Synchronous activation of distinct cell ensembles in …Pre-exposure CFC Test 2 Test 3 Optical...
Transcript of Synchronous activation of distinct cell ensembles in …Pre-exposure CFC Test 2 Test 3 Optical...
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
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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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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
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exon of NR1
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Time (min)161~180101~12041~60
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KA1::Cre
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KA1::Cre CA3-NR1-KO
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Scale bar, 500 µm
n = 4 mice/group; * p < 0.05, unpaired t-test; Error bars, mean ± SEM
Arrows: the tip of the electrode