Synchronous activation of distinct cell ensembles in CA3

integrates two memories

○ Naoya Oishi1, 2, Masanori Nomoto1, 2, Noriaki Ohkawa1, 2, Yoshito Saitoh1,2, Yoshitake Sano3, Shuhei Tsujimura1, 2, Hirofumi Nishizono2,4,

Mina Matsuo4, Shin-Ichi Muramatsu5,6, Kaoru Inokuchi1,2

1 Department of Biochemistry, Faculty of Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Japan. 2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST),

University of Toyama, Japan. 3 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Japan. 4 Division of Animal Experimental Laboratory, Life Science Research Center, University of Toyama, Japan. 5

Division of Neurology, Department of Medicine, Jichi Medical University, Japan. 6 Center for Gene and Cell Therapy, The Institute of Medical Science, The University of Tokyo, Japan.

海馬CA3領域の二つの異なるセルアンサンブルの同期活性化による記憶の統合

The hippocampal area CA3 forms CA3-CA3 recurrent excitatory circuits. Previous studies showed that

this circuit is important for the paired association learning or sequence learning. However, whether the

recurrent CA3 network plays an important role in the interaction between previously stored information is still

unclear. To address this question, we focus on whether the coincident firing of distinct pre-stored ensembles

in CA3 induce integration of these memories by using optogenetics.

We induced the expression of ChR2-mCherry to CA3 cell ensembles responded to “context

exploration” and “contextual fear conditioning (CFC) in a different context” using tet-tag system and Cre-loxP

system. Then, we induced coactivation of ChR2-mCherry expressing neurons by 20 Hz laser stimulation

followed by memory tests. In “the context which mice didn’t received CFC”, laser on group showed

significantly higher freezing than that of laser OFF control group, indicating that the two context memories

were associated. However, there was no difference between the two groups in freezing in “the context in

which mice received CFC” and “another new context”, suggesting that the memory had a context specificity.

Also, we checked whether 20 Hz optical stimulation can induce long-term potentiation (LTP) in CA3-

CA3 synapses to elucidate the mechanisms of the observed memory integration. Using in vivo excitatory post

synaptic potential (fEPSP) recording, LTP was observed after 20 Hz laser stimulation in the control mice but

not in the CA3 pyramidal cell-restricted N-methyl-D-aspartate (NMDA) receptor knock out mice, which lack

NMDA receptor function at CA3-CA3 synapses.

Taken together, our results suggest that the strengthening of synaptic efficacy between CA3

ensembles connected by recurrent circuit associate two memories.

Abstract

Conclusion

Introduction

A system for the activity-dependent labeling

of the hippocampal CA3 neurons1.

Questions

❖ A system for the activity-dependent labeling of the hippocampal CA3 neurons is

established.

❖ Synchronous activation of distinct cell ensembles in CA3 induce memory

integration.

❖ 20 Hz laser stimulation induce LTP in CA3-CA3 synapses.

Taken together, the strengthening of synaptic efficacy between CA3

ensembles connected by recurrent circuit associate two memories.

●We declare no Conflict of Interest (COI).

▪ Recurrent circuits in the CA3 is important for

paired association learning or sequence learning.

In vivo field excitatory post synaptic potential (fEPSP) recording

in the hippocampal CA3

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Test 1

(Context A)

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E F GTest 2

(Context B)

Test 3

(Context C)

n.s.

n.s.

n.s.

Pre-exposure

(Context A)

n.s.

n.s.

CFC

Pre-foot shock

(Context B)

CFC

Foot shock

(Context B)

B C D

Context A Context B Context C

ON-Dox OFF-Dox ON-Dox

1 day5 weeks ~

AAV injection

1 day 1 day1 day

Test 1

1 day

Context BContext A Context CContext BContext A

Labeling of ensemble

+/-

Test 2 Test 3Pre-exposure CFC

Optical

stimulation

in home cage

Laser stimulation of distinct cell ensembles

in the hippocampal CA3 induces memory integration

3.

2.

▪ However, the role for the interaction between

previously stored information is still unclear.

Does the coincident firing of distinct pre-

stored ensembles in the CA3 induce

integration of memories?

DAPI

ChR2-mCherry

Laser ON, n = 12; Laser OFF, n=10; ** p < 0.01, unpaired t-test; n.s., no significant difference; Error bars, mean ± SEM

Laser stimulation: 10 trains of light (20 Hz, 100 pulses of light, 500 µsec each, 473 nm, 10 mW) at 45 sec inter-train intervals

n = 4 mice/group; p < 0.01, one-way ANOVA; * p < 0.05, ** p < 0.01, Tukey's post hoc multiple comparisons test; Error bars, mean ± SEM

DAPI

ChR2-mCherry

DAPI

ChR2-mCherry

Scale bar, 500 µm

Scale bar, 300 µm

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KA1::Cre CA3-NR1 KO

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-21~0

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8 weeks ~

Tungsten electrode Optic fiber

In vivo extracellular recording from

CA3

473 nm laser

Under urethane anesthesia

EF1α

ChR2-mCherry

20 Hz optical

stimulation

AAV injection

KA1::Cre CA3-NR1 KO

DAPI

ChR2-mCherry

exon of NR1

or

KA1 Cre

KA1 Cre

Time (min)161~180101~12041~60

KA1::Cre CA3-NR1 KO

DAPI

ChR2-mCherry

KA1::Cre

CA3-NR1-KO

KA1::Cre CA3-NR1-KO

D

E F

Scale bar, 500 µm

n = 4 mice/group; * p < 0.05, unpaired t-test; Error bars, mean ± SEM

Arrows: the tip of the electrode