Relative Magnitude 0 1 2 3 4 Relative Magnitude 0 1 2 3 4 Matching Score (MS) Relative Session A Engram Non-engram Sessions C B A D E 0 0.2 0.4 0.6 0.8 1 F # n.s. Learning (A) REM (D) Retrieval (E) 20 40 60 80 100 0 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 Ca 2+ 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 0 10 20 30 40 50 Compared Time [s] 60 1 Dept Biochem, Grad Sch Med Pharm, Univ Toyama, Japan, 2 CREST, JST, 3 PRESTO, JST, 4 RIKEN BSI, 5 Fac Informatics, Kogakuin Univ, 6 WPI-IIIS, Univ Tsukuba, 7 Life Sci Res Cent, Univ Toyama, 8 Grad Sch Sci Eng, Saitama Univ, 9 Brain Body Sys Sci Inst, Saitama Univ, 10 Grad Sch Med, Kyoto Univ, 11 Tohoku Univ Grad Sch Med, 12 Grad Sch Biomed Eng, Tohoku Univ Compared Session Reference Session C B A D E C B A D E F F 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 N =6, ** P < 0.01; *** P<0.001; # P<0.05 between Engram and Non-engram, Paired t test (one-tailed) Poulation Vector Distance (PVD) Relative to Session A 0 1 2 3 4 Difference in PVD between Engram and Non-engram cells 0 0.5 1 1.5 2 Sessions C B A D E F Sessions C B A D E F *** *** *** ** ** # Engram Non-engram Engram cells original data maintain higher MS across sessions compared to shuffled data Matching Score (MS) Relative Session A Sessions C B A D E 0 0.2 0.4 0.6 0.8 1 F Original data of engram Shuffled data of engram Difference in MS between Original and Shuffled data Sessions C B A 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 sessions ⑥ Freezing (%) * ** 0 10 20 30 40 50 EYFP Paired ChR2 Paired ChR2 Unpaired 60
Transcript of Integrating Neuronal Ensembles Constitute and …Itishypothesizedthatamemoryisencodedinasubsetof...
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
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②
③
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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
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Laser OFF Laser ONLaserON/ OFF
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Habituation inNew Cage with bedding
Memory test inContext A
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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
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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
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KiKGR=Engram
①
KiKGR
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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
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Data (D)Non-negativeMatrixFactorization(NMF)
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The number of patterns are determined by AICc.
(= AICc)The data matrix (D) is approximatedby two matrices of Patterns (B) andSynchrony (C).
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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
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tens
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tens
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Overlay of Pattern #5 and #9
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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
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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
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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
