Artificial association of pre-stored information to generate a qualitatively new memory○Noriaki Ohkawa1,2, Yoshito Saitoh1,2, Akinobu Suzuki1,2, Shuhei Tsujimura1,2, Emi Murayama1,2, Sakurako Kosugi1,2, Hirofumi Nishizono3,3
44
44,54,51,2
Mina Matsuo , Yukari Takahashi , Masashi Nagase , Yae K. Sugimura , Ayako M. Watabe , Fusao Kato , and Kaoru Inokuchi
1Dept Biochem, Grad Sch Med Pharm, Univ Toyama, Toyama, Japan, 2JST, CREST, 3Div Animal Exp Lab, Life Sci Res Cent, Univ Toyama, Toyama, Japan,4Dept Neurosci, Jikei Univ, Tokyo, Japan, 5Nagoya Univ Grad Sch Med, Nagoya, Japan②• Activity-dependent targeting ofneurons in c-fos::tTA mice

LVs

BLABLA

ChR2-EYFP

0CeA

-20

BLA

Cell BgSam
plin

da
ydaym
in

da
ys

130

1sda
y
230

**
**daym
in

130

da
ys
2Freezing (%)

dayda
y

1

1OFF Dox +Home cage

OFF Dox +“Unpaired”

ON Dox +“Unpaired”*30
20
10Test-1 inContext B

Test-2 inContext A

ChR2 + light** P < 0.02, paired t test, two-tailed

P < 0.01, one-way ANOVA;*P < 0.05, Scheffe’s post-hoc testion

PBS +Vehicle ON Dox

CA1

Test inContext B

BLA

AP5

AP5 +CA1

BLAAniso

Aniso +CA1

BLA

ChR2
ON Dox+light10

P < 0.004, one-way ANOVA;*P < 0.05, **P < 0.01,
Scheffe’s post-hoc test

AP5Aniso30

The pLenti-TRE::EYFP and pLenti-TRE::ChR2-EYFP plasmids were constructed and used for LV preparation as described previously {Goshen, I.
et al., (2011) Cell 147, p678-}. The LVs were stereotaxically injected into CA1 and the BLA of the right hemisphere of c-fos::tTA mice purchased from theMMRRC.
➢Ex vivo electrophysiology
Transverse brain slices were prepared from, and the membrane potential of ChR2-EYFP-expressing neurons in the CA1 and BLA regions wasrecorded under whole-cell current-clamp mode. Light stimulation (465 nm, 20 Hz, 5 msec, 40-pulse) was delivered with a high-power LED
illumination system.

➢Histology
An arc catFISH analysis was performed according to previous studies {Guzowski, J. F. et al., (1999) Nat. Neurosci. 2, p1120-}. For immunohistochemistry, brain
sections were incubated with anti-GFP (1:1000, Molecular Probes, A11122) and/or anti-c-Fos (1:1000, Santa Cruz, sc-52-G) antibodies followed
by treatments with secondary antibodies and then subjected to confocal microscopy.

➢Behavioural analysis
Mice were pre-exposed to context for 6 min, returned to the home cage for 30 min, and then given a 0.8 mA foot shock for 2 sec in context A
after 5 sec of acclimation. The ‘unpaired’ paradigm used distinct contexts for pre-exposure (context B) and the IS in context A. The ‘paired’paradigm used the same context (context A) for pre-exposure and the IS. The memory tests of the ‘unpaired’ and ‘paired’ conditions wereperformed in contexts B and A, respectively. Cannula-implanted and LV-injected c-fos::tTA mice were maintained on Dox food pellets (40 mg/kg).
The mice were taken OFF Dox for 2 days and then trained with the ‘unpaired’ paradigm. After 24 h, the mice were anaesthetised for ~4 min andoptic fibres were attached through the cannula. The mice were returned to the home cage for 20-25 min before optical stimulation (473 nm light,20 Hz, 10 msec) was delivered in the home cage for 2 min. Five minutes after the end of the stimulation, the optic fibre was detached, and themice were returned to the home cage. The mice were put into context B for the fear memory test.

n.s.
30

30

20100

Remote memory
Test-2 (Familiar)n.s.
Freezing (%)

20

Remote memory
Test-1 (Novel)n.s.

VehicleDay 31
Remote memory
Test-2 (Familiar)

BLA

Recent memory
Test30

0CA1Freezing (%)*28 day

Day 30
Remote memoryTest-1 (Novel)

Naive
PairedFreezing (%)*

Optical stimulation
in home cageDay 1Day 2
Optical stimulation Recent memory
in home cage
Test

Naive
Paired

ChR2+lightP > 0.4,
unpaired Student’s t-test, two tailed

Freezing (%)

Paired1040

Day 0Conditioning

ct20

ChR2 + light5

ChR2+light

300⑥

Temporal changes in artificial associative memory andphysiological associative memory are comparable• Recent and Remote memory test of artificial and physiological associative memory

Conditioning

Unpairedc-Fos+ BLA cells / ROIChR2 + light⑤Artificial association requires NMDAR activity andde novo protein synthesis during optical stimulation

40

ChR2 No light

ChR2 No light

100 ms**50

EYFP + light

PairedUnpairedarc+ BLA cells (%)

Context A
Memory test-2

**-20

*

Cell A

Synchronous optical stimulation of
CA1 and BLA in home cageContext A
Immediate shock

P < 0.001, one-way ANOVA;*P < 0.05, **P < 0.01, Scheffe’s post-hoc test

➢ Virus injection
corresponding to specific event

Context B
Memory test-10• D-AP5 and Anisomycin injection to
CA1 and the BLA**P < 0.01, unpaired Student’s t test, two-tailed****** P < 0.001,
Welch’s t-test, two-tailedContext B
pre-exposure

40BLA

20

**

Cyto+ Nuc+

100 μm

100LVinjection

0400CA130

5

ChR2 NoS+light

20

Nuc arc

1015ON-Dox

c-fos::tTATg

BLA

1010 μm

Paired15

Paired10

EYFP + light

100 msLED

100 ms200

ChR2+ light(( (-6030** P < 0.01,
unpaired Student’s t test,
two-tailed: Cyto: Nuc

BLA

40

BLA-40-60

Unpaired

BLAFreezing?

0BLA
UnpairedinLA

20

Paired

50 μm

Paired10*** P < 0.001,
unpaired Student’s t test,
two-tailed

Cyto arcm40

Both the artificial and physiological association increase
the representation of the sensory input in the BLA circuit

Paired20c-Fos
Nucleus (DAPI)**

Unpaired30

0Cyto+ Nuc+

***

50

Unpaired40***

Unpaired

arc+ CA1 cells (%)

Nuc arc50

Cyto+ Nuc+ CA1 cells (%): Cyto: Nuc

CA1Cyto arc

EYFP+ BLA cells (%)

40• Endogenous c-Fos expression 90 min after
the test session in the BLA

Cyto+ Nuc+ BLA cells (%)

CA1• ChR2 +light group show specific fear memory,
linking the original context infomation

CA1Inje④• Arc catFISH in Unpired and Paired conditions

Vm (mV)60

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

-20

60

-40

20 μm

Paired

ChR2+light

ChR2+lightBLA

Unpaired

CA1

ChR2+BLA light0LED

Vm (mV)Vm (mV)

CeA

20

DG

100 msc-Fos+ BLA cells / ROI

2Vm (mV)

ysinm30da**• Paired and Unpaired for context pre-exposure and IS paradigm• Different cell staining profiles
detected by arc catFISH

ChR2No light

ChR2+CA1 light-60-60

LAP < 0.004, one-way ANOVA;*P < 0.05, ** P < 0.01, Scheffe’s post-hoc test

-40

LED

**P < 0.006,
unpaired Student’s t-test, two-tailedContext C

**

-20

BLA

**

30

2520

15

1050Context A

040-40

IS only

PairedCA1CA1Context B**

Freezing (%)

Test

Context A

5CA1

Pyr

0BLA

EYFP+light• TRE::ChR2-EYFP LV-injected c-fos::tTA mice

20CA1*PyrDG

Context A**P < 0.001, one-way ANOVA; **P < 0.01, Scheffe’s post-hoc test

40CA1Context A

OFF Dox +Home cage

50 μm

0OFF Dox +“Unpaired”

BLA

5ON Dox +“Unpaired”

BLA

P > 0.9, One-way ANOVAContext A

LV

BLA
EYFP+ CA1 cells (%)

CA1

10**

10Context A(( (tTA RNA
arc RNA**

Paired*

BLA

**

1540

88.5 ± 5.0Test**

83.9 ± 5.7

Optical stimulation
in home cage

Conditioning

IS30

CA1BLAFreezing (%)

Freezing (%)
20Dentalcement

CA1Test inContext
B or C

Context
pre-exposure40LV

Injection cannula
Dummy cannula
Guide cannulaContext C

ISP > 0.8, Welch’s t-test, two-tailed

30

tTA+arc+ / tTA+x 100 (%)

473 nm Laser• TRE::EYFP LV-injected c-fos::tTA miceContext B

ChR2

orSynchronous optical stimulation ofCA1 and BLA in home cageContext C

n.s.

UnpairedContext
pre-exposure

Context A
Immediate shock• Blue LED light (20 Hz, 5 msec)-dependent spikes from the cells in CA1 and the BLA

Context A• Context pre-exposure
facilitates effect of the IS

Context B
pre-exposure

20in c-fos::tTABLA

PairedContext B

10• Double-targeting of
CA1 and the BLA

7.5

5.0

2.5

Context
pre-exposure

EYFP0• Double-FISH to detect both of tTA and arc
RNAs in c-fos::tTA mice

LV or solution Optical stimulation
injection

0.0

Context C

Context
pre-exposure• Control experiments in naive mice

Freezing (%)

Context B

Memory test inContext B

LV
injection

100 μmEYFP• Contexts A-C have the same impact on freezing levels at the first exposure
Context A

c-fos::tTATg

1 mmTRE(( (ON-DoxCA1OFF DoxTRE

EYFP

2tTA

OFF Dox +Home cage

OFF Dox +“Unpaired”

n.s.

ON Dox +“Unpaired”

Freezing (%)

Freezing?

7.5

ON-Dox

5.0

tTA

CA1

“Unpaired”
conditioning

10.0

CA1infect
ioninfection

2d1d

ON Dox• Freezing responses in TRE::EYFP LV- andTRE::ChR2-EYFP LV-injected c-fos::tTA mice incontext B at the first exposure

10①• Optical stimulation (20 Hz, 10 msec, 2 min) of cell assemblies in
CA1 and the BLA generates artificial association

2.5c-fos promoter

CA1

Cell assemblies corresponding to the pre-exposedcontext and the immediate shock (IS) in
CA1 and the BLA• Activity and OFF Dox-dependent expression of
EYFP+ cells in CA1 and the BLA ofTRE::EYFP LV-infected c-fos::tTA mice

0.0c-fos::tTATg

LV• Representative labeling pattern of
CA1 and BLA cells in c-fos::tTA mice(( (Neuronalactivation③

Synchronous activation of distinct cell ensembles creates
an artificial association of corresponding information

0Memory is assumed to be stored in the brain as a cellular ensemble consisting of aset of neurons that is activated during learning. Although optical stimulation of acellular ensemble is known to trigger the retrieval of the corresponding memory, it isunclear how the association of distinct information occurs at the cell ensemble level.
Here, we show in mice that activation of a cell ensemble corresponding to two distinct
memory events generates an artificial association between initially non-related events.
In the context pre-exposure and immediate shock (IS) paradigm, mice failed toassociate the shock with the pre-exposed context when the IS was delivered to theirfoot in a different context. Cells activated during the context pre-exposure and the IS in
hippocampal CA1 and the basolateral amygdala (BLA) were targeted withchannelrhodopsin-2, a light-activated cation channel. These cells were later
simultaneously activated by optical stimulation in the mice's home cage. The next day,these mice exhibited freezing behaviour, an indicator of a fear response, in the preexposed context that was not originally associated with the shock. This artificial
association shared characteristics with physiologically associated memories, such asN-methyl-D-aspartate receptor activity- and protein synthesis-dependence. Thus, theartificial activation of distinct cell ensembles, without any sensory input, is capable ofgenerating an artificially associated memory. Furthermore, our finding suggests thatthe association of distinct units of information is achieved through the synchronousactivity of distinct cell ensembles. This mechanism may underlie memory update byincorporating novel information into pre-existing networks to form qualitatively newmemories.

A system for the simultaneous targeting and activation ofcell assemblies in CA1 and the BLA2010020

100
Naive
Paired

ChR2+lightP > 0.9,
unpaired Student’s t-test, two tailed

Naive
Paired

ChR2+lightP > 0.6,
unpaired Student’s t-test, two tailed➢ The arc catFISH revealed expression of neuronal assemblies that were activated during the context pre-exposure andthe IS in hippocampal CA1 and the BLA.
➢ The entirely artificial activation of the two separate sets of neuronal assemblies, without any new sensory input fromthe conditioned and unconditioned stimuli (the context pre-exposure and foot shock), was capable of connecting twoindependent events.
➢ This artificially associated memory shared several characteristics with a physiologically associated memory: Bothdepended on NMDAR activity and protein synthesis, both were long lasting (at least four weeks), and both underwent
generalization.
➢ This result suggests that synchronous activation of cell assemblies
corresponding to two distinct events could generate an artificial link
between these memory episodes.
➢ This mechanism may underlie memory updating by incorporating novel
information into pre-existing networks to form qualitatively new memories.