一般口演
学習、記憶および可塑性 2
Learning, Memory and Plasticity 2
座長：内田 周作（京都大学） |
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| 2022年7月3日 10:00～10:15 沖縄コンベンションセンター 会議場A2 第7会場
4O07m2-01
ショウジョウバエの匂い連合学習における2つのドパミン放出様式
Two different dopamine release modes during olfactory associative learning in Drosophila
*上野 耕平(1)、長野 慎太郎(1)、齊藤 実(1)
1. （公財）東京都医学総合研究所
*Kohei Ueno(1), Shintaro Naganos(1), Minoru Saitoe(1)
1. Tokyo Metropolitan Institute of Medical Science
Keyword: dopamine, Drosophila, memory, plasticity
Dopamine (DA) is an important neurotransmitter for memory formation in many animals from mammals to insects. Although neurotransmitter release is regulated by activity of the presynaptic neurons, using ex vivo imaging analysis of isolated Drosophila brain, we found a novel mode of DA release that is regulated by activity of the postsynaptic mushroom body neurons (MBNs) where associative memory is formed. When MBNs receive coincident inputs from odor sensory pathways and somatosensory pathways, they generate carbon monoxide (CO), as a retrograde messenger for DA release, to activate Ca2+ efflux through ryanodine receptors in the presynaptic DA release sites (Ueno et al., 2018, 2020). While we named this DA release mode as the on-demand release, it is unclear whether it is taken place during olfactory aversive conditioning. In Drosophila olfactory aversive conditioning, DA released onto the γ1 compartment of the γ lobe of the MB plays an essential role. Using thermogenetic procedures, we determined that DA release onto this region after conditioning but not during conditioning is critically required for memory formation. In in vivo imaging analysis of DA release, we found two different phases of DA release in the γ1 during conditioning and after conditioning. While DA release during conditioning is correlatively occurred upon electrical shock presentation and requires the machinery of the active zone of the release sites, Drosophila CAST homolog (bruchpilot, brp) and voltage-gated Ca2+ channel (cacophony, cac), DA release after conditioning is dependent on the association of odor and electrical shock and requires ryanodine receptors in the DA neurons and CO synthase, heme oxygenase, in the γ lobe. Importantly, suppressing the expression of either heme oxygenase in the γ lobe of the MB or ryanodine receptors in the dopaminergic neurons that innervate γ1 significantly disrupted olfactory aversive conditioning. In contrast, suppressing the expression of neither brp nor cac disrupted the conditioning. These results support the idea that on-demand DA release is critical for associative learning. | | 2022年7月3日 10:15～10:30 沖縄コンベンションセンター 会議場A2 第7会場
4O07m2-02
恐怖記憶消去における前辺縁皮質神経細胞群の再活性化
Neuronal reactivation in the prelimbic cortex during fear memory extinction
*近藤 勇人(1)、金 亮(1,2)、下田 藍丸(1)、山下 りえ(1)、小針 成敬(1)、富山 優介(1)、金 正珉(1)、杓野 拓光(1)、Brebner Leonie(1)、宋 浩博(1)、横山 達士(1,3)、井ノ口 霞(1)、近藤 弥生(1)、岡村 理子(1)、坂本 雅行(3)、奥野 浩行(4)、太田 桂輔(1)、藤井 哉(1)、尾藤 晴彦(1)
1. 東京大学 大学院医学系研究科 神経生化学分野、2. テキサス大学サウスウェスタン医療センター 精神医学科、3. 京都大学 大学院生命科学研究科 光神経分子生理学分野、4. 鹿児島大学 大学院医歯学総合研究科 生化学・分子生物学分野
*Hayato Kondo(1), Ryang Kim(1,2), Ranmaru Shimoda(1), Rie Yamashita(1), Shigetaka Kobari(1), Yusuke Tomiyama(1), Jungmin Kim(1), Takumitsu Shakuno(1), Leonie S Brebner(1), Haobo Song(1), Tatsushi Yokoyama(1,3), Kasumi Inokuchi(1), Yayoi Kondo(1), Michiko Okamura(1), Masayuki Sakamoto(3), Hiroyuki Okuno(4), Keisuke Ota(1), Hajime Fujii(1), Haruhiko Bito(1)
1. Dept of Neurochem, Grad Sch of Med, Univ Tokyo, Japan, 2. Dept of Psychiatry, U Texas Southwest Med Cntr, Dallas, USA, 3. Dept of Adv Imaging, Grad Sch of Biostud, Kyoto Univ, Japan, 4. Dept of Biochem & Mol Biol, Kagoshima Univ Grad Sch Med & Dent Stud, Japan
Keyword: fear memory extinction, immediate early gene, prelimbic cortex, 2-photon calcium imaging
Immunohistological and in situ hybridization analyses of immediate early gene (IEG) expression have previously revealed differential dynamics of engram cell recruitment across a wide variety of neuronal populations in the brain during distinct phases of fear memory. Due to the low temporal resolution of these studies, however, little is known about the spatio-temporal ensemble codes of neuronal activity that directly underlie encoding, retrieval, and extinction of fear memory. Furthermore, how the population activity dynamics change over time during learning remains unclear.
In this study, we first performed a dual IEG activation mapping to direct compare, within the same individual animals, the neural activity during fear encoding and during fear extinction. We combined a newly generated Arc hybrid promoter-based reporter Tg mice, capable of activity-dependent Venus expression, with c-Fos-based activity mapping, and computed the reactivation rate in multiple brain regions. Among several areas examined, we found that the rate of reactivation was augmented in one prefrontal area, the prelimbic cortex (PrL), in the extinction-induced group compared to the retrieval-only group.
To investigate the specific time course and physiological significance of this reactivation, we next established an in vivo 2-photon imaging paradigm to simultaneously monitor the ongoing neural activity and behavioral representation during fear extinction. Somatic and dendritic activities were recorded in the PrL via a preimplanted GRIN lens in head-fixed mice undergoing auditory fear conditioning, using virally expressed XCaMP-G. These direct recordings will shed new light on neural mechanisms essential for emotional behavior of negative valence, and may provide useful insights on stress disorders such as PTSD. | | 2022年7月3日 10:30～10:45 沖縄コンベンションセンター 会議場A2 第7会場
4O07m2-03
成熟前脳神経細胞特異的CFC症候群モデルマウスの解析
Analysis of matured forebrain neuron-specific CFC syndrome model mice
*森谷 晃(1)、井上 晋一(2)、齋藤 文仁(3)、青木 洋子(2)、大西 浩史(1)
1. 群馬大学大学院保健学研究科生体情報検査科学講座、2. 東北大学大学院医学系研究科遺伝医療学分野、3. 日本医科大学大学院医学研究科薬理学分野
*Akira Moriya(1), Shin-ichi Inoue(2), Fumihito Saitow(3), Yoko Aoki(2), Hiroshi Ohnishi(1)
1. Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences, 2. Department of Medical Genetics, Tohoku University School of Medicine, 3. Department of Pharmacology, Graduate School of Medicine, Nippon Medical School
Keyword: RAS/MAPK pathway
Germline mutations in genes that encode components of RAS/MAPK signaling pathway has been identified in a group of congenital malformation syndromes known as RASopathies (RAS/MAPK syndromes). RASopathies are phenotypically overlapping syndromes characterized by common pathologies such as cardiac malformations, skeletal abnormalities, a distinctive facial appearance, and neurocognitive impairment. Cardio-Facio-Cutaneous (CFC) syndrome is a malformation syndrome included in RASopathies, and in individuals with this syndrome, Braf gene mutations that result in activation of BRAF, a component of the RAS/MAPK signaling system, are frequently identified. Knock-in (KI) mice (CFC mice) having mutant Braf gene identified in human CFC syndrome showed a variety of pathological phenotypes similar to those in the human syndrome. These CFC-like phenotypes included neurocognitive impairment that was evaluated by behavioral analyses. However, systematic malformations of the mutant mice made it difficult to evaluate the results of behavioral analyses correctly. Thus, we generated conditional KI mice expressing the mutant BRAF only in matured postmitotic forebrain neurons to avoid abnormal development that cause CFC-like systemic malformation. Matured postmitotic forebrain neuron-specific Braf mutant mice (MFN-CFC mice) grew normally and no malformations were found throughout the body including the brain. In contrast, MFN-CFC mice showed reduced spontaneous locomotor activity and learning deficits, as observed in systemic CFC mice. Biochemical analysis revealed that active form (phosphorylated) extracellular signal-regulated kinase (ERK), downstream signal of RAS/MAPK pathway, was increased in the hippocampal tissue collected from MFN-CFC mice. In addition, electrophysiological data showed that steady state synaptic transmission was normal in the hippocampal slice prepared from the mutant mice, while long-term potentiation (LTP) of synaptic transmission was significantly increased in the slice from mutant mice. Our results suggest that BRAF contributes to the regulation of ERK activation and synaptic plasticity in matured neurons. Abnormal activation of BRAF not only causes malformation through the disturbance of developmental processes, but can also disrupt neural function in the matured brain and may be responsible for neurocognitive impairment in CFC syndrome. | | 2022年7月3日 10:45～11:00 沖縄コンベンションセンター 会議場A2 第7会場
4O07m2-04
２光子生体脳スパインイメージングで明らかになった恐怖条件付け記憶回路におけるスパイン新生
Fear conditioning induces spine formation in Arc-expressing neurons revealed by in vivo two-photon imaging
*湊原 圭一郎(1,2,3)、大波 壮一郎(3,4)、岡部 繁男(1)、尾藤 晴彦(5)、奥野 浩行(2,3)
1. 東京大学大学院医学系研究科神経細胞生物学、2. 鹿児島大学大学院医歯学総合研究科生化学・分子生物学、3. 京都大学大学院医学研究科SKプロジェクト、4. 塩野義製薬（株）創薬疾患研究所ニューロサイエンス部門 、5. 東京大学大学院医学系研究科神経生化学
*Keiichiro Minatohara(1,2,3), Soichiro Ohnami(3,4), Shigeo Okabe(1), Haruhiko Bito(5), Hiroyuki Okuno(2,3)
1. Cellular Neurobiol, Grad Sch Med, Univ of Tokyo, Tokyo, Japan, 2. Biochem & Mol Biol, Grad Sch Med and Dent Sci, Kagoshima Univ, Kagoshima, Japan, 3. SK Proj, MIC, Grad Sch Med, Kyoto Univ, Kyoto, Japan, 4. Neuroscience Dept, Drug Discovery & Disease Research Lab, SHIONOGI & CO., LTD., Osaka, Japan, 5. Neurochem, Grad Sch Med, Univ of Tokyo, Tokyo, Japan
Keyword: memory trace, dendritic spine, two photon imaging, immediate-early gene
Contextual fear conditioning is a learning paradigm that involves the association of spatial cue and electrical stimulus. Neuronal circuits in the brain are thought to be changed by the association learning. Exposure to a novel context induces the immediate-early gene expression in a subset of neurons that constitute neuronal networks for memory trace. These neural ensembles may be modulated by memory updating such as fear association through synaptic reorganization. However, the steps of the circuit modulation have yet to be visualized at the level of individual synapses. To visualize and manipulate neural circuits activated during behaviors by recombinant adeno-associated virus vectors, we developed a doxycycline-inducible reporter expression system (Tet-ON) under the control of enhanced synaptic activity response elements (E-SARE) from the Arc promoter. By using this method, we first expressed channelrodopsin-2 (ChR2) into neuronal ensembles in the retrosplenial cortex (RSC) of the mouse brain activated during exposure to a neutral context, and then tested whether light-induced activation of these ensembles could elicit fear responses 1 day after the fear conditioning. We found that ensembles recruited by a preceding exposure to the same context with the fear conditioning could induce fear responses (*P = 0.013, paired t-test, n = 8) whereas neurons recruited by a distinct context exposure could not (P = 0.49, paired t-test, n = 8). Next, by using 2-photon microscopy, we visualized and monitored dendritic spines of the context-activated RSC neurons that were labeled with enhanced green fluorescent protein using the E-SARE Tet-On system. We detected dendritic spines newly formed after the fear conditioning in the neuronal ensembles recruited by the preceding exposure to the same context rather than by the exposure to a distinct context (**P < 0.01, one-way ANOVA post hoc turkey’s test, n = 6). Finally, we found that differential distributions of nascent spines between fear-memory-allocated or non-allocated neurons by calculating the nearest neighbor distances of the observed nascent spines. These results suggest that, accompanied with synapse formation, the RSC neuronal ensembles responding to the neutral context are incorporated into fear memory traces after subsequent fear conditioning training in the same context. | |
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