Synchronous activation of distinct cell ensembles in CA3
integrates two memories
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○ 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. 5Division 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.

Laser stimulation of distinct cell ensembles
in the hippocampal CA3 induces memory integration

2.

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 therecurrent 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 “contextexploration” and “contextual fear conditioning (CFC) in a different context” using tet-tag system and Cre-loxPsystem. 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 showedsignificantly 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 inwhich 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 CA3CA3 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.

AContext A

Context C

Labeling of ensemble

AAV injection

Context A

Context B

Pre-exposure

CFC

5 weeks ~1 day

ON-Dox

OpticalContext C
Context B
Context A
stimulation
Test 1
Test 3
Test 2in home cage+/1 day
1 day
1 day
1 day

ON-DoxOFF-Dox
CB

D
CFC
Foot shock(Context B)

CFC
Pre-foot shock(Context B)

Pre-exposure(Context A)
30

30

60

Freezing (%)

Freezing (%)

n.s.

20

20

Introduction

40

n.s.
n.s.

▪ However, the role for the interaction between
previously stored information is still unclear.

▪ Recurrent circuits in the CA3 is important for
paired association learning or sequence learning.

Context B

Freezing (%)

Abstract

1010Questions
Does the coincident firing of distinct prestored ensembles in the CA3 induce
integration of memories?

2000Laser ON

E0Laser ON

Laser OFFF

Test 1(Context A)
30

Laser OFF

Laser ONG

Test 2(Context B)

80

Test 3(Context C)

30n.s.

✱✱

Laser OFF60

2020Freezing (%)

A system for the activity-dependent labelingof the hippocampal CA3 neurons

Freezing (%)

1.

Freezing (%)

n.s.

40

10102000
Laser ON

0Laser OFF

Laser ON

Laser OFF

Laser ON

Laser OFFLaser 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
ChR2-mCherry
ChR2-mCherry
DAPI
DAPI

3.

In vivo field excitatory post synaptic potential (fEPSP) recording
in the hippocampal CA3A

In vivo extracellular recording fromCA3 Tungsten electrode Optic fiber

AAV injection

ChR2-mCherry

EF1αKA1
Scale bar, 500 µm

Cre8 weeks ~or
KA1

ChR2-mCherry
DAPI

473 nm laser

Creexon of NR1
BUnder urethane anesthesia
ChR2-mCherry
CA3-NR1 KODAPIChR2-mCherry
DAPI

KA1::Cre

Scale bar, 300 µm

Arrows: the tip of the electrode
Scale bar, 500 µm
KA1::CreC

D

KA1::Cre

CA3-NR1
KA1::Cre KO

CA3-NR1-KO

CA3-NR1-KO

0.5 mVPre 20 Hz stimulationn = 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 ± SEMEEDF

5 ms

Post 20 Hz stimulation

140130Conclusion

Taken together, the strengthening of synaptic efficacy between CA3ensembles connected by recurrent circuit associate two memories.

fEPSP Slope (% baseline)

120

fEPSP Slope (% baseline)❖ A system for the activity-dependent labeling of the hippocampal CA3 neurons isestablished.
❖ Synchronous activation of distinct cell ensembles in CA3 induce memory
integration.
❖ 20 Hz laser stimulation induce LTP in CA3-CA3 synapses.

KA1::Cre20 Hz optical130
stimulation

110100CA3-NR1 KO✱

1201101009080
9070

KA1::Cre

CA3-NR1 KO
80

60-30

03060

90

120

150

180Time (min)-21..0-21~041..60
101..120
161..180101~120161~18041~60
Time (min)n = 4 mice/group; * p < 0.05, unpaired t-test; Error bars, mean ± SEM● We declare no Conflict of Interest (COI).