論文 - 田中　謙二

Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

Aida Tomomi, Chiyo Keiho, Usami Takako, Ishikubo Harumi, Imahashi Risa, Wada Yusaku, Tanaka Kenji F., Sakuma Tetsushi, Yamamoto Takashi, Tanaka Kohichi

Genome Biology 16 （ 1 ）  2015年04月

ISSN  1474-7596

　概要を見る

<p>Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.</p>

• Access to Document (DOI)

Optogenetic activation of CA1 pyramidal neurons at the dorsal and ventral hippocampus evokes distinct brain-wide responses revealed by mouse fMRI

Takata Norio, Yoshida Keitaro, Komaki Yuji, Xu Ming, Sakai Yuki, Hikishima Keigo, Mimura Masaru, Okano Hideyuki, Tanaka Kenji F.

PLoS One 10 （ 3 ）  2015年03月

　概要を見る

<p>The dorsal and ventral hippocampal regions (dHP and vHP) are proposed to have distinct functions. Electrophysiological studies have revealed intra-hippocampal variances along the dorsoventral axis. Nevertheless, the extra-hippocampal influences of dHP and vHP activities remain unclear. In this study, we compared the spatial distribution of brain-wide responses upon dHP or vHP activation and further estimate connection strengths between the dHP and the vHP with corresponding extra-hippocampal areas. To achieve this, we first investigated responses of local field potential (LFP) and multi unit activities (MUA) upon light stimulation in the hippocampus of an anesthetized transgenic mouse, whose CA1 pyramidal neurons expressed a step-function opsin variant of channelrhodopsin-2 (ChR2). Optogenetic stimulation increased hippocampal LFP power at theta, gamma, and ultra-fast frequency bands, and augmented MUA, indicating light-induced activation of CA1 pyramidal neurons. Brain-wide responses examined using fMRI revealed that optogenetic activation at the dHP or vHP caused blood oxygenation level-dependent (BOLD) fMRI signals in situ. Although activation at the dHP induced BOLD responses at the vHP, the opposite was not observed. Outside the hippocampal formation, activation at the dHP, but not the vHP, evoked BOLD responses at the retrosplenial cortex (RSP), which is in line with anatomical evidence. In contrast, BOLD responses at the lateral septum (LS) were induced only upon vHP activation, even though both dHP and vHP send axonal fibers to the LS. Our findings suggest that the primary targets of dHP and vHP activation are distinct, which concurs with attributed functions of the dHP and RSP in spatial memory, as well as of the vHP and LS in emotional responses.</p>

• Access to Document (DOI)

Astroglial glutamate transporter deficiency increases synaptic excitability and leads to pathological repetitive behaviors in mice

Aida Tomomi, Yoshida Junichi, Nomura Masatoshi, Tanimura Asami, Iino Yusuke, Soma Miho, Bai Ning, Ito Yukiko, Cui Wanpeng, Aizawa Hidenori, Yanagisawa Michiko, Nagai Terumi, Takata Norio, Tanaka Kenji F., Takayanagi Ryoichi, Kano Masanobu, Götz Magdalena, Hirase Hajime, Tanaka Kohichi

Neuropsychopharmacology 40 （ 7 ） 1569 - 1579 2015年02月

ISSN  0893-133X

　概要を見る

<p>An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD), and Tourette's syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLAST^CreERT2/+ /GLT1^flox/flox, iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 has a critical role in controlling the synaptic efficacy at corticostriatal synapses and its dysfunction causes pathological repetitive behaviors.</p>

• Access to Document (DOI)

Optogenetics research using the mouse as a model system

Tanaka Kenji F.

  227 - 240 2015年01月

　概要を見る

<p>Smooth progress in the field of optogenetic research can be, as the suffix ‘-genetics’ indicates, expected using biological models that provide conditions suitable for genetic control or gene manipulation. The artificial expression of opsin molecules is expected to be carried out smoothly in mice compared with rats or non-human primates because of established inheritable genetic modification technologies in this species. However, when compared with the nematode or the fly, there are still many difficulties in the expression of opsin in mice if flexible gene manipulation is attempted.</p>

• Access to Document (DOI)

Optogenetic activation of dorsal raphe serotonin neurons enhances patience for future rewards

Miyazaki Kayoko W., Miyazaki Katsuhiko, Tanaka Kenji F., Yamanaka Akihiro, Takahashi Aki, Tabuchi Sawako, Doya Kenji

Current Biology 24 （ 17 ） 2033 - 2040 2014年09月

ISSN  0960-9822

　概要を見る

<p>Serotonin is a neuromodulator that is involved extensively in behavioral, affective, and cognitive functions in the brain. Previous recording studies of the midbrain dorsal raphe nucleus (DRN) revealed that the activation of putative serotonin neurons correlates with the levels of behavioral arousal [1], rhythmic motor outputs [2], salient sensory stimuli [3-6], reward, and conditioned cues [5-8]. The classic theory on serotonin states that it opposes dopamine and inhibits behaviors when aversive events are predicted [9-14]. However, the therapeutic effects of serotonin signal-enhancing medications have been difficult to reconcile with this theory [15, 16]. In contrast, a more recent theory states that serotonin facilitates long-term optimal behaviors and suppresses impulsive behaviors [17-21]. To test these theories, we developed optogenetic mice that selectively express channelrhodopsin in serotonin neurons and tested how the activation of serotonergic neurons in the DRN affects animal behavior during a delayed reward task. The activation of serotonin neurons reduced the premature cessation of waiting for conditioned cues and food rewards. In reward omission trials, serotonin neuron stimulation prolonged the time animals spent waiting. This effect was observed specifically when the animal was engaged in deciding whether to keep waiting and was not due to motor inhibition. Control experiments showed that the prolonged waiting times observed with optogenetic stimulation were not due to behavioral inhibition or the reinforcing effects of serotonergic activation. These results show, for the first time, that the timed activation of serotonin neurons during waiting promotes animals patience to wait for a delayed reward.</p>

• Access to Document (DOI)

In Vivo visualization of subtle, transient, and local activity of astrocytes using an ultrasensitive Ca²⁺ indicator

Kanemaru Kazunori, Sekiya Hiroshi, Xu Ming, Satoh Kaname, Kitajima Nami, Yoshida Keitaro, Okubo Yohei, Sasaki Takuya, Moritoh Satoru, Hasuwa Hidetoshi, Mimura Masaru, Horikawa Kazuki, Matsui Ko, Nagai Takeharu, Iino Masamitsu, Tanaka Kenji F.

Cell Reports 8 （ 1 ） 311 - 318 2014年07月

　概要を見る

<p>Astrocytes generate local calcium (Ca²⁺) signals that are thought to regulate their functions. Visualization of these signals in the intact brain requires an imaging method with high spatiotemporal resolution. Here, we describe such a method using transgenic mice expressing the ultrasensitive ratiometric Ca²⁺ indicator yellow Cameleon-Nano 50 (YC-Nano50) inastrocytes. In these mice, we detected a unique pattern of Ca²⁺ signals. These occur spontaneously, predominantly in astrocytic fine processes, but not the cell body. Upon sensory stimulation, astrocytes initially responded with Ca²⁺ signals at fine processes, which then propagated to the cell body. These observations suggest that astrocytic fine processes function as a high-sensitivity detector of neuronal activities. Thus, the method provides a useful tool for studying the activity of astrocytes in brain physiology and pathology. © 2014 The Authors.</p>

• Access to Document (DOI)

Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis

Denny Christine A., Kheirbek Mazen A., Alba Eva L., Tanaka Kenji F., Brachman Rebecca A., Laughman Kimberly B., Tomm Nicole K., Turi Gergely F., Losonczy Attila, Hen René

Neuron 83 （ 1 ） 189 - 201 2014年07月

ISSN  0896-6273

　概要を見る

<p>Memory traces are believed to be ensembles of cells used to store memories. To visualize memory traces, we created a transgenic line that allows for the comparison between cells activated during encoding and expression of a memory. Mice re-exposed to a fear-inducing context froze more and had a greater percentage of reactivated cells in the dentate gyrus (DG) and CA3 than mice exposed to a novel context. Over time, these differences disappeared, in keeping with the observation that memories become generalized. Optogenetically silencing DG or CA3 cells that were recruited during encoding of a fear-inducing context prevented expression of the corresponding memory. Mice with reduced neurogenesis displayed less contextual memory and less reactivation in CA3but, surprisingly, normal reactivation in the DG. These studies suggest that distinct memory traces are located in the DG and in CA3 but that the strength of the memory is related to reactivation in CA3. © 2014 Elsevier Inc.</p>

• Access to Document (DOI)

Optogenetic activation of serotonergic neurons enhances anxiety-like behaviour in mice

Ohmura Yu, Tanaka Kenji F., Tsunematsu Tomomi, Yamanaka Akihiro, Yoshioka Mitsuhiro

International Journal of Neuropsychopharmacology 17 （ 11 ） 1777 - 1783 2014年05月

ISSN  1461-1457

　概要を見る

<p>Whether increased serotonin (5-HT) release in the forebrain attenuates or enhances anxiety has been controversial for over 25 yr. Although there is considerable indirect evidence, there is no direct evidence that indicates a relationship between acute 5-HT release and anxiety. In particular, there is no known method that can reversibly, selectively, and temporally control serotonergic activity. To address this issue, we generated transgenic animals to manipulate the firing rates of central 5-HT neurons by optogenetic methods. Activation of serotonergic neurons in the median raphe nucleus was correlated to enhanced anxiety-like behaviour in mice, whereas activation of serotonergic neurons in the dorsal raphe nucleus had no effect on anxiety-like behaviour. These results indicate that an acute increase in 5-HT release from the median raphe nucleus enhances anxiety.</p>

• Access to Document (DOI)

Optogenetic countering of glial acidosis suppresses glial glutamate release and ischemic brain damage

Beppu Kaoru, Sasaki Takuya, Tanaka Kenji F., Yamanaka Akihiro, Fukazawa Yugo, Shigemoto Ryuichi, Matsui Ko

Neuron 81 （ 2 ） 314 - 320 2014年01月

ISSN  0896-6273

　概要を見る

<p>The brain demands high-energy supply and obstruction of blood flow causes rapid deterioration of the healthiness of brain cells. Two major events occur upon ischemia: acidosis and liberation of excess glutamate, which leads to excitotoxicity. However, cellular source of glutamate and its release mechanism upon ischemia remained unknown. Here we show a causal relationship between glial acidosis and neuronal excitotoxicity. As the major cation that flows through channelrhodopsin-2 (ChR2) is proton, this could be regarded as an optogenetic tool for instant intracellular acidification. Optical activation of ChR2 expressed in glial cells led to glial acidification and to release of glutamate. On the other hand, glial alkalization via optogenetic activation of a proton pump, archaerhodopsin (ArchT), led to cessation of glutamate release and to the relief of ischemic brain damage in vivo. Our results suggest that controlling glial pH may be an effective therapeutic strategy for intervention of ischemic brain damage. © 2014 Elsevier Inc.</p>

• Access to Document (DOI)

Optogenetic activation of serotonergic neurons enhances anxiety-like behaviour in mice.

田中 謙二

Int J Neuropsychopharmacol 14   1777 - 1783 2014年

研究論文（学術雑誌）, 共著, 査読有り

• Access to Document (DOI)

Optogenetic activation of dorsal raphe serotonin neurons enhances patience for future rewards

田中 謙二

Curr Biol 24   2033 - 2040 2014年

研究論文（学術雑誌）, 共著, 査読有り

• Access to Document (DOI)

Short- and long-term functional plasticity of white matter induced by oligodendrocyte depolarization in the hippocampus

田中 謙二

Glia 62   1299 - 1312 2014年

研究論文（学術雑誌）, 共著, 査読有り

• Access to Document (DOI)

In vivo visualization of subtle, transient, and local activity of astrocytes using an ultrasensitive Ca(2+) indicator

田中 謙二

Cell Rep 8   311 - 318 2014年

研究論文（学術雑誌）, 共著

• Access to Document (DOI)

Increased adenosine levels in mice expressing mutant glial fibrillary acidic protein in astrocytes result in failure of induction of LTP reversal (depotentiation) in hippocampal CA1 neurons

田中 謙二

Brain Res 1578   1 - 13 2014年

研究論文（学術雑誌）, 共著, 査読有り

• Access to Document (DOI)

Hippocampal memory traces are differentially modulated by experience, time, and adult neurogenesis

田中 謙二

Neuron 83   189 - 201 2014年

研究論文（学術雑誌）, 共著, 査読有り

• Access to Document (DOI)

Optogenetic manipulation of activity and temporally controlled cell-specific ablation reveal a role for MCH neurons in sleep/wake regulation

田中 謙二

J Neurosci 34   6896 - 6909 2014年

研究論文（学術雑誌）, 共著, 査読有り

• Access to Document (DOI)

Optogenetic countering of glial acidosis suppresses glial glutamate release and ischemic brain damage

田中 謙二

Neuron 81   314 - 320 2014年

研究論文（学術雑誌）, 共著, 査読有り

• Access to Document (DOI)

Optogenetic manipulation of activity and temporally controlled cell-specific ablation reveal a role for MCH neurons in sleep/wake regulation

Tsunematsu Tomomi, Ueno Takafumi, Tabuchi Sawako, Inutsuka Ayumu, Tanaka Kenji F., Hasuwa Hidetoshi, Kilduff Thomas S., Terao Akira, Yamanaka Akihiro

Journal of Neuroscience 34 （ 20 ） 6896 - 6909 2014年

ISSN  0270-6474

　概要を見る

<p>Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generated using the recently developed knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states. Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance of NREM sleep. © 2014 the authors.</p>

• Access to Document (DOI)

Short- and long-term functional plasticity of white matter induced by oligodendrocyte depolarization in the hippocampus

Yamazaki Yoshihiko, Fujiwara Hiroki, Kaneko Kenya, Hozumi Yasukazu, Xu Ming, Ikenaka Kazuhiro, Fujii Satoshi, Tanaka Kenji F.

GLIA 62 （ 8 ） 1299 - 1312 2014年

ISSN  0894-1491

　概要を見る

<p>Plastic changes in white matter have received considerable attention in relation to normal cognitive function and learning. Oligodendrocytes and myelin, which constitute the white matter in the central nervous system, can respond to neuronal activity with prolonged depolarization of membrane potential and/or an increase in the intracellular Ca²⁺ concentration. Depolarization of oligodendrocytes increases the conduction velocity of an action potential along axons myelinated by the depolarized oligodendrocytes, indicating that white matter shows functional plasticity, as well as structural plasticity. However, the properties and mechanism of oligodendrocyte depolarization-induced functional plastic changes in white matter are largely unknown. Here, we investigated the functional plasticity of white matter in the hippocampus using mice with oligodendrocytes expressing channelrhodopsin-2. Using extracellular recordings of compound action potentials at the alveus of the hippocampus, we demonstrated that light-evoked depolarization of oligodendrocytes induced early- and late-onset facilitation of axonal conduction that was dependent on the magnitude of oligodendrocyte depolarization; the former lasted for approximately 10 min, whereas the latter continued for up to 3 h. Using whole-cell recordings from CA1 pyramidal cells and recordings of antidromic action potentials, we found that the early-onset short-lasting component included the synchronization of action potentials. Moreover, pharmacological analysis demonstrated that the activation of Ba²⁺-sensitive K⁺ channels was involved in early- and late-onset facilitation, whereas 4-aminopyridine-sensitive K⁺ channels were only involved in the early-onset component. These results demonstrate that oligodendrocyte depolarization induces short- and long-term functional plastic changes in the white matter of the hippocampus and plays active roles in brain functions. GLIA 2014;62:1299-1312 Main Points: Transcription factor Fosb gene products regulate C5ar1 and C5ar2 gene expression in microglia. In the neurodegenerative state, microglial activation was attenuated in Fosb-null microglia with decreased expression of C5aR1. © 2014 Wiley Periodicals, Inc.</p>

• Access to Document (DOI)

Development of transgenic animals in optogenetics

Tanaka Kenji

Folia Pharmacologica Japonica 143 （ 4 ） 193 - 197 2014年

ISSN  0015-5691

• Access to Document (DOI)