Neuronal ensemble orchestrated intersection of two distinct emotional memory traces〇 Jun Yokose 1,2, Okubo-Suzuki Reiko 1,2, Masanori Nomoto 1,2, Noriaki Ohkawa 1,2, Hirofumi Nishizono 2,3, Akinobu Suzuki 1,2, Mina Matsuo 3,Shuhei Tsujimura1,2, Yukari Takahashi 5, Masashi Nagase 5, Ayako M. Watabe 5, Masakiyo Sasahara 4, Fusao Kato 5, and Kaoru Inokuchi 1,2
1 Department of Biochemistry, Faculty of Medicine, Graduate School of Medicine & Pharmaceutical Sciences, University of Toyama, Toyama,
2 CREST, Japan Science and Technology Agency 3 Division of Animal Experimental Laboratory, Life Science Research Center, University of Toyama, Toyama
4 Department of Pathology, Faculty of Medicine, University of Toyama, Toyama 5 Department of Neuroscience, Jikei University School of Medicine, Tokyo

Disruption of CTA memory retrieval by blockade of protein synthesis
in BLA impairs original AFC memory

Synchronous activity of distinct neuronal ensembles during the RCS
generates a link between CTA and AFC memory◆ Checking for disruption of a single component
in CTA memory could render labile of the other24 hcomponent in AFC memory
CTAtrainingAFCtraining

AFC

24 h

111213 (Days)RCS TEST1 TEST2(CTA) (AFC)CTA AFC
training training(RCS; Co-retrieval of CTA memory and AFC memory)

RCS(3 sessions; ITI 1h )24 hCTATEST

1 session (x 3 times)

Homecage
5min
5min
6min

Double+active cellat CTA- and AFCretrieval

INF Arc+
active cellat AFC-retrieval

Sync
…..

Co-retrieval:

5s

5s

5s
…..

Control:

Lentivirus:

c-fos

tTA

tTA
TRE3G

ArchT-EYFP WPRE

2w

CTA
training

AFChabituation

Dox +BLA◆ ArchT-expressing neuron elicited a robust andsustained hyperpolarizing current that effectively
blocked action potentials evoked by somatic2s
current injections (ex vivo)【AFC to CTA group】

5 min
CTA-CS

5 min
AFC-CSAFC
2 min

CTA

ArchT OFF

40s

CTA(CTA) (AFC)

AFC

1110

12

EYFP ON●: Evoked-response during
light illumination

RCS TEST1 TEST2(+or-) (CTA) (AFC)○: Evoked-response after
light illumination

12□ Pre-tone■ Tone**ArchT ON
ArchT OFFEYFP ONc-Fos+ cells / DAPI (%)
Constant light illumination(5 min)(p=0.53,t19=0.63,
unpaired Student’s t-test)(*p< 0.05,t21=2.55
unpaired Student’s t-test)

Reference: 1. D. O. Hebb, The Organization of Behavior: A Neuropsychological approach (John Wiley and Sons, New York, 1949).

(p=0.53,t20=0.63,unpaired Student’s t-test)

Acknowledgments:

2. J. R. Anderson, G. H. Bower, Human Associative Memory. (Wiley, New York, 1973). 3. X. Liu et al., Nature 484 , 381-385 (2012).
4. S. A. Josselyn, S. Kohler, P. W. Frankland, Nat. Rev. Neurosci. 16, 521-534 (2015). 5. S. Tonegawa, X. Liu, S. Ramirez, R. Redondo, Neuron 87, 918-931 (2015).

-Dox

ArchT-EYFP

WPRE
CreERT2 (+) & H2B-mcherry(+)(Ready for activity-dependent ArchT-expression)

48 h

24 h

100

24 h100mm(5h before TEST1)①24 h

24 hor②TEST4(AFC)TEST3

Dox -【①. Optical silencing procedure during the CTA TEST 】

Overlapping ensemble

IHC
TEST2(CTA)

CTA-retrieval
Overlapping ensemble
active ensemble ( ArchT (+) ensemble )TEST5(CTA)

(13)
(12)

ArchT OFF

80
N.S.

60
40200

Opticalsilencing(Optical silencing)

ArchT ON13 12Pre tone
Tone
TEST4( AFC-retrieval TEST2:
after light illumination)【②. Optical silencing procedure during the AFC TEST 】

ArchTEYFP

H2B mcherry

CreERT2Merge

100mm(inlet 20mm)

Opticalsilencing

(8)
(7)
N.S.

87

Pre toneTone
TEST4( AFC-retrieval TEST2:
after light illumination)

Conclusions

10Co-retrieval

ESARE▲ Representative image of overlapping ensemble expressing the ArchT-EYFP with orwithout co-reactivation. Yellow arrow head indicates ArchT-EYFP+, H2B-mcherry+ neuron
as overlapping ensemble.

5s 5s 5s 5s11+Dox

OpticalsilencingLaser
Illumination(4 min)ArchT-EYFP

WPRE

AFC-retrieval CTA-retrieval
ensembleensemblelabelinglabeling

Dox +Saline Saline

RCS TEST1 TEST2

H2B -mcherry

ESARE

TEST1
4-OHT i.p. (AFC)Dox OFF 2d / AFC training (+)
EYFP / DAPIIHC●: Evoked-response before
light illumination…..

H2B - mcherry

RCS

H2Bmcherry

CreERT2【+ or – ; for IHC】

(n=12, *p<0.05, t22=2.23,unpaired Student’s t-test)

LiCl

WPRE

4-OHT

AFC-retrieval
active ensemble(3 sessions; ITI 1h)【No CTA-US group】
LiCl

ESAREOptical silencing
experiments

CreERT2

EYFP / c-Fos / DAPI

c-Fos+ cells / DAPI (%)

ITI; 30 min

Rosa26STOP

LV cocktail
inoculation

90 minSaccharin-inducedfreezing (%)(4-6 min; *p<0.05, z=-2.51, 7-9 min; *p<0.05, z=-2.04,Wilcoxon rank-sum test)【Unpaired group】

AFC-training
ensemblelabeling
48 h
24 h

ArchT ON

ArchT-EYFP

×WPRE◆ Behavioral schedule for manipulation of the overlapping ensemble◆ ArchT-mediated inhibition of BLA neurons
prevented the c-fos expression and freezing
response (in vivo)(LED light stim)

Yellow arrow shows longer latency to move fromthe corner in the co-retrieval group. The filled yellow
circles indicate the nozzle positions for water andsaccharin.

60.5▲ Scaled Venn diagrams
representing the size andproportion of overlap in theactivated BLA ensembles.

One way ANOVA followed by Tukey-Kramer multiple
comparisons (Co-retrieval vs Control, **p<0.05, Coretrieval vs No React, **p<0.05, F2,13=23.07)

Dox -BLASTOP

H2B- mcherry

CreERT2

CreERT2
Rosa26

Electrophysiology(ex vivo)

Dox OFF121231.3LV-inoculation~15 min(c-fos tTA Tet-tag)

tTAtTA

CTA AFCc-fos::tTA /R26R::H2B-mcherry training training

CreERT2tTA
TRE 3GCTATESTRCS+ 4 -OHTAFCCTA

5 min

Rosa26

Optical silencing of AFC-training ensemble
suppresses freezing response
Transgenic mouse:

For 5 min immediately after
the mouse finished the firstlick of the saccharin nozzle
during the CTA test session(TEST1), with the time spentin consumption subtracted.

c - fos

63.4

8.2

(4)

◆ Schematic illustration of optogenetic experiments◆ A cross-modal association between CTA and AFC memories as saccharin-induced freezing

1 session (x3)

27.5

4

LV1; TRE3G::CreERT2
LV2; ESARE::DIO-ArchT-EYFP
Dox OFFtTA

9.1

Result ④

Saccharin-induced freezing is generated through repetitive memory retrieval

Co-retrieval (12)Control (12)(5)

Representative images
for catFISH staining in BLA.
The yellow arrows indicate
the INF Arc+ H1a+ doublepositive cells.

Result ①
Representative behavioural tracing
and occupancy plots during TEST1Control

5

One way ANOVA followed by Tukey-Kramermultiple comparisons (ArchT ON vs ArchT
OFF, **p<0.01, ArchT ON vs EYFP ON,**p<0.01, F2,19=15.08)

AFCTEST

Lentivirus vector (cocktail):c-fos::tTA / R26R::H2B-mcherry

No React(5)(A) Combination of two amygdala-dependent behavioral paradigms, conditioned taste aversion task (CTA) andauditory cued fear conditioning (AFC). (B),(C) Both original CTA and AFC memory were not impaired after
repeated co-retrieval session (RCS). Control mice received no tone when they licked the saccharin solution inthe RCS session. Aversion index, which was defined as [Water (ml) / Total intake (Water + Saccharin sol.)(ml)] x 100, for CTA memory performance in the RCS and TEST1 sessions, AFC memory performance forfreezing level (%) in TEST2.

【Schematic diagram of labeling the overlapping ensemble】 LV cocktail-inoculation
Double transgenic mouse:

5(p=0.463, t22=0.746,unpaired Student’s t-test)(p=0.189, t22=1.354,unpaired Student’s t-test)

59.2

25.6

Arc / H1a / DAPI

RCS36

◆ A critical question: does neuronal activity of the overlapping ensemble mediate the saccharin-induced freezing?

▲Sync Sync

RCS2

One way ANOVA followed by Tukey-Kramer multiple
comparisons (ArchT ON vs ArchT OFF, *p<0.05, ArchT
ON vs EYFP ON, **p<0.01, F2,19=6.82)

7Overlapping ensemble links two distinct emotional memories
15.2

Saccharin sol.

RCS1

67

Result ⑥

Co-retrieval

AFCTEST Sacrifice

30min

INF H1a+
active cellat CTA-retrieval

Water

8

Saccharin-inducedfreezing (%)(TEST2; p=0.81N.S, F2,19= 0.21,
One way ANOVA)(TEST1; p=0.89 N.S, F2,19=0.12, One way ANOVA)◆ catFISH analysis in BLA after the reactivation (RCS)

Repeated Co-retrieval Session

5 min

6(p=0.74, N.S., t19=0.33, unpaired
Student’s t-test)

Repetitive retrieval-induced plasticity triggers
neuronal convergence in the BLA(catFISH procedure)

ITI ; 1 h8

Freezing (%)

CTA

10

(TEST1 vs TEST2 in Ani;*p<0.05, t9=3.01,
co-retrieval group vs control group inTEST2; **p<0.01, t19=3.96, unpaired
Student’s t-test)**Freezing (%)

9catFISH analysis**

Saccharin-inducedfreezing (%)

8Laser illumination(5 min/session)

TEST2(AFC)－Chance level

10Co-retrieval (+)

7TEST1(CTA)

6Control (-)

611

INF Arc+ H1a+ cells/ DAPI (%)

5Co-retrieval (12)Control (12)Dox -N.S.

Result ③

6 min
Co-retrieval (12)Control (12)(3 sessions ; ITI 1h)□ Pre-tone■ Tone12Freezing (%)

415 minDox +RCS【Original CTA memory test 】【Original AFC memory test 】

AniVehN.S.

2 min 30s30s30s30s

AFC
habituation

Expression

Ani (10)Veh (11)

AniVehC (TEST2: AFC test)

DW and saccharin sol.

CTA
training

24 h

24 h

Saccharin-inducedfreezing (%)

Ani (12)Veh (13)

IEG+ cells / DAPI (%)

3

TEST3

ArchT-EYFP

2

TEST2

ArchT-EYFP

1

TEST1

24 h2w

OpticalSilencing

AFC-training
ensemblelabeling
48 h
24 h■ No Reactivated group■ Reactivated group(TEST1 vs TEST2 in Ani group; **p<0.01,(*p<0.05, t23=2.68, unpaired Student’s t-test)
t11=3.27,
co-retrieval group vs control group in TEST2;**p<0.01, t23=3.60,unpaired Student’s t-test)

B (TEST1: CTA test) Free access toTone: 7kHz, 75db, 30s
Shock: 0.4mA, Last 1s x 4 times
CTA-CS: Presentation of water and saccharin sol.

(0.15M) (0.15M)

24 h6◆ Schematic diagram of the experimental design(CTA and AFC combination behavioral paradigm)
LiCl(3 sessions ; ITI 1h)

No Reactivation

13Methodology
LiCl(BLA: AP1.5 ML 3.3 DV 4.9)

(Home cage)Our findings provide new insights
into the shared neuronal ensemble isindispensable for the association of two
emotional memories but is not required
for the retrieval of the original memories.

A◆ Suppression of the AFC-training
ensemble activity during the RCS
impaired the condition response of
AFC such as saccharin-induced
freezing and the increase of co-sharing
neuronal subpopulation

Ani or Veh

24 hRCS

LV-inoculation~Memories are not stored in isolation from other memories but are integrated into associative networks1,2.
Although recent progresses reveal that a specific neuronal ensemble represents a given memory3-5,
mechanisms underlying the memory association remains elusive. Using two amygdala-dependent
behavioural paradigms, conditioned taste aversion (CTA) and auditory-cued fear conditioning (AFC) in mice,
we find that presenting the conditioned stimulus used for the CTA task triggers the conditioned response of
the AFC task, that is, freezing behaviour, after natural co-reactivation of their memory retrievals, which isaccompanied by an increase in the ratio of the co-shared neuronal subpopulation, an overlapping ensemble,
in the basolateral amygdala. Optical silencing of the overlapping ensemble suppresses CTA retrievalinduced freezing. However, retrieval of the original CTA or AFC memory is not affected. These results
indicate the existence of a small population of co-shared neurons that mediates the link between memories
but is unnecessary for recalling individual memories.

Result ⑤Freezing (%)

SummaryResult ②▲ Long-lasting optical silencing prevents
spontaneous spiking in the ArchT-EYFPexpressing cells.

One way ANOVA followed by Tukey-Kramer multiple comparisons (c-fos analysis: ArchT
ON vs ArchT OFF, **p<0.01, ArchT ON vs EYFP ON, **p<0.01, F2,15=14.11, Freezing(%):
ArchT ON vs ArchT OFF, **p<0.01, ArchT ON vs EYFP ON, *p<0.05, F2,15=9.90,
Spearman’s rank test *p<0.05, rs=0.647, z=2.506)

We thank K. Deisseroth (Stanford University) for the ArchT 3.0-EYFP cDNA, H. Hioki and T. Kaneko (Kyoto University) for the STB vector, and H. Bito (Tokyo University)
for the ESARE cDNA. All members of the Inokuchi laboratory supported and discussed this study. This work was supported by the Core Research for Evolutional Scienceand Technology (CREST) program of the Japan Science and Technology Agency (JST), JSPS KAKENHI grant number JP23220009,①． We show a pronounced property of the sharedensemble. We provide evidence that its neuronal
activity is indispensable for the association of two
emotional memories but is not required for theretrieval of the original memories.
②. Generation of the shared ensemble is not
restricted to the memory acquisition, as shown in
the aforementioned studies, but is also formed bysynchronous and repeated retrieval of onceconsolidated memories.
③． Our findings provide a fundamental
principle for the basis of dissociating
unwanted connected memories without
affecting the individual original memories.

yokose@med.u-toyama.ac.jp