研究者詳細 - 田中　謙二

論文 - 田中　謙二

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A rapidly progressive multiple system atrophy-cerebellar variant model presenting marked glial reactions with inflammation and spreading of α-synuclein oligomers and phosphorylated α-synuclein aggregates.

Yamaguchi H, Nishimura Y, Matsuse D, Sekiya H, Masaki K, Tanaka T, Saiga T, Harada M, Kira YI, Dickson DW, Fujishima K, Matsuo E, Tanaka KF, Yamasaki R, Isobe N, Kira JI

Brain, behavior, and immunity 121 122 - 141 2024年07月

ISSN 0889-1591

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Glutamatergic signaling from melanin-concentrating hormone-producing neurons: A requirement for memory regulation, but not for metabolism control.

Pham XT, Abe Y, Mukai Y, Ono D, Tanaka KF, Ohmura Y, Wake H, Yamanaka A

PNAS nexus 3 （ 7 ） pgae275 2024年07月

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Neurogenesis-independent mechanisms of MRI-detectable hippocampal volume increase following electroconvulsive stimulation.

Abe Y, Yokoyama K, Kato T, Yagishita S, Tanaka KF, Takamiya A

Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 49 （ 8 ） 1236 - 1245 2024年07月

ISSN 0893-133X

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Optogenetic activation of dorsal raphe serotonin neurons induces brain-wide activation.

Hamada HT, Abe Y, Takata N, Taira M, Tanaka KF, Doya K

Nature communications 15 （ 1 ） 4152 2024年05月

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Brain-derived neurotrophic factor from microglia regulates neuronal development in the medial prefrontal cortex and its associated social behavior.

Komori T, Okamura K, Ikehara M, Yamamuro K, Endo N, Okumura K, Yamauchi T, Ikawa D, Ouji-Sageshima N, Toritsuka M, Takada R, Kayashima Y, Ishida R, Mori Y, Kamikawa K, Noriyama Y, Nishi Y, Ito T, Saito Y, Nishi M, Kishimoto T, Tanaka KF, Hiroi N, Makinodan M

Molecular psychiatry 29 （ 5 ） 1338 - 1349 2024年05月

ISSN 1359-4184

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Neurobiological mechanisms of electroconvulsive therapy for depression: Insights into hippocampal volumetric increases from clinical and preclinical studies.

Abe Y, Erchinger VJ, Ousdal OT, Oltedal L, Tanaka KF, Takamiya A

Journal of neurochemistry 2024年01月

ISSN 0022-3042

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Metabolites for monitoring symptoms and predicting remission in patients with depression who received electroconvulsive therapy: a pilot study

Uchida T., Sugiura Y., Sugiyama E., Maeda R., Tanaka K.F., Suematsu M., Mimura M., Uchida H.

Scientific Reports （Scientific Reports） 13 （ 1 ） 13218 2023年12月

The lack of biomarkers to monitor and predict the efficacy of electroconvulsive therapy (ECT) has hindered its optimal use. To establish metabolomic markers for monitoring and predicting the treatment efficacy of ECT, we comprehensively evaluated metabolite levels in patients with major depressive disorder (MDD) by performing targeted and non-targeted metabolomic analyses using plasma samples before and after the first, third, and final ECT sessions, and 3–7 days after the final session. We compared the plasma metabolomes of age- and sex-matched healthy controls (HCs). Thirteen hospitalized patients with MDD and their corresponding HCs were included in this study. We observed that patients with MDD exhibited lower levels of amino acids, including gamma-aminobutyric acid (GABA), and metabolites involved in tryptophan metabolism and the kynurenine pathway, and higher levels of cortisol at baseline. Furthermore, we investigated the relationship between metabolite levels and depression severity across seven measurement timepoints along with one correlation analysis and found that amino acids, including GABA and tryptophan catabolites, were significantly correlated with the severity of depression. Despite the exploratory nature of this study due to the limited sample size necessitating further validation, our findings suggest that the blood metabolic profile has potential as a biomarker for ECT.

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Prostaglandin E<inf>2</inf> Induces Long-Lasting Inhibition of Noradrenergic Neurons in the Locus Coeruleus and Moderates the Behavioral Response to Stressors

Mukai Y., Okubo T.S., Lazarus M., Ono D., Tanaka K.F., Yamanaka A.

Journal of Neuroscience （Journal of Neuroscience） 43 （ 47 ） 7982 - 7999 2023年11月

ISSN 02706474

Neuronal activity is modulated not only by inputs from other neurons but also by various factors, such as bioactive substances. Noradrenergic (NA) neurons in the locus coeruleus (LC-NA neurons) are involved in diverse physiological functions, including sleep/wakefulness and stress responses. Previous studies have identified various substances and receptors that modulate LC-NA neuronal activity through techniques including electrophysiology, calcium imaging, and single-cell RNA sequencing. However, many substances with unknown physiological significance have been overlooked. Here, we established an efficient screening method for identifying substances that modulate LC-NA neuronal activity through intracellular calcium ([Ca21]i) imaging using brain slices. Using both sexes of mice, we screened 53 bioactive substances, and identified five novel substances: gastrin-releasing peptide, neuromedin U, and angiotensin II, which increase [Ca21]i, and pancreatic polypeptide and prostaglandin D2, which decrease [Ca21]i. Among them, neuromedin U induced the greatest response in female mice. In terms of the duration of [Ca21]i change, we focused on prostaglandin E2 (PGE2), since it induces a long-lasting decrease in [Ca21]i via the EP3 receptor. Conditional knock-out of the receptor in LC-NA neurons resulted in increased depression-like behavior, prolonged wakefulness in the dark period, and increased [Ca21]i after stress exposure. Our results demonstrate the effectiveness of our screening method for identifying substances that modulate a specific neuronal population in an unbiased manner and suggest that stress-induced prostaglandin E2 can suppress LC-NA neuronal activity to moderate the behavioral response to stressors. Our screening method will contribute to uncovering previously unknown physiological functions of uncharacterized bioactive substances in specific neuronal populations.

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Shared GABA transmission pathology in dopamine agonist- and antagonist-induced dyskinesia

Abe Y., Yagishita S., Sano H., Sugiura Y., Dantsuji M., Suzuki T., Mochizuki A., Yoshimaru D., Hata J., Matsumoto M., Taira S., Takeuchi H., Okano H., Ohno N., Suematsu M., Inoue T., Nambu A., Watanabe M., Tanaka K.F.

Cell Reports Medicine （Cell Reports Medicine） 4 （ 10 ） 101208 2023年10月

Dyskinesia is involuntary movement caused by long-term medication with dopamine-related agents: the dopamine agonist 3,4-dihydroxy-L-phenylalanine (L-DOPA) to treat Parkinson's disease (L-DOPA-induced dyskinesia [LID]) or dopamine antagonists to treat schizophrenia (tardive dyskinesia [TD]). However, it remains unknown why distinct types of medications for distinct neuropsychiatric disorders induce similar involuntary movements. Here, we search for a shared structural footprint using magnetic resonance imaging-based macroscopic screening and super-resolution microscopy-based microscopic identification. We identify the enlarged axon terminals of striatal medium spiny neurons in LID and TD model mice. Striatal overexpression of the vesicular gamma-aminobutyric acid transporter (VGAT) is necessary and sufficient for modeling these structural changes; VGAT levels gate the functional and behavioral alterations in dyskinesia models. Our findings indicate that lowered type 2 dopamine receptor signaling with repetitive dopamine fluctuations is a common cause of VGAT overexpression and late-onset dyskinesia formation and that reducing dopamine fluctuation rescues dyskinesia pathology via VGAT downregulation.

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Starburst amacrine cells amplify optogenetic visual restoration through gap junctions.

Katada Y, Kunimi H, Serizawa N, Lee D, Kobayashi K, Negishi K, Okano H, Tanaka KF, Tsubota K, Kurihara T

Molecular therapy. Methods & clinical development （Molecular Therapy Methods and Clinical Development） 30 1 - 13 2023年09月

ISSN 2329-0501

Ectopic induction of optogenetic actuators, such as channelrhodopsin, is a promising approach to restoring vision in the degenerating retina. However, the cell type-specific response of ectopic photoreception has not been well understood. There are limits to obtaining efficient gene expression in a specifically targeted cell population by a transgenic approach. In the present study, we established a murine model with high efficiency of gene induction to retinal ganglion cells (RGCs) and amacrine cells using an improved tetracycline transactivator-operator bipartite system (KENGE-tet system). To investigate the cell type-specific visual restorative effect, we expressed the channelrhodopsin gene into RGCs and amacrine cells using the KENGE-tet system. As a result, enhancement in the visual restorative effect was observed to RGCs and starburst amacrine cells. In conclusion, a photoresponse from amacrine cells may enhance the maintained response of RGCs and further increase or improve the visual restorative effect.

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Brain Response to Interferential Current Compared with Alternating Current Stimulation

Xin Z., Abe Y., Kuwahata A., Tanaka K.F., Sekino M.

Brain Sciences （Brain Sciences） 13 （ 9 ） 2023年09月

ISSN 2076-3425

Temporal interference (TI) stimulation, which utilizes multiple external electric fields with amplitude modulation for neural modulation, has emerged as a potential noninvasive brain stimulation methodology. However, the clinical application of TI stimulation is inhibited by its uncertain fundamental mechanisms, and research has previously been restricted to numerical simulations and immunohistology without considering the acute in vivo response of the neural circuit. To address the characterization and understanding of the mechanisms underlying the approach, we investigated instantaneous brainwide activation patterns in response to invasive interferential current (IFC) stimulation compared with low-frequency alternative current stimulation (ACS). Results demonstrated that IFC stimulation is capable of inducing regional neural responses and modulating brain networks; however, the activation threshold for significantly recruiting a neural response using IFC was higher (at least twofold) than stimulation via alternating current, and the spatial distribution of the activation signal was restricted. A distinct blood oxygenation level-dependent (BOLD) response pattern was observed, which could be accounted for by the activation of distinct types of cells, such as inhibitory cells, by IFC. These results suggest that IFC stimulation might not be as efficient as conventional brain modulation methods, especially when considering TI stimulation as a potential alternative for stimulating subcortical brain areas. Therefore, we argue that a future transcranial application of TI on human subjects should take these implications into account and consider other stimulation effects using this technique.

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The gut microbiota-induced kynurenic acid recruits GPR35-positive macrophages to promote experimental encephalitis

Miyamoto K., Sujino T., Harada Y., Ashida H., Yoshimatsu Y., Yonemoto Y., Nemoto Y., Tomura M., Melhem H., Niess J.H., Suzuki T., Suzuki T., Suzuki S., Koda Y., Okamoto R., Mikami Y., Teratani T., Tanaka K., Yoshimura A., Sato T., Kanai T.

Cell Reports （Cell Reports） 42 （ 8 ） 113005 2023年08月

The intricate interplay between gut microbes and the onset of experimental autoimmune encephalomyelitis (EAE) remains poorly understood. Here, we uncover remarkable similarities between CD4+ T cells in the spinal cord and their counterparts in the small intestine. Furthermore, we unveil a synergistic relationship between the microbiota, particularly enriched with the tryptophan metabolism gene EC:1.13.11.11, and intestinal cells. This symbiotic collaboration results in the biosynthesis of kynurenic acid (KYNA), which modulates the recruitment and aggregation of GPR35-positive macrophages. Subsequently, a robust T helper 17 (Th17) immune response is activated, ultimately triggering the onset of EAE. Conversely, modulating the KYNA-mediated GPR35 signaling in Cx3cr1+ macrophages leads to a remarkable amelioration of EAE. These findings shed light on the crucial role of microbial-derived tryptophan metabolites in regulating immune responses within extraintestinal tissues.

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Inhibition of the dorsomedial striatal direct pathway is essential for the execution of action sequences.

Kono A, Shikano Y, Tanaka KF, Yamaura K, Tsutsui-Kimura I

Neuropsychopharmacology reports （Neuropsychopharmacology Reports） 43 （ 3 ） 414 - 424 2023年08月

Contrary to the previous notion that the dorsomedial striatum (DMS) is crucial for acquiring new learning, accumulated evidence has suggested that the DMS also plays a role in the execution of already learned action sequences. Here, we examined how the direct and indirect pathways in the DMS regulate action sequences using a task that requires animals to press a lever consecutively. Cell-type-specific bulk Ca2+ recording revealed that the direct pathway was inhibited at the time of sequence execution. The sequence-related response was blunted in trials where the sequential behaviors were disrupted. Optogenetic activation at the sequence start caused distraction of action sequences without affecting motor function or memory of the task structure. By contrast with the direct pathway, the indirect pathway was slightly activated at the start of the sequence, but the optogenetic suppression of such sequence-related signaling did not impact the behaviors. These results suggest that the inhibition of the DMS direct pathway promotes sequence execution potentially by suppressing the formation of a new association.

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Acceleration of the Development of Microcirculation Embolism in the Brain due to Capillary Narrowing.

Murata J, Unekawa M, Kudo Y, Kotani M, Kanno I, Izawa Y, Tomita Y, Tanaka KF, Nakahara J, Masamoto K

Stroke （Stroke） 54 （ 8 ） 2135 - 2144 2023年08月

ISSN 0039-2499

BACKGROUND: Cerebral microvascular obstruction is critically involved in recurrent stroke and decreased cerebral blood flow with age. The obstruction must occur in the capillary with a greater resistance to perfusion pressure through the microvascular networks. However, little is known about the relationship between capillary size and embolism formation. This study aimed to determine whether the capillary lumen space contributes to the development of microcirculation embolism. METHODS: To spatiotemporally manipulate capillary diameters in vivo, transgenic mice expressing the light-gated cation channel protein ChR2 (channelrhodopsin-2) in mural cells were used. The spatiotemporal changes in the regional cerebral blood flow in response to the photoactivation of ChR2 mural cells were first characterized using laser speckle flowgraphy. Capillary responses to optimized photostimulation were then examined in vivo using 2-photon microscopy. Finally, microcirculation embolism due to intravenously injected fluorescent microbeads was compared under conditions with or without photoactivation of ChR2 mural cells. RESULTS: Following transcranial photostimulation, the stimulation intensity-dependent decrease in cerebral blood flow centered at the irradiation was observed (14%-49% decreases relative to the baseline). The cerebrovascular response to photostimulation showed significant constriction of the cerebral arteries and capillaries but not of the veins. As a result of vasoconstriction, a temporal stall of red blood cell flow occurred in the capillaries of the venous sides. The 2-photon excitation of a single ChR2 pericyte demonstrated the partial shrinkage of capillaries (7% relative to the baseline) around the stimulated cell. With the intravenous injection of microbeads, the occurrence of microcirculation embolism was significantly enhanced (11% increases compared to the control) with photostimulation. CONCLUSIONS: Capillary narrowing increases the risk of developing microcirculation embolism in the venous sides of the cerebral capillaries.

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Pupil Dynamics-derived Sleep Stage Classification of a Head-fixed Mouse Using a Recurrent Neural Network.

Kobayashi G, Tanaka KF, Takata N

The Keio journal of medicine （Keio Journal of Medicine） 72 （ 2 ） 44 - 59 2023年06月

ISSN 0022-9717

The standard method for sleep state classification is thresholding the amplitudes of electroencepha-lography (EEG) and electromyography (EMG) data, followed by manual correction by an expert. Al-though popular, this method has some shortcomings: (1) the time-consuming manual correction by human experts is sometimes a bottleneck hindering sleep studies, (2) EEG electrodes on the skull interfere with wide-field imaging of the cortical activity of a head-fixed mouse under a microscope, (3) invasive surgery to fix the electrodes on the thin mouse skull risks brain tissue injury, and (4) metal electrodes for EEG and EMG recording are difficult to apply to some experimental apparatus such as that for functional magnetic resonance imaging. To overcome these shortcomings, we propose a pupil dynamics-based vigilance state classification method for a head-fixed mouse using a long short-term memory (LSTM) model, a variant of a recurrent neural network, for multi-class labeling of NREM, REM, and WAKE states. For supervisory hypnography, EEG and EMG recording were performed on head-fixed mice. This setup was combined with left eye pupillometry using a USB camera and a markerless tracking toolbox, DeepLabCut. Our open-source LSTM model with feature inputs of pupil diameter, pupil location, pupil velocity, and eyelid opening for 10 s at a 10 Hz sampling rate achieved vigilance state estimation with a higher classification performance (macro F1 score, 0.77; accuracy, 86%) than a feed-forward neural network. Findings from a diverse range of pupillary dynamics implied possible subdivi-sion of the vigilance states defined by EEG and EMG. Pupil dynamics-based hypnography can expand the scope of alternatives for sleep stage scoring of head-fixed mice.

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Mesolimbic dopamine release precedes actively sought aversive stimuli in mice.

Yawata Y, Shikano Y, Ogasawara J, Makino K, Kashima T, Ihara K, Yoshimoto A, Morikawa S, Yagishita S, Tanaka KF, Ikegaya Y

Nature communications （Nature Communications） 14 （ 1 ） 2433 2023年04月

In some models, animals approach aversive stimuli more than those housed in an enriched environment. Here, we found that male mice in an impoverished and unstimulating (i.e., boring) chamber without toys sought aversive air puffs more often than those in an enriched chamber. Using this animal model, we identified the insular cortex as a regulator of aversion-seeking behavior. Activation and inhibition of the insular cortex increased and decreased the frequencies of air-puff self-stimulation, respectively, and the firing patterns of insular neuron ensembles predicted the self-stimulation timing. Dopamine levels in the ventrolateral striatum decreased with passive air puffs but increased with actively sought puffs. Around 20% of mice developed intense self-stimulation despite being offered toys, which was prevented by administering opioid receptor antagonists. This study establishes a basis for comprehending the neural underpinnings of usually avoided stimulus-seeking behaviors.

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Separate optogenetic manipulation of Nerve/glial antigen 2 (NG2) glia and mural cells using the NG2 promoter

Oishi M., Passlick S., Yamazaki Y., Unekawa M., Adachi R., Yamada M., Imayoshi I., Abe Y., Steinhäuser C., Tanaka K.F.

GLIA （GLIA） 71 （ 2 ） 317 - 333 2023年02月

ISSN 08941491

Nerve/glial antigen 2 (NG2) is a protein marker of NG2 glia and mural cells, and NG2 promoter activity is utilized to target these cells. However, the NG2 promoter cannot target NG2 glia and mural cells separately. This has been an obstacle for NG2 glia-specific manipulation. Here, we developed transgenic mice in which either cell type can be targeted using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type-specific transgene expression, and generated NG2-tetracycline transactivator (tTA) transgenic lines. We crossed tTA lines with the tetO-ChR2 (channelrhodopsin-2)-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA mouse lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in NG2 glia-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that tTA-mediated ChR2 expression was sufficient to impact cellular function. ChR2-mediated depolarization was observed in NG2 glia in acute hippocampal slices. In addition, ChR2-mediated depolarization of NG2 glia inhibited their proliferation but promoted their differentiation in juvenile mice. Since the tTA–tetO combination is expandable, the mural cell–specific NG2-tTA line and the NG2 glia-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.

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Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle.

Natsubori A, Hirai S, Kwon S, Ono D, Deng F, Wan J, Miyazawa M, Kojima T, Okado H, Karashima A, Li Y, Tanaka KF, Honda M

iScience （iScience） 26 （ 1 ） 105830 2023年01月

The central serotonergic system has multiple roles in animal physiology and behavior, including sleep-wake control. However, its function in controlling brain energy metabolism according to the state of animals remains undetermined. Through in vivo monitoring of energy metabolites and signaling, we demonstrated that optogenetic activation of raphe serotonergic neurons increased cortical neuronal intracellular concentration of ATP, an indispensable cellular energy molecule, which was suppressed by inhibiting neuronal uptake of lactate derived from astrocytes. Raphe serotonergic neuronal activation induced cortical astrocytic Ca2+ and cAMP surges and increased extracellular lactate concentrations, suggesting the facilitation of lactate release from astrocytes. Furthermore, chemogenetic inhibition of raphe serotonergic neurons partly attenuated the increase in cortical neuronal intracellular ATP levels as arousal increased in mice. Serotonergic neuronal activation promoted an increase in cortical neuronal intracellular ATP levels, partly mediated by the facilitation of the astrocyte-neuron lactate shuttle, contributing to state-dependent optimization of neuronal intracellular energy levels.

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Slow-rising and fast-falling dopaminergic dynamics jointly adjust negative prediction error in the ventral striatum

Shikano Y., Yagishita S., Tanaka K.F., Takata N.

European Journal of Neuroscience （European Journal of Neuroscience） 58 （ 12 ） 4502 - 4522 2023年

ISSN 0953816X

The greater the reward expectations are, the more different the brain's physiological response will be. Although it is well-documented that better-than-expected outcomes are encoded quantitatively via midbrain dopaminergic (DA) activity, it has been less addressed experimentally whether worse-than-expected outcomes are expressed quantitatively as well. We show that larger reward expectations upon unexpected reward omissions are associated with the preceding slower rise and following larger decrease (DA dip) in the DA concentration at the ventral striatum of mice. We set up a lever press task on a fixed ratio (FR) schedule requiring five lever presses as an effort for a food reward (FR5). The mice occasionally checked the food magazine without a reward before completing the task. The percentage of this premature magazine entry (PME) increased as the number of lever presses approached five, showing rising expectations with increasing proximity to task completion, and hence greater reward expectations. Fibre photometry of extracellular DA dynamics in the ventral striatum using a fluorescent protein (genetically encoded GPCR activation-based DA sensor: GRABDA2m) revealed that the slow increase and fast decrease in DA levels around PMEs were correlated with the PME percentage, demonstrating a monotonic relationship between the DA dip amplitude and degree of expectations. Computational modelling of the lever press task implementing temporal difference errors and state transitions replicated the observed correlation between the PME frequency and DA dip amplitude in the FR5 task. Taken together, these findings indicate that the DA dip amplitude represents the degree of reward expectations monotonically, which may guide behavioural adjustment.

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A reinforcement learning model with choice traces for a progressive ratio schedule.

Ihara K, Shikano Y, Kato S, Yagishita S, Tanaka KF, Takata N

Frontiers in behavioral neuroscience 17 1302842 2023年

ISSN 1662-5153

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Dexamethasone induces sleep disturbance in a dose-dependent manner in mice.

Kato T, Okawa G, Tanaka KF, Mitsukura Y

PloS one 18 （ 12 ） e0296028 2023年

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Optogenetic demonstration of the involvement of SMA-negative mural cells in the regulation of cerebral blood flow.

Iba C, Abe Y, Tanaka KF

Frontiers in physiology 14 1322250 2023年

ISSN 1664-042X

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Dopamine Receptor Type 2-Expressing Medium Spiny Neurons in the Ventral Lateral Striatum Have a Non-REM Sleep-Induce Function

Kato T., Tanaka K.F., Natsubori A.

eNeuro （eNeuro） 10 （ 9 ） 2023年

Dopamine receptor type 2-expressing medium spiny neurons (D2-MSNs) in the medial part of the ventral striatum (VS) induce non-REM (NREM) sleep from the wake state in animals. However, it is unclear whether D2-MSNs in the lateral part of the VS (VLS), which is anatomically and functionally different from the medial part of the VS, contribute to sleep-wake regulation. This study aims to clarify whether and how D2-MSNs in the VLS are involved in sleep-wake regulation. Our study found that specifically removing D2-MSNs in the VLS led to an increase in wakefulness time in mice during the dark phase using a diphtheria toxin-mediated cell ablation/dysfunction technique. D2-MSN ablation throughout the VS further increased dark phase wakefulness time. These findings suggest that VLS D2-MSNs may induce sleep during the dark phase with the medial part of the VS. Next, our fiber photometric recordings revealed that the population intracellular calcium (Ca2+) signal in the VLS D2-MSNs increased during the transition from wake to NREM sleep. The mean Ca2+ signal level of VLS D2-MSNs was higher during NREM and REM sleep than during the wake state, supporting their sleep-inducing role. Finally, optogenetic activation of the VLS D2-MSNs during the wake state always induced NREM sleep, demonstrating the causality of VLS D2-MSNs activity with sleep induction. Additionally, activation of the VLS D1-MSNs, counterparts of D2-MSNs, always induced wake from NREM sleep, indicating a wake-promoting role. In conclusion, VLS D2-MSNs could have an NREM sleep-inducing function in coordination with those in the medial VS.

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Synaptic pruning through glial synapse engulfment upon motor learning

Morizawa Y.M., Matsumoto M., Nakashima Y., Endo N., Aida T., Ishikane H., Beppu K., Moritoh S., Inada H., Osumi N., Shigetomi E., Koizumi S., Yang G., Hirai H., Tanaka K., Tanaka K.F., Ohno N., Fukazawa Y., Matsui K.

Nature Neuroscience （Nature Neuroscience） 25 （ 11 ） 1458 - 1469 2022年11月

ISSN 10976256

Synaptic pruning is a fundamental process of neuronal circuit refinement in learning and memory. Accumulating evidence suggests that glia participates in sculpting the neuronal circuits through synapse engulfment. However, whether glial involvement in synaptic pruning has a role in memory formation remains elusive. Using newly developed phagocytosis reporter mice and three-dimensional ultrastructural characterization, we found that synaptic engulfment by cerebellar Bergmann glia (BG) frequently occurred upon cerebellum-dependent motor learning in mice. We observed increases in pre- and postsynaptic nibbling by BG along with a reduction in spine volume after learning. Pharmacological blockade of engulfment with Annexin V inhibited both the spine volume reduction and overnight improvement of motor adaptation. These results indicate that BG contribute to the refinement of the mature cerebellar cortical circuit through synaptic engulfment during motor learning.

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Oscillatory Population-Level Activity of Dorsal Raphe Serotonergic Neurons Is Inscribed in Sleep Structure

Kato T., Mitsukura Y., Yoshida K., Mimura M., Takata N., Tanaka K.F.

Journal of Neuroscience （Journal of Neuroscience） 42 （ 38 ） 7244 - 7255 2022年09月

ISSN 02706474

Dorsal raphe (DR) 5-HT neurons regulate sleep–wake transitions. Previous studies demonstrated that single-unit activity of DR 5-HT neurons is high during wakefulness, decreases during non-rapid eye movement (NREM) sleep, and ceases during rapid eye movement (REM) sleep. However, characteristics of the population-level activity of DR 5-HT neurons, which influence the entire brain, are largely unknown. Here, we measured population activities of 5-HT neurons in the male and female mouse DR across the sleep–wake cycle by ratiometric fiber photometry. We found a slow oscillatory activity of compound intracellular Ca21 signals during NREM sleep. The trough of the concave 5-HT activity increased across sleep progression, but 5-HT activity always returned to that seen during the wake period. When the trough reached a minimum and remained there, REM sleep was initiated. We also found a unique coupling of the oscillatory 5-HT activity and wideband EEG power fluctuation. Furthermore, optogenetic activation of 5-HT neurons during NREM sleep triggered a high EMG power and induced wakefulness, demonstrating a causal role of 5-HT neuron activation. Optogenetic inhibition induced REM sleep or sustained NREM, with an EEG power increase and EEG fluctuation, and pharmacological silencing of 5-HT activity using a selective serotonin reuptake inhibitor led to sustained NREM, with an EEG power decrease and EEG fluctuation. These inhibitory manipulations supported the association between oscillatory 5-HT activity and EEG fluctuation. We propose that NREM sleep is not a monotonous state, but rather it contains dynamic changes that coincide with the oscillatory population-level activity of DR 5-HT neurons.

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Ratio-metric measurement of intracellular calcium in visceral muscles via selective expression of a yellow cameleon calcium sensor

Takai C., Iwata N., Kanemaru K., Tanaka K.F., Yu Y., Iino S., Nakayama S.

Sensors and Actuators B: Chemical （Sensors and Actuators B: Chemical） 364 2022年08月

ISSN 09254005

Visceral muscles exhibit various patterns of contractions depending on their locations and functions. Most visceral muscles are comprised of smooth muscle, and an increase in the intracellular Ca2+ concentration ([Ca2+]i) induces contraction, as in striated muscle. In order to critically assess the mechanisms underlying a variety of visceral movements, it is important to measure [Ca2+]i. However, due to limitations of sensing methods, it is difficult to compare [Ca2+]i between different organs and individuals. In this study, we thus measured [Ca2+]i in visceral muscles using transgenic mice that selective expressed a genetically encoded ratiometric Ca2+ indicator (YC-Nano50) in both skeletal and smooth muscles. We acquired CFP and YFP fluorescence images of YC-Nano50 separately, to evaluate the fluorescence resonance energy transfer (FRET), which reflects [Ca2+]i. In the resting condition, the YFP/CFP ratio measured in isolated muscularis was lower in smooth muscle than in skeletal muscle, corresponding to [Ca2+]i in the range of approximately 20–40 nM and 50–80 nM, respectively. Among visceral smooth muscles, the YFP/CFP ratio was highest in the urinary bladder, and lowest in the stomach (antrum). In the esophagus, comprised of skeletal muscle, the YFP/CFP ratio was higher than in other visceral smooth muscles, and was comparable to that in abdominal wall and diaphragm skeletal muscles. In addition, we were able to measure Ca2+ transients and oscillations in muscle sheets and intact segments isolated from the intestine even during movement. This method is able to elucidate the biological significance of Ca2+ signaling in diverse functions of visceral muscles.

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Lateral habenula glutamatergic neurons projecting to the dorsal raphe nucleus promote aggressive arousal in mice.

Takahashi A, Durand-de Cuttoli R, Flanigan ME, Hasegawa E, Tsunematsu T, Aleyasin H, Cherasse Y, Miya K, Okada T, Keino-Masu K, Mitsui K, Li L, Patel V, Blitzer RD, Lazarus M, Tanaka KF, Yamanaka A, Sakurai T, Ogawa S, Russo SJ

Nature communications （Nature Communications） 13 （ 1 ） 4039 2022年07月

The dorsal raphe nucleus (DRN) is known to control aggressive behavior in mice. Here, we found that glutamatergic projections from the lateral habenula (LHb) to the DRN were activated in male mice that experienced pre-exposure to a rival male mouse (“social instigation”) resulting in heightened intermale aggression. Both chemogenetic and optogenetic suppression of the LHb-DRN projection blocked heightened aggression after social instigation in male mice. In contrast, inhibition of this pathway did not affect basal levels of aggressive behavior, suggesting that the activity of the LHb-DRN projection is not necessary for the expression of species-typical aggressive behavior, but required for the increase of aggressive behavior resulting from social instigation. Anatomical analysis showed that LHb neurons synapse on non-serotonergic DRN neurons that project to the ventral tegmental area (VTA), and optogenetic activation of the DRN-VTA projection increased aggressive behaviors. Our results demonstrate that the LHb glutamatergic inputs to the DRN promote aggressive arousal induced by social instigation, which contributes to aggressive behavior by activating VTA-projecting non-serotonergic DRN neurons as one of its potential targets.

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Rhythmic activation of excitatory neurons in the mouse frontal cortex improves the prefrontal cortex-mediated cognitive function.

Hazra D, Yoshinaga S, Yoshida K, Takata N, Tanaka KF, Kubo KI, Nakajima K

Cerebral cortex (New York, N.Y. : 1991) （Cerebral cortex (New York, N.Y. : 1991)） 32 （ 23 ） 5243 - 5258 2022年02月

ISSN 1047-3211

The prefrontal cortex (PFC) plays essential roles in cognitive processes. Previous studies have suggested the layer and the cell type-specific activation for cognitive enhancement. However, the mechanism by which a temporal pattern of activation affects cognitive function remains to be elucidated. Here, we investigated whether the specific activation of excitatory neurons in the superficial layers mainly in the PFC according to a rhythmic or nonrhythmic pattern could modulate the cognitive functions of normal mice. We used a C128S mutant of channelrhodopsin 2, a step function opsin, and administered two light illumination patterns: (i) alternating pulses of blue and yellow light for rhythmic activation or (ii) pulsed blue light only for nonrhythmic activation. Behavioral analyses were performed to compare the behavioral consequences of these two neural activation patterns. The alternating blue and yellow light pulses, but not the pulsed blue light only, significantly improved spatial working memory and social recognition without affecting motor activity or the anxiety level. These results suggest that the rhythmic, but not the nonrhythmic, activation could enhance cognitive functions. This study indicates that not only the population of neurons that are activated but also the pattern of activation plays a crucial role in the cognitive enhancement.

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Downregulation of Bdnf Expression in Adult Mice Causes Body Weight Gain.

Suzuki T, Tanaka KF

Neurochemical research 2022年01月

ISSN 0364-3190

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The Mlc1 Promoter Directs Müller Cell-specific Gene Expression in the Retina.

Danjo Y, Shinozaki Y, Natsubori A, Kubota Y, Kashiwagi K, Tanaka KF, Koizumi S

Translational vision science & technology 11 （ 1 ） 25 2022年01月

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Dysfunction of parvalbumin-expressing cells in the thalamic reticular nucleus induces cortical spike-and-wave discharges and an unconscious state.

Abdelaal MS, Midorikawa M, Suzuki T, Kobayashi K, Takata N, Miyata M, Mimura M, Tanaka KF

Brain communications （Brain Communications） 4 （ 2 ） fcac010 2022年

Spike-and-wave discharges and an accompanying loss of consciousness are hallmarks of absence seizure, which is a childhood generalized epilepsy disorder. In absence seizure, dysfunction of the cortico-thalamo-cortico circuitry is thought to engage in abnormal cortical rhythms. Previous studies demonstrated that the thalamic reticular nucleus has a critical role in the formation of normal cortical rhythms; however, whether thalamic reticular nucleus dysfunction leads directly to abnormal rhythms, such as epilepsy, is largely unknown. We found that expressing the inhibitory opsin, archaerhodopsin, including in the thalamic reticular nucleus, caused abnormal cortical rhythms in Pvalb-tetracycline transactivator::tetO-ArchT (PV-ArchT) double transgenic mice. We validated the PV-ArchT line as a new mouse model of absence seizure through physiological and pharmacological analyses, as well as through examining their behavioural features. We then discovered that archaerhodopsin expression exclusively in thalamic reticular nucleus parvalbumin-positive neurons was sufficient to induce cortical spike-and-wave discharges using adeno-associated virus-mediated thalamic reticular nucleus targeting. Furthermore, we found that archaerhodopsin expression impaired rebound burst firing and T-current in thalamic reticular nucleus parvalbumin-positive cells by slice physiology. Although T-current in the thalamic reticular nucleus was impaired, the T-current blocker ethosuximide still had a therapeutic effect in PV-ArchT mice, suggesting a gain of function of T-type calcium channels in this absence seizure model. However, we did not find any over- or misexpression of T-type calcium channel genes in the thalamus or the cortex. Thus, we demonstrated that thalamic reticular nucleus dysfunction led to an absence seizure-like phenotype in mice. In a final set of experiments, we showed that the archaerhodopsin-mediated absence seizure-like phenotype disappeared after the removal of archaerhodopsin by using a time-controllable transgenic system. These data may provide a hint as to why many absence seizures naturally regress.

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A gradual temporal shift of dopamine responses mirrors the progression of temporal difference error in machine learning

Amo R., Matias S., Yamanaka A., Tanaka K.F., Uchida N., Watabe-Uchida M.

Nature Neuroscience （Nature Neuroscience） 25 （ 8 ） 1082 - 1092 2022年

ISSN 10976256

A large body of evidence has indicated that the phasic responses of midbrain dopamine neurons show a remarkable similarity to a type of teaching signal (temporal difference (TD) error) used in machine learning. However, previous studies failed to observe a key prediction of this algorithm: that when an agent associates a cue and a reward that are separated in time, the timing of dopamine signals should gradually move backward in time from the time of the reward to the time of the cue over multiple trials. Here we demonstrate that such a gradual shift occurs both at the level of dopaminergic cellular activity and dopamine release in the ventral striatum in mice. Our results establish a long-sought link between dopaminergic activity and the TD learning algorithm, providing fundamental insights into how the brain associates cues and rewards that are separated in time.

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Optogenetic stimulus-triggered acquisition of seizure resistance.

Shimoda Y, Beppu K, Ikoma Y, Morizawa YM, Zuguchi S, Hino U, Yano R, Sugiura Y, Moritoh S, Fukazawa Y, Suematsu M, Mushiake H, Nakasato N, Iwasaki M, Tanaka KF, Tominaga T, Matsui K

Neurobiology of disease 163 105602 2021年12月

ISSN 0969-9961

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Sustained ErbB Activation Causes Demyelination and Hypomyelination by Driving Necroptosis of Mature Oligodendrocytes and Apoptosis of Oligodendrocyte Precursor Cells.

Hu X, Xiao G, He L, Niu X, Li H, Lou T, Hu Q, Yang Y, Xu Q, Wei Z, Qiu M, Tanaka KF, Shen Y, Tao Y

The Journal of neuroscience : the official journal of the Society for Neuroscience 41 （ 48 ） 9872 - 9890 2021年12月

ISSN 0270-6474

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Global knockdown of glutamate decarboxylase 67 elicits emotional abnormality in mice

Miyata S., Kakizaki T., Fujihara K., Obinata H., Hirano T., Nakai J., Tanaka M., Itohara S., Watanabe M., Tanaka K.F., Abe M., Sakimura K., Yanagawa Y.

Molecular Brain （Molecular Brain） 14 （ 1 ） 5 2021年12月

Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by the Gad1 gene, is a consistent finding in postmortem brains of patients with several psychiatric disorders, including schizophrenia, bipolar disorder and major depressive disorder. The dysfunction of GAD67 in the brain is implicated in the pathophysiology of these psychiatric disorders; however, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous Gad1 knockout is lethal in newborn mice. We hypothesized that the tetracycline-controlled gene expression/suppression system could be applied to develop global GAD67 knockdown mice that would survive into adulthood. In addition, GAD67 knockdown mice would provide new insights into the neurobiological impact of GAD67 dysfunction. Here, we developed Gad1tTA/STOP−tetO biallelic knock-in mice using Gad1STOP−tetO and Gad1tTA knock-in mice, and compared them with Gad1+/+ mice. The expression level of GAD67 protein in brains of Gad1tTA/STOP−tetO mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated Gad1tTA/STOP−tetO mice. In the open-field test, Dox-treated Gad1tTA/STOP−tetO mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated Gad1tTA/STOP−tetO mice. These results suggest that global reduction in GAD67 elicits emotional abnormalities in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.

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Flexible annotation atlas of the mouse brain: combining and dividing brain structures of the Allen Brain Atlas while maintaining anatomical hierarchy

Takata N., Sato N., Komaki Y., Okano H., Tanaka K.F.

Scientific Reports （Scientific Reports） 11 （ 1 ） 6234 2021年12月

A brain atlas is necessary for analyzing structure and function in neuroimaging research. Although various annotation volumes (AVs) for the mouse brain have been proposed, it is common in magnetic resonance imaging (MRI) of the mouse brain that regions-of-interest (ROIs) for brain structures (nodes) are created arbitrarily according to each researcher’s necessity, leading to inconsistent ROIs among studies. One reason for such a situation is the fact that earlier AVs were fixed, i.e. combination and division of nodes were not implemented. This report presents a pipeline for constructing a flexible annotation atlas (FAA) of the mouse brain by leveraging public resources of the Allen Institute for Brain Science on brain structure, gene expression, and axonal projection. A mere two-step procedure with user-specified, text-based information and Python codes constructs FAA with nodes which can be combined or divided objectively while maintaining anatomical hierarchy of brain structures. Four FAAs with total node count of 4, 101, 866, and 1381 were demonstrated. Unique characteristics of FAA realized analysis of resting-state functional connectivity (FC) across the anatomical hierarchy and among cortical layers, which were thin but large brain structures. FAA can improve the consistency of whole brain ROI definition among laboratories by fulfilling various requests from researchers with its flexibility and reproducibility.

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Oligodendrocytic Na+-K+-Cl– co-transporter 1 activity facilitates axonal conduction and restores plasticity in the adult mouse brain

Yamazaki Y., Abe Y., Fujii S., Tanaka K.F.

Nature Communications （Nature Communications） 12 （ 1 ） 5146 2021年12月

The juvenile brain presents plasticity. Oligodendrocytes are the myelinating cells of the central nervous system and myelination can be adaptive. Plasticity decreases from juvenile to adulthood. The mechanisms involving oligodendrocytes underlying plasticity are unclear. Here, we show Na+-K+-Cl– co-transporter 1 (NKCC1), highly expressed in the juvenile mouse brain, regulates the oligodendrocyte activity from juvenile to adulthood in mice, as shown by optogenetic manipulation of oligodendrocytes. The reduced neuronal activity in adults was restored by Nkcc1 overexpression in oligodendrocytes. Moreover, in adult mice overexpressing Nkcc1, long-term potentiation and learning were facilitated compared to age-matched controls. These findings demonstrate that NKCC1 plays a regulatory role in the age-dependent activity of oligodendrocytes, furthermore inducing activation of NKCC1 in oligodendrocytes can restore neuronal plasticity in the adult mouse brain.

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Optogenetic activation of DRN 5-HT neurons induced active wakefulness, not quiet wakefulness.

Moriya R, Kanamaru M, Okuma N, Yoshikawa A, Tanaka KF, Hokari S, Ohshima Y, Yamanaka A, Honma M, Onimaru H, Kikuchi T, Izumizaki M

Brain research bulletin （Brain Research Bulletin） 177 129 - 142 2021年09月

ISSN 0361-9230

There has been a long-standing controversy regarding the physiological role of serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) in sleep/wake architecture. Some studies have reported that 5-HT acts as a sleep-promoting agent, but several studies have suggested that DRN 5-HT neurons function predominantly to promote wakefulness and inhibit rapid eye movement (REM) sleep. Furthermore, recent studies have reported that there is a clear neurobiological difference between a waking state that includes alertness and active exploration (i.e., active wakefulness) and a waking state that is devoid of locomotion (i.e., quiet wakefulness). These states have also been shown to differ clinically in terms of memory consolidation. However, the effects of 5-HT neurons on the regulation of these two different waking states have not been fully elucidated. In the present study, we attempted to examine the physiological role of DRN 5-HT neurons in various sleep/wake states using optogenetic methods that allowed manipulation of cell-type specific neuronal activation with high temporal and anatomical precision. We crossed TPH2-tTA and TetO-ChR2(C128S) mice to obtain mice with channelrhodopsin-2 (ChR2) [C128S]-expressing central 5-HT neurons, and we activated DRN-5HT neurons or medullary 5-HT neurons. Optogenetic activation of DRN 5-HT neurons caused rapid transition from non-REM sleep to active wakefulness, not quiet wakefulness, whereas activation of medullary 5-HT neurons did not appear to affect sleep/wake states or locomotor activity. Our results may shed light on the physiological role of DRN 5-HT neurons in sleep/wake architecture and encourage further investigations of the cortical functional connectivity involved in sleep/wake state regulation.

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Mice with reduced glutamate transporter GLT1 expression exhibit behaviors related to attention-deficit/hyperactivity disorder.

Hiraoka Y, Sugiyama K, Nagaoka D, Tsutsui-Kimura I, Tanaka KF, Tanaka K

Biochemical and biophysical research communications （Biochemical and Biophysical Research Communications） 567 161 - 165 2021年08月

ISSN 0006-291X

Attention-deficit/hyperactivity disorder (ADHD) is a common neuropsychiatric disorder in children. Although animal models and human brain imaging studies indicate a significant role for glutamatergic dysfunction in ADHD, there is no direct evidence that glutamatergic dysfunction is sufficient to induce ADHD-like symptoms. The glial glutamate transporter GLT1 plays a critical role in glutamatergic neurotransmission. We report here the generation of mice expressing only 20% of normal levels of the GLT1. Unlike conventional GLT1 knockout mice, these mice survive to adulthood and exhibit ADHD-like phenotypes, including hyperactivity, impulsivity and impaired memory. These findings indicate that glutamatergic dysfunction due to GLT1 deficiency, a mechanism distinct from the dopaminergic deficit hypothesis of ADHD, underlies ADHD-like symptoms.

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Chronic social defeat stress impairs goal-directed behavior through dysregulation of ventral hippocampal activity in male mice

Yoshida K., Drew M.R., Kono A., Mimura M., Takata N., Tanaka K.F.

Neuropsychopharmacology （Neuropsychopharmacology） 46 （ 9 ） 1606 - 1616 2021年08月

ISSN 0893133X

Chronic stress is a risk factor for a variety of psychiatric disorders, including depression. Although impairments to motivated behavior are a major symptom of clinical depression, little is known about the circuit mechanisms through which stress impairs motivation. Furthermore, research in animal models for depression has focused on impairments to hedonic aspects of motivation, whereas patient studies suggest that impairments to appetitive, goal-directed motivation contribute significantly to motivational impairments in depression. Here, we characterized goal-directed motivation in repeated social defeat stress (R-SDS), a well-established mouse model for depression in male mice. R-SDS impaired the ability to sustain and complete goal-directed behavior in a food-seeking operant lever-press task. Furthermore, stress-exposed mice segregated into susceptible and resilient subpopulations. Interestingly, susceptibility to stress-induced motivational impairments was unrelated to stress-induced social withdrawal, another prominent effect of R-SDS in mouse models. Based on evidence that ventral hippocampus (vHP) modulates sustainment of goal-directed behavior, we monitored vHP activity during the task using fiber photometry. Successful task completion was associated with suppression of ventral hippocampal neural activity. This suppression was diminished after R-SDS in stress-susceptible but not stress-resilient mice. The serotonin selective reuptake inhibitor (SSRI) escitalopram and ketamine both normalized vHP activity during the task and restored motivated behavior. Furthermore, optogenetic vHP inhibition was sufficient to restore motivated behavior after stress. These results identify vHP hyperactivity as a circuit mechanism of stress-induced impairments to goal-directed behavior and a putative biomarker that is sensitive to antidepressant treatments and that differentiates susceptible and resilient individuals.

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Optical manipulation of local cerebral blood flow in the deep brain of freely moving mice.

Abe Y, Kwon S, Oishi M, Unekawa M, Takata N, Seki F, Koyama R, Abe M, Sakimura K, Masamoto K, Tomita Y, Okano H, Mushiake H, Tanaka KF

Cell reports （Cell Reports） 36 （ 4 ） 109427 2021年07月

An artificial tool for manipulating local cerebral blood flow (CBF) is necessary for understanding how CBF controls brain function. Here, we generate vascular optogenetic tools whereby smooth muscle cells and endothelial cells express optical actuators in the brain. The illumination of channelrhodopsin-2 (ChR2)-expressing mice induces a local reduction in CBF. Photoactivated adenylyl cyclase (PAC) is an optical protein that increases intracellular cyclic adenosine monophosphate (cAMP), and the illumination of PAC-expressing mice induces a local increase in CBF. We target the ventral striatum, determine the temporal kinetics of CBF change, and optimize the illumination intensity to confine the effects to the ventral striatum. We demonstrate the utility of this vascular optogenetic manipulation in freely and adaptively behaving mice and validate the task- and actuator-dependent behavioral readouts. The development of vascular optogenetic animal models will help accelerate research linking vasculature, circuits, and behavior to health and disease.

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Region-Specific and State-Dependent Astrocyte Ca2+ Dynamics during the Sleep-Wake Cycle in Mice.

Tsunematsu T, Sakata S, Sanagi T, Tanaka KF, Matsui K

The Journal of neuroscience : the official journal of the Society for Neuroscience （Journal of Neuroscience） 41 （ 25 ） 5440 - 5452 2021年06月

ISSN 0270-6474

Neural activity is diverse, and varies depending on brain regions and sleep/wakefulness states. However, whether astrocyte activity differs between sleep/wakefulness states, and whether there are differences in astrocyte activity among brain regions remain poorly understood. Therefore, in this study, we recorded astrocyte intracellular calcium (Ca21) concentrations of mice during sleep/wakefulness states in the cortex, hippocampus, hypothalamus, cerebellum, and pons using fiber photometry. For this purpose, male transgenic mice expressing the genetically encoded ratiometric Ca21 sensor YCnano50 specifically in their astrocytes were used. We demonstrated that Ca21 levels in astrocytes substantially decrease during rapid eye movement (REM) sleep, and increase after the onset of wakefulness. In contrast, differences in Ca21 levels during non-REM (NREM) sleep were observed among the different brain regions, and no significant decrease was observed in the hypothalamus and pons. Further analyses focusing on the transition between sleep/wakefulness states and correlation analysis with the duration of REM sleep showed that Ca21 dynamics differs among brain regions, suggesting the existence of several clusters, i.e., the first comprising the cortex and hippocampus, the second comprising the hypothalamus and pons, and the third comprising the cerebellum. Our study thus demonstrated that astrocyte Ca21 levels change substantially according to sleep/wakefulness states. These changes were consistent in general unlike neural activity. However, we also clarified that Ca21 dynamics varies depending on the brain region, implying that astrocytes may play various physiological roles in sleep.

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Exacerbation of epilepsy by astrocyte alkalization and gap junction uncoupling

Onodera M., Meyer J., Furukawa K., Hiraoka Y., Aida T., Tanaka K., Tanaka K.F., Rose C.R., Matsui K.

Journal of Neuroscience （Journal of Neuroscience） 41 （ 10 ） 2106 - 2118 2021年03月

ISSN 02706474

Seizures invite seizures. At the initial stage of epilepsy, seizures intensify with each episode; however, the mechanisms underlying this exacerbation remain to be solved. Astrocytes have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K1 clearance relies on the K1 uptake by astrocytes and the subsequent diffusion of K1 through the astrocyte network. When astrocytes become uncoupled, K1 clearance becomes hindered. Accumulation of extracellular K1 leads to hyperexcitability of neurons. Here, using acute hippocampal slices from mice, we uncovered that brief periods of epileptiform activity result in gap junction uncoupling. In slices that experienced short-term epileptiform activity, extracellular K1 transients in response to glutamate became prolonged. Na1 imaging with a fluorescent indicator indicated that intercellular diffusion of small cations in the astrocytic syncytium via gap junctions became rapidly restricted after epileptiform activity. Using a transgenic mouse with astrocyte-specific expression of a pH sensor (Lck-E2GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na1/HCO3-cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification both in vitro and in vivo. Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.

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Astrocytic cAMP modulates memory via synaptic plasticity

Zhou Z., Okamoto K., Onodera J., Hiragi T., Andoh M., Ikawa M., Tanaka K.F., Ikegaya Y., Koyama R.

Proceedings of the National Academy of Sciences of the United States of America （Proceedings of the National Academy of Sciences of the United States of America） 118 （ 3 ） 2021年01月

ISSN 00278424

Astrocytes play a key role in brain homeostasis and functions such as memory. Specifically, astrocytes express multiple receptors that transduce signals via the second messenger cAMP. However, the involvement of astrocytic cAMP in animal behavior and the underlying glial-neuronal interactions remains largely unknown. Here, we show that an increase in astrocytic cAMP is sufficient to induce synaptic plasticity and modulate memory. We developed a method to increase astrocytic cAMP levels in vivo using photoactivated adenylyl cyclase and found that increased cAMP in hippocampal astrocytes at different time points facilitated memory formation but interrupted memory retention via NMDA receptor-dependent plasticity. Furthermore, we found that the cAMP-induced modulation of memory was mediated by the astrocyte-neuron lactate shuttle. Thus, our study unveils a role of astrocytic cAMP in brain function by providing a tool to modulate astrocytic cAMP in vivo.

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Ninjin'yoeito, a traditional Japanese Kampo medicine, suppresses the onset of anhedonia induced by dysfunction in the striatal dopamine receptor type 2-expressing medium spiny neurons

Oizumi H., Imai R., Suzuki T., Omiya Y., Tanaka K.F., Mimura M., Mizoguchi K.

NeuroReport （NeuroReport） 32 （ 10 ） 869 - 874 2021年

ISSN 09594965

Objective Recent studies have suggested that ninjin'yoeito (NYT), a traditional Japanese Kampo medicine, improves diminished motivation in humans and animals, rendering it a novel therapeutic option for impaired motivation. To better characterize the effect of NYT on motivation, we examined its effect on motivated behaviors in mice. Methods Mouse models of neurodegeneration-related apathy, in which striatal dopamine receptor type 2-expressing medium spiny neurons (D2-MSNs) were progressively damaged by diphtheria toxin expression, were chosen. Results The decrease in effort-based operant responding for rewards (sucrose pellets), indicative of the mouse's motivated behavior, in the affected mice was not suppressed by chronic treatment with NYT suspended in drinking water at 1% (w/v). Mice were then subjected to a sucrose preference test, wherein they freely chose to ingest tap water and a sucrose solution without being required to exert effort. The affected mice showed a decline in preference for sucrose over tap water, relative to nonaffected controls, indicating anhedonia-like traits. In contrast to the diminished operant behavior, the anhedonic behavior in the affected mice was prevented by the NYT administration. Furthermore, NYT did not affect the size of Drd2 mRNA disappearance in the striatum of affected mice, suggesting that the NYT effect was unrelated to DTA-mediated neurodegeneration. Conclusion These results demonstrate that the beneficial effect of NYT on motivation is mediated, at least in part, through the potentiation of hedonic capacity by certain neuromodulatory pathways.

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Differential pial and penetrating arterial responses examined by optogenetic activation of astrocytes and neurons

Hatakeyama N., Unekawa M., Murata J., Tomita Y., Suzuki N., Nakahara J., Takuwa H., Kanno I., Matsui K., Tanaka K.F., Masamoto K.

Journal of Cerebral Blood Flow and Metabolism （Journal of Cerebral Blood Flow and Metabolism） 41 （ 10 ） 271678X211010355 - 2689 2021年

ISSN 0271678X

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

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Nitric oxide-mediated signal transmission in bladder vasculature as a therapeutic target of PDE5 inhibitors: Rodent model studies.

Tanaka H, Mitsui R, Oishi M, Passlick S, Jabs R, Steinhäuser C, Tanaka KF, Hashitani H

British journal of pharmacology （British Journal of Pharmacology） 178 （ 5 ） 1073 - 1094 2020年12月

ISSN 0007-1188

Background and Purpose: While the bladder vasculature is considered as a target of PDE5 inhibitors to improve bladder storage dysfunctions, its characteristics are largely unknown. Thus, the functional and morphological properties of arteries/arterioles of the bladder focusing on the NO-mediated signal transmission were explored. Experimental Approach: Diameter changes in rat bladder arteries/arterioles were measured using a video-tracking system. Intercellular Ca2+ dynamics in pericytes or smooth muscle cells (SMCs) of suburothelial arterioles were visualised using transgenic mice expressing GCaMP6 under control of the NG2- or parvalbumin-promoter. The perivascular innervation was investigated using fluorescence immunohistochemistry. Key Results: In rat suburothelial arterioles and vesical arteries, tadalafil (100 nM) attenuated nerve-evoked sympathetic vasoconstrictions. In both vascular segments, tadalafil-induced inhibition of sympathetic vasoconstriction was prevented by N ω-propyl-l-arginine hydrochloride (l-NPA, 1 μM), an nNOS inhibitor or N ω-nitro-l-arginine (l-NA, 100 μM). Both vascular segments were densely innervated with nNOS-positive nitrergic nerves in close apposition to tyrosine hydroxylase-immunoreactive sympathetic nerves. In pericyte-covered pre-capillary arterioles of the mouse bladder where sympathetic nerves were absent, nerve stimulation evoked transient reductions in pericyte Ca2+ levels that were shortened by l-NPA and abolished by l-NA. In SMC-containing arterioles, tadalafil (10 nM) caused a l-NPA-sensitive suppression of sympathetic Ca2+ transients. In mice, nitrergic perivascular nerves were distributed in the arterioles and the pre-capillary arterioles. Conclusion and Implications: Both nitrergic nerve and nerve-evoked endothelial NO release appear to be involved in vasodilatory signal transmission in bladder vasculature. The NO-mediated signal transmission is a potential target for PDE5 inhibitor therapy in bladder dysfunctions.

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Intracellular ATP levels in mouse cortical excitatory neurons varies with sleep–wake states

Natsubori A., Tsunematsu T., Karashima A., Imamura H., Kabe N., Trevisiol A., Hirrlinger J., Kodama T., Sanagi T., Masamoto K., Takata N., Nave K.A., Matsui K., Tanaka K.F., Honda M.

Communications Biology （Communications Biology） 3 （ 1 ） 491 2020年12月

© 2020, The Author(s). Whilst the brain is assumed to exert homeostatic functions to keep the cellular energy status constant under physiological conditions, this has not been experimentally proven. Here, we conducted in vivo optical recordings of intracellular concentration of adenosine 5’-triphosphate (ATP), the major cellular energy metabolite, using a genetically encoded sensor in the mouse brain. We demonstrate that intracellular ATP levels in cortical excitatory neurons fluctuate in a cortex-wide manner depending on the sleep-wake states, correlating with arousal. Interestingly, ATP levels profoundly decreased during rapid eye movement sleep, suggesting a negative energy balance in neurons despite a simultaneous increase in cerebral hemodynamics for energy supply. The reduction in intracellular ATP was also observed in response to local electrical stimulation for neuronal activation, whereas the hemodynamics were simultaneously enhanced. These observations indicate that cerebral energy metabolism may not always meet neuronal energy demands, consequently resulting in physiological fluctuations of intracellular ATP levels in neurons.

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Essential role of microglial transforming growth factor-β1 in antidepressant actions of (R)-ketamine and the novel antidepressant TGF-β1

Zhang K., Yang C., Chang L., Sakamoto A., Suzuki T., Fujita Y., Qu Y., Wang S., Pu Y., Tan Y., Wang X., Ishima T., Shirayama Y., Hatano M., Tanaka K.F., Hashimoto K.

Translational Psychiatry （Translational Psychiatry） 10 （ 1 ） 32 2020年12月

© 2020, The Author(s). In rodent models of depression, (R)-ketamine has greater potency and longer-lasting antidepressant effects than (S)-ketamine; however, the precise molecular mechanisms underlying the antidepressant actions of (R)-ketamine remain unknown. Using RNA-sequencing analysis, we identified novel molecular targets that contribute to the different antidepressant effects of the two enantiomers. Either (R)-ketamine (10 mg/kg) or (S)-ketamine (10 mg/kg) was administered to susceptible mice after chronic social defeat stress (CSDS). RNA-sequencing analysis of prefrontal cortex (PFC) and subsequent GSEA (gene set enrichment analysis) revealed that transforming growth factor (TGF)-β signaling might contribute to the different antidepressant effects of the two enantiomers. (R)-ketamine, but not (S)-ketamine, ameliorated the reduced expressions of Tgfb1 and its receptors (Tgfbr1 and Tgfbr2) in the PFC and hippocampus of CSDS susceptible mice. Either pharmacological inhibitors (i.e., RepSox and SB431542) or neutralizing antibody of TGF-β1 blocked the antidepressant effects of (R)-ketamine in CSDS susceptible mice. Moreover, depletion of microglia by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 blocked the antidepressant effects of (R)-ketamine in CSDS susceptible mice. Similar to (R)-ketamine, the recombinant TGF-β1 elicited rapid and long-lasting antidepressant effects in animal models of depression. Our data implicate a novel microglial TGF-β1-dependent mechanism underlying the antidepressant effects of (R)-ketamine in rodents with depression-like phenotype. Moreover, TGF-β1 and its receptor agonists would likely constitute a novel rapid-acting and sustained antidepressant in humans.

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Diffusion functional MRI reveals global brain network functional abnormalities driven by targeted local activity in a neuropsychiatric disease mouse model

Abe Y., Takata N., Sakai Y., Hamada H.T., Hiraoka Y., Aida T., Tanaka K., Bihan D.L., Doya K., Tanaka K.F.

NeuroImage （NeuroImage） 223 117318 2020年12月

ISSN 10538119

© 2020 The Authors Diffusion functional magnetic resonance imaging (DfMRI) has been proposed as an alternative functional imaging method to detect brain activity without confounding hemodynamic effects. Here, taking advantage of this DfMRI feature, we investigated abnormalities of dynamic brain function in a neuropsychiatric disease mouse model (glial glutamate transporter-knockdown mice with obsessive–compulsive disorder [OCD]-related behavior). Our DfMRI approaches consisted of three analyses: resting state brain activity, functional connectivity, and propagation of neural information. We detected hyperactivation and biased connectivity across the cortico-striatal-thalamic circuitry, which is consistent with known blood oxygen-level dependent (BOLD)-fMRI patterns in OCD patients. In addition, we performed ignition-driven mean integration (IDMI) analysis, which combined activity and connectivity analyses, to evaluate neural propagation initiated from brain activation. This analysis revealed an unbalanced distribution of neural propagation initiated from intrinsic local activation to the global network, while these were not detected by the conventional method with BOLD-fMRI. This abnormal function detected by DfMRI was associated with OCD-related behavior. Together, our comprehensive DfMRI approaches can successfully provide information on dynamic brain function in normal and diseased brains.

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Chd8 mutation in oligodendrocytes alters microstructure and functional connectivity in the mouse brain.

Kawamura A, Abe Y, Seki F, Katayama Y, Nishiyama M, Takata N, Tanaka KF, Okano H, Nakayama KI

Molecular brain （Molecular Brain） 13 （ 1 ） 160 2020年11月

CHD8 encodes a chromatin-remodeling factor and is one of the most recurrently mutated genes in individuals with autism spectrum disorder (ASD). Although we have recently shown that mice heterozygous for Chd8 mutation manifest myelination defects and ASD-like behaviors, the detailed mechanisms underlying ASD pathogenesis have remained unclear. Here we performed diffusion tensor imaging (DTI) and resting-state functional magnetic resonance imaging (rsfMRI) in oligodendrocyte lineage-specific Chd8 heterozygous mutant mice. DTI revealed that ablation of Chd8 specifically in oligodendrocytes of mice was associated with microstructural changes of specific brain regions including the cortex and striatum. The extent of these changes in white matter including the corpus callosum and fornix was correlated with total contact time in the reciprocal social interaction test. Analysis with rsfMRI revealed changes in functional brain connectivity in the mutant mice, and the extent of such changes in the cortex, hippocampus, and amygdala was also correlated with the change in social interaction. Our results thus suggest that changes in brain microstructure and functional connectivity induced by oligodendrocyte dysfunction might underlie altered social interaction in mice with oligodendrocyte-specific CHD8 haploinsufficiency.

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Serotonergic projections to the orbitofrontal and medial prefrontal cortices differentially modulate waiting for future rewards.

Miyazaki K, Miyazaki KW, Sivori G, Yamanaka A, Tanaka KF, Doya K

Science advances （Science Advances） 6 （ 48 ） 2020年11月

Optogenetic activation of serotonergic neurons in the dorsal raphe nucleus (DRN) enhances patience when waiting for future rewards, and this effect is maximized by both high probability and high timing uncertainty of reward. Here, we explored which serotonin projection areas contribute to these effects using optogenetic axon terminal stimulation. We found that serotonin stimulation in the orbitofrontal cortex (OFC) is nearly as effective as that in the DRN for promoting waiting, while in the nucleus accumbens, it does not promote waiting. We also found that serotonin stimulation in the medial prefrontal cortex (mPFC) promotes waiting only when the timing of future rewards is uncertain. Our Bayesian decision model of waiting assumed that the OFC and mPFC calculate the posterior probability of reward delivery separately. These results suggest that serotonin in the mPFC affects evaluation of time committed, while serotonin in the OFC is responsible for overall valuation of delayed rewards.

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Opposing Ventral Striatal Medium Spiny Neuron Activities Shaped by Striatal Parvalbumin-Expressing Interneurons during Goal-Directed Behaviors

Yoshida K., Tsutsui-Kimura I., Kono A., Yamanaka A., Kobayashi K., Watanabe M., Mimura M., Tanaka K.F.

Cell Reports （Cell Reports） 31 （ 13 ） 107829 2020年06月

© 2020 The Authors Medium spiny neurons (MSNs) of mice show opposing activities upon the initiation of a food-seeking lever press task. Ventromedial striatal (VMS)-MSNs are inhibited but ventrolateral striatal (VLS)-MSNs are activated; these activities mediate action selection and action initiation, respectively. To understand what input shapes the opposing MSN activities, here, we monitor cortical input activities at the cell population level and artificially reverse them. We demonstrate that the ventral hippocampus (vHP) and the insular cortex (IC) are major inputs to the VMS and VLS, both projections show silencing at the trial start time, and the vHP-VMS and IC-VLS pathways form functionally coupled input-output units during the task. Of note, the upstream IC silencing is converted to the downstream VLS-MSN activation. We find biased localization of striatal parvalbumin-expressing interneurons (PV INs) and verify PV IN-dependent feedforward architecture in the VLS. Our results reveal a distinct mode of cortico-striatal signal conveyance via feedforward disinhibition in behaving animals.

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Behavioral and electrophysiological evidence for a neuroprotective role of aquaporin-4 in the 5xFAD transgenic mice model

Abe Y., Ikegawa N., Yoshida K., Muramatsu K., Hattori S., Kawai K., Murakami M., Tanaka T., Goda W., Goto M., Yamamoto T., Hashimoto T., Yamada K., Shibata T., Misawa H., Mimura M., Tanaka K.F., Miyakawa T., Iwatsubo T., Hata J.I., Niikura T., Yasui M.

Acta Neuropathologica Communications （Acta Neuropathologica Communications） 8 （ 1 ） 67 2020年05月

© 2020 The Author(s). Aquaporin-4 (AQP4) has been suggested to be involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD), which may be due to the modulation of neuroinflammation or the impairment of interstitial fluid bulk flow system in the central nervous system. Here, we show an age-dependent impairment of several behavioral outcomes in 5xFAD AQP4 null mice. Twenty-four-hour video recordings and computational analyses of their movement revealed that the nighttime motion of AQP4-deficient 5xFAD mice was progressively reduced between 20 and 36 weeks of age, with a sharp deterioration occurring between 30 and 32 weeks. This reduction in nighttime motion was accompanied by motor dysfunction and epileptiform neuronal activities, demonstrated by increased abnormal spikes by electroencephalography. In addition, all AQP4-deficient 5xFAD mice exhibited convulsions at least once during the period of the analysis. Interestingly, despite such obvious phenotypes, parenchymal amyloid β (Aβ) deposition, reactive astrocytosis, and activated microgliosis surrounding amyloid plaques were unchanged in the AQP4-deficient 5xFAD mice relative to 5xFAD mice. Taken together, our data indicate that AQP4 deficiency greatly accelerates an age-dependent deterioration of neuronal function in 5xFAD mice associated with epileptiform neuronal activity without significantly altering Aβ deposition or neuroinflammation in this mouse model. We therefore propose that there exists another pathophysiological phase in AD which follows amyloid plaque deposition and neuroinflammation and is sensitive to AQP4 deficiency.

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Different roles of distinct serotonergic pathways in anxiety-like behavior, antidepressant-like, and anti-impulsive effects

Ohmura Y., Tsutsui-Kimura I., Sasamori H., Nebuka M., Nishitani N., Tanaka K.F., Yamanaka A., Yoshioka M.

Neuropharmacology （Neuropharmacology） 167 107703 2020年05月

ISSN 00283908

© 2019 Elsevier Ltd Serotonergic agents have been widely used for treatment of psychiatric disorders, but the therapeutic effects are insufficient and these drugs often induce severe side effects. We need to specify the distinct serotonergic pathways underlying each mental function to overcome these problems. Preclinical studies have demonstrated that the central serotonergic system is involved in several emotional/cognitive functions including anxiety, depression, and impulse control, but it remains unclear whether each function is regulated by a different serotonergic system. We used optogenetic strategy to increase central serotonergic activity in mice and evaluated the behavioral consequences. Pharmacological and genetic tools were used to determine the subtype of 5-HT receptors responsible for the observed effects. We demonstrated that the serotonergic activation in the median raphe nucleus enhanced anxiety-like behavior, the serotonergic activation in the dorsal raphe nucleus exerted antidepressant-like effects, and the serotonergic activation in the median or dorsal raphe nucleus suppressed impulsive action. We also found that different serotonergic terminals, ventral hippocampus, ventral tegmental area/substantia nigra, and subthalamic/parasubthalamic nucleus, are involved in regulating anxiety-like behavior, antidepressant-like, and anti-impulsive effects, respectively. Furthermore, we found, using triple-transgenic mice, that the stimulation of the 5-HT2C receptor is required to evoke anxiety-like behavior, but not to exert anti-impulsive effects. These results suggest the need for pathway-specific treatments and provide important insights that will help the development of more effective and safer therapeutics. This article is part of the special issue entitled ‘Serotonin Research: Crossing Scales and Boundaries’.

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Identification of Lacrimal Gland Postganglionic Innervation and Its Regulation of Tear Secretion

Jin K., Imada T., Hisamura R., Ito M., Toriumi H., Tanaka K.F., Nakamura S., Tsubota K.

American Journal of Pathology （American Journal of Pathology） 190 （ 5 ） 1068 - 1079 2020年05月

ISSN 00029440

© 2020 American Society for Investigative Pathology Tear fluid secreted from the exocrine lacrimal gland (LG) has an essential role in maintaining a homeostatic environment for a healthy ocular surface. Tear secretion is regulated by the sympathetic and parasympathetic components of the autonomic nervous system, although the contribution of each component is not fully understood. To investigate LG innervation, we identified sympathetic and parasympathetic postganglionic nerves, specifically innervating the mouse LG, by injecting a retrograde neuronal tracer into the LG. Interruption of neural stimuli to the LG by the denervation of these postganglionic nerves immediately and chronically decreased tear secretion, leading to LG atrophy along with destruction of the lobular structure. This investigation also found that parasympathetic, but not sympathetic, innervation was involved in these alterations.

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Direct Impact of Motor Cortical Stimulation on the Blood Oxygen-level Dependent Response in Rats.

Xin Z, Abe Y, Liu S, Tanaka KF, Hosomi K, Saitoh Y, Sekino M

Magnetic resonance in medical sciences : MRMS : an official journal of Japan Society of Magnetic Resonance in Medicine （Magnetic Resonance in Medical Sciences） 20 （ 1 ） 83 - 90 2020年04月

ISSN 1347-3182

Purpose: Neuropathic pain is a complex and distressing chronic illness in modern medicine. Since 1990s, motor cortex stimulation (MCS) has emerged as a potential treatment for chronic neuropathic pain; however, the precise mechanisms underlying analgesia induced by MCS are not completely understood. The purpose of the present study was to investigate the blood oxygen-level dependent (BOLD) response in the brain during MCS. Methods: We inserted a bipolar tungsten electrode into the primary motor cortex (M1) of adult male Wistar rats. Functional magnetic resonance imaging (fMRI) scans were implemented simultaneously with the electrical stimulation of M1 and the BOLD signals taken from the fMRI were used as an index to reflect the response against MCS. Results: Our results demonstrated that the bilateral M1, ipsilateral caudate-putamen, and ipsilateral primary somatosensory cortex to the stimulation spot were activated after the onset of MCS. The BOLD signal time courses were analysed in these regions and similar temporal characteristics were found. Conclusion: By conducting direct cortical stimulation of the rodent brain to investigate its instant effect using fMRI, we identified encephalic regions directly involved in the instant motor cortical stimulation effects in healthy rat models. This result may be essential in establishing a foundation for further research on the underlying neuropathways associated with the MCS effects.

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Activation of ventral CA1 hippocampal neurons projecting to the lateral septum during feeding

Kosugi K., Yoshida K., Suzuki T., Kobayashi K., Yoshida K., Mimura M., Tanaka K.F.

Hippocampus （Hippocampus） 31 （ 3 ） 294 - 304 2020年

ISSN 10509631

© 2020 The Authors. Hippocampus published by Wiley Periodicals LLC A number of studies have reported the involvement of the ventral hippocampus (vHip) and the lateral septum (LS) in negative emotional responses. Besides these well-documented functions, they are also thought to control feeding behavior. In particular, optogenetic and pharmacogenetic interventions to LS-projecting vHip neurons have demonstrated that the vHip→LS neural circuit exerts an inhibition on feeding behavior. However, there have been no reports of vHip neuronal activity during feeding. Here, we focused on LS-projecting vCA1 neurons (vCA1→LS) and monitored their activity during feeding behaviors in mice. vCA1→LS neurons were retrogradely labeled with adeno-associated virus carrying a ratiometric Ca2+ indicator and measured compound Ca2+ dynamics by fiber photometry. We first examined vCA1→LS activity in random food-exploring behavior and found that vCA1→LS activation seemed to coincide with food intake; however, our ability to visually confirm this during freely moving behaviors was not sufficiently reliable. We next examined vCA1→LS activity in a goal-directed, food-seeking lever-press task which temporally divided the mouse state into preparatory, effort, and consummatory phases. We observed vCA1→LS activation in the postprandial period during the consummatory phase. Such timing- and pathway-specific activation was not observed from pan-vCA1 neurons. In contrast, reward omission eliminated this activity, indicating that vCA1→LS activation is contingent on the food reward. Sated mice pressed the lever significantly fewer times but still ate food; however, vCA1→LS neurons were not activated, suggesting that vCA1→LS neurons did not respond to habitual behavior. Combined, these results suggest that gastrointestinal interoception rather than food-intake motions or external sensations are likely to coincide with vCA1→LS activity. Accordingly, we propose that vCA1→LS neurons discriminate between matched or unmatched predictive bodily states in which incoming food will satisfy an appetite. We also demonstrate that vCA1→LS neurons are activated in aversive/anxious situations in an elevated plus maze and tail suspension test. Future behavioral tests utilizing anxious conflict and food intake may reconcile the multiple functions of vCA1→LS neurons.

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Varying perivascular astroglial endfoot dimensions along the vascular tree maintain perivascular-interstitial flux through the cortical mantle

Wang M.X., Ray L., Tanaka K.F., Iliff J.J., Heys J.

GLIA （GLIA） 69 （ 3 ） 715 - 728 2020年

ISSN 08941491

© 2020 Wiley Periodicals LLC The glymphatic system is a recently defined brain-wide network of perivascular spaces along which cerebrospinal fluid (CSF) and interstitial solutes exchange. Astrocyte endfeet encircling the perivascular space form a physical barrier in between these two compartments, and fluid and solutes that are not taken up by astrocytes move out of the perivascular space through the junctions in between astrocyte endfeet. However, little is known about the anatomical structure and the physiological roles of the astrocyte endfeet in regulating the local perivascular exchange. Here, visualizing astrocyte endfoot–endfoot junctions with immunofluorescent labeling against the protein megalencephalic leukoencephalopathy with subcortical cysts-1 (MLC1), we characterized endfoot dimensions along the mouse cerebrovascular tree. We observed marked heterogeneity in endfoot dimensions along vessels of different sizes, and of different types. Specifically, endfoot size was positively correlated with the vessel diameters, with large vessel segments surrounded by large endfeet and small vessel segments surrounded by small endfeet. This association was most pronounced along arterial, rather than venous segments. Computational modeling simulating vascular trees with uniform or varying endfeet dimensions demonstrates that varying endfoot dimensions maintain near constant perivascular-interstitial flux despite correspondingly declining perivascular pressures along the cerebrovascular tree through the cortical depth. These results describe a novel anatomical feature of perivascular astroglial endfeet and suggest that endfoot heterogeneity may be an evolutionary adaptation to maintain perivascular CSF-interstitial fluid exchange through deep brain structures.

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Compartmentalized Input–Output Organization of Lugaro Cells in the Cerebellar Cortex

Miyazaki T., Yamasaki M., Tanaka K.F., Watanabe M.

Neuroscience （Neuroscience） 462 89 - 105 2020年

ISSN 03064522

© 2020 IBRO Purkinje cells (PCs) are principal cerebellar neurons, and several classes of interneurons modulate their activity. Lugaro cells (LCs) are one such inhibitory interneuron with distinctive cytology and location, but still most enigmatic among cerebellar neurons. Here we serendipitously produced a novel transgenic mouse line, where a half of Yellow Cameleon (YC)(+) cells in the cerebellar cortex were judged to be LCs, and YC(+) LCs were estimated to constitute one-third of the total LC populations. Neurochemically, two-thirds of YC(+) LCs were dually GABAergic/glycinergic, with the rest being GABAergic. Beneath the PC layer, they extended a sheet of somatodendritic meshwork interconnected with neighboring LCs by adherens junctions, and received various inputs from climbing fibers, mossy fibers, granule cell axons, recurrent PC axons, Golgi cell axons, LC axons, and serotonergic fibers. Intriguingly, somatodendritic elements of individual LCs preferentially extended within a given cerebellar compartment defined by aldolase C expression. In turn, YC(+) LCs projected a dense lattice of ascending and transverse axons to the molecular layer, and innervated molecular layer interneurons (basket and stellate cells) and Golgi cells, but not PCs. Of note, ascending axons profusely innervated individual targets within a cerebellar compartment, while transverse axons ran across several compartments and innervated targets sparsely. This unique circuit configuration highlights that LCs integrate various excitatory, inhibitory, and modulatory inputs coming to the belonging cerebellar compartment and that, as an interneuron-selective interneuron, LCs can effectively disinhibit cerebellar cortical activities in a compartment-dependent manner through inhibition of inhibitory interneurons selectively targeting PCs and granule cells.

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Enriched environment alleviates stress-induced dry-eye through the BDNF axis

Sano K., Kawashima M., Imada T., Suzuki T., Nakamura S., Mimura M., Tanaka K.F., Tsubota K.

Scientific Reports （Scientific Reports） 9 （ 1 ） 3422 2019年12月

© 2019, The Author(s). The number of patients with dry eye disease (DED) is increasing, and DED has become an urgent public health problem. A comorbidity of mental disorders has been reported in DED patients. We hypothesized that physical and psychological stressors impair tear secretion. To examine the relationship between stress loading and decreased tear secretion, we established a stress-induced DED mouse model, which permitted us to address the underlying mechanism of pathogenesis and resilience. Enriched environment (EE) was an effective intervention to prevent and alleviate stress-induced decreased tear secretion. Because stress loading resulted in decreased brain-derived neurotrophic factor (BDNF) expression while EE resulted in increased expression, we focused on the role of BDNF in tear secretion. Using two distinct Bdnf gene knockdown mice, we evaluated whether BDNF was a deterministic factor in regulating tear secretion in healthy and stressed conditions. Bdnf knockdown mice showed decreased basal tear secretion and loss of stress tolerance by EE for tear secretion. These results suggest that BDNF expression is related to tear secretion and to the pathology of DED.

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The physiology and pathophysiology of basal ganglia: From signal transduction to circuits

Chuhma N., Tanaka K.F., Nagai T.

Neurochemistry International （Neurochemistry International） 131 104544 2019年12月

ISSN 01970186

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Neuroprotective effects of microglial P2Y<inf>1</inf> receptors against ischemic neuronal injury

Fukumoto Y., Tanaka K.F., Parajuli B., Shibata K., Yoshioka H., Kanemaru K., Gachet C., Ikenaka K., Koizumi S., Kinouchi H.

Journal of Cerebral Blood Flow and Metabolism （Journal of Cerebral Blood Flow and Metabolism） 39 （ 11 ） 2144 - 2156 2019年11月

ISSN 0271678X

© The Author(s) 2018. Extracellular ATP, which is released from damaged cells after ischemia, activates P2 receptors. P2Y1 receptors (P2Y1R) have received considerable attention, especially in astrocytes, because their activation plays a central role in the regulation of neuron-to-glia communication. However, the functions or even existence of P2Y1R in microglia remain unknown, despite the fact that many microglial P2 receptors are involved in several brain diseases. Herein, we demonstrate the presence and functional capability of microglial P2Y1R to provide neuroprotective effects following ischemic stress. Cerebral ischemia resulted in increased microglial P2Y1R expression. The number of injured hippocampal neurons was significantly higher in P2Y1 R knockout (KO) mice than wildtype mice after forebrain ischemia. Propidium iodide (PI) uptake, a marker for dying cells, was significantly higher in P2Y1R KO hippocampal slices compared with wildtype hippocampal slices at 48 h after 40-min oxygen–glucose deprivation (OGD). Furthermore, increased PI uptake following OGD was rescued by ectopic overexpression of P2Y1R in microglia. In summary, these data suggest that microglial P2Y1R mediate neuroprotective effects against ischemic stress and OGD insult.

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Translational approach to apathy-like behavior in mice: From the practical point of view

Tanaka K.F., Hamaguchi T.

Psychiatry and Clinical Neurosciences （Psychiatry and Clinical Neurosciences） 73 （ 11 ） 685 - 689 2019年11月

ISSN 13231316

© 2019 The Authors. Psychiatry and Clinical Neurosciences © 2019 Japanese Society of Psychiatry and Neurology Apathy is a pervasive clinical phenomenon that deserves more attention at the translational and pre-clinical levels. To study apathy-like behavior in mice, we relied on an operational definition of apathy: the quantitative reduction of voluntary, goal-directed behaviors. We recently found that the chronic loss of function of a specific cell type (striatal dopamine receptor type 2-expressing medium spiny neurons, D2-MSN) in a restricted region (the ventrolateral striatum, VLS) was sufficient to induce apathy-like behavior in a food-seeking operant task. We further showed that VLS D2-MSN are activated at the preparatory period, and that optogenetic inhibition of VLS D2-MSN during that period resulted in transient decreases in instrumental motivation, strengthening the hypothesis that VLS D2-MSN mediate apathy-like behavior in mice. Mice bearing VLS D2-MSN dysfunction can thus be regarded as an apathy model for future translational studies.

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Detection of a High-Turnover Serotonin Circuit in the Mouse Brain Using Mass Spectrometry Imaging

Sugiyama E., Guerrini M.M., Honda K., Hattori Y., Abe M., Källback P., Andrén P.E., Tanaka K.F., Setou M., Fagarasan S., Suematsu M., Sugiura Y.

iScience （iScience） 20 359 - 372 2019年10月

© 2019 The Author(s) Monoamine neurotransmitters are released by specialized neurons regulating behavioral, motor, and cognitive functions. Although the localization of monoaminergic neurons in the brain is well known, the distribution and kinetics of monoamines remain unclear. Here, we generated a murine brain atlas of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) levels using mass spectrometry imaging (MSI). We found several nuclei rich in both 5-HT and a catecholamine (DA or NE) and identified the paraventricular nucleus of the thalamus (PVT), where 5-HT and NE are co-localized. The analysis of 5-HT fluctuations in response to acute tryptophan depletion and infusion of isotope-labeled tryptophan in vivo revealed a close kinetic association between the raphe nuclei, PVT, and amygdala but not the other nuclei. Our findings imply the existence of a highly dynamic 5-HT-mediated raphe to PVT pathway that likely plays a role in the brain monoamine system.

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AppNL-G-F/NL-G-F mice overall do not show impaired motivation, but cored amyloid plaques in the striatum are inversely correlated with motivation

Hamaguchi T., Tsutsui-Kimura I., Mimura M., Saito T., Saido T.C., Tanaka K.F.

Neurochemistry International （Neurochemistry International） 129 104470 2019年10月

ISSN 01970186

© 2019 Elsevier Ltd Apathy is clinically defined as lack of motivation. Apathy is a frequent symptom in patients with Alzheimer's disease (AD). It is unclear whether amyloid β (Aβ)pathology is associated with apathy. To address this question, we employed the AppNL-G-F/NL-G-F mouse, an Aβ deposition-bearing mouse without neurofibrillary tangles and neuronal cell death throughout the lifespan and used a progressive-ratio (PR)task to monitor instrumental motivation between the ages of 16 and 39 weeks. In the PR task, the number of lever presses to receive one reward increases and the number of active lever presses in the final trial a mouse completes represents a break point, which is an index of motivation. During the observation period, AppNL-G-F/NL-G-F mice overall did not show impaired motivation. However, AppNL-G-F/NL-G-F mice showed a dispersion of the break point at 39 weeks of age within the group. Therefore, we examined the association between the degree of the break point and Aβ pathology; the number of cored amyloid plaques in the striatum was inversely correlated with the degree of motivation. Furthermore, we measured the dopamine transporter (DAT)levels in the subcortical tissues including the striatum using western blot analysis and showed that AppNL-G-F/NL-G-F mice have lower DAT levels than do C57BL/6J mice. Although we could not directly determine the effect of core amyloid plaques on the DAT, the results of this study suggest a pathway through which cored amyloid plaques damage the DAT and cause impaired motivation. These results will draw attention to cored amyloid plaques and will aid researchers searching for new strategies that are effective for the prevention and treatment of impaired motivation.

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大縫線核セロトニン神経による疼痛抑制の光遺伝学的解析

三橋学, 金丸みつ子, 田中謙二, 吉川輝, 稲垣克記, 久光正, 砂川正隆, 泉崎雅彦

昭和学士会雑誌（昭和大学学士会） 79 （ 4 ） 483 - 491 2019年08月

ISSN 2187-719X

延髄大縫線核のセロトニン(5-hydroxytryptamine、5-HT)神経は、下行性疼痛抑制系として鎮痛作用を発揮する。一方で、痛みを増強させるという報告もあり、セロトニン・ノルアドレナリン再取り込み阻害薬の鎮痛薬としての使用が広まるなか、5-HTの疼痛制御に関する検討が必要である。近年、光遺伝学的手法によって大縫線核の5-HT神経を選択的に刺激することが可能になった。本研究では、5-HT系下行性疼痛抑制系の障害が示唆されている間欠的寒冷ストレス(intermittent cold stress、ICS)モデルのマウスを用い、光遺伝学的手法による大縫線核の5-HT神経の選択的刺激が鎮痛作用を発揮するか検討した。青色光照射で大縫線核の5-HT神経を刺激するため、光感受性チャネルを5-HT神経細胞に発現させた遺伝子改変マウス(Tph2-tTA::tetO-ChR2(C128S))に対し、大縫線核直上に光ファイバーを刺入、留置した。このマウスにICSを与えてICS群とし、青色光照射による大縫線核5-HT神経への刺激が疼痛閾値へ与える効果を行動学的手法で評価した。機械刺激性疼痛試験としてvon Frey test、熱刺激性疼痛試験としてHot plate testを用いた。対照群にはSham ICS処置を行った。ICS群とSham ICS処置によるマウス群を比較検討したところ、ICS処置はvon Frey testによる疼痛閾値を低下させた。しかし、遺伝子改変マウスに青色光照射で刺激をしても、von Frey testによる疼痛閾値の変化は認めなかった。一方、Hot plate testで疼痛閾値を評価すると、Sham ICS処置による疼痛閾値の変化とICS処置による疼痛閾値の変化に有意な差はなかった。しかし、曝露処置(ICS処置か、Sham ICS処置か)と時期(処置前か、処置後か)に関わらず、青色光照射で疼痛閾値が上昇した。つまり、ICS処置は、von Frey testによる疼痛閾値を低下させたが、Hot plate testによる疼痛閾値を変化させなかった。一方、青色光照射による大縫線核5-HT神経への刺激は、Hot plate testによる疼痛閾値を上昇させたが、von Frey testによる疼痛閾値を変化させなかった。以上より、大縫線核の5-HT神経への刺激は、熱刺激性疼痛に対する鎮痛作用を発揮した。一方、ICS処置で機械刺激性疼痛に対する疼痛閾値は低下したが、その機序に大縫線核の5-HT神経の積極的な関与は示唆されなかった。(著者抄録)

Region- and cell type-specific facilitation of synaptic function at destination synapses induced by oligodendrocyte depolarization

Yamazaki Y., Abe Y., Shibata S., Shindo T., Fujii S., Ikenaka K., Tanaka K.F.

Journal of Neuroscience （Journal of Neuroscience） 39 （ 21 ） 4036 - 4050 2019年05月

ISSN 02706474

© 2019 the authors. The axonal conduction of action potentials affects the absolute time it takes to transmit nerve impulses as well as temporal summation at destination synapses. At the physiological level, oligodendrocyte depolarization facilitates axonal conduction along myelinated fibers in the hippocampus; however, the functional significance of this facilitation is largely unknown. In this study, we examined the physiology of the facilitation of axonal conduction by investigating the changes in synaptic responses at destination synapses using male and female mice in which channelrhodopsin-2 expression was restricted to oligodendrocytes. The subiculum, one of the projection areas of the examined axons at the alveus of the hippocampus, is divided into three regions (proximal, mid, and distal) and contains two types of principal neurons: regular firing and bursting pyramidal cells.Wefound a significant increase in excitatory synaptic responses following optogenetic oligodendrocyte depolarization in bursting neurons at two of the three regions, but not in regular firing neurons at any region. The long-term potentiation (LTP) induced by theta burst stimulation at the synapses showing a significant increase was also enhanced after oligodendrocyte depolarization. Conversely, the reduction of oligodendrocyte depolarization during theta burst stimulation, which was achieved by photostimulation of archaerhodopsin-T expressed selectively on oligodendrocytes, reduced the magnitude of LTP. These results show that oligodendrocyte depolarization contributes to the fine control of synaptic activity between the axons they myelinate and targets subicular cells in a region- and cell type-specific manner, and suggest that oligodendrocyte depolarization during conditioning of stimuli is involved in the induction of LTP.

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Serotonin-mediated inhibition of ventral hippocampus is required for sustained goal-directed behavior

Yoshida K., Drew M., Mimura M., Tanaka K.

Nature Neuroscience （Nature Neuroscience） 22 （ 5 ） 770 - 777 2019年05月

ISSN 10976256

© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. The ability to sustain goal-directed action is essential for success in many domains, but little is known about the corresponding neural substrates. Using fiber photometry to monitor population neural activity, we demonstrate that engagement in sustained food- or punishment-motivated behavior is associated with suppression of ventral but not dorsal hippocampal activity. Using optogenetic stimulation, we demonstrate that this suppression is required for goal-directed behavior, whereas optogenetic suppression of the ventral hippocampus (vHP) enhances the ability to sustain goal-directed behavior. Suppression of vHP during sustained goal-directed behavior was accompanied by increased activity in median but not dorsal raphe, implicating serotonergic signaling through Htr3a as a mechanism of vHP suppression during successful goal-directed behavior. Sustainment of goal-directed action may require suppression of vHP because of the structure’s well-documented role in behavioral inhibition.

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Correlative study using structural MRI and super-resolution microscopy to detect structural alterations induced by long-term optogenetic stimulation of striatal medium spiny neurons

Abe Y., Komaki Y., Seki F., Shibata S., Okano H., Tanaka K.F.

Neurochemistry International （Neurochemistry International） 125 163 - 174 2019年05月

ISSN 01970186

© 2019 Elsevier Ltd Striatal medium spiny neurons (MSNs) control motor function. Hyper- or hypo-activity of MSNs coincides with basal ganglia-related movement disorders. Based on the assumption that lasting alterations in neuronal activity lead to structural changes in the brain, understanding these structural alterations may be used to infer MSN functional abnormalities. To infer MSN function from structural data, understanding how long-lasting alterations in MSN activity affect brain morphology is essential. To address this, we utilized a simplified model of functional induction by stimulating MSNs expressing channelrhodopsin 2 (ChR2). Subsequent structural alterations which induced long-term activity changes in these MSNs were investigated in the striatal pathway and its associated regions by diffusion tensor imaging (DTI) and histological assessment with super-resolution microscopy. DTI detected changes in the striatum, substantia nigra, and motor cortex. Histological assessment found a reduction in the diameter of myelinated cortical axons as well as MSN dendrites and axons. The structural changes showed a high correlation between DTI parameters and histological data. These results demonstrated that long-term neural activation in the MSNs alters the diameter of MSN and cortical neurons fibers. This study provides a tool for understanding the causal relationship between functional and structural alterations.

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Loss of adult 5-HT1A autoreceptors results in a paradoxical anxiogenic response to antidepressant treatment

Turcotte-Cardin V., Vahid-Ansari F., Luckhart C., Daigle M., Geddes S.D., Tanaka K.F., Hen R., James J., Merali Z., Béïque J.C., Albert P.R.

Journal of Neuroscience （Journal of Neuroscience） 39 （ 8 ） 1334 - 1346 2019年02月

ISSN 02706474

© 2019 the authors. Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are first-line antidepressants but require several weeks to elicit their actions. Chronic SSRI treatment induces desensitization of 5-HT1A autoreceptors to enhance 5-HT neurotransmission. Mice (both sexes) with gene deletion of 5-HT1A autoreceptors in adult 5-HT neurons (1AcKO) were tested for response to SSRIs. Tamoxifen-induced recombination in adult 1AcKO mice specifically reduced 5-HT1A autoreceptor levels. The 1AcKO mice showed a loss of 5-HT1A autoreceptor-mediated hypothermia and electrophysiological responses, but no changes in anxiety-or depression-like behavior. Subchronic fluoxetine (FLX) treatment induced an unexpected anxiogenic effect in 1AcKO mice in the novelty suppressed feeding and elevated plus maze tests, as did escitalopram in the novelty suppressed feeding test. No effect was seen in wild-type (WT) mice. Subchronic FLX increased 5-HT metabolism in prefrontal cortex, hippocampus, and raphe of 1AcKO but not WT mice, suggesting hyperactivation of 5-HT release. To detect chronic cellular activation, FosB + cells were quantified. FosB + cells were reduced in entorhinal cortex and hippocampus (CA2/3) and increased in dorsal raphe 5-HT cells of 1AcKO mice, suggesting increased raphe activation. In WT but not 1AcKO mice, FLX reduced FosB + cells in the median raphe, hippocampus, entorhinal cortex, and median septum, which receive rich 5-HT projections. Thus, in the absence of 5-HT1A autoreceptors, SSRIs induce a paradoxical anxiogenic response. This may involve imbalance in activation of dorsal and median raphe to regulate septohippocampal or fimbria-fornix pathways. These results suggest that markedly reduced 5-HT1A autoreceptors may provide a marker for aberrant response to SSRI treatment.

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Cathepsin C modulates myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis

Durose W., Shimizu T., Li J., Abe M., Sakimura K., Chetsawang B., Tanaka K., Suzumura A., Tohyama K., Ikenaka K.

Journal of Neurochemistry （Journal of Neurochemistry） 148 （ 3 ） 413 - 425 2019年02月

ISSN 00223042

© 2018 International Society for Neurochemistry Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated inflammation, which attacks the myelin sheath. MS pursues a relapsing and remitting course with varying intervals between symptoms. The main clinical pathological features include inflammation, myelin sheath destruction and plaque formation in the central nervous system (CNS). We previously reported that cystatin F (CysF) expression is induced in demyelinating lesions that are accompanied by active remyelination (referred to as shadow plaques) but is down-regulated in chronic demyelinated lesions (plaques) in the spinal cord of MS patients and in several murine models of demyelinating disease. CysF is a cathepsin protease inhibitor whose major target is cathepsin C (CatC), which is co-expressed in demyelinating regions in Plp 4e/− mice, a model of chronic demyelination. Here, we report the time course of CatC and CysF expression and describe the symptoms in a mouse experimental autoimmune encephalomyelitis (EAE) model using CatC knockdown (KD) and CatC over-expression (OE) mice. In myelin oligodendrocyte glycoprotein (MOG)-EAE, CatC positive cells were found to infiltrate the CNS at an early stage prior to any clinical signs, in comparison to WT mice. CysF expression was not observed at this early stage, but appeared later within shadow plaques. CatC expression was found in chronic demyelinated lesions but was not associated with CysF expression, and CatCKD EAE mouse showed delayed demyelination. Whereas, CatCOE in microglia significantly increased severity of demyelination in the MOG-EAE model. Thus, these results demonstrate that CatC plays a major role in MOG-EAE. (Figure presented.).

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Mechanical regulation of oligodendrocyte morphology and maturation by the mechanosensor p130Cas

Shimizu T., Osanai Y., Tanaka K., Thai T., Abe M., Natsume R., Sakimura K., Ikenaka K.

Journal of Neurochemistry （Journal of Neurochemistry） 150 （ 2 ） 158 - 172 2019年

ISSN 00223042

© 2018 International Society for Neurochemistry Oligodendrocytes (OLs) are myelinating cells of the central nervous system. Recent studies have shown that mechanical factors influence various cell properties. Mechanical stimulation can be transduced into intracellular biochemical signals through mechanosensors, such as integrin, p130Cas, talin and vinculin. However, the molecular mechanisms underlying the mechanical regulation of OLs by mechanosensors remain largely unknown. We found that morphology of OL was affected by knockdown of the mechanosensors p130Cas or talin1. Stretching of OL precursor cells induced the phosphorylation of p130Cas and talin-associated assembly of vinculin. Shear stress decreased the number of OL processes, whereas these effects were mechanically suppressed by dominant-negative (DN) p130Cas, but not by DN-talin1. To investigate the roles of p130Cas in post-natal OLs in vivo, we constructed a novel p130Cas knock-in mouse and found overexpression of p130Cas in vivo affected the number of mature OLs in the cortex. These results indicate that the mechanosensor p130Cas controls both OL morphogenesis and maturation. (Figure presented.).

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Reward probability and timing uncertainty alter the effect of dorsal raphe serotonin neurons on patience

Miyazaki K., Miyazaki K., Yamanaka A., Tokuda T., Tanaka K., Doya K.

Nature Communications （Nature Communications） 9 （ 1 ） 2048 2018年12月

© 2018 The Author(s). Recent experiments have shown that optogenetic activation of serotonin neurons in the dorsal raphe nucleus (DRN) in mice enhances patience in waiting for future rewards. Here, we show that serotonin effect in promoting waiting is maximized by both high probability and high timing uncertainty of reward. Optogenetic activation of serotonergic neurons prolongs waiting time in no-reward trials in a task with 75% food reward probability, but not with 50 or 25% reward probabilities. Serotonin effect in promoting waiting increases when the timing of reward presentation becomes unpredictable. To coherently explain the experimental data, we propose a Bayesian decision model of waiting that assumes that serotonin neuron activation increases the prior probability or subjective confidence of reward delivery. The present data and modeling point to the possibility of a generalized role of serotonin in resolving trade-offs, not only between immediate and delayed rewards, but also between sensory evidence and subjective confidence.

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Visualization of myelinated fiber bundles orientation during brain slice preparation by reflection polarized light microscopy

Takata N., Kusayanagi K., Maruyama H., Eitai K., Abo M., Akiyama F., Tajima R., Inaba S., Tanaka K., Mimura M., Sanaka K.

Microscopy Research and Technique （Microscopy Research and Technique） 81 （ 12 ） 1366 - 1373 2018年12月

ISSN 1059910X

© 2018 Wiley Periodicals, Inc. Polarized light imaging (PLI) enables detecting the orientation of myelinated axon bundles in brain slices at microscopic resolution without histological staining. However, standard PLI requires labor-intensive procedures such as mounting brain cryosections on slide glasses. We developed an optical system that does not require a mounting procedure for PLI. Specifically, we developed an optical system to perform PLI in reflection mode (rPLI) instead of employing transmitted light as in standard PLI. We integrated this rPLI system with a conventional vibratome slicer whose cutting blade surface is a mirror. This combination allowed PLI measurements directly during the slicing procedure at room temperature. Thus, mounting procedure for PLI is not necessary. As a proof-of-concept experiment, a perfusion-fixed brain of a mouse was embedded in gelatin-containing agar and cut serially at 40~200 μm intervals. The slicing procedure was temporarily halted after each cut to capture the PLI images of the slice on the reflecting blade surface while the slice was still held up by the agar block. The orientation of the fiber bundle estimated with this method agreed with the results obtained from previous reports. Combination of a popular vibratome slicer and our rPLI system that uses versatile and inexpensive optical components would increase popularity of PLI and facilitates connectome studies at microscopic resolution. Research Highlights: Polarized light imaging (PLI) of brain slices was realized by using reflected light (rPLI) instead of transmitted light. The rPLI method allows detecting the myelinated fiber bundle orientation during slice preparation.

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Ectopic positioning of Bergmann glia and impaired cerebellar wiring in Mlc1-over-expressing mice

Kikuchihara S., Sugio S., Tanaka K., Watanabe T., Kano M., Yamazaki Y., Watanabe M., Ikenaka K.

Journal of Neurochemistry （Journal of Neurochemistry） 147 （ 3 ） 344 - 360 2018年11月

ISSN 00223042

© 2018 International Society for Neurochemistry Mlc1 is a causative gene for megalencephalic leukoencephalopathy with subcortical cysts, and is expressed in astrocytes. Mlc1-over-expressing mice represent an animal model of early-onset leukoencephalopathy, which manifests as astrocytic swelling followed by myelin membrane splitting in the white matter. It has been previously reported that Mlc1 is highly expressed in Bergmann glia, while the cerebellar phenotypes of Mlc1-over-expressing mouse have not been characterized. Here, we examined the cerebellum of Mlc1-over-expressing mouse and found that the distribution of Bergmann glia (BG) was normally compacted along the Purkinje cell (PC) layer until postnatal day 10 (P10), while most BG were dispersed throughout the molecular layer by P28. Ectopic BG were poorly wrapped around somatodendritic elements of PCs and exhibited reduced expression of the glutamate transporter glutamate-aspartate transporter. Extraordinarily slow and small climbing fiber (CF)-mediated excitatory post-synaptic currents, which are known to be elicited under accelerated glutamate spillover, emerged at P20-P28 when BG ectopia was severe, but not at P9-P12 when ectopia was mild. Furthermore, maturation of CF wiring, which translocates the site of innervation from somata to proximal dendrites, was also impaired. Manipulations that restricted the Mlc1-over-expressing period successfully generated mice with and without BG ectopia, depending on the over-expressing period. Together, these findings suggest that there is a critical time window for mechanisms that promote the positioning of BG in the PC layer. Once normal positioning of BG is affected, the differentiation of BG is impaired, leading to insufficient glial wrapping, exacerbated glutamate spillover, and aberrant synaptic wiring in PCs. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/. (Figure presented.). Cover Image for this issue: doi: 10.1111/jnc.14199.

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Heparan Sulfate Organizes Neuronal Synapses through Neurexin Partnerships

Zhang P., Lu H., Peixoto R., Pines M., Ge Y., Oku S., Siddiqui T., Xie Y., Wu W., Archer-Hartmann S., Yoshida K., Tanaka K., Aricescu A., Azadi P., Gordon M., Sabatini B., Wong R., Craig A.

Cell （Cell） 174 （ 6 ） 1450 - 1464.e23 2018年09月

ISSN 00928674

© 2018 Elsevier Inc. Synapses are fundamental units of communication in the brain. The prototypical synapse-organizing complex neurexin-neuroligin mediates synapse development and function and is central to a shared genetic risk pathway in autism and schizophrenia. Neurexin's role in synapse development is thought to be mediated purely by its protein domains, but we reveal a requirement for a rare glycan modification. Mice lacking heparan sulfate (HS) on neurexin-1 show reduced survival, as well as structural and functional deficits at central synapses. HS directly binds postsynaptic partners neuroligins and LRRTMs, revealing a dual binding mode involving intrinsic glycan and protein domains for canonical synapse-organizing complexes. Neurexin HS chains also bind novel ligands, potentially expanding the neurexin interactome to hundreds of HS-binding proteins. Because HS structure is heterogeneous, our findings indicate an additional dimension to neurexin diversity, provide a molecular basis for fine-tuning synaptic function, and open therapeutic directions targeting glycan-binding motifs critical for brain development. Neurexins, major synaptic-organizing proteins, are heparan sulfate (HS) proteoglycans, and HS modification is required for neurexin functions in synaptic transmission, development, and behavior.

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5-HT<inf>3</inf> antagonists decrease discounting rate without affecting sensitivity to reward magnitude in the delay discounting task in mice

Mori M., Tsutsui-Kimura I., Mimura M., Tanaka K.

Psychopharmacology （Psychopharmacology） 235 （ 9 ） 2619 - 2629 2018年09月

ISSN 00333158

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. Rationale: Impulsive choice has often been evaluated in rodents according to the proportion of choices for the delayed large magnitude reinforcer (%large choice) in a delay-discounting task (DDT). However, because %large choice is influenced by both sensitivity to reinforcer magnitude and sensitivity to delayed reinforcement (i.e., discounting rate), distinctively evaluating such discounting parameters represents a critical issue demanding methods to determine each parameter in rats. The serotonin (5-HT) system is well known to be involved in impulsive choice; nevertheless, only a few studies have distinguished discounting parameters and investigated how 5-HT modulators affect discounting rate. Objective: Here, we performed a discounting parameter analysis in mice and examined the effects of various 5-HT modulators on discounting rate. Methods: We set up DDTs with different delay schedules to determine which schedule could address delay-discounting rates in mice. We examined the effect of the following drugs on impulsive choice: a 5-HT reuptake inhibitor (paroxetine), a 5-HT1A receptor agonist (8-OH-DPAT), and two 5-HT3 receptor antagonists (granisetron and ondansetron). Results: Mice showed typical delay discounting at the shorter delay schedules (up to 4 s delay). The %large choice under shorter, but not longer, schedules followed an exponential function and allowed us to derive discounting rates. We selected a DDT with a 4-s delay schedule for further experiments. Granisetron and ondansetron, but not paroxetine or 8-OH-DPAT, decreased discounting rates without affecting sensitivity to reinforcer magnitude. Conclusion: We found that a method to calculate discounting rates in rats is also applicable to mouse models. We also provided evidence that 5-HT3 antagonism controls impulsive choice in mice.

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Optogenetic astrocyte activation evokes BOLD fMRI response with oxygen consumption without neuronal activity modulation

Takata N., Sugiura Y., Yoshida K., Koizumi M., Hiroshi N., Honda K., Yano R., Komaki Y., Matsui K., Suematsu M., Mimura M., Okano H., Tanaka K.

GLIA （GLIA） 66 （ 9 ） 2013 - 2023 2018年09月

ISSN 08941491

© 2018 Wiley Periodicals, Inc. Functional magnetic resonance imaging (fMRI) based on the blood oxygenation level-dependent (BOLD) signal has been used to infer sites of neuronal activation in the brain. A recent study demonstrated, however, unexpected BOLD signal generation without neuronal excitation, which led us to hypothesize the presence of another cellular source for BOLD signal generation. Collective assessment of optogenetic activation of astrocytes or neurons, fMRI in awake mice, electrophysiological measurements, and histochemical detection of neuronal activation, coherently suggested astrocytes as another cellular source. Unexpectedly, astrocyte-evoked BOLD signal accompanied oxygen consumption without modulation of neuronal activity. Imaging mass spectrometry of brain sections identified synthesis of acetyl-carnitine via oxidative glucose metabolism at the site of astrocyte-, but not neuron-evoked BOLD signal. Our data provide causal evidence that astrocytic activation alone is able to evoke BOLD signal response, which may lead to reconsideration of current interpretation of BOLD signal as a marker of neuronal activation.

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Calcium Transient Dynamics of Neural Ensembles in the Primary Motor Cortex of Naturally Behaving Monkeys

Kondo T., Saito R., Otaka M., Yoshino-Saito K., Yamanaka A., Yamamori T., Watakabe A., Mizukami H., Schnitzer M., Tanaka K., Ushiba J., Okano H.

Cell Reports （Cell Reports） 24 （ 8 ） 2191 - 2195.e4 2018年08月

© 2018 The Authors To understand brain circuits of cognitive behaviors under natural conditions, we developed techniques for imaging neuronal activities from large neuronal populations in the deep layer cortex of the naturally behaving common marmoset. Animals retrieved food pellets or climbed ladders as a miniature fluorescence microscope monitored hundreds of calcium indicator-expressing cortical neurons in the right primary motor cortex. This technique, which can be adapted to other brain regions, can deepen our understanding of brain circuits by facilitating longitudinal population analyses of neuronal representation associated with cognitive naturalistic behaviors and their pathophysiological processes. Kondo et al. demonstrate calcium imaging from neuronal populations using a miniature fluorescence microscope in naturally behaving non-human primates (NHPs), common marmosets. This technique marks an advance beyond methods that use fixed head positioning, which limits the study of complex, self-determined behaviors such as social interactions or fear and anxiety.

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Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents

Tainaka K., Murakami T., Susaki E., Shimizu C., Saito R., Takahashi K., Hayashi-Takagi A., Sekiya H., Arima Y., Nojima S., Ikemura M., Ushiku T., Shimizu Y., Murakami M., Tanaka K., Iino M., Kasai H., Sasaoka T., Kobayashi K., Miyazono K., Morii E., Isa T., Fukayama M., Kakita A., Ueda H.

Cell Reports （Cell Reports） 24 （ 8 ） 2196 - 2210.e9 2018年08月

© 2018 The Author(s) We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues. Tainaka et al. describe a strategy for developing hydrophilic tissue-clearing reagents based on comprehensive chemical profiling. The strategic integration of optimal chemical cocktails provided a series of CUBIC protocols, which enable the 3D imaging of the mammalian body and large primate and human tissues.

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Anti-Depressant Fluoxetine Reveals its Therapeutic Effect Via Astrocytes

Kinoshita M., Hirayama Y., Fujishita K., Shibata K., Shinozaki Y., Shigetomi E., Takeda A., Le H., Hayashi H., Hiasa M., Moriyama Y., Ikenaka K., Tanaka K., Koizumi S.

EBioMedicine （EBioMedicine） 32 72 - 83 2018年06月

© 2018 The Authors Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y 11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.

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Aberrant astrocyte Ca 2+ signals “AxCa signals” exacerbate pathological alterations in an Alexander disease model

Saito K., Shigetomi E., Yasuda R., Sato R., Nakano M., Tashiro K., Tanaka K., Ikenaka K., Mikoshiba K., Mizuta I., Yoshida T., Nakagawa M., Mizuno T., Koizumi S.

GLIA （GLIA） 66 （ 5 ） 1053 - 1067 2018年05月

ISSN 08941491

© 2018 Wiley Periodicals, Inc. Alexander disease (AxD) is a rare neurodegenerative disorder caused by gain of function mutations in the glial fibrillary acidic protein (GFAP) gene. Accumulation of GFAP proteins and formation of Rosenthal fibers (RFs) in astrocytes are hallmarks of AxD. However, malfunction of astrocytes in the AxD brain is poorly understood. Here, we show aberrant Ca 2+ responses in astrocytes as playing a causative role in AxD. Transcriptome analysis of astrocytes from a model of AxD showed age-dependent upregulation of GFAP, several markers for neurotoxic reactive astrocytes, and downregulation of Ca 2+ homeostasis molecules. In situ AxD model astrocytes produced aberrant extra-large Ca 2+ signals “AxCa signals”, which increased with age, correlated with GFAP upregulation, and were dependent on stored Ca 2+ . Inhibition of AxCa signals by deletion of inositol 1,4,5-trisphosphate type 2 receptors (IP3R2) ameliorated AxD pathogenesis. Taken together, AxCa signals in the model astrocytes would contribute to AxD pathogenesis.

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A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing

Murakami T., Mano T., Saikawa S., Horiguchi S., Shigeta D., Baba K., Sekiya H., Shimizu Y., Tanaka K., Kiyonari H., Iino M., Mochizuki H., Tainaka K., Ueda H.

Nature Neuroscience （Nature Neuroscience） 21 （ 4 ） 625 - 637 2018年04月

ISSN 10976256

© 2018 The Author(s). A three-dimensional single-cell-resolution mammalian brain atlas will accelerate systems-level identification and analysis of cellular circuits underlying various brain functions. However, its construction requires efficient subcellular-resolution imaging throughout the entire brain. To address this challenge, we developed a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion protocol based on an aqueous chemical solution (CUBIC-X). The expanded, well-cleared brain enabled us to construct a point-based mouse brain atlas with single-cell annotation (CUBIC-Atlas). CUBIC-Atlas reflects inhomogeneous whole-brain development, revealing a significant decrease in the cerebral visual and somatosensory cortical areas during postnatal development. Probabilistic activity mapping of pharmacologically stimulated Arc-dVenus reporter mouse brains onto CUBIC-Atlas revealed the existence of distinct functional structures in the hippocampal dentate gyrus. CUBIC-Atlas is shareable by an open-source web-based viewer, providing a new platform for whole-brain cell profiling.

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日中の運動によりもたらされる「良質な睡眠」の脳内機序の解明　脳内アストロサイトの細胞内エネルギー状態に着目して

夏堀晃世, 辛島彰洋, 山本正道, 小島崇, 児玉亨, 高田則雄, 田中謙二, 本多真

若手研究者のための健康科学研究助成成果報告書（(公財)明治安田厚生事業団）（ 33 ） 88 - 92 2018年04月

Near-infrared deep brain stimulation via upconversion nanoparticle–mediated optogenetics

Chen S., Weitemier A., Zeng X., He L., Wang X., Tao Y., Huang A., Hashimotodani Y., Kano M., Iwasaki H., Parajuli L., Okabe S., Loong Teh D., All A., Tsutsui-Kimura I., Tanaka K., Liu X., McHugh T.

Science （Science） 359 （ 6376 ） 679 - 684 2018年02月

ISSN 00368075

2017 © The Authors. Optogenetics has revolutionized the experimental interrogation of neural circuits and holds promise for the treatment of neurological disorders. It is limited, however, because visible light cannot penetrate deep inside brain tissue. Upconversion nanoparticles (UCNPs) absorb tissue-penetrating near-infrared (NIR) light and emit wavelength-specific visible light. Here, we demonstrate that molecularly tailored UCNPs can serve as optogenetic actuators of transcranial NIR light to stimulate deep brain neurons. Transcranial NIR UCNP-mediated optogenetics evoked dopamine release from genetically tagged neurons in the ventral tegmental area, induced brain oscillations through activation of inhibitory neurons in the medial septum, silenced seizure by inhibition of hippocampal excitatory cells, and triggered memory recall. UCNP technology will enable less-invasive optical neuronal activity manipulation with the potential for remote therapy.

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Role of purinergic receptor P2Y1 in spatiotemporal Ca2+ dynamics in astrocytes

Shigetomi E., Hirayama Y., Ikenaka K., Tanaka K., Koizumi S.

Journal of Neuroscience （Journal of Neuroscience） 38 （ 6 ） 1383 - 1395 2018年02月

ISSN 02706474

© 2018 the authors. Fine processes of astrocytes enwrap synapses and are well positioned to sense neuronal information via synaptic transmission. In rodents, astrocyte processes sense synaptic transmission via Gq-protein coupled receptors (GqPCR), including the P2Y1 receptor (P2Y1R), to generate Ca2+signals. Astrocytes display numerous spontaneous microdomain Ca2+signals; however, it is not clear whether such signals are due to local synaptic transmission and/or in what timeframe astrocytes sense local synaptic transmission. To ask whether GqPCRs mediate microdomain Ca2+ signals, we engineered mice (both sexes) to specifically overexpress P2Y1Rs in astrocytes, and we visualized Ca2+ signals via a genetically encoded Ca2+ indicator, GCaMP6f, in astrocytes from adult mice. Astrocytes overexpressing P2Y1Rs showed significantly larger Ca2+ signals in response to exogenously applied ligand and to repetitive electrical stimulation of axons compared with controls. However, we found no evidence of increased microdomain Ca2+signals. Instead, Ca2+waves appeared and propagated to occupy areas that were up to 80-fold larger than microdomain Ca2+ signals. These Ca2+ waves accounted for only 2% of total Ca2+ events, but they were 1.9-fold larger and 2.9-fold longer in duration than microdomain Ca2+ signals at processes. Ca2+ waves did not require action potentials for their generation and occurred in a probenecid-sensitive manner, indicating that the endogenous ligand for P2Y1R is elevated independently of synaptic transmission. Our data suggest that spontaneous microdomain Ca2+ signals occur independently of P2Y1R activation and that astrocytes may not encode neuronal information in response to synaptic transmission at a point source of neurotransmitter release.

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Amplitude and frequency feature extraction of neural activity in mouse ventrolateral striatum under different motivational states using fiber photometric system

Imai S., Mitsukura Y., Yoshida K., Tsutsui-Kimura I., Takata N., Tanaka K.

2017 IEEE Signal Processing in Medicine and Biology Symposium, SPMB 2017 - Proceedings （2017 IEEE Signal Processing in Medicine and Biology Symposium, SPMB 2017 - Proceedings） 2018-January 1 - 6 2018年01月

ISSN 9781538648735

© 2017 IEEE. In this paper, we focused on the motivation of mice and aimed to extract neural activity features of D2 medium spiny neurons (D2-MSNs) in the ventrolateral striatum of mice under different motivational states. Motivated behavior is defined as the activation of goal-directed behavior, and this enables actions such as ingestion, sleeping and reproduction, which are essential for living. In human society, motivation allows us to participate in society, improving our quality of life. Loss of motivation has been causing problems such as withdrawal from society. If the symptom is heavy, it can even threaten our lives, and treatment is necessary. The mechanisms which lead to loss of motivation are yet to be understood, and an effective treatment does not exist. To solve these problems, we decided to find features in the neural activity which affect motivation, because understanding the mechanism may contribute to the establishment of treatment. In the experiment, the neural activity was recorded using gene-encoded ratio metric calcium ion (Ca2+) indicator and by constructing a fiber photometric system, which enabled recording of neural activity at specific brain region for specific type of neurons. While the recording took place, mice performed food-incentive leverpressing tasks, which were used to define motivational states. The experimental results show that both the amplitude and frequency components of Ca2+ fluctuation have features which are good predictors of motivational states.

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Hippocampal activity during serotonergic neuronal activity manipulation using optogenetics

Kondo D., Mitsukura Y., Yoshida K., Tsutsui-Kimura I., Takata N., Tanaka K.

ISSN 9781538648735

© 2017 IEEE. In order to improve brain dysfunction such as mental disease, it is necessary to clarify the mechanism of activity in the brain. To clarify the relationship between the brain region and the neurotransmitter is important for elucidating the brain. Especially investigating the relationship between serotonin and hippocampus leads to elucidate of the mechanism of depression and sleep. The technique called optogenetics enabled only specific cells to be manipulated by light stimulation in recent years. This technique is a useful technique for clarifying the function of the brain. Because instead of stimulating multiple cells such as electrical stimulation, optogenetics can stimulate only specific cells, so that its function can be clarified. This study focused on neurotransmitter serotonin and the brain region called the hippocampus. For these purpose we can clarify the relationship between serotonin and hippocampus by manipulating serotonin neural activity using optogenetics while invasively measuring the ventral hippocampus of the mouse. In addition, identification of pyramidal cell layer is necessary to clarify hippocampal activity. Currently, visual estimation is performed based on the histology that dissected the brain, it still has a problem. Now this technique can't estimate ambiguous of the brain part and measure in an online manner. For this problem, we focus on the clarity of the brain part estimation when we used optogenetics. Therefore, we could estimate the pyramidal cell layer of the hippocampus by analyzing nerve activity and thought that the effect of serotonin activation or suppression on the hippocampus could be clarified. As a result, we showed that pyramidal cell layer can be identified by using principal component analysis, and could clarify the variation of nerve activity in the pyramidal cell layer of hippocampus during serotonin operation.

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Abrogated freud-1/Cc2d1a repression of 5-HT1A autoreceptors induces fluoxetine-resistant anxiety/ depression-like behavior

Vahid-Ansari Faranak, Daigle Mireille, Manzini M. Chiara, Tanaka Kenji F., Hen René, Geddes Sean D., Béïque Jean Claude, James Jonathan, Merali Zul, Albert Paul R.

Journal of Neuroscience 37 （ 49 ） 11967 - 11978 2017年12月

ISSN 0270-6474

Freud-1/Cc2d1a represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo, we generated mice with adulthood conditional knock-out of Freud-1 in 5-HT neurons (cF1ko). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knock-out (cF1/1A dko) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behavior was seen upon knock-out of Freud-1 on the 5-HT1A autoreceptor-negative background; rather, a reduction in depression-like behavior emerged. These studies implicate transcriptional dysregulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors, such as Freud-1, to restore transcriptional balance may augment response to antidepressant treatment.

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Time-controllable Nkcc1 knockdown replicates reversible hearing loss in postnatal mice

Watabe Takahisa, Xu Ming, Watanabe Miho, Nabekura Junichi, Higuchi Taiga, Hori Karin, Sato Mitsuo P., Nin Fumiaki, Hibino Hiroshi, Ogawa Kaoru, Masuda Masatsugu, Tanaka Kenji F.

Scientific Reports 7 （ 1 ） 2017年12月

ISSN 2045-2322

Identification of the causal effects of specific proteins on recurrent and partially reversible hearing loss has been difficult because of the lack of an animal model that provides reversible gene knockdown. We have developed the transgenic mouse line Actin-tTS::Nkcc1^tetO/tetO for manipulatable expression of the cochlear K⁺ circulation protein, NKCC1. Nkcc1 transcription was blocked by the binding of a tetracycline-dependent transcriptional silencer to the tetracycline operator sequences inserted upstream of the Nkcc1 translation initiation site. Administration of the tetracycline derivative doxycycline reversibly regulated Nkcc1 knockdown. Progeny from pregnant/lactating mothers fed doxycycline-free chow from embryonic day 0 showed strong suppression of Nkcc1 expression (∼90% downregulation) and Nkcc1 null phenotypes at postnatal day 35 (P35). P35 transgenic mice from mothers fed doxycycline-free chow starting at P0 (delivery) showed weaker suppression of Nkcc1 expression (∼70% downregulation) and less hearing loss with mild cochlear structural changes. Treatment of these mice at P35 with doxycycline for 2 weeks reactivated Nkcc1 transcription to control levels and improved hearing level at high frequency; i.e., these doxycycline-treated mice exhibited partially reversible hearing loss. Thus, development of the Actin-tTS::Nkcc1^tetO/tetO transgenic mouse line provides a mouse model for the study of variable hearing loss through reversible knockdown of Nkcc1.

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Serotonin hormonally regulates lacrimal gland secretory function via the serotonin type 3a receptor

Imada Toshihiro, Nakamura Shigeru, Hisamura Ryuji, Izuta Yusuke, Jin Kai, Ito Masataka, Kitamura Naoki, Tanaka Kenji F., Mimura Masaru, Shibuya Izumi, Tsubota Kazuo

Scientific Reports 7 （ 1 ） 2017年12月

ISSN 2045-2322

Tears are extracellular fluid secreted from the lacrimal gland (LG). Tears consist of a dynamic tri-layered film composed of secretions from the LG, Meibomian gland, and conjunctival goblet cells. The LG secretes the aqueous component of the tear, the Meibomian gland secretes the lipid component, and conjunctival goblet cells secrete mucin. The regulation of LG activity via the autonomic nervous system has been recognized as fundamental to maintaining aqueous tear flow. Here, we describe the role of a hormone, peripheral serotonin, in tear secretion. We found that blood serotonin concentration, changed by feeding a diet deprived of the serotonin precursor tryptophan, correlated with tear secretion, and that a sustained decrease in serotonin resulted in LG atrophy and autophagy. The combination of a decrease in serotonin with the interruption of autonomic neural stimuli to the LG preceded these alterations. Furthermore, we found that the serotonin type 3a receptor expressed in LG acinar cells is involved in tear secretion via intracellular calcium mobilization. Our findings demonstrate that hormonal regulation by serotonin, in cooperation with the autonomic nervous system, regulates tear secretion.

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Maturation of Cerebellar Purkinje Cell Population Activity during Postnatal Refinement of Climbing Fiber Network

Good Jean Marc, Mahoney Michael, Miyazaki Taisuke, Tanaka Kenji F., Sakimura Kenji, Watanabe Masahiko, Kitamura Kazuo, Kano Masanobu

Cell Reports 21 （ 8 ） 2066 - 2073 2017年11月

ISSN 2211-1247

Neural circuits undergo massive refinements during postnatal development. In the developing cerebellum, the climbing fiber (CF) to Purkinje cell (PC) network is drastically reshaped by eliminating early-formed redundant CF to PC synapses. To investigate the impact of CF network refinement on PC population activity during postnatal development, we monitored spontaneous CF responses in neighboring PCs and the activity of populations of nearby CF terminals using in vivo two-photon calcium imaging. Population activity is highly synchronized in newborn mice, and the degree of synchrony gradually declines during the first postnatal week in PCs and, to a lesser extent, in CF terminals. Knockout mice lacking P/Q-type voltage-gated calcium channel or glutamate receptor δ2, in which CF network refinement is severely impaired, exhibit an abnormally high level of synchrony in PC population activity. These results suggest that CF network refinement is a structural basis for developmental desynchronization and maturation of PC population activity. In the cerebellum, the climbing fiber to Purkinje cell network is extensively remodeled during postnatal development. Good et al. show that in vivo population activity of Purkinje cells in response to climbing fiber synaptic inputs is highly synchronized in newborn mice and massively desynchronized due to climbing fiber network refinement.

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Mitochondrial DNA double-strand breaks in oligodendrocytes cause demyelination, axonal injury, and CNS inflammation

Madsen Pernille M., Pinto Milena, Patel Shreyans, McCarthy Stephanie, Gao Han, Taherian Mehran, Karmally Shaffiat, Pereira Claudia V., Dvoriantchikova Galina, Ivanov Dmitry, Tanaka Kenji F., Moraes Carlos T., Brambilla Roberta

Journal of Neuroscience 37 （ 42 ） 10185 - 10199 2017年10月

ISSN 0270-6474

Mitochondrial dysfunction has been implicated in the pathophysiology of neurodegenerative disorders, including multiple sclerosis (MS). To date, the investigation of mitochondrial dysfunction in MS has focused exclusively on neurons, with no studies exploring whether dysregulation of mitochondrial bioenergetics and/or genetics in oligodendrocytes might be associated with the etiopathogenesis of MS and other demyelinating syndromes. To address this question, we established a mouse model where mitochondrial DNA (mtDNA) double-strand breaks (DSBs) were specifically induced in myelinating oligodendrocytes (PLP:mtPstI mice) by expressing a mitochondrial-targeted endonuclease, mtPstI, starting at 3 weeks of age. In both female and male mice, DSBs of oligodendroglial mtDNA caused impairment of locomotor function, chronic demyelination, glial activation, and axonal degeneration, which became more severe with time of induction. In addition, after short transient induction of mtDNA DSBs, PLP:mtPstI mice showed an exacerbated response to experimental autoimmune encephalomyelitis. Together, our data demonstrate that mtDNA damage can cause primary oligodendropathy, which in turn triggers demyelination, proving PLP:mtPstI mice to be a useful tool to study the pathological consequences of mitochondrial dysfunction in oligodendrocytes. In addition, the demyelination and axonal loss displayed by PLP:mtPstI mice recapitulate some of the key features of chronic demyelinating syndromes, including progressive MS forms, which are not accurately reproduced in the models currently available. For this reason, the PLP: mtPstI mouse represents a unique and much needed platform for testing remyelinating therapies.

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Distinct Roles of Ventromedial versus Ventrolateral Striatal Medium Spiny Neurons in Reward-Oriented Behavior

Tsutsui-Kimura Iku, Natsubori Akiyo, Mori Marina, Kobayashi Kenta, Drew Michael R., de Kerchove d'Exaerde Alban, Mimura Masaru, Tanaka Kenji F.

Current Biology 27 （ 19 ） 3042 - 3048.e4 2017年10月

ISSN 0960-9822

The ventral striatum (VS) is a key brain center regulating reward-oriented behavior [1–4]. The VS can be anatomically divided into medial (VMS) and lateral (VLS) portions based on cortical input patterns. The VMS receives inputs from medial pallium-originated limbic structures (e.g., the medial prefrontal cortex [mPFC]), and the VLS receives inputs from the lateral pallium-originated areas (e.g., the insula) [5, 6]. This anatomical feature led us to hypothesize a functional segregation within the VS in terms of the regulation of reward-oriented behavior. Here, we engineered a fiber photometry system [4] and monitored population-level Ca²⁺ activities of dopamine D2-receptor-expressing medium spiny neurons (D2-MSNs), one of the major cell types in the striatum, during a food-seeking discrimination task. We found that VLS D2-MSNs were activated at the time of cue presentation. In stark contrast, VMS D2-MSNs were inhibited at this time point. Optogenetic counteraction of those changes in the VLS and VMS impaired action initiation and increased responding toward non-rewarded cues, respectively. During lever-press reversal training, VMS inhibition at the time of cue presentation temporarily ceased and optogenetic activation of VMS D2-MSNs facilitated acquisition of the new contingency. These data indicate that the opposing inhibition and excitation in VMS and VLS are important for selecting and initiating a proper action in a reward-oriented behavior. We propose distinct subregional roles within the VS in the execution of successful reward-oriented behavior. Tsutsui-Kimura, Natsubori, et al. find opposing activity patterns of medium spiny neurons at the medial and lateral portions of the ventral striatum during reward-oriented behavior. Each activity mediates distinct function but cooperates with successful reward-oriented behavior, proposing a new functional dichotomy of the ventral striatum.

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Hearing loss controlled by optogenetic stimulation of nonexcitable nonglial cells in the cochlea of the inner ear

Sato Mitsuo P., Higuchi Taiga, Nin Fumiaki, Ogata Genki, Sawamura Seishiro, Yoshida Takamasa, Ota Takeru, Hori Karin, Komune Shizuo, Uetsuka Satoru, Choi Samuel, Masuda Masatsugu, Watabe Takahisa, Kanzaki Sho, Ogawa Kaoru, Inohara Hidenori, Sakamoto Shuichi, Takebayashi Hirohide, Doi Katsumi, Tanaka Kenji F., Hibino Hiroshi

Frontiers in Molecular Neuroscience 10 2017年09月

ISSN 1662-5099

Light-gated ion channels and transporters have been applied to a broad array of excitable cells including neurons, cardiac myocytes, skeletal muscle cells and pancreatic β-cells in an organism to clarify their physiological and pathological roles. Nonetheless, among nonexcitable cells, only glial cells have been studied in vivo by this approach. Here, by optogenetic stimulation of a different nonexcitable cell type in the cochlea of the inner ear, we induce and control hearing loss. To our knowledge, deafness animal models using optogenetics have not yet been established. Analysis of transgenic mice expressing channelrhodopsin-2 (ChR2) induced by an oligodendrocyte-specific promoter identified this channel in nonglial cells—melanocytes—of an epithelial-like tissue in the cochlea. The membrane potential of these cells underlies a highly positive potential in a K⁺-rich extracellular solution, endolymph; this electrical property is essential for hearing. Illumination of the cochlea to activate ChR2 and depolarize the melanocytes significantly impaired hearing within a few minutes, accompanied by a reduction in the endolymphatic potential. After cessation of the illumination, the hearing thresholds and potential returned to baseline during several minutes. These responses were replicable multiple times. ChR2 was also expressed in cochlear glial cells surrounding the neuronal components, but slight neural activation caused by the optical stimulation was unlikely to be involved in the hearing impairment. The acute-onset, reversible and repeatable phenotype, which is inaccessible to conventional gene-targeting and pharmacological approaches, seems to at least partially resemble the symptom in a population of patients with sensorineural hearing loss. Taken together, this mouse line may not only broaden applications of optogenetics but also contribute to the progress of translational research on deafness.

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Yokukansankachimpihange increased body weight but not food-incentive motivation in wild-type mice

Hamaguchi Takuya, Tsutsui-Kimura Iku, Tanaka Kenji F., Mimura Masaru

Nagoya Journal of Medical Science 79 （ 3 ） 351 - 362 2017年08月

ISSN 0027-7622

Yokukansankachimpihange (YKSCH), a traditional Japanese medicine, is widely used for the amelioration of the behavioral and psychological symptoms of dementia with digestive dysfunction. Regardless of its successful use for digestive dysfunction, the effect of YKSCH on body weight was unknown. Furthermore, if YKSCH increased body weight, it might increase motivation according to Kampo medicine theory. Therefore, we investigated whether YKSCH had the potential to increase body weight and enhance motivation in mice. To address this, C57BL/6J mice were used to evaluate the long-term effect of YKSCH on body weight and food-incentive motivation. As part of the evaluation, we optimized an operant test for use over the long-term. We found that feeding mice YKSCH-containing chow increased body weight, but did not increase their motivation to food reward. We propose that YKSCH may be a good treatment option for preventing decrease in body weight in patients with dementia.

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A New Paradigm for Evaluating Avoidance/Escape Motivation

Tsutsui-Kimura Iku, Bouchekioua Youcef, Mimura Masaru, Tanaka Kenji F.

International Journal of Neuropsychopharmacology 20 （ 7 ） 593 - 601 2017年07月

ISSN 1461-1457

Organisms have evolved to approach pleasurable opportunities and to avoid or escape from aversive experiences. These 2 distinct motivations are referred to as approach and avoidance/escape motivations and are both considered vital for survival. Despite several recent advances in understanding the neurobiology of motivation, most studies addressed approach but not avoidance/escape motivation. Here we develop a new experimental paradigm to quantify avoidance/escape motivation and examine the pharmacological validity. Methods: We set up an avoidance variable ratio 5 task in which mice were required to press a lever for variable times to avoid an upcoming aversive stimulus (foot shock) or to escape the ongoing aversive event if they failed to avoid it. We i.p. injected ketamine (0, 1, or 5 mg/kg) or buspirone (0, 5, or 10 mg/kg) 20 or 30 minutes before the behavioral task to see if ketamine enhanced avoidance/escape behavior and buspirone diminished it as previously reported. Results: We found that the performance on the avoidance variable ratio 5 task was sensitive to the intensity of the aversive stimulus. Treatment with ketamine increased while that with buspirone decreased the probability of avoidance from an aversive stimulus in the variable ratio 5 task, being consistent with previous reports. Conclusion: Our new paradigm will prove useful for quantifying avoidance/escape motivation and will contribute to a more comprehensive understanding of motivation.

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High-Speed and Scalable Whole-Brain Imaging in Rodents and Primates

Seiriki Kaoru, Kasai Atsushi, Hashimoto Takeshi, Schulze Wiebke, Niu Misaki, Yamaguchi Shun, Nakazawa Takanobu, Inoue Ken Ichi, Uezono Shiori, Takada Masahiko, Naka Yuichiro, Igarashi Hisato, Tanuma Masato, Waschek James A., Ago Yukio, Tanaka Kenji F., Hayata-Takano Atsuko, Nagayasu Kazuki, Shintani Norihito, Hashimoto Ryota, Kunii Yasuto, Hino Mizuki, Matsumoto Junya, Yabe Hirooki, Nagai Takeharu, Fujita Katsumasa, Matsuda Toshio, Takuma Kazuhiro, Baba Akemichi, Hashimoto Hitoshi

Neuron 94 （ 6 ） 1085 - 1100 2017年06月

ISSN 0896-6273

Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of light-sheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases.

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OCD candidate gene SLC1A1/EAAT3 impacts basal ganglia-mediated activity and stereotypic behavior

Zike Isaac D., Chohan Muhammad O., Kopelman Jared M., Krasnow Emily N., Flicker Daniel, Nautiyal Katherine M., Bubser Michael, Kellendonk Christoph, Jones Carrie K., Stanwood Gregg, Tanaka Kenji Fransis, Moore Holly, Ahmari Susanne E., Veenstra-VanderWeele Jeremy

Proceedings of the National Academy of Sciences of the United States of America 114 （ 22 ） 5719 - 5724 2017年05月

ISSN 0027-8424

Obsessive-compulsive disorder (OCD) is a chronic, disabling condition with inadequate treatment options that leave most patients with substantial residual symptoms. Structural, neurochemical, and behavioral findings point to a significant role for basal ganglia circuits and for the glutamate system in OCD. Genetic linkage and association studies in OCD point to SLC1A1, which encodes the neuronal glutamate/aspartate/cysteine transporter excitatory amino acid transporter 3 (EAAT3)/excitatory amino acid transporter 1 (EAAC1). However, no previous studies have investigated EAAT3 in basal ganglia circuits or in relation to OCD-related behavior. Here, we report a model of Slc1a1 loss based on an excisable STOP cassette that yields successful ablation of EAAT3 expression and function. Using amphetamine as a probe, we found that EAAT3 loss prevents expected increases in (i) locomotor activity, (ii) stereotypy, and (iii) immediate early gene induction in the dorsal striatum following amphetamine administration. Further, Slc1a1-STOP mice showed diminished grooming in an SKF-38393 challenge experiment, a pharmacologic model of OCDlike grooming behavior. This reduced grooming is accompanied by reduced dopamine D₁ receptor binding in the dorsal striatum of Slc1a1-STOP mice. Slc1a1-STOP mice also exhibit reduced extracellular dopamine concentrations in the dorsal striatum both at baseline and following amphetamine challenge. Viral-mediated restoration of Slc1a1/EAAT3 expression in the midbrain but not in the striatum results in partial rescue of amphetamine-induced locomotion and stereotypy in Slc1a1-STOP mice, consistent with an impact of EAAT3 loss on presynaptic dopaminergic function. Collectively, these findings indicate that the most consistently associated OCD candidate gene impacts basal ganglia-dependent repetitive behaviors.

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Transformation of Astrocytes to a Neuroprotective Phenotype by Microglia via P2Y₁ Receptor Downregulation

Shinozaki Youichi, Shibata Keisuke, Yoshida Keitaro, Shigetomi Eiji, Gachet Christian, Ikenaka Kazuhiro, Tanaka Kenji F., Koizumi Schuichi

Cell Reports 19 （ 6 ） 1151 - 1164 2017年05月

ISSN 2211-1247

Microglia and astrocytes become reactive following traumatic brain injury (TBI). However, the coordination of this reactivity and its relation to pathophysiology are unclear. Here, we show that microglia transform astrocytes into a neuroprotective phenotype via downregulation of the P2Y₁ purinergic receptor. TBI initially caused microglial activation in the injury core, followed by reactive astrogliosis in the peri-injured region and formation of a neuroprotective astrocyte scar. Equivalent changes to astrocytes were observed in vitro after injury. This change in astrocyte phenotype resulted from P2Y₁ receptor downregulation, mediated by microglia-derived cytokines. In mice, astrocyte-specific P2Y₁ receptor overexpression (Astro-P2Y₁OE) counteracted scar formation, while astrocyte-specific P2Y₁ receptor knockdown (Astro-P2Y₁KD) facilitated scar formation, suggesting critical roles of P2Y₁ receptors in the transformation. Astro-P2Y₁OE and Astro-P2Y₁KD mice showed increased and reduced neuronal damage, respectively. Altogether, our findings indicate that microglia-astrocyte interaction, involving a purinergic signal, is essential for the formation of neuroprotective astrocytes.

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Ventrolateral striatal medium spiny neurons positively regulate food-incentive, goal-directed behavior independently of D1 and D2 selectivity

Natsubori Akiyo, Tsutsui-Kimura Iku, Nishida Hiroshi, Bouchekioua Youcef, Sekiya Hiroshi, Uchigashima Motokazu, Watanabe Masahiko, De Kerchove D’Exaerde Alban, Mimura Masaru, Takata Norio, Tanaka Kenji F.

Journal of Neuroscience 37 （ 10 ） 2723 - 2733 2017年03月

ISSN 0270-6474

The ventral striatum is involved in motivated behavior. Akin to the dorsal striatum, the ventral striatum contains two parallel pathways: the striatomesencephalic pathway consisting of dopamine receptor Type 1-expressing medium spiny neurons (D1-MSNs) and the striatopallidal pathway consisting of D2-MSNs. These two genetically identified pathways are thought to encode opposing functions in motivated behavior. It has also been reported that D1/D2 genetic selectivity is not attributed to the anatomical discrimination of two pathways. We wanted to determine whether D1-and D2-MSNs in the ventral striatum functioned in an opposing manner as previous observations claimed, and whether D1/D2 selectivity corresponded to a functional segregation in motivated behavior of mice. To address this question, we focused on the lateral portion of ventral striatum as a region implicated in food-incentive, goal-directed behavior, and recorded D1 or D2-MSN activity by using a gene-encoded ratiometric Ca²⁺ indicator and by constructing a fiberphotometry system, and manipulated their activities via optogenetic inhibition during ongoing behaviors. We observed concurrent event-related compound Ca²⁺ elevations in ventrolateral D1-and D2-MSNs, especially at trial start cue-related and first lever press-related times. D1 or D2 selective optogenetic inhibition just after the trial start cue resulted in a reduction of goal-directed behavior, indicating a shared coding of motivated behavior by both populations at this time. Only D1-selective inhibition just after the first lever press resulted in the reduction of behavior, indicating D1-MSN-specific coding at that specific time. Our data did not support opposing encoding by both populations in food-incentive, goal-directed behavior.

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Dysfunction of ventrolateral striatal dopamine receptor type 2-expressing medium spiny neurons impairs instrumental motivation

Tsutsui-Kimura Iku, Takiue Hiroyuki, Yoshida Keitaro, Xu Ming, Yano Ryutaro, Ohta Hiroyuki, Nishida Hiroshi, Bouchekioua Youcef, Okano Hideyuki, Uchigashima Motokazu, Watanabe Masahiko, Takata Norio, Drew Michael R., Sano Hiromi, Mimura Masaru, Tanaka Kenji F.

Nature Communications 8 2017年02月

ISSN 2041-1723

Impaired motivation is present in a variety of neurological disorders, suggesting that decreased motivation is caused by broad dysfunction of the nervous system across a variety of circuits. Based on evidence that impaired motivation is a major symptom in the early stages of Huntington's disease, when dopamine receptor type 2-expressing striatal medium spiny neurons (D2-MSNs) are particularly affected, we hypothesize that degeneration of these neurons would be a key node regulating motivational status. Using a progressive, time-controllable, diphtheria toxin-mediated cell ablation/dysfunction technique, we find that loss-of-function of D2-MSNs within ventrolateral striatum (VLS) is sufficient to reduce goal-directed behaviours without impairing reward preference or spontaneous behaviour. Moreover, optogenetic inhibition and ablation of VLS D2-MSNs causes, respectively, transient and chronic reductions of goal-directed behaviours. Our data demonstrate that the circuitry containing VLS D2-MSNs control motivated behaviours and that VLS D2-MSN loss-of-function is a possible cause of motivation deficits in neurodegenerative diseases.

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YAP functions as a mechanotransducer in oligodendrocyte morphogenesis and maturation

Shimizu Takeshi, Osanai Yasuyuki, Tanaka Kenji F., Abe Manabu, Natsume Rie, Sakimura Kenji, Ikenaka Kazuhiro

GLIA 65 （ 2 ） 360 - 374 2017年02月

ISSN 0894-1491

Oligodendrocytes (OLs) are myelinating cells of the central nervous system. Recent studies have shown that mechanical factors influence various cell properties. Mechanical stimuli can be transduced into intracellular biochemical signals through mechanosensors and intracellular mechanotransducers, such as YAP. However, the molecular mechanisms underlying mechanical regulation of OLs by YAP remain unknown. We found that OL morphology and interactions between OLs and neuronal axons were affected by knocking down YAP. Mechanical stretching of OL precursor cells induced nuclear YAP accumulation and assembly of focal adhesion, key platforms for mechanotransduction. Shear stress decreased the number of OL processes, whereas a dominant-negative form of YAP suppressed these effects. To investigate the roles of YAP in postnatal OLs in vivo, we constructed a novel YAP knock-in mouse and found that in vivo overexpression of YAP widely affected OL maturation. These results indicate that YAP regulates OL morphology and maturation in response to mechanical factors. GLIA 2017;65:360–374.

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The balance between cathepsin C and cystatin F controls remyelination in the brain of Plp1-overexpressing mouse, a chronic demyelinating disease model

Shimizu Takahiro, Wisessmith Wilaiwan, Li Jiayi, Abe Manabu, Sakimura Kenji, Chetsawang Banthit, Sahara Yoshinori, Tohyama Koujiro, Tanaka Kenji F., Ikenaka Kazuhiro

GLIA 2017年

ISSN 0894-1491

In demyelinating diseases such as multiple sclerosis (MS), an imbalance between the demyelination and remyelination rates underlies the degenerative processes. Microglial activation is observed in demyelinating lesions; however, the molecular mechanism responsible for the homeostatic/environmental change remains elusive. We previously found that cystatin F (CysF), a cysteine protease inhibitor, is selectively expressed in microglia only in actively demyelinating/remyelinating lesions but ceases expression in chronic lesions, suggesting its role in remyelination. Here, we report the effects of manipulating the expression of CysF and cathepsin C (CatC), a key target of CysF, in a murine model of transgenic demyelinating disease, Plp^4e/-. During the active remyelinating phase, both CysF knockdown (CysFKD) and microglial-selective CatC overexpression (CatCOE) showed a worsening of the demyelination in Plp^4e/- transgenic mice. Conversely, during the chronic demyelinating phase, CatC knockdown (CatCKD) ameliorated the demyelination. Our results suggest that the balance between CatC and CysF expression controls the demyelination and remyelination process.

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Quantitative temporal changes in DTI values coupled with histological properties in cuprizone-induced demyelination and remyelination

Yano R., Hata J., Abe Y., Seki F., Yoshida K., Komaki Y., Okano H., Tanaka K.

Neurochemistry International （Neurochemistry International） 119 151 - 158 2017年

ISSN 0197-0186

© 2017 Elsevier Ltd Diffusion tensor imaging (DTI) is widely used to evaluate microstructural variations in brain tissue. In particular, fractional anisotropy (FA), reflecting the magnitude and orientation of anisotropic water diffusion, allows us to detect pathological events in white matter. An ex vivo DTI study coupled with histological assessment is an efficient strategy to evaluate the myelination process, i.e. demyelination and remyelination. The relationship between DTI values and myelin content or the individual cellular components such as oligodendrocytes, microglia, and astrocytes during both processes of demyelination and remyelination are not well-understood. To address this issue, we employed a cuprizone-inducible demyelination mouse model. Demyelination can be induced in this model during cuprizone exposure and termination of cuprizone exposure induces remyelination. We fed the mice cuprizone-containing chow for 4 weeks and then normal chow for an additional 4 weeks. The ex vivo DTI was performed to evaluate the white matter profiles observed by FA, mean diffusivity (MD), and radial diffusivity (RD) at both demyelinating and remyelinating time points, and then we evaluated histological properties at the same time points. The results indicated a gradual FA decrease during the cuprizone treatment (0, 2, 3, 4 weeks). A lower peak was seen at 1 week after the normal chow was resumed, with recovery to baseline at 2 and 4 weeks. MD and RD showed an opposing pattern to that of FA. These DTI values were positively or negatively correlated with myelin content regardless of the status of the white matter. The RD value was more sensitive to myelination status than FA and MD. We have clarified the temporal changes in the DTI values coupled with histological properties over both the demyelination and remyelination processes.

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Striatonigral direct pathway activation is sufficient to induce repetitive behaviors

Bouchekioua Y., Tsutsui-Kimura I., Sano H., Koizumi M., Tanaka K., Yoshida K., Kosaki Y., Watanabe S., Mimura M.

Neuroscience Research （Neuroscience Research） 132 53 - 57 2017年

ISSN 0168-0102

© 2017 Pharmacological intervention in the substantia nigra is known to induce repetitive behaviors in rodents, but a direct causal relationship between a specific neural circuit and repetitive behavior has not yet been established. Here we demonstrate that optogenetic activation of dopamine D1 receptor-expressing MSNs terminals in the substantia nigra pars reticulata resulted in sustained and chronic repetitive behaviors. These data show for the first time that activation of the striatonigral direct pathway is sufficient to generate motor stereotypies.

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Physiological effects of a habituation procedure for functional MRI in awake mice using a cryogenic radiofrequency probe

Yoshida Keitaro, Mimura Yu, Ishihara Ryosuke, Nishida Hiroshi, Komaki Yuji, Minakuchi Tomohito, Tsurugizawa Tomokazu, Mimura Masaru, Okano Hideyuki, Tanaka Kenji F., Takata Norio

Journal of Neuroscience Methods 274 38 - 48 2016年12月

ISSN 0165-0270

Background Functional magnetic resonance imaging (fMRI) in mice is typically performed under anesthesia due to difficulties in holding the head of awake mice stably with a conventional three-point fixation method that uses a tooth-bar and earplugs. Although some studies have succeeded in fMRI in awake mice by attaching a head-post on the skull, this cannot be applied to fMRI using a high signal-to-noise ratio (SNR) cryogenic MRI-detector, CryoProbe, because it covers the head of a mouse closely. New method We developed head-fixation implements for awake mice that are applicable to fMRI using CryoProbe. Results A head-bar was surgically attached to the skull of a mouse that was then habituated to a mock fMRI-environment, two hours/day for eight days with physiological examinations of body-weight, fecal weight, electromyogram (EMG), and electrocardiogram. EMG power decreased with just one day of habituation, whereas heart rate decreased after at least seven days of habituation. Estimated head motions of awake mice during fMRI were significantly smaller than a voxel size. Unexpectedly, temporal SNR of fMRI signals for awake mice was higher than that for anesthetized mice held by a conventional method. Functional connectivity in the brain of both anesthetized and awake mice showed bilateral and unilateral networks. Comparison with existing method(s) fMRI using CryoProbe had been performed on anesthetized mice previously. Our method does not use anesthetics during habituation or fMRI. Conclusion Our method would be beneficial for translational research using fMRI in mice and humans because human fMRI is typically performed without anesthetics.

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Subcellular calcium dynamics during juvenile development in mouse hippocampal astrocytes

Nakayama Ryota, Sasaki Takuya, Tanaka Kenji F., Ikegaya Yuji

European Journal of Neuroscience 43 （ 7 ） 923 - 932 2016年04月

ISSN 0953-816X

Astrocytes generate calcium signals throughout their fine processes, which are assumed to locally regulate neighbouring neurotransmission and blood flow. The intercellular morphological relationships mature during juvenile periods when astrocytes elongate highly ramified processes. In this study, we examined developmental changes in calcium activity patterns of single hippocampal astrocytes using a transgenic mouse line in which astrocytes selectively express a genetically encoded calcium indicator, Yellow Cameleon-Nano50. Compared with postnatal day 7, astrocytes at postnatal day 30 showed larger subcellular calcium events and a greater proportion of somatic events. At both ages, the calcium activity was abolished by removal of extracellular calcium ions. Calcium events in late juvenile astrocytes were not affected by spontaneously occurring sharp waves that trigger synchronized neuronal spikes, implying the independence of astrocyte calcium signals from neuronal synchronization. These results demonstrate that astrocytes undergo dynamic changes in their activity patterns during juvenile development.

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Immune modulation of the T cell response in asthma through Wnt10b

Trischler Jordis, Shiomi Takayuki, Turner Damian L., Sklepkiewicz Piotr L., Goldklang Monica P., Tanaka Kenji F., Xu Ming, Farber Donna L., D'Armiento Jeanine M.

American Journal of Respiratory Cell and Molecular Biology 54 （ 4 ） 584 - 593 2016年04月

ISSN 1044-1549

Asthma is a chronic inflammatory disease, which is characterized by activation of CD4⁺ T helper 2 cells orchestrating an allergic airway response.Whereas the role ofWnt family members in regulating T cell maintenance and maturation is established, their contribution to T cell activation in allergic asthma is not known.We hypothesized that Wnt10b plays a role in the modulation of the allergic airway response and affectsTcell activation and polarization.Using an in vivo house dust mite asthma model, Wnt10b-deficient (Wnt10b^-/-) mice were allergen-sensitized and inflammation, as well as T cell activation, was studied in vivo and in vitro. Wnt10b^-/- mice exhibited an augmented inflammatory phenotype with an increase in eosinophils in the bronchoalveolar lavage and IL-4 and IL-13 in the lungs when compared with wild-type mice. In vitro studies confirmed an increased T helper type 2 polarization and increased T cell activation ofWnt10b^-/- cells. Accordingly, the percentage of naive T cells was elevated by the addition of recombinantWnt10b protein. Finally,Wnt10b^-/- mice exhibited an increase in the percentage of effector T cells in the lungs after house dust mite sensitization, which indicated a heightened activation state, measured by an increased percentage of CD69^hiCD11a^hi cells. These findings suggest thatWnt10b plays an important role in regulating asthmatic airway inflammation through modification of the T cell response and is a prospective target in the disease process.

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Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis

Zhou Zhiwen, Tanaka Kenji F., Matsunaga Shigeru, Iseki Mineo, Watanabe Masakatsu, Matsuki Norio, Ikegaya Yuji, Koyama Ryuta

Scientific Reports 5 2016年01月

ISSN 2045-2322

Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways.

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Microglial phospholipase D4 deficiency influences myelination during brain development

Chiba Terumasa, Otani Yoshinori, Yamaguchi Yoshihide, Ishibashi Tomoko, Hayashi Akiko, Tanaka Kenji F., Yamazaki Maya, Sakimura Kenji, Baba Hiroko

Proceedings of the Japan Academy Series B: Physical and Biological Sciences 92 （ 7 ） 237 - 254 2016年

ISSN 0386-2208

Phospholipase D4 (PLD4) is expressed in activated microglia that transiently appear in white matter during postnatal brain development. Previous knockdown experiments using cultured microglia showed PLD4 involvement in phagocytosis and proliferation. To elucidate the role of PLD4 in vivo, PLD4-deficient mice were generated and the cerebella were examined at postnatal day 5 (P5) and P7, when PLD4 expression is highest in microglia. Wild type microglia showed strong immunoreactivity for microglial marker CD68 at P5, whereas CD68 signals were weak in PLD4-deficient microglia, suggesting that loss of PLD4 affects microglial activation. At P5 and P7, immunostaining for anti-myelin basic protein (MBP) antibody indicated a mild but significant delay in myelination in PLD4-deficient cerebellum. Similar change was also observed in the corpus callosum at P7. However, this difference was not apparent at P10, suggesting that microglial PLD4-deficiency primarily influences the early myelination stage. Thus, microglia may have a transient role in myelination via a PLD4-related mechanism during development.

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Concentration-Dependent Dual Mode of Zn Action at Serotonin 5-HT1A Receptors

Satała Grzegorz, Duszyńska Beata, Stachowicz Katarzyna, Rafalo Anna, Pochwat Bartlomiej, Luckhart Christine, Albert Paul R., Daigle Mireille, Tanaka Kenji F., Hen René, Lenda Tomasz, Nowak Gabriel, Bojarski Andrzej J., Szewczyk Bernadeta

Molecular Neurobiology 1 - 13 2015年12月

ISSN 0893-7648

Recent data has indicated that Zn can modulate serotonergic function through the 5-HT_1A receptor (5-HT_1AR); however, the exact mechanisms are unknown. In the present studies, radioligand binding assays and behavioural approaches were used to characterize the pharmacological profile of Zn at 5-HT_1ARs in more detail. The influence of Zn on agonist binding to 5-HT_1ARs stably expressed in HEK293 cells was investigated by in vitro radioligand binding methods using the agonist [³H]-8-OH-DPAT. The in vivo effects of Zn were compared with those of 8-OH-DPAT in hypothermia, lower lip retraction (LLR), 5-HT behavioural syndrome and the forced swim (FST) tests. In the in vitro studies, biphasic effects, which involved allosteric potentiation of agonist binding at sub-micromolar Zn concentrations and inhibition at sub-millimolar Zn concentrations, were found. The in vivo studies showed that Zn did not induce LLR or elements of 5-HT behavioural syndrome but blocked such effects induced by 8-OH-DPAT. Zn decreased body temperature in rats and mice; however, Zn failed to induce hypothermia in the 5-HT_1A autoreceptor knockout mice. In the FST, Zn potentiated the effect of 8-OH-DPAT. However, in the FST performed with the 5-HT_1A autoreceptor knockout mice, the anti-immobility effect of Zn was partially blocked. Both the binding and behavioural studies suggest a concentration-dependent dual mechanism of Zn action at 5-HT_1ARs, with potentiation at low dose and inhibition at high dose. Moreover, the in vivo studies indicate that Zn can modulate both presynaptic and postsynaptic 5-HT_1ARs; however, Zn’s effects at presynaptic receptors seem to be more potent.

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5-HT1A receptors on mature dentate gyrus granule cells are critical for the antidepressant response

Samuels Benjamin Adam, Anacker Christoph, Hu Alice, Levinstein Marjorie R., Pickenhagen Anouchka, Tsetsenis Theodore, Madroñal Noelia, Donaldson Zoe R., Drew Liam John, Dranovsky Alex, Gross Cornelius T., Tanaka Kenji F., Hen René

Nature Neuroscience 18 （ 11 ） 1606 - 1616 2015年11月

ISSN 1097-6256

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants, but the mechanisms by which they influence behavior are only partially resolved. Adult hippocampal neurogenesis is necessary for some of the responses to SSRIs, but it is not known whether mature dentate gyrus granule cells (DG GCs) also contribute. We deleted the serotonin 1A receptor (5HT1AR, a receptor required for the SSRI response) specifically from DG GCs and found that the effects of the SSRI fluoxetine on behavior and the hypothalamic-pituitary-adrenal (HPA) axis were abolished. By contrast, mice lacking 5HT1ARs only in young adult-born GCs (abGCs) showed normal fluoxetine responses. Notably, 5HT1AR-deficient mice engineered to express functional 5HT1ARs only in DG GCs responded to fluoxetine, indicating that 5HT1ARs in DG GCs are sufficient to mediate an antidepressant response. Taken together, these data indicate that both mature DG GCs and young abGCs must be engaged for an antidepressant response.

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Neuronal heterotopias affect the activities of distant brain areas and lead to behavioral deficits

Ishii Kazuhiro, Kubo Ken Ichiro, Endo Toshihiro, Yoshida Keitaro, Benner Seico, Ito Yukiko, Aizawa Hidenori, Aramaki Michihiko, Yamanaka Akihiro, Tanaka Kohichi, Takata Norio, Tanaka Kenji F., Mimura Masaru, Tohyama Chiharu, Kakeyama Masaki, Nakajima Kazunori

Journal of Neuroscience 35 （ 36 ） 12432 - 12445 2015年09月

ISSN 0270-6474

Neuronal heterotopia refers to brain malformations resulting from deficits of neuronal migration. Individuals with heterotopias show a high incidence of neurological deficits, such as epilepsy. More recently, it has come to be recognized that focal heterotopiasmayalso show a range of psychiatric problems, including cognitive and behavioral impairments. However, because focal heterotopias are not always located in the brain areas responsible for the symptoms, the causal relationship between the symptoms and heterotopias remains elusive. In this study, we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited spatial working memory deficit and low competitive dominance behavior, which have been shown to be closely associated with the activity of the medial prefrontal cortex (mPFC) in rodents. Analysis of the mPFC activity revealed that the immediate-early gene expression was decreased and the local field potentials of the mPFC were altered in the mice with heterotopias compared with the control mice. Moreover, activation of these ectopic and overlying sister neurons using the DREADD (designer receptor exclusively activated by designer drug) system improved the working memory deficits. These findings suggest that cortical regions containing focal heterotopias can affect distant brain regions and give rise to behavioral abnormalities.

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Identification of the extent of cortical spreading depression propagation by Npas4 mRNA expression

Yoshida Keitaro, Xu Ming, Natsubori Akiyo, Mimura Masaru, Takata Norio, Tanaka Kenji F.

Neuroscience Research 98 1 - 8 2015年09月

ISSN 0168-0102

Cortical spreading depression (CSD) is a phenomenon associated with a propagating large shift in direct current (DC) potential followed by suppression of electrophysiological activity. For temporal analysis of CSD propagation, electrophysiological recording is the most reliable tool. However, it is difficult to completely identify the spatial area of the brain influenced by CSD, because recording sites are technically limited. Histological post hoc identification of activated neurons by labeling the induction of an immediate early gene (IEG) could determine areas of CSD propagation. We found that cortical application of potassium chloride induced expression of Npas4 IEG mRNA in the ipsilateral dorsal cortex. Interestingly, induction of Npas4 was never observed in the ipsilateral hippocampus and there was a clear boundary to the area of Npas4 expression. To determine whether the boundary of the area of Npas4 mRNA expression was the limit of CSD propagation, we recorded local field potentials from multiple sites that crossed the boundary of Npas4 expression. We found that the area of Npas4 mRNA expression coincided with the area of DC-potential shift propagation. We propose that induction of Npas4 identifies the area influenced by CSD propagation.

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Adult hippocampal neurogenesis modulates fear learning through associative and nonassociative mechanisms

Seo Dong Oh, Carillo Mary Ann, Lim Sean Chih Hsiung, Tanaka Kenji F., Drew Michael R.

Journal of Neuroscience 35 （ 32 ） 11330 - 11345 2015年08月

ISSN 0270-6474

Adult hippocampal neurogenesis is believed to support hippocampus-dependent learning and emotional regulation. These putative functions of adult neurogenesis have typically been studied in isolation, and little is known about how they interact to produce adaptive behavior. We used trace fear conditioning as a model system to elucidate mechanisms through which adult hippocampal neurogenesis modulates processing of aversive experience. To achieve a specific ablation of neurogenesis, we generated transgenic mice that express herpes simplex virus thymidine kinase specifically in neural progenitors and immature neurons. Intracerebroventricular injection of the prodrug ganciclovir caused a robust suppression of neurogenesis without suppressing gliogenesis. Neurogenesis ablation via this method or targeted x-irradiation caused an increase in context conditioning in trace but not delay fear conditioning. Data suggest that this phenotype represents opposing effects of neurogenesis ablation on associative and nonassociative components of fear learning. Arrest of neurogenesis sensitizes mice to nonassociative effects of fear conditioning, as evidenced by increased anxiety-like behavior in the open field after (but not in the absence of) fear conditioning. In addition, arrest of neurogenesis impairs associative trace conditioning, but this impairment can be masked by nonassociative fear. The results suggest that adult neurogenesis modulates emotional learning via two distinct but opposing mechanisms: it supports associative trace conditioning while also buffering against the generalized fear and anxiety caused by fear conditioning.

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Unveiling astrocytic control of cerebral blood flow with optogenetics

Masamoto Kazuto, Unekawa Miyuki, Watanabe Tatsushi, Toriumi Haruki, Takuwa Hiroyuki, Kawaguchi Hiroshi, Kanno Iwao, Matsui Ko, Tanaka Kenji F., Tomita Yutaka, Suzuki Norihiro

Scientific Reports 5 2015年06月

Cortical neural activities lead to changes in the cerebral blood flow (CBF), which involves astrocytic control of cerebrovascular tone. However, the manner in which astrocytic activity specifically leads to vasodilation or vasoconstriction is difficult to determine. Here, cortical astrocytes genetically expressing a light-sensitive cation channel, channelrhodopsin-2 (ChR2), were transcranially activated with a blue laser while the spatiotemporal changes in CBF were noninvasively monitored with laser speckle flowgraphy in the anesthetised mouse cortex. A brief photostimulation induced a fast transient increase in CBF. The average response onset time was 0.7 ± 0.7 sec at the activation foci, and this CBF increase spread widely from the irradiation spot with an apparent propagation speed of 0.8-1.1 mm/sec. The broad increase in the CBF could be due to a propagation of diffusible vasoactive signals derived from the stimulated astrocytes. Pharmacological manipulation showed that topical administration of a K + channel inhibitor (BaCl<inf>2</inf>; 0.1-0.5 mM) significantly reduced the photostimulation-induced CBF responses, which indicates that the ChR2-evoked astrocytic activity involves K + signalling to the vascular smooth muscle cells. These findings demonstrate a unique model for exploring the role of the astrocytes in gliovascular coupling using non-invasive, time-controlled, cell-type specific perturbations.

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Observation and manipulation of glial cell function by virtue of sufficient probe expression

Natsubori Akiyo, Takata Norio, Tanaka Kenji F.

Frontiers in Cellular Neuroscience 9 （ MAY ） 2015年05月

ISSN 1662-5102

The development of gene-encoded indicators and actuators to observe and manipulate cellular functions is being advanced and investigated. Expressing these probe molecules in glial cells is expected to enable observation and manipulation of glial cell activity, leading to elucidate the behaviors and causal roles of glial cells. The first step toward understanding glial cell functions is to express the probes in sufficient amounts, and the Knockin-mediated ENhanced Gene Expression (KENGE)-tet system provides a strategy for achieving this. In the present article, three examples of KENGE-tet system application are reviewed: depolarization of oligodendrocytes, intracellular acidification of astrocytes, and observation of intracellular calcium levels in the fine processes of astrocytes.

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Distinct Circuits Underlie the Effects of 5-HT1B Receptors on Aggression and Impulsivity

Nautiyal Katherine M., Tanaka Kenji F., Barr Mary M., Tritschler Laurent, Le Dantec Yannick, David Denis J., Gardier Alain M., Blanco Carlos, Hen René, Ahmari Susanne E.

Neuron 86 （ 3 ） 813 - 827 2015年05月

ISSN 0896-6273

Impulsive and aggressive behaviors are both modulated by serotonergic signaling, specifically through the serotonin 1B receptor (5-HT1BR). 5-HT1BR knockout mice show increased aggression and impulsivity, and 5-HT1BR polymorphisms are associated with aggression and drug addiction in humans. To dissect the mechanisms by which the 5-HT1BR affects these phenotypes, we developed a mouse model to spatially and temporally regulate 5-HT1BR expression. Our results demonstrate that forebrain 5-HT1B heteroreceptors expressed during an early postnatal period contribute to the development of the neural systems underlying adult aggression. However, distinct heteroreceptors acting during adulthood are involved in mediating impulsivity. Correlating with the impulsivity, dopamine inthe nucleus accumbens is elevated in the absence of 5-HT1BRs and normalized following adult rescue of the receptor. Overall, these data show that while adolescent expression of 5-HT1BRs influences aggressive behavior, a distinct set of 5-HT1B receptors modulates impulsive behavior during adulthood. Nautiyal etal. show that serotonin 1B receptors modulate aggressive and impulsive behavior through distinct neural circuits. Adolescent expression of forebrain heteroreceptors influences aggressive behavior, while a separate population affects impulsive behavior during adulthood.

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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

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.

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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

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.

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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

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.

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Optogenetics research using the mouse as a model system

Tanaka Kenji F.

227 - 240 2015年01月

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.

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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

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.

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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

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.

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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

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.

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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

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.

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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

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.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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.

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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

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.

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Development of transgenic animals in optogenetics

Tanaka Kenji

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

ISSN 0015-5691

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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

Fujii Satoshi, Tanaka Kenji F., Ikenaka Kazuhiro, Yamazaki Yoshihiko

Brain Research 1578 （ 1 ） 1 - 13 2014年

ISSN 0006-8993

Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters, including ATP. Adenosine levels in the cerebrospinal fluidof mice thatexpress amutanthuman glial fibrillary acidic protein in astrocytes are slightly elevated compared to those in wild type mice and this might result from the observed increased release by mutant astrocytes of ATP, which can be used to produce adenosine. Using hippocampal slices from these mutant mice, we examined whether the increased endogenous adenosine levels in the hippocampus modulate the reversal of long-term potentiation (LTP), i.e. depotentiation (DP), in CA1 neurons. In hippocampal slices from wild type mice, a stable LTP was induced by tetanic stimulation consisting of 100 pulses at 100 Hz, and this was reversed by a train of low frequency stimulation (LFS) of 500 pulses at 1 Hz applied 30 min later. This induction of DP was inhibited by application of either 100 nM adenosine or 0.5 nM N⁶-cyclopentyladenosine, an adenosine A₁ receptor agonist, during LFS, indicating that the increase in extracellular adenosine levels attenuated DP induction by acting on adenosine A1 receptors. In contrast, although a stable LTP was also induced in hippocampal slices from mutant mice, induction of DP was inhibited, but DP could be induced by application, during LFS, of 50 nM 8-cyclopentyltheophylline, an adenosine A1 receptor antagonist. These results suggest that a small increase in extracellular adenosine levels resulting from increased ATP release by astrocytes resultsinattenuationofDPinhippocampal CA1neurons inthemutantmice.

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Long-lasting silencing of orexin/hypocretin neurons using archaerhodopsin induces slow-wave sleep in mice

Tsunematsu Tomomi, Tabuchi Sawako, Tanaka Kenji F., Boyden Edward S., Tominaga Makoto, Yamanaka Akihiro

Behavioural Brain Research 255 64 - 74 2013年10月

ISSN 0166-4328

Orexin/hypocretin neurons have a crucial role in the regulation of sleep and wakefulness. Recent optogenetic studies revealed that the activation or inhibition of orexin neuronal activity affects the probability of sleep/wakefulness transition in the acute phase. To expand our understanding of how orexin neurons maintain wakefulness, we generated new transgenic mice in which orexin neurons expressed archaerhodopsin from Halorubrum strain TP009 (ArchT), a green light-driven neuronal silencer, using the tet-off system (orexin-tTA; TetO ArchT mice). Slice patch clamp recordings of ArchT-expressing orexin neurons demonstrated that long-lasting photic illumination was able to silence the activity of orexin neurons. We further confirmed that green light illumination for 1. h in the dark period suppressed orexin neuronal activity in vivo using c-Fos expression. Continuous 1. h silencing of orexin neurons in freely moving orexin-tTA; TetO ArchT mice during the night (the active period, 20:00-21:00) significantly increased total time spent in slow-wave sleep (SWS) and decreased total wake time. Additionally, photic inhibition increased sleep/wakefulness state transitions, which is also evident in animals lacking the prepro-orexin gene, orexin neurons, or functional orexin-2 receptors. However, continuous 1. h photic illumination produced little effect on sleep/wakefulness states during the day (the inactive period, 12:00-13:00). These results suggest that orexin neuronal activity plays a crucial role in the maintenance of wakefulness especially in the active phase in mice. © 2013 The Authors.

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Influence of inhibitory serotonergic inputs to orexin/hypocretin neurons on the diurnal rhythm of sleep and wakefulness

Tabuchi Sawako, Tsunematsu Tomomi, Kilduff Thomas S., Sugio Shouta, Xu Ming, Tanaka Kenji F., Takahashi Satoru, Tominaga Makoto, Yamanaka Akihiro

Sleep 36 （ 9 ） 1391 - 1404 2013年09月

ISSN 0161-8105

Study Objective: Serotonergic (5HT) neurons of the dorsal raphe nuclei receive excitatory input from hypothalamic orexin (hypocretin) neurons and reciprocally inhibit orexin neurons through the 5HT1A receptor. However, the physiological significance of this negative feedback circuit for sleep/wakefulness regulation is little understood. Design: 5HT1A receptor expression level was specifically and reversibly controlled in the orexin neurons using the Tet-off system. The responsiveness of orexin neurons to 5HT in vitro and the sleep/wakefulness patterns were compared between 5HT1A-overexpressing and control mice. Measurements and Results: When the 5HT1A receptor was overexpressed in orexin neurons of Orexin-EGFP; orexin-tTA; TetO Htr1a mice, 5HTinduced inhibition of orexin neurons was prolonged. In the absence of doxycycline, Orexin-tTA; TetO Htr1a mice exhibited severe fragmentation of sleep/wakefulness during the first half of the dark period-the time of maximal activity in nocturnal rodents-without affecting sleep/wakefulness during the light period when sleep time is maximal. However, when the 5HT1A receptor in orexin neurons was reduced to basal expression levels in the presence of doxycycline, sleep/wakefulness patterns in Orexin-tTA; TetO Htr1a mice during the early active period were indistinguishable from those of littermate TetO Htr1a mice. These results strongly suggest that enhancement of inhibitory serotonergic input to orexin neurons caused fragmentation of wakefulness. In contrast, sleep/wakefulness architecture in the light period was unaffected by 5HT1A receptor overexpression in the orexin neurons. Conclusion: Inhibitory serotonergic input likely functions as negative feedback to orexin neurons in the early dark period and helps stabilize wakefulness bouts, thereby contributing to the diurnal rhythm of sleep and wakefulness. © 2013 Associated Professional Sleep Societies, LLC.

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Increased astrocytic ATP release results in enhanced excitability of the hippocampus

Lee Hae Ung, Yamazaki Yoshihiko, Tanaka Kenji F., Furuya Kishio, Sokabe Masahiro, Hida Hideki, Takao Keizo, Miyakawa Tsuyoshi, Fujii Satoshi, Ikenaka Kazuhiro

GLIA 61 （ 2 ） 210 - 224 2013年02月

ISSN 0894-1491

Astrocytes, a major subtype of glia, interact with neurons as a supportive partner supplying energy sources and growth factors. Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters (gliotransmitters). However, the precise role of gilotransmitters in regulating neuronal activity is still under debate. Here, we report that a subtle enhancement in the release of one gliotransmitter, ATP, affects synaptic potentiation from an analysis of mice containing an astrocyte-selective (GFAP) mutation. We found that, relative to normal mice, weaker stimulation induced long-term potentiation (LTP) in mutant mice, indicating that the threshold to induce LTP was lowered in the mutant. While excitatory transmission was normal in the mutant, inhibitory GABAergic transmission was suppressed. We found that a low concentration of adenosine selectively attenuated inhibitory neuronal activity and lowered the threshold to induce LTP in wild type mice. In comparison, adenosine A₁ receptor antagonism reversed the lowered LTP threshold back to normal in the mutant mouse. We verified that adenosine levels in the cerebrospinal fluid of mutant mice were slightly elevated compared to wild type mice. This was apparently caused by an increase in ATP release from mutant astrocytes that could provide a source of augmented adenosine levels in the mutant. ATP is thought to suppress the excitability of neuronal circuits; however, a small increase in ATP release can result in a suppressed inhibitory tone and enhanced excitability of neuronal circuitry. These findings demonstrate that ATP released from astrocytes acts in a bidirectional fashion to regulate neuronal excitability depending on concentration. © 2012 Wiley Periodicals, Inc.

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Olig2-lineage cells preferentially differentiate into oligodendrocytes but their processes degenerate at the chronic demyelinating stage of proteolipid protein-overexpressing mouse

Shimizu Takahiro, Tanaka Kenji F., Takebayashi Hirohide, Higashi Mikito, Wisesmith Wilaiwan, Ono Katsuhiko, Hitoshi Seiji, Ikenaka Kazuhiro

Journal of Neuroscience Research 91 （ 2 ） 178 - 186 2013年02月

ISSN 0360-4012

In chronic demyelinating lesions of the central nervous system, insufficient generation of oligodendrocytes (OLs) is not due to a lack of oligodendrocyte precursor cells (OPCs), because the accumulation of OPCs and premyelinating OLs can be observed within these lesions. Here we sought to identify the basis for the failure of OLs to achieve terminal differentiation in chronic demyelinating lesions through the utilization of plp1-overexpressing (Plp^tg/-) mice. These mice are characterized by progressive demyelination in young adults and chronic demyelinating lesions at more mature stages. We show that neural stem cells, which are the precursors of OL-lineage cells, are present in the Plp^tg/- mouse brain and that their multipotentiality and ability to self-renew are comparable to those of wild-type adults in culture. Lineage-tracing experiments using a transgenic mouse line, in which an inducible Cre recombinase is knocked in at the Olig2 locus, revealed that Olig2-lineage cells preferentially differentiated into OPCs and premyelinating OLs, but not into astrocytes, in the Plp^tg/- mouse brain. These Olig2-lineage cells matured to express myelin basic protein but after that their processes degenerated in the chronic demyelinating lesions of the Plp^tg/- brain. These results indicate that in chronic demyelinated lesions more OL-lineage cells are produced as part of the repair process, but their processes degenerate after maturation. © 2012 Wiley Periodicals, Inc.

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The manipulation of neural and cellular activities by ectopic expression of melanopsin

Koizumi Amane, Tanaka Kenji F., Yamanaka Akihiro

Neuroscience Research 75 （ 1 ） 3 - 5 2013年01月

ISSN 0168-0102

Melanopsin (OPN4) is a photosensitive pigment originally found in a subtype of retinal ganglion cells and is a 7-transmembrane G-protein-coupled receptor (GPCR). Several previous reports showed that ectopic expression of OPN4 can be used as an optogenetic tool to control neural and cellular activities in various tissues. Compared with other optogenetic pigments, OPN4 is more sensitive to light, shows long-lasting activation, and can also control intracellular Ca²⁺ dynamics. Here, we review how the ectopic expression of OPN4 enables the control of neural and cellular activities in vivo. In the retina, the ectopic expression of melanopsin in retinal ganglion cells successfully restored the vision of blind mice. It has also been reported that ectopic expression of melanopsin in orexin/hypocretin neurons enabled control of wakefulness in mice by blue light. In addition to neural activity, the ectopic expression of OPN4 has been reported to enable circuit control of the nuclear factor of activated T cells (NFAT) to enhance blood-glucose homeostasis in mice. We discuss the possibility of optogenetic control of other systems through the ectopic expression of OPN4. © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society.

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Ectopic expression of melanopsin in orexin/hypocretin neurons enables control of wakefulness of mice in vivo by blue light

Tsunematsu Tomomi, Tanaka Kenji F., Yamanaka Akihiro, Koizumi Amane

Neuroscience Research 75 （ 1 ） 23 - 28 2013年01月

ISSN 0168-0102

Melanopsin (OPN4) is a photosensitive G-protein-coupled photopigment and its ectopic expression enables control of neural activity induced by blue light. Here we report that we successfully expressed OPN4 in hypothalamic orexin/hypocretin neurons of double-transgenic mice (orexin-tTA; Bitet-O human OPN4 [hOPN4]/mCherry mice). In the double-transgenic mice, hypothalamic orexin neurons selectively expressed hOPN4 as well as mCherry as a reporter. We conducted slice patch-clamp recordings on hOPN4/mCherry-expressing orexin neurons, which showed long-lasting activation initiated by blue light even after the light was switched off. Optical fiber-guided blue light stimulation in the hypothalamus successfully initiated the electroencephalography pattern that reflects long-lasting wakefulness in the mice in vivo. Taken together, the results indicate that ectopic expression of hOPN4 in orexin neurons enables long-lasting activation of orexin neurons by blue light to control sleep/wakefulness of the mice. © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society.

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[Optogenetics in biological psychiatry].

Tanaka Kenji F., Takata Norio, Mimura Masaru

Unknown Journal 115 （ 12 ） 1211 - 1215 2013年

The manipulation of neural and cellular activities by ectopic expression of melanopsin.

田中謙二

Neurosci　Res. 75 3 - 5 2013年

研究論文（学術雑誌）, 共著, 査読有り

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Ectopic expression of melanopsin in orexin/hypocretin neurons enables control of wakefulness of mice in vivo by blue light.

田中謙二

Neurosci Res. 75 23 - 28 2013年

研究論文（学術雑誌）, 共著, 査読有り

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Olig2-lineage cells preferentially differentiate into oligodendrocytes but their processes degenerate at the chronic demyelinating stage of proteolipid protein-overexpressing mouse.

田中謙二

J Neurosci Res. 91 178 - 186 2013年

研究論文（学術雑誌）, 共著, 査読有り

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Increased astrocytic ATP release results in enhanced excitability of the hippocampus.

田中謙二

Glia. 61 210 - 214 2013年

研究論文（学術雑誌）, 共著, 査読有り

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Influence of inhibitory serotonergic inputs to orexin/hypocretin neurons on the diurnal rhythm of sleep and wakefulness.

田中謙二

Sleep. 36 1391 - 1404 2013年

研究論文（学術雑誌）, 共著

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Long-lasting silencing of orexin/hypocretin neurons using archaerhodopsin induces slow-wave sleep in mice.

田中謙二

Behav Brain Res. 255 64 - 74 2013年

研究論文（学術雑誌）, 共著, 査読有り

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Identification of Optogenetically Activated Striatal Medium Spiny Neurons by Npas4 Expression

Bepari Asim K., Sano Hiromi, Tamamaki Nobuaki, Nambu Atsushi, Tanaka Kenji F., Takebayashi Hirohide

PLoS One 7 （ 12 ） 2012年12月

ISSN 1932-6203

Optogenetics is a powerful neuromodulatory tool with many unique advantages to explore functions of neuronal circuits in physiology and diseases. Yet, interpretation of cellular and behavioral responses following in vivo optogenetic manipulation of brain activities in experimental animals often necessitates identification of photoactivated neurons with high spatial resolution. Although tracing expression of immediate early genes (IEGs) provides a convenient approach, neuronal activation is not always followed by specific induction of widely used neuronal activity markers like c-fos, Egr1 and Arc. In this study we performed unilateral optogenetic stimulation of the striatum in freely moving transgenic mice that expressed a channelrhodopsin-2 (ChR2) variant ChR2(C128S) in striatal medium spiny neurons (MSNs). We found that in vivo blue light stimulation significantly altered electrophysiological activity of striatal neurons and animal behaviors. To identify photoactivated neurons we then analyzed IEG expression patterns using in situ hybridization. Upon light illumination an induction of c-fos was not apparent whereas another neuronal IEG Npas4 was robustly induced in MSNs ipsilaterally. Our results demonstrate that tracing Npas4 mRNA expression following in vivo optogenetic modulation can be an effective tool for reliable and sensitive identification of activated MSNs in the mouse striatum. © 2012 Bepari et al.

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Application of an optogenetic byway for perturbing neuronal activity via glial photostimulation.

Sasaki Takuya, Beppu Kaoru, Tanaka Kenji F., Fukazawa Yugo, Shigemoto Ryuichi, Matsui K.

Proceedings of the National Academy of Sciences of the United States of America 109 （ 50 ） 20720 - 20725 2012年12月

ISSN 0027-8424

Dynamic activity of glia has repeatedly been demonstrated, but if such activity is independent from neuronal activity, glia would not have any role in the information processing in the brain or in the generation of animal behavior. Evidence for neurons communicating with glia is solid, but the signaling pathway leading back from glial-to-neuronal activity was often difficult to study. Here, we introduced a transgenic mouse line in which channelrhodopsin-2, a light-gated cation channel, was expressed in astrocytes. Selective photostimulation of these astrocytes in vivo triggered neuronal activation. Using slice preparations, we show that glial photostimulation leads to release of glutamate, which was sufficient to activate AMPA receptors on Purkinje cells and to induce long-term depression of parallel fiber-to-Purkinje cell synapses through activation of metabotropic glutamate receptors. In contrast to neuronal synaptic vesicular release, glial activation likely causes preferential activation of extrasynaptic receptors that appose glial membrane. Finally, we show that neuronal activation by glial stimulation can lead to perturbation of cerebellar modulated motor behavior. These findings demonstrate that glia can modulate the tone of neuronal activity and behavior. This animal model is expected to be a potentially powerful approach to study the role of glia in brain function.

Shared synaptic pathophysiology in syndromic and nonsyndromic rodent models of autism

Baudouin Stéphane J., Gaudias Julien, Gerharz Stefan, Hatstatt Laetitia, Zhou Kuikui, Punnakkal Pradeep, Tanaka Kenji F., Spooren Will, Hen Rene, De Zeeuw Chris I., Vogt Kaspar, Scheiffele Peter

Science 338 （ 6103 ） 128 - 132 2012年10月

ISSN 0036-8075

The genetic heterogeneity of autism poses a major challenge for identifying mechanism-based treatments. A number of rare mutations are associated with autism, and it is unclear whether these result in common neuronal alterations. Monogenic syndromes, such as fragile X, include autism as one of their multifaceted symptoms and have revealed specific defects in synaptic plasticity. We discovered an unexpected convergence of synaptic pathophysiology in a nonsyndromic form of autism with those in fragile X syndrome. Neuroligin-3 knockout mice (a model for nonsyndromic autism) exhibited disrupted heterosynaptic competition and perturbed metabotropic glutamate receptor - dependent synaptic plasticity, a hallmark of fragile X. These phenotypes could be rescued by reexpression of neuroligin-3 in juvenile mice, highlighting the possibility of reverting neuronal circuit alterations in autism after the completion of development.

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Preinspiratory calcium rise in putative pre-Bötzinger complex astrocytes

Okada Yasumasa, Sasaki Takuya, Oku Yoshitaka, Takahashi Naoya, Seki Megumi, Ujita Sakiko, Tanaka Kenji F., Matsuki Norio, Ikegaya Yuji

Journal of Physiology 590 （ 19 ） 4933 - 4944 2012年10月

ISSN 0022-3751

The neural inspiratory activity originates from a ventrolateral medullary region called the pre-Bötzinger complex (preBötC), yet the mechanism underlying respiratory rhythmogenesis is not completely understood. Recently, the role of not only neurons but astrocytes in the central respiratory control has attracted considerable attention. Here we report our discovery that an intracellular calcium rise in a subset of putative astrocytes precedes inspiratory neuronal firing in rhythmically active slices. Functional calcium imaging from hundreds of preBötC cells revealed that a subset of putative astrocytes exhibited rhythmic calcium elevations preceding inspiratory neuronal activity with a time lag of approximately 2 s. These preinspiratory putative astrocytes maintained their rhythmic activities even during the blockade of neuronal activity with tetrodotoxin, whereas the rhythm frequency was lowered and the intercellular phases of these rhythms were decoupled. In addition, optogenetic stimulation of preBötC putative astrocytes induced firing of inspiratory neurons. These findings raise the possibility that astrocytes in the preBötC are actively involved in respiratory rhythm generation in rhythmically active slices. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.

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Serotonin receptor expression along the dorsal-ventral axis of mouse hippocampus

Tanaka Kenji F., Samuels Benjamin Adam, Hen René

Philosophical Transactions of the Royal Society B: Biological Sciences 367 （ 1601 ） 2395 - 2401 2012年09月

ISSN 0962-8436

Using in situ hybridization, we describe, for the first time, the profiles of expression of serotonin receptors (Htr/5-HTR) along the dorsal-ventral axis of mouse hippocampus. cRNA probes for most Htrs, excluding Htr6, were used. All hippocampal subregions and the entorhinal cortex cells providing input into the hippocampus were examined. The study shows that some, but not all, Htrs are expressed in the cells of the hippocampal circuitry. At both the subfield and the cell type levels, a somewhat overlapping pattern is observed. Four serotonin receptors, Htr1a, Htr2a, Htr2c and Htr7, display an expression pattern that changes along the dorsal-ventral axis of the hippocampus. Given the proposed functional differentiation of the hippocampus along its long axis, with the dorsal pole more involved in cognitive functions and the ventral pole more involved in mood and anxiety, our results suggest that serotonin receptors enriched in the ventral pole probably contribute to mood- and anxiety-related behaviours. © 2012 The Royal Society.

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Expanding the Repertoire of Optogenetically Targeted Cells with an Enhanced Gene Expression System

Tanaka Kenji F., Matsui Ko, Sasaki Takuya, Sano Hiromi, Sugio Shouta, Fan Kai, Hen René, Nakai Junichi, Yanagawa Yuchio, Hasuwa Hidetoshi, Okabe Masaru, Deisseroth Karl, Ikenaka Kazuhiro, Yamanaka Akihiro

Cell Reports 2 （ 2 ） 397 - 406 2012年08月

Drosophila neuroblasts (NBs) have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. Here, we describe a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. We determine transcriptional profiles by mRNA sequencing and identify 28 predicted NB-specific transcription factors that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. We show that loss of Klumpfuss function causes premature differentiation and that overexpression results in the formation of transplantable brain tumors. Our data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well.

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Challenge to understand the neurobiology of obsessive-compulsive disorder

Tanaka Kenji

Brain and Nerve 64 （ 8 ） 903 - 909 2012年08月

ISSN 1881-6096

Obsessive-compulsive disorder (OCD) is characterized by obsession (recurrent intrusive thoughts) and compulsion (repetitive behaviors or mental acts). There is no consensus regarding the pathogenesis of OCD, which could support the idea that this disorder is heterogeneous. However, functional imaging data and surgical findings in humans suggest that the hyperactivity of the specific circuit including the striatum, called the cortico-striato-thalamo- cortical circuit, plays a role in OCD pathogenesis. Recently, validated animal models of OCD have been established, and they provide us the opportunity to address how the altered functional activity of this circuit contributes to the repetitive behavior in OCD. To test the causal relationship between the altered function of the circuit and behavioral abnormalities in animals, cell-type-specific manipulation and detection of changes in the circuit will be required. Optogenetic approaches may be used as tools to dissect the complex circuit. Moreover, mouse functional magnetic resonance imaging may yield data comparable to human imaging data.

Gene induction in mature oligodendrocytes with a PLP-tTA mouse line

Inamura Naoko, Sugio Shouta, Macklin Wendy B., Tomita Koichi, Tanaka Kenji F., Ikenaka Kazuhiro

Genesis 50 （ 5 ） 424 - 428 2012年05月

ISSN 1526-954X

Mature oligodendrocytes are critical for myelin maintenance. To understand the molecular basis for this, genetic manipulation of mature oligodendrocytes is needed. Here we generated a mature oligodendrocyte tTA (tetracycline-controlled transcriptional activator) mouse line which, in combination with a tTA-dependent promoter line driving the expression of the desired transgene, can be used for gain-of-function studies. We used an oligodendrocyte promoter, the mouse proteolipid protein (PLP) promoter, to express mammalianized tTA, and generated a PLP-mtTA mouse line. In adults, mtTA mRNA was predominantly detected in brain white matter where it co-localized with PLP mRNA. mtTA-mediated gene induction was confirmed by crossing to mice with a tTA-dependent promoter driving expression of yellow fluorescent protein (tetO-YFP mice). YFP induction in PLP-mtTA::tetO-YFP mice was consistent with PLP expression in adult mature oligodendrocytes and premyelinating-stage myelinating oligodendrocytes. This PLP-mtTA mouse line is the first to enable gain-of-function studies in mature oligodendrocytes with the tet system. © 2011 Wiley Periodicals, Inc.

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Mitral cells in the olfactory bulb are mainly excited through a multistep signaling path

Gire David H., Franks Kevin M., Zak Joseph D., Tanaka Kenji F., Whitesell Jennifer D., Mulligan Abigail A., Hen René, Schoppa Nathan E.

Journal of Neuroscience 32 （ 9 ） 2964 - 2975 2012年02月

ISSN 0270-6474

Within the olfactory system, information flow from the periphery onto output mitral cells (MCs) of the olfactory bulb (OB) has been thought to be mediated by direct synaptic inputs from olfactory sensory neurons (OSNs). Here, we performed patch-clamp measurements in rat and mouse OB slices to investigate mechanisms of OSN signaling onto MCs, including the assumption of a direct path, using electrical and optogenetic stimulation methods that selectively activated OSNs. We found that MCs are in fact not typically activated by direct OSN inputs and instead require a multistep, diffuse mechanism involving another glutamatergic cell type, the tufted cells. The preference for a multistep mechanism reflects the fact that signals arising from direct OSN inputs are drastically shunted by connexin 36-mediated gap junctions on MCs, but not tufted cells. An OB circuit with tufted cells intermediate between OSNs and MCs suggests that considerable processing of olfactory information occurs before its reaching MCs. © 2012 the authors.

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Mitral cells in the olfactory bulb are mainly excited through a multistep signaling path.

田中謙二

J Neurosci. 32 2964 - 2975 2012年

研究論文（学術雑誌）, 査読有り

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Application of an optogenetic byway for perturbing neuronal activity via glial photostimulation.

田中謙二

Proc Natl Acad Sci U S A 109 20720 - 20725 2012年

研究論文（学術雑誌）, 共著, 査読有り

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Identification of optogenetically activated striatal medium spiny neurons by npas4 expression.

田中謙二

PLoS One 7 e52783 2012年

査読有り

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Preinspiratory calcium rise in putative pre-Botzinger complex astrocytes.

田中謙二

J Physiol 590 4933 - 4944 2012年

共著, 査読有り

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Shared Synaptic Pathophysiology in Syndromic and Nonsyndromic Rodent Models of Autism.

田中謙二

Science 338 128 - 132 2012年

研究論文（学術雑誌）, 共著, 査読有り

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Serotonin receptor expression along the dorsal-ventral axis of mouse hippocampus.

田中謙二

Philos Trans R Soc Lond B Biol Sci 367 2395 - 2401 2012年

研究論文（学術雑誌）, 共著, 査読有り

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Expanding the Repertoire of Optogenetically Targeted Cells with an Enhanced Gene Expression System.

田中謙二

Cell Rep 2 397 - 406 2012年

研究論文（学術雑誌）, 共著, 査読有り

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Gene induction in mature oligodendrocytes with a PLP-tTA mouse line.

田中謙二

Genesis 50 424 - 428 2012年

研究論文（学術雑誌）, 共著, 査読有り

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Mammalian Gcm genes induce Hes5 expression by active DNA demethylation and induce neural stem cells

Hitoshi Seiji, Ishino Yugo, Kumar Akhilesh, Jasmine Salma, Tanaka Kenji F., Kondo Takeshi, Kato Shigeaki, Hosoya Toshihiko, Hotta Yoshiki, Ikenaka Kazuhiro

Nature Neuroscience 14 （ 8 ） 957 - 964 2011年08月

ISSN 1097-6256

Signaling mediated by Notch receptors is crucial for the development of many organs and the maintenance of various stem cell populations. The activation of Notch signaling is first detectable by the expression of an effector gene, Hes5, in the neuroepithelium of mouse embryos at embryonic day (E) 8.0-8.5, and this activation is indispensable for the generation of neural stem cells. However, the molecular mechanism by which Hes5 expression is initiated in stem-producing cells remains unknown. We found that mammalian Gcm1 and Gcm2 (glial cells missing 1 anD₂) are involved in the epigenetic regulation of Hes5 transcription by DNA demethylation independently of DNA replication. Loss of both Gcm genes and subsequent lack of Hes5 upregulation in the neuroepithelium of E7.5-8.5 Gcm1 ^-/-; Gcm2 ^-/- mice resulted in the impaired induction of neural stem cells. Our data suggest that Hes5 expression is serially activated first by Gcms and later by the canonical Notch pathway. © 2011 Nature America, Inc. All rights reserved.

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Microglial cystatin F expression is a sensitive indicator for ongoing demyelination with concurrent remyelination

Ma Jianmei, Tanaka Kenji F., Shimizu Takahiro, Bernard Claude C A, Kakita Akiyoshi, Takahashi Hitoshi, Pfeiffer Steven E., Ikenaka Kazuhiro

Journal of Neuroscience Research 89 （ 5 ） 639 - 649 2011年05月

ISSN 0360-4012

Demyelination coincides with numerous changes of gene expression in the central nervous system (CNS). Cystatin F, which is a papain-like lysosomal cysteine proteinase inhibitor that is normally expressed by immune cells and not in the brain, is massively induced in the CNS during acute demyelination. We found that microglia, which are monocyte/macrophage-lineage cells in the CNS, express cystatin F only during demyelination. By using several demyelinating animal models and the spinal cord tissues from multiple sclerosis (MS) patients, we examined spatiotemporal expression pattern of cystatin F by in situ hybridization and immunohistochemistry. We found that the timing of cystatin F induction matches with ongoing demyelination, and the places with cystatin F expression overlapped with the remyelinating area. Most interestingly, cystatin F induction ceased in chronic demyelination, in which remyelinating ability was lost. These findings demonstrate that the expression of cystatin F indicates the occurrence of ongoing demyelination/remyelination and the absence of cystatin F expression indicates the cessation of remyelination in the demyelinating area. © 2011 Wiley-Liss, Inc.

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Leptin-dependent serotonin control of appetite

Yadav Vijay K., Oury Franck, Tanaka Kenji F., Thomas Tiffany, Wang Ying, Cremers Serge, Hen Rene, Krust Andree, Chambon Pierre, Karsenty Gerard

Journal of Experimental Medicine 208 （ 2 ） 413 2011年02月

ISSN 0022-1007

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Functional connectome of the striatal medium spiny neuron

Chuhma Nao, Tanaka Kenji F., Hen René, Rayport Stephen

Journal of Neuroscience 31 （ 4 ） 1183 - 1192 2011年01月

ISSN 0270-6474

Dopamine system disorders ranging from movement disorders to addiction and schizophrenia involve striatal medium spiny neurons (MSNs), yet their functional connectivity has been difficult to determine comprehensively. We generated a mouse with conditional channelrhodopsin-2 expression restricted to medium spiny neurons and assessed the specificity and strength of their intrinsic connections in the striatum and their projections to the globus pallidus and the substantia nigra. In the striatum, medium spiny neurons connected with other MSNs and tonically active cholinergic interneurons, but not with fast-spiking GABA interneurons. In the globus pallidus, medium spiny neurons connected strongly with one class of electrophysiologically identified neurons, but weakly with the other. In the substantia nigra, medium spiny neurons connected strongly with GABA, but not with dopamine neurons. Projections to the globus pallidus showed solely D2-mediated presynaptic inhibition, whereas projections to the substantia nigra showed solely D1-mediated presynaptic facilitation. This optogenetic approach defines the functional connectome of the striatal medium spiny neuron. Copyright © 2011 the authors.

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Leptin-dependent serotonin control of appetite

Yadav Vijay K., Oury Franck, Tanaka Kenji, Thomas Tiffany, Wang Ying, Cremers Serge, Hen Rene, Krust Andree, Chambon Pierre, Karsenty Gerard

Journal of Experimental Medicine 208 （ 1 ） 41 - 52 2011年01月

ISSN 0022-1007

Recent evidence indicates that leptin regulates appetite and energy expenditure, at least in part by inhibiting serotonin synthesis and release from brainstem neurons. To demonstrate that this pathway works postnatally, we used a conditional, brainstem-specific mouse CreER^T2 driver to show that leptin signals in brainstem neurons after birth to decrease appetite by inhibiting serotonin synthesis. Cell-specific gene deletion experiments and intracerebroventricular leptin infusions reveal that serotonin signals in arcuate nuclei of the hypothalamus through the Htr1a receptor to favor food intake and that this serotonin function requires the expression of Creb, which regulates the expression of several genes affecting appetite. Accordingly, a specific antagonist of the Htr1a receptor decreases food intake in leptin-deficient but not in Htr1a^-/- mice. Collectively, these results establish that leptin inhibition of serotonin is necessary to inhibit appetite postnatally and provide a proof of principle that selective inhibition of this pathway may be a viable option to treat appetite disorders. © 2011 Yadav et al.

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Increased numbers of oligodendrocyte lineage cells in the optic nerves of cerebroside sulfotransferase knockout mice

Kajigaya Hitoshi, Tanaka Kenji F., Hayashi Akiko, Suzuki Ayaka, Ishibashi Tomoko, Ikenaka Kazuhiro, Baba Hiroko

Proceedings of the Japan Academy Series B: Physical and Biological Sciences 87 （ 7 ） 415 - 424 2011年

ISSN 0386-2208

Sulfatide is a myelin glycolipid that functions in the formation of paranodal axoglial junctions in vivo and in the regulation of oligodendrocyte differentiation in vitro. Cerebroside sulfotransferase (CST) catalyzes the production of two sulfated glycolipids, sulfatide and proligodendroblast antigen, in oligodendrocyte lineage cells. Recent studies have demonstrated significant increases in oligodendrocytes from the myelination stage through adulthood in brain and spinal cord under CST-deficient conditions. However, whether these result from excess migration or in situ proliferation during development is undetermined. In the present study, CST-deficient optic nerves were used to examine migration and proliferation of oligodendrocyte precursor cells (OPCs) under sulfated glycolipid-deficient conditions. In adults, more NG2-positive OPCs and fully differentiated cells were observed. In developing optic nerves, the number of cells at the leading edge of migration was similar in CST-deficient and wild-type mice. However, BrdUD proliferating OPCs were more abundant in CST-deficient mice. These results suggest that sulfated glycolipids may be involved in proliferation of OPCs in vivo. ©2011 The Japan Academy.

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Flexible Accelerated STOP Tetracycline Operator-Knockin (FAST)

Tanaka Kenji F., Ahmari Susanne E., Leonardo E. David, Richardson-Jones Jesse W., Budreck Elaine C., Scheiffele Peter, Sugio Shouta, Inamura Naoko, Ikenaka Kazuhiro, Hen René

Biological Psychiatry 67 （ 8 ） 770 - 773 2010年04月

ISSN 0006-3223

We created the Flexible Accelerated STOP Tetracycline Operator (tetO)-knockin (FAST) system, an efficient method for manipulating gene expression in vivo to rapidly screen animal models of disease. A single gene targeting event yields two distinct knockin mice-STOP-tetO and tetO knockin-that permit generation of multiple strains with variable expression patterns: 1) knockout, 2) Cre-mediated rescue, 3) tetracycline-controlled transcriptional activator (tTA)-mediated misexpression, 4) tetracycline-controlled transcriptional activator (tTA)-mediated overexpression, and 5) tetracycline-controlled transcriptional silencer (tTS)-mediated conditional knockout/knockdown. Using the FAST system, multiple gain-of-function and loss-of-function strains can therefore be generated on a time scale not previously achievable. These strains can then be screened for clinically relevant abnormalities. We demonstrate the flexibility and broad applicability of the FAST system by targeting several genes encoding proteins implicated in neuropsychiatric disorders: Mlc1, neuroligin 3, the serotonin 1A receptor, and the serotonin 1B receptor. © 2010 Society of Biological Psychiatry.

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Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro

Moritoh Satoru, Tanaka Kenji F., Jouhou Hiroshi, Ikenaka Kazuhiro, Koizumi Amane

PLoS One 5 （ 9 ） 2010年

ISSN 1932-6203

Background: Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. Methodology/Principal Findings: We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age) which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames' medium (>26 mL) per retina, a higher speed (constant 55 rpm) of agitation by rotary shaker, and a greater concentration (10%) of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age). The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP) into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons. Conclusions/Significance: This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing molecular biological bioassays that used to be conducted in isolated cultured cells. © 2010 Moritoh et al.

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A Serotonin-Dependent Mechanism Explains the Leptin Regulation of Bone Mass, Appetite, and Energy Expenditure

Yadav Vijay K., Oury Franck, Suda Nina, Liu Zhong Wu, Gao Xiao Bing, Confavreux Cyrille, Klemenhagen Kristen C., Tanaka Kenji F., Gingrich Jay A., Guo X. Edward, Tecott Laurence H., Mann J. John, Hen Rene, Horvath Tamas L., Karsenty Gerard

Cell 138 （ 5 ） 976 - 989 2009年09月

ISSN 0092-8674

Leptin inhibition of bone mass accrual requires the integrity of specific hypothalamic neurons but not expression of its receptor on these neurons. The same is true for its regulation of appetite and energy expenditure. This suggests that leptin acts elsewhere in the brain to achieve these three functions. We show here that brainstem-derived serotonin (BDS) favors bone mass accrual following its binding to Htr2c receptors on ventromedial hypothalamic neurons and appetite via Htr1a and 2b receptors on arcuate neurons. Leptin inhibits these functions and increases energy expenditure because it reduces serotonin synthesis and firing of serotonergic neurons. Accordingly, while abrogating BDS synthesis corrects the bone, appetite and energy expenditure phenotypes caused by leptin deficiency, inactivation of the leptin receptor in serotonergic neurons recapitulates them fully. This study modifies the map of leptin signaling in the brain and identifies a molecular basis for the common regulation of bone and energy metabolisms. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online. © 2009 Elsevier Inc. All rights reserved.

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Synergistic neurochemical and behavioural effects of acute intrahippocampal injection of brain-derived neurotrophic factor and antidepressants in adult mice

Deltheil Thierry, Tanaka Kenji, Reperant Christelle, Hen René, David Denis J., Gardier Alain M.

International Journal of Neuropsychopharmacology 12 （ 7 ） 905 - 915 2009年08月

ISSN 1461-1457

Preclinical data support the view that brain-derived neurotrophic factor (BDNF) and serotonergic systems regulate circuits involved in affective disorders. The present study examined neurochemical and behavioural consequences of an acute intrahippocampal injection of BDNF combined with an antidepressant by using in-vivo intracerebral microdialysis in the ventral hippocampus (vHi) in conscious mice and behavioural paradigms predictive of antidepressant and anxiolytic-like effects [the mouse forced swim test (FST), the open-field (OF) paradigm and the elevated plus maze (EPM)]. Neurochemical data revealed that BDNF (100 ng) potentiated the effects of the systemic administration of a serotonin selective reuptake inhibitor (SSRI; paroxetine 4 mg/kg i.p.) and that of a locally applied citalopram perfusion on dialysate 5-HT levels in the vHi. These neurochemical changes correlated with behavioural data since, in the FST, antidepressant-like activity of paroxetine as measured on swimming behaviour was potentiated by BDNF. These data suggest an interesting synergy between BDNF and SSRI on antidepressant-like activity. Furthermore, in both the OF and EPM paradigms BDNF induced an anxiogenic-like activity, whereas paroxetine prevented this effect. Finally, the neurochemical and behavioural effects of BDNF on the serotonergic system might occur at both pre- and post-synaptic levels since by using in-situ hybridization, we showed that TrkB-R mRNA was expressed in the hippocampus and the dorsal raphe nucleus in adult mice. Taken together the neurochemical and behavioural effects of BDNF suggest that these behavioural changes were mediated by increases in 5-HT neurotransmission in vHi. Thus a BDNF+SSRI combination may offer new alternatives to treat mood disorders. Copyright © 2009 CINP.

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Mice with altered myelin proteolipid protein gene expression display cognitive deficits accompanied by abnormal neuron-glia interactions and decreased conduction velocities

Tanaka Hisataka, Ma Jianmei, Tanaka Kenji F., Takao Keizo, Komada Munekazu, Tanda Koichi, Suzuki Ayaka, Ishibashi Tomoko, Baba Hiroko, Isa Tadashi, Shigemoto Ryuichi, Ono Katsuhiko, Miyakawa Tsuyoshi, Ikenaka Kazuhiro

Journal of Neuroscience 29 （ 26 ） 8363 - 8371 2009年07月

ISSN 0270-6474

Conduction velocity (CV) of myelinated axons has been shown to be regulated by oligodendrocytes even after myelination has been completed. However, how myelinating oligodendrocytes regulate CV, and what the significance of this regulation is for normal brain function remain unknown. To address these questions, we analyzed a transgenic mouse line harboring extra copies of the myelin proteolipid protein 1 (plp1) gene (plp1^tg/- mice) at 2 months of age. At this stage, the plp1^tg/- mice have an unaffected myelin structure with a normally appearing ion channel distribution, but the CV in all axonal tracts tested in the CNS is greatly reduced. We also found decreased axonal diameters and slightly abnormal paranodal structures, both of which can be a cause for the reduced CV. Interestingly the plp1^tg/- mice showed altered anxiety-like behaviors, reduced prepulse inhibitions, spatial learning deficits and working memory deficit, all of which are schizophrenia-related behaviors. Our results implicate that abnormalities in the neuron-glia interactions at the paranodal junctions can result in reduced CV in the CNS, which then induces behavioral abnormalities related to schizophrenia. Copyright © 2009 Society for Neuroscience.

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LDL-receptor related protein five controls bone formation by inhibiting serotonin synthesis in the duodenum

Yadav Vijay K., Ryu Je Hwang, Suda Nina, Tanaka Kenji F., Gingrich Jay A., Schütz Günther, Glorieux Francis H., Chiang Cherie Y., Zajac Jeffrey D., Insogna Karl L., John Mann J., Hen Rene, Ducy Patricia, Karsenty Gerard

Obstetrical and Gynecological Survey 64 （ 4 ） 240 - 242 2009年04月

ISSN 0029-7828

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Lrp5 Controls Bone Formation by Inhibiting Serotonin Synthesis in the Duodenum

Yadav Vijay K., Ryu Je Hwang, Suda Nina, Tanaka Kenji F., Gingrich Jay A., Schütz Günther, Glorieux Francis H., Chiang Cherie Y., Zajac Jeffrey D., Insogna Karl L., Mann J. John, Hen Rene, Ducy Patricia, Karsenty Gerard

Cell 135 （ 5 ） 825 - 837 2008年11月

ISSN 0092-8674

Loss- and gain-of-function mutations in the broadly expressed gene Lrp5 affect bone formation, causing osteoporosis and high bone mass, respectively. Although Lrp5 is viewed as a Wnt coreceptor, osteoblast-specific disruption of β-Catenin does not affect bone formation. Instead, we show here that Lrp5 inhibits expression of Tph1, the rate-limiting biosynthetic enzyme for serotonin in enterochromaffin cells of the duodenum. Accordingly, decreasing serotonin blood levels normalizes bone formation and bone mass in Lrp5-deficient mice, and gut- but not osteoblast-specific Lrp5 inactivation decreases bone formation in a β-Catenin-independent manner. Moreover, gut-specific activation of Lrp5, or inactivation of Tph1, increases bone mass and prevents ovariectomy-induced bone loss. Serotonin acts on osteoblasts through the Htr1b receptor and CREB to inhibit their proliferation. By identifying duodenum-derived serotonin as a hormone inhibiting bone formation in an Lrp5-dependent manner, this study broadens our understanding of bone remodeling and suggests potential therapies to increase bone mass. © 2008 Elsevier Inc. All rights reserved.

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Behavioral and serotonergic consequences of decreasing or increasing hippocampus brain-derived neurotrophic factor protein levels in mice

Deltheil T., Guiard B. P., Cerdan J., David D. J., Tanaka K. F., Repérant C., Guilloux J. P., Coudoré F., Hen R., Gardier A. M.

Neuropharmacology 55 （ 6 ） 1006 - 1014 2008年11月

ISSN 0028-3908

Antidepressants such as Selective Serotonin Reuptake Inhibitors (SSRI) act as indirect agonists of serotonin (5-HT) receptors. Although these drugs produce a rapid blockade of serotonin transporters (SERTs) in vitro, several weeks of treatment are necessary to observe clinical benefits. This paradox has not been solved yet. Recent studies have identified modifications of intracellular signaling proteins and target genes that could contribute to antidepressant-like activity of SSRI (e.g., increases in neurogenesis and BDNF protein levels), and may explain, at least in part, their long delay of action. Although these data suggest a positive regulation of 5-HT on the expression of the gene coding for BDNF, the reciprocal effects of BDNF on brain 5-HT neurotransmission remains poorly documented. To study the impact of BDNF on serotonergic activity, a dual experimental strategy was used to analyze neurochemical and behavioral consequences of its decrease (strategy 1) or increase (strategy 2) in the brain of adult male mice. (1) In heterozygous BDNF+/- mice in which brain BDNF protein levels were decreased by half, an enhancement of basal extracellular 5-HT levels (5-HText) that induced a down-regulation of SERT, i.e., a decrease in its capacity to reuptake 5-HT, was found in the hippocampus. In addition, the SSRI, paroxetine, failed to increase hippocampal 5-HText in BDNF+/- mice, while it produces robust effects in wild-type littermates. Thus, BDNF+/- mice can be viewed as an animal model of genetic resistance to serotonergic antidepressant drugs. (2) In wild-type BDNF+/+ mice, the effects of intra-hippocampal (vHi) injection of BDNF (100 ng) in combination with a SSRI was examined by using intracerebral microdialysis and behavioral paradigms that predict an antidepressant- and anxiolytic-like activity of a molecule [the forced swim test (FST) and the open field paradigm (OF) respectively]. BDNF induced a rapid and transient increase in paroxetine response on 5-HText in the adult hippocampus, which was correlated with a potentiation of its antidepressant-like activity in the FST. The effects of BDNF were selectively blocked by K252a, an antagonist of its high-affinity TrkB receptor. Such a correlation between neurochemical and behavioral effects of [BDNF + SSRI] co-administration suggests that its antidepressant-like activity is linked to the activation of 5-HT neurotransmission in the adult hippocampus. BDNF also had a facilitatory effect on anxiety-like behavior in the OF test, and paroxetine prevented this anxiogenesis. What was the mechanism by which BDNF exerted these latter effects? Surprisingly, by using zero net flux method of quantitative microdialysis in vivo, we found that an intra-hippocampal BDNF injection in wild-type mice decreased the functional activity of SERT as observed in BDNF+/- mice. However, the decreased capacity of SERT to reuptake 5-HT was not associated to an increase in basal 5-HText in the hippocampus of WT mice. Interestingly, using in situ hybridization experiments indicated that TrkB receptor mRNA was expressed in the hippocampus and dorsal raphe nucleus in adult mice suggesting that the neurochemical and behavioral effects of intra-hippocampal BDNF injection can mobilize both pre- and post-synaptic elements of the brain 5-HT neurotransmission. Taken together, these set of experiments unveiled a relative opposition of neurochemical and behavioral responses following either a decrease (in BDNF+/- mutant mice) or an increase in brain BDNF levels (bilateral intra-hippocampal injection) in adult mice. In view of developing new antidepressant drug strategy, a poly-therapy combining BDNF with a chronic SSRI treatment could thus improve the efficacy of current medications. © 2008 Elsevier Ltd. All rights reserved.

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Termination of lesion-induced plasticity in the mouse barrel cortex in the absence of oligodendrocytes

Toda Tomohisa, Hayakawa Itaru, Matsubayashi Yutaka, Tanaka Kenji, Ikenaka Kazuhiro, Lu Qing Richard, Kawasaki Hiroshi

Molecular and Cellular Neuroscience 39 （ 1 ） 40 - 49 2008年09月

ISSN 1044-7431

Termination of developmental plasticity occurs at specific points in development, and the mechanisms responsible for it are not well understood. One hypothesis that has been proposed is that oligodendrocytes (OLs) play an important role. Consistent with this, we found that OLs appeared in the mouse somatosensory cortex at the end of the critical period for whisker lesion-induced barrel structural plasticity. To test this hypothesis, we used two mouse lines with defective OL differentiation: Olig1-deficient and jimpy. In Olig1-deficient mice, although OLs were totally absent, the termination of lesion-induced plasticity was not delayed. The timing was normal even when the cytoarchitectonic barrel formation was temporarily blocked by pharmacological treatment in Olig1-deficient mice. Furthermore, the termination was not delayed in jimpy mice. These results demonstrate that, even though OLs appear at the end of the critical period, OLs are not intrinsically necessary for the termination of lesion-induced plasticity. Our findings underscore a mechanistic distinction between the termination of thalamocortical axonal plasticity in the barrel cortex and that in the visual cortex, in which OL-derived Nogo-A/B was recently suggested to be essential. © 2008 Elsevier Inc. All rights reserved.

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GFAP aggregates in the cochlear nerve increase the noise vulnerability of sensory cells in the organ of Corti in the murine model of Alexander disease

Masuda Masatsugu, Tanaka Kenji F., Kanzaki Sho, Wakabayashi Kenichiro, Oishi Naoki, Suzuki Takafumi, Ikenaka Kazuhiro, Ogawa Kaoru

Neuroscience Research 62 （ 1 ） 15 - 24 2008年09月

ISSN 0168-0102

Outer hair cell (OHC) loss in the auditory sensory epithelium is a primary cause of noise-induced sensory-neural hearing loss (SNHL). To clarify the participation of glial cells in SNHL, we used an Alexander disease (AxD) mouse model. These transgenic mice harbor the AxD causal mutant of the human glial fibrillary acidic protein (GFAP) under the control of the mouse GFAP promoter. It is thought that GFAP aggregates compromise the function of astrocytes. In the auditory pathway, the formation of GFAP aggregates was observed only in GFAP-positive cells of the cochlear nerve. The presence of GFAP aggregates did not change auditory function at the threshold level. To assess the change in vulnerability to auditory excitotoxicity, both transgenic and control mice were treated with intense noise exposure. Auditory threshold shifts were assessed by auditory brainstem responses (ABR) at 1 and 4 weeks after noise exposure, and OHC damage was analyzed by quantitative histology at 4 weeks after exposure. Transgenic mice showed more severe ABR deficits and OHC damage, suggesting that cochlear nerve glial cells with GFAP aggregates play a role in noise susceptibility. Thus, we should focus more on the roles of cochlear nerve glial cells in SNHL. © 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society.

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Genetic fate mapping of Olig2 progenitors in the injured adult cerebral cortex reveals preferential differentiation into astrocytes

Tatsumi Kouko, Takebayashi Hirohide, Manabe Takayuki, Tanaka Kenji F., Makinodan Manabu, Yamauchi Takahira, Makinodan Eri, Matsuyoshi Hiroko, Okuda Hiroaki, Ikenaka Kazuhiro, Wanaka Akio

Journal of Neuroscience Research 86 （ 16 ） 3494 - 3502 2008年

ISSN 0360-4012

Olig2 is a basic helix-loop-helix (bHLH) transcription factor essential for development of motoneurons and oligodendrocytes. It is known that Olig2⁺ cells persist in the central nervous system (CNS) from embryonic to adult stages and that the number of Olig2⁺ progenitors increases in the injured adult CNS. Recent studies have demonstrated an inhibitory action of Olig2 on neurogenesis in adult CNS, but the fate of Olig2⁺ cells in the injured state remains largely unknown. To trace directly the fate of Olig2 cells in the adult cerebral cortex after injury, we employed the CreER/loxP system to target the olig2 locus. In this genetic tracing study, green fluorescent protein (GFP) reporter-positive cells labeled after cryoinjury coexpressed glial fibrillary acidic protein (GFAP), an astrocytic marker. Electron microscopy also showed that GFP⁺ cells have the ultrastructural characteristics of astrocytes. Furthermore, GFP⁺ cells labeled before injury, most of which had been NG2 cells, also produced bushy astrocytes. Here we show direct evidence that Olig2⁺ cells preferentially differentiate into astrocytes, which strongly express GFAP, in response to injury in the adult cerebral cortex. These results suggest that reactive astrocytes, known to be the main contributors to glial scars, originate, at least in part, from Olig2⁺ cells. © 2008 Wiley-Liss, Inc.

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Generation of mice with glial cell dysfunction

Tanaka Kenji, Hae Ung Lee, Ikenaka Kazuhiro

Brain and Nerve 59 （ 7 ） 747 - 753 2007年07月

ISSN 0006-8969

To examine astrocytic function, we have developed model mice harboring astrocyte-specific disease causal gene and tried to examine astrocytic function in vivo. Alexander disease, megalencephalic leukodystrophy with subcortical cysts (MLC), and autistic spectrum disorder with neuroligin 3/4 mutations are known to be astrocyte-specific disease so far. First of all, we have established Alexander disease model mouse. Alexander disease is caused by coding mutation in glial fibrillary acidic protein (GFAP) and mutant GFAP forms inclusion bodies, called Rosenthal fibers, in astrocytes. Its pathophysiology is still unknown. We generated transgenic mice that express human GFAP R239H mutant under the control of mouse GFAP promoter. Lines with single copy exhibited weak human GFAP expression in astrocytes that did not produce aggregates despite the existence of mutation, whereas lines with multi copies exhibited strong expression and the formation of aggregates, starting at P14. The line with aggregates showed higher sensitivity to kainate than the line without them, whose sensitivity was not different from the wild type mouse, suggesting that the presence of GFAP aggregates but not the presence of mutant GFAP altered the sensitivity. Changes in several electrophysiological parameters, including facilitation of LTP, were also observed in this model mouse. We believe that this transgenic line is a useful tool to study astrocytic function in vivo.

Murine model of Alexander disease

Tanaka Kenji F., Takebayashi Hirohide, Yamazaki Yoshihiko, Ono Katsuhiko, Naruse Masae, Iwasato Takuji, Itohara Shigeyoshi, Kato Hiroshi, Ikenaka Kazuhiro

GLIA 55 （ 6 ） 617 - 631 2007年04月

ISSN 0894-1491

Alexander disease is caused by a coding mutation in the glial fibrillary acidic protein (GFAP) gene. The pathological hallmark is the formation of cytoplasmic inclusions within astrocytes known as Rosenthal fibers (RFs), which primarily consist of GFAP and several heat shock proteins. The presence of mutant GFAP would appear to be involved in RF formation; however, overproduction of wild type human GFAP in mouse brain also results in RF formation. Here, we investigated the in vivo conditions leading to formation of RF-like aggregates. We used transgenic mice (mouse GFAP promoter-human GFAP cDNA with R239H mutation) in which the dosage of the GFAP transgene could be manipulated within the same genetic locus. We found that the presence of mutant GFAP per se was insufficient for aggregate formation. Instead, a 30% increase in GFAP content over that in wild type was also required. GFAP aggregates upregulated endogenous GFAP and nestin gene expression, and intermediate filament structure revealed by immunostaining was fragmented under these conditions. However, overall morphology of astrocytes, including their fine processes, was unaffected. In this transgenic animal model, mice did not show megalencephaly, leukodystrophy, or seizure characteristic of Alexander disease with R239H mutation. Nevertheless, their mortality after kainate challenge was dramatically increased, whereas transgenic mice lacking aggregates exhibited mortality similar to that of wild type mice. These results indicate that the presence of GFAP aggregates containing mutant GFAP is not sufficient to induce a major phenotype of Alexander disease, even though it causes some abnormalities in the mouse. © 2007 Wiley-Liss, Inc.

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Induced expression of cathepsins and cystatin C in a murine model of demyelination

Ma Jianmei, Tanaka Kenji F., Yamada Gen, Ikenaka Kazuhiro

Neurochemical Research 32 （ 2 ） 311 - 320 2007年02月

ISSN 0364-3190

While proteolytic enzymes are involved in the pathogenesis of multiple sclerosis (MS), the involvement of cathepsins has not been characterized in detail. To better understand the role of cathepsins, cDNA microarray analysis was used to study the brains of proteolipid protein transgenic (plp^tg /-) mice, an animal model that closely mimics the failure of remyelination in MS. Analysis revealed upregulated expression of cathepsins L, H and B and their inhibitor, cystatin C. By in situ hybridization, the induction of cathepsins was primarily limited to microglia/macrophages of the white matter, with continuous expression from 2 to 8 months of age. Elevated protein level of cathepsins was confirmed at 4 months of age. In contrast, elevated expression of cystatin C was found in astrocytes. The ratio of microglia/macrophages to astrocytes increased throughout the course of demyelination, suggesting that the ratio of secreted cathepsins to cystatin C increased during that period. We propose that in MS, remyelination may be impaired by increasing activity of cathepsins inadequately controlled by cystatin C. © 2006 Springer Science+Business Media, LLC.

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Fluoro-Jade

Tanaka Kenji F., Ochi Nobuhiko, Hayashi Toshiyuki, Ikeda Eiji, Ikenaka Kazuhiro

Neuroscience Letters 407 （ 2 ） 127 - 130 2006年10月

ISSN 0304-3940

Rosenthal fibers are homogeneous eosinophilic masses found in astrocytes that are composed of glial fibrillary acidic protein (GFAP) aggregates along with chaperone proteins and other unknown components. Rosenthal fiber formation is a pathological hallmark of Alexander disease and its detection is diagnostically significant. However, the lack of a specific fluorescent marker has greatly limited the histochemical characterization of Rosenthal fibers. Here, we report for the first time a fluorescent marker of Rosenthal fibers called Fluoro Jade. Fluoro Jade-positive masses were seen in samples of Alexander disease brain, pilocytic astrocytoma, and in brain tissue from a mouse model of Alexander disease. Fluoro Jade co-labeled tissue samples stained with GFAP immunofluorescence. Our results indicated that Fluoro Jade labeled Rosenthal fibers, and that Rosenthal fibers could be labeled with antibodies of interest in combination with Fluoro Jade staining. © 2006 Elsevier Ireland Ltd. All rights reserved.

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An animal model for late onset chronic demyelination disease caused by failed terminal differentiation of oligodendrocytes

Ma Jianmei, Matsumoto Michio, Tanaka Kenji F., Takebayashi Hirohide, Ikenaka Kazuhiro

Neuron Glia Biology 2 （ 2 ） 81 - 91 2006年05月

ISSN 1740-925X

Various animal models are available for studying human multiple sclerosis (MS). Most of them model the initial phase of MS, including the immune-triggered attack of the myelin membrane and/or oligodendrocytes and, occasionally, demonstrate the remission and relapsing phases. However, few mimic the late chronic demyelinating phase. Overexpression of the proteolipid protein gene (Plp) causes a unique demyelinating disorder in mice in which normal-appearing myelin forms early in life and chronic demyelination occurs later. We found that remyelination is severely affected in this late demyelinating phase, but is not caused by deprivation of oligodendrocyte progenitors expressing PDGF receptor alpha (PDGFRα) and Olig2, which are present at an even higher number in the demyelinated white matter of the mutants than in wild-type controls. Furthermore, mature oligodendrocytes containing PLP were observed, but failed to remyelinate. The ability of oligodendrocytes from older transgenic animals to produce a myelin membrane-like structure was not impaired when cultured in vitro, which indicates that the lack of remyelination is not simply caused by changes in the intrinsic properties of the oligodendrocytes. Glial activation also occurred much earlier than active demyelination in mutant mice. Thus, in addition to intrinsic mechanisms, extrinsic mechanisms might also have an important role in defects of remyelination. These features are also observed in patients at a late stage of MS, leading to chronic demyelinating lesions. Thus, this mouse model partly mimics the late stage of MS and can be used to study the cause of inhibition of remyelination. © 2005 Cambridge University Press.

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Short-term lineage analysis of dorsally derived Olig3 cells in the developing spinal cord

Ding Lei, Takebayashi Hirohide, Watanabe Keisuke, Ohtsuki Toshiaki, Tanaka Kenji F., Nabeshima Yo Ichi, Chisaka Osamu, Ikenaka Kazuhiro, Ono Katsuhiko

Developmental Dynamics 234 （ 3 ） 622 - 632 2005年11月

ISSN 1058-8388

We examined the migration and differentiation of cells expressing Olig3, a basic helix-loop-helix transcriptional factor, in the developing spinal cord. Distribution of Olig3 lineage cells was demonstrated with in situ hybridization and X-gal staining in an Olig3-lacZ knock-in mouse. Olig3-positive cells first appeared in the dorsal spinal cord, except for the roof plate. Some of the dorsal Olig3 lineage cells co-expressed Islet1/2, Math1, or Brn3a, markers for dorsal interneuron. LacZ-positive cells were observed in the ventral-most part of the E10.5 spinal cord, suggesting that some dorsal Olig3 lineage cells migrate into the ventral-most part by E10.5. Ventral-ward migration of dorsal cells and contribution to commissural interneurons were substantiated by electroporation of EGFP expression plasmid in the dorsal spinal cord of chick embryo. Dorsal midline cells were also LacZ-positive during development. These findings suggest that dorsal Olig3 cells contribute to dorsal midline cells and commissural interneurons at intermediate and ventral levels. © 2005 Wiley-Liss, Inc.

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Revising polypharmacy to a single antipsychotic regimen for patients with chronic schizophrenia

Suzuki Takefumi, Uchida Hiroyuki, Tanaka Kenji F., Nomura Kensuke, Takano Harumasa, Tanabe Akira, Watanabe Koichiro, Yagi Gohei, Kashima Haruo

International Journal of Neuropsychopharmacology 7 （ 2 ） 133 - 142 2004年06月

ISSN 1461-1457

Antipsychotic polypharmacy has been empirically used and a recent trend in favour of that mode of therapy has been suggested for the treatment of schizophrenia. The clinical efficacy, however, still remains to be clarified. In order to critically evaluate the usefulness of such kind of psychopharmacotherapy, antipsychotic combination regimen (polypharmacy) was switched to a treatment with the single main antipsychotic (monotherapy) in cross-tapered fashion, while approximately maintaining the total amount, for patients with chronic schizophrenia. Patients had been treated with an average of three antipsychotics and maintained with the same antipsychotic polypharmacy regimen for more than 6 months before the entry. They were followed up with an antipsychotic monopharmacy and evaluated at 24 wk after completion of switching. Forty-seven patients were recruited for this study. Of 44 patients for whom evaluation was possible, 24 (54.5%) remained stable, while 10 (22.7%) showed improvement and the same number of patients ended in a deleterious status. Twenty-two patients were converted to antipsychotic monotherapy, while another 12 needed minimal dosing of low-potency agents. Overall, social functioning, evaluated by the Global Assessment of Functioning and the Clinical Global Impression, remained unchanged. Eighteen of 34 successful patients showed adverse effects of the main antipsychotic medication, which necessitated a significant dose reduction. Nine out of 10 deteriorating patients had been treated with a combination of low- and high-potency antipsychotics. It is suggested that many instances of antipsychotic polypharmacy is avoidable. The result is compatible with the current treatment recommendations, which dictate the use of a single antipsychotic agent.

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Reducing the dose of antipsychotic medications for those who had been treated with high-dose antipsychotic polypharmacy

Suzuki Takefumi, Uchida Hiroyuki, Tanaka Kenji F., Tomita Masayuki, Tsunoda Kenichi, Nomura Kensuke, Takano Harumasa, Tanabe Akira, Watanabe Koichiro, Yagi Gohei, Kashima Haruo

International Clinical Psychopharmacology 18 （ 6 ） 323 - 329 2003年11月

ISSN 0268-1315

Antipsychotic medications are often used at higher than the recommended dose and sometimes in a combination regimen to treat schizophrenia. However, in general, high-dose therapies have been abandoned in recent clinical studies. In this study, dose reduction of antipsychotic medication was implemented for patients with chronic schizophrenia, most of whom (81%) had been treated with an antipsychotic high-dose polypharmacy regimen consisting of more than 1000 mg/day in total amount. The results show that merely reducing the amount of antipsychotic led to favourable outcome in 23 out of 41 cases (56%), with another 13 cases (32%) showing no change. Dose reduction ended in failure in only five subjects (12%). Overall, the amount as well as the number of antipsychotic medications was significantly reduced from 1984 mg to 812 mg per day (reductions of 59% and from 3.6 to 2.2, respectively; both P<0.0001). The Global Assessment of Functioning scale improved from 30.6 to 37.2, which reached significance (P<0.001). Accordingly, the Severity of Illness improved from 4.7 to 4.2, and was also significant (P<0.01). Dose reduction is an encouraging strategy to consider for those patients with schizophrenia who have chronically been treated with high-dose antipsychotic polypharmacy, even if judged unavoidable in the past. © 2003 Lippincott Williams & Wilkins.

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Recurrent episodes of perceptual alteration in patients treated with antipsychotic agents

Uchida Hiroyuki, Suzuki Takefumi, Tanaka Kenji F., Watanabe Koichiro, Yagi Gohei, Kashima Haruo

Journal of Clinical Psychopharmacology 23 （ 5 ） 496 - 499 2003年10月

ISSN 0271-0749

Antipsychotics have been sporadically reported to induce the brief recurrent episodes characterized by hypersensitivity of the visual perception, which is known as paroxysmal perceptual alteration (PPA) mainly in Japan. PPA is characterized by hypersensitivity of perception mainly in visual modalities, which could occur in patients treated with antipsychotics. PPA is occasionally known to be accompanied by an oculogyric crisis (OGC). In this study, we examine the prevalence and the characteristics of patients with PPA. Three hundred thirty-eight patients who were treated with antipsychotics and diagnosed with schizophrenia, mood disorders, or neurotic disorders (International Classification of Disease, or ICD-10) were interviewed. We compared the characteristics between subjects with and without PPA. The mean overall prevalence of PPA was 3.25%, which was higher among patients treated with high-potency antipsychotics (3.91%) than in subjects treated with mid-potency or low-potency drugs (1.16%). PPA occurred simultaneously with OGC at the rate of 36.4%. We also found much similarity between PPA and OGC in terms of phenomenology. We suggest that PPA could manifest itself as an undesirable effect of antipsychotics, especially those of high potency. PPA and OGC may a share common underlying mechanism.

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Preservation of hematopoietic properties in transplanted bone marrow cells in the brain

Ono Kenji, Yoshihara Ken, Suzuki Hiromi, Tanaka Kenji F., Takii Takemasa, Onozaki Kikuo, Sawada Makoto

Journal of Neuroscience Research 72 （ 4 ） 503 - 507 2003年05月

ISSN 0360-4012

Recent studies have described the possible transdifferentiation of bone marrow cells (BMC) into neurons and glia when they migrate to the brain. However, we have reported that some immature BMC migrating into the brain parenchyma after bone marrow transplantation express early hematopoietic markers but not neural or glial markers. The present study further characterizes transplanted BMC that migrate to the brain. Double immunolabeling confirmed that BMC migrating to the brain expressed hematopoietic but not neural markers, such as nestin, microtubule-associated protein-2 and glial fibrillary acidic protein, even 4 and 18 weeks after bone marrow transplantation. BMC that expressed green fluorescent protein also expressed hematopoietic but not neural markers when cultured with mixed brain cells according to double immunolabeling and single-cell dissection using a laser. Analysis of the DNA content indicated that most of the migrated BMC were arrested at the G0/G1 phase, and aneuploidy or tetraploidy was undetectable. Thus, BMC that migrate to the brain probably have preserved hematopoietic properties under physiological conditions. © 2003 Wiley-Liss, Inc.

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Existence of functional β₁- and β₂-adrenergic receptors on microglia

Tanaka Kenji F., Kashima Haruo, Suzuki Hiromi, Ono Kenji, Sawada Makoto

Journal of Neuroscience Research 70 （ 2 ） 232 - 237 2002年10月

ISSN 0360-4012

We examined the expression and function of β-adrenergic receptor (β-AR) subtypes in both isolated primary rat microglia and a rat microglial cell line. RT-PCR analyses revealed that microglia expressed β₁- and β₂-ARs but not β₃-ARs, whereas rat primary peritoneal macrophages expressed only β₂-ARs. Stimulation of β-ARs on microglia by norepinephrine (NE) resulted in an increase in the level of intracellular cAMP and the subsequent expression of interleukin-1β mRNA. These effects were prevented by propranolol. Similar results were obtained with other selective β₁-AR agonists and antagonists. β₂-ARs on microglia were also functional. It is possible that noradrenergic innervations participate in the control of microglial functions via β₁-ARs on microglia in the brain, because NE has high affinity for β₁- and β₃-ARs but little or no affinity for β₂-ARs. It seems physiologically significant that microglia can be controlled by NE, which predominates over epinephrine in the brain, whereas macrophages in peripheral tissues can be controlled by epinephrine, which is at higher levels in peripheral tissues. © 2002 Wiley-Liss, Inc.

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Effect of intracerebral 6-nitronoradrenaline, an endogenous catechol-O-methyltransferase (COMT) inhibitor, on striatal dopamine metabolism in anaesthetised rats

Huotari M., Passlin M., Nordberg H. L., Forsberg M., Kotisaari S., Tuomisto L., Shintani F., Tanaka K. F., Reenilä I., Laitinen K., Männistö P. T.

Journal of Neuroscience Methods 109 （ 1 ） 47 - 52 2001年08月

ISSN 0165-0270

6-Nitronoradrenaline, a bioactive compound recently identified in the brain, is known to inhibit catechol-O-methyltransferase. To study its effect on dopamine metabolism, it was administered into rat striatum via a microdialysis probe. Other nitrated catechols (6-nitrodopamine, 6-nitro-DOPAC and 5-nitro-HVA) were studied for comparison. Tolcapone, a selective catechol-O-methyltransferase inhibitor, was used as a positive reference compound. Both 6-nitronoradrenaline and tolcapone increased striatal extracellular dopamine levels during the perfusion (at 100 μM concentration but not at 10 μM) and decreased the efflux of homovanillic acid. Tolcapone, but not other nitrated catechols, increased 3,4-dihydroxyphenylacetic acid efflux. None of the compounds inhibited MAO-B activity at 100 μM or lower. At 1 mM, 6-nitrodopamine inhibited MAO-B by 60%. Compared to tolcapone, other nitrated catechols were very weak COMT inhibitors in vitro. Neither tolcapone nor 6-nitronoradrenaline modified the metabolism of L-dopa which was given peripherally. In binding studies, both 6-nitronoradrenaline and other nitrocatechols failed to affect the dopamine transporter even at high μmolar concentrations. In conclusion, exogenous 6-nitronoradrenaline can act as a COMT inhibitor in the striatum and elevate striatal dopamine levels without inhibiting dopamine reuptake. Whether endogenous 6-nitronoradrenaline can be formed also in vivo in the striatum and act as a regulator of dopaminergic tone remains to be determined

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Effect of intracerebral 6-nitronoradrenaline, an endogenous catechol-O-methyltransferase (COMT) inhibitor, on striatal dopamine metaolism in anesthetized rats

Huotari M, Passlin M, Nordberg HL, Forsberg M, Kotisaari S, Tuomisto L, Shintani Futoshi, Tanaka Kenji, F Reenila I, Laitinen K, Mannisto PT

Journal of Neuroscience Methods 109 （ 1 ） 47-52 2001年08月

研究論文（学術雑誌）, 共著

Elevated serum levels of interleukin-18 in schizophrenia

Shintani Futoshi, Tanaka Kenji, Fujii Yasuo, Yagi Gohei, Asai Masahiro

Psychiatry and Clinical Neuroscience 54 （ 6 ） S5-55 2000年12月

研究論文（学術雑誌）, 共著

Switching Neuroleptics polypharmacy into monopharmacy in Chronic schizophrenic patients

Watanabe Koichiro, Tanaka Kenji, Arai Tatsuyoshi, Hosogane Nana, Oda Kenichi, Tsunoda Kenichi, Uchida Hiroyuki, Inagaki Ataru, Yagi Gouhei

International Clinical Psychopharmacology 15 （ 6 ） 374 2000年11月

研究論文（学術雑誌）, 共著

Serum interleukin-18 levels are elevated in schizophrenia

Tanaka Kenji F., Shintani Futoshi, Fujii Yasuo, Yagi Gohei, Asai Masahiro

Psychiatry Research 96 （ 1 ） 75 - 80 2000年09月

ISSN 0165-1781

Interleukin-18 (IL-18) is a recently discovered proinflammatory cytokine which plays a pivotal role in T helper 1 (Th1) responses. IL-18 is produced by macrophage-like cells, and inappropriate IL-18 production has been known to be involved in immunological disturbances. Schizophrenia is a common disease whose pathogenesis is still unclear; however, an activation of the inflammatory response system, including the Th1 cytokine response, may be related to the pathophysiology of schizophrenia. We measured the serum IL-18 levels of 66 schizophrenics and age- and sex-matched control subjects by using an ELISA assay. We found significantly increased serum IL-18 levels in the schizophrenic patients (P = 0.0002). This finding supports the hypothesis that immune activation is involved in the pathophysiology of schizophrenia. (C) 2000 Elsevier Science Ireland Ltd.

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Serum interleukin-18 levels are elevated in schizophrenia

Tanaka Kenji, Shintani Futoshi, Fujii Yasuo, Yagi Gohei, Asai Masahiro

Psychiatry Research 96 （ 1 ） 75-80 2000年09月

研究論文（学術雑誌）, 共著

脳内インターロイキン―1受容体アンタゴニストによるストレス反応の調節機構の解明

新谷太，田中謙二，脇田哲矢，八木剛平，浅井昌弘

精神薬研究年報 32 30-32 2000年03月

研究論文（学術雑誌）, 共著

精神分裂病の慢性重症例に対する抗精神病薬療法と血中モノアミン代謝産物の変動

八木剛平，田中謙二

臨床薬理 31 （ 1 ） 47-48 2000年01月

研究論文（学術雑誌）, 共著

Awakenings（めざめ現象）と非定型抗精神病薬への切り替え

田中謙二，藤井康男

臨床精神薬理 2 （ 8 ） 859-866 1999年08月

研究論文（学術雑誌）, 共著

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