論文 - 田中　謙二

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

　概要を見る

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

　概要を見る

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

　概要を見る

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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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

　概要を見る

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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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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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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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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