Biology subjects

Kobayashi, K.

Publications and source records attributed to Kobayashi, K..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

A Checking Model of Obsessive-Compulsive Disorder Based on the Hypothalamic Defensive System Against a Potential Threat

BACKGROUNDObsessive-compulsive disorder (OCD) is a psychiatric disorder characterized by obsessive thoughts and compulsive behavior. While some theories imply that OCD patients have cognitive biases and dysfunctional motivation regarding a potential threat, these views are not adequately supported by neurological evidence. Hypothalamic perifornical (PeF) urocortin-3 (UCN3) neurons are involved in defensive responses to a potential threat, and the activation of these neurons in mice induces repetitive and excessive checking and burying of novel objects. In this study, we evaluated the hypothesis that mice in which PeF UCN3 neurons are activated can serve as an OCD model. METHODSPeF UCN3 neurons were chemogenetically activated with clozapine-N oxide (CNO) in Ucn3-Cre mice. Marble-burying activity, repetitive/stereotypic behaviors in the homecage, and excessive responses to a novel object were measured as OCD-like behaviors. The effects of clinically used drugs for treating OCD on these behaviors were evaluated. The effect of CNO on neural activity in the cortico-striato-thalamo-cortical loop (which is regarded as an OCD circuit) was assessed with c-Fos immunolabeling. RESULTSCNO increased marble-burying activity, evoked homecage-specific repetitive/stereotypic behaviors that probably aimed to seal entrances, and induced repetitive and excessive checking and burying of novel objects. These behaviors were suppressed by selective serotonin-reuptake inhibitors but not by diazepam. CNO increased neural activity in the cortico-striato-thalamo-cortical loop. CONCLUSIONSThese results indicated that mice whose PeF UCN3 neurons are activated can serve as a model of OCD, particularly as a checking model. This supports theories concerning the role of potential threats in the pathophysiology of OCD.

animal behavior and cognition↗

bioRxiv2022

Two cyclic electron flows around photosystem I differentially participate in C4 photosynthesis

C4 plants assimilate CO2 more efficiently than C3 plants because of their C4 cycle that concentrates CO2. However, the C4 cycle requires additional ATP molecules, which may be supplied by the cyclic electron flow around photosystem I. One cyclic electron flow route, which depends on a chloroplast NADH dehydrogenase-like (NDH) complex, is suggested to be crucial for C4 plants despite the low activity in C3 plants. The other route depends on proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic phenotype 1 (PGRL1), which is considered a major cyclic electron flow route to generate the proton gradient across the thylakoid membrane in C3 plants. However, its contribution to C4 photosynthesis is still unclear. In this study, we investigated the contribution of the two cyclic electron flow routes to the NADP-malic enzyme subtype of C4 photosynthesis in Flaveria bidentis. We observed that the suppression of the NDH-dependent route drastically delayed growth and decreased the CO2 assimilation rate to approximately 30% of the wild-type rate. On the other hand, the suppression of the PGR5/PGRL1-dependent route did not affect plant growth and resulted in a CO2 assimilation rate that was approximately 80% of the wild-type rate. Our data indicate that the NDH-dependent cyclic electron flow substantially contributes to the NADP-malic enzyme subtype of C4 photosynthesis and that the PGR5/PGRL1-dependent route cannot complement the NDH-dependent route in F. bidentis. These findings support the fact that during the C4 evolution, the photosynthetic electron flow may be optimized to provide the energy required for C4 photosynthesis.

plant biology↗

bioRxiv2022

Cold-induced Suspension and Resetting of Ca2+ and Transcriptional Rhythms in the Suprachiasmatic Nucleus Neurons

Mammalian circadian rhythms are coordinated by the master clock located in the hypothalamic suprachiasmatic nucleus (SCN). Under severe environmental conditions, such as during the harsh winter season for food, certain mammalian species reduce their basal metabolism and thermogenesis, thereby undergoing torpor, a controlled state of hypothermia, which naturally returns to the normothermic state. A long-lasting debate focused on whether the SCN with a temperature-compensated clock remains functional during hypothermia. However, so far, no direct and quantitative evidence has been reported of temperature sensitivity in living SCN neurons. In this study, we performed dual-color fluorescence imaging of clock gene transcriptions and intracellular Ca2+ in mouse SCN neurons, using slices at various temperatures. We demonstrated that the Bmal1 transcription and Ca2+ circadian rhythms persisted at 22{degrees}C-28{degrees}C, although the two rhythms underwent temporal dissociation at 22 {degrees}C. Notably, Ca2+, Bmal1, and Per2 rhythms were suspended at 15{degrees}C, coupled with a significant Ca2+ increase, and all rhythms were reset by rewarming to 35{degrees}C. Upon rewarming, the Ca2+ rhythm showed stable oscillations immediately, whereas the Bmal1 and Per2 rhythms took several days to reach stable oscillations and recover their phase relationship with the Ca2+ rhythm. Taken together, we concluded that SCN neurons remain functional under moderate hypothermic conditions at approximately 22{degrees}C-28{degrees}C but stop ticking time in deep hypothermia at 15{degrees}C and that the rhythms reset after deep hypothermia. These data also indicate that the stable Ca2+ oscillation precedes clock gene transcriptional rhythms in the SCN neurons.

neuroscience↗

bioRxiv2022

A unique case in which Kimoto-style fermentation was completed with Leuconostoc as the dominant genus without transitioning to Lactobacillus.

The Kimoto-style fermentation starter is a traditional preparation method of sake brewing. In this process, specific microbial transition patterns have been observed within nitrate-reducing bacteria and lactic acid bacteria during the production process of the fermentation starter. We have characterized phylogenetic compositions and diversity of the bacterial community in a sake brewery performing the Kimoto-style fermentation. Comparing the time-series changes with other sake breweries previously reported, we found a novel type of Kimoto-style fermentation in which the microbial transition differed significantly from other breweries during the fermentation step. Specifically, the lactic acid bacteria, Leuconostoc spp. was a predominant species in the late stage in the preparation process of fermentation starter, on the other hand, Lactobacillus spp., which plays a pivotal role in other breweries, was not detected in this analysis. The discovery of this new variation of microbiome transition in Kimoto-style fermentation has further deepened our understanding of the diversity of sake brewing.

microbiology↗

bioRxiv2022

Dissection of insular cortex layer 5 reveals sublayers with opposing modulatory roles in appetitive behavior

The insular cortex (insula) is known to play a modulatory role in motivated behaviors including feeding and drinking. Previous studies have revealed that the anterior and posterior subregions of the insula have differential subcortical efferents and roles, yet the anatomical and functional heterogeneity among the cortical layers remains poorly understood. Here, we show that layer 5 of the mouse dysgranular insula has two distinct neuronal subpopulations along the entire anterior-posterior axis: the upper layer (L5a) population, expressing NECAB1, projects bilaterally to the lateral and capsular divisions of the central amygdala, and the deeper layer (L5b) population, expressing CTIP2, projects ipsilaterally to the parasubthalamic nucleus and the medial division of the central amygdala. Optogenetically activating L5a and L5b neuronal populations in thirsty mice led to suppressed and facilitated water spout licking, respectively, in a single-spout test. However, the opto-activation induced no avoidance against or preference for the spout paired with the opto-stimulation in a two-spout choice test, indicating no induction of emotional valences by the opto-activation per se. Our results suggest sublayer-specific bidirectional modulatory roles of insula layer 5 in the motivational aspect of appetitive behavior.

neuroscience↗

bioRxiv2022

Transcription factors that specify cell fate activate cell cycle regulator genes to determine cell numbers in ascidian larval tissues

In animal development, most cell types stop dividing before terminal differentiation; thus, cell cycle control is tightly linked to cell differentiation programs. Although cell cycle control in animal development has been studied extensively, such links are not well understood. In ascidian embryos, cell lineages do not vary among individuals, and rounds of the cell cycle are determined according to cell lineages. In the present study, we first showed that maternal factors promote approximately 10 rounds of cell division without zygotic gene expression. Notochord and muscle cells stop dividing after fewer than 10 rounds of cell division, and we show that a Cdk inhibitor (Cdkn1.b) is responsible. Cdkn1.b is also necessary for epidermal cells to stop dividing. In contrast, mesenchymal and endodermal cells divided more than 10 times, and Myc, which encodes a proto-oncogenic transcription factor, is responsible for maintaining cell cycle progression in these tissues. Expression of Cdkn1.b in notochord and muscle is controlled by the same developmental programs that specify the developmental fate of notochord and muscle. Likewise, expression of Myc in mesenchyme and endoderm was under control of the same developmental programs that specify the developmental fate of mesenchyme and endoderm. Because these transcription factors that regulate Cdkn1.b and Myc are essential factors for fate specification of these tissues, cell fate specification and cell cycle control are linked by those transcription factors. In other words, ectodermal, mesodermal, and endodermal tissues in ascidian embryos control the cell cycle through Cdkn1.b and Myc, which are under the control of transcription factors that specify cell fate.

developmental biology↗

bioRxiv2022

5' transgenes drive leaky expression of 3' transgenes in inducible bicistronic vectors

Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences in a cell type of interest provide an invaluable tool for understanding the molecular regulation of cellular functions. DNA recombination employing the Cre-lox system is commonly used as a molecular switch for inducing the expression of recombinant proteins encoded within a bicistronic cassette. In such an approach, the two protein-coding sequences are separated by a 2A peptide or internal ribosome entry site (IRES), and expression is designed to be strictly Cre-dependent by using a lox-STOP-lox cassette or flip-excision (FLEX) switch. However, low-level or leaky expression of recombinant proteins is often observed in the absence of Cre activity, potentially compromising the utility of this approach. To investigate the mechanism of leaky gene expression, we generated pCAG-lox-GFP-STOP-lox-Transgene A-2A-Transgene B vectors, which are designed to express nuclear-targeted GFP in the absence of Cre, and express both transgenes A and B after Cre-mediated recombination. We found that cells transfected with these bicistronic vectors exhibited low-level Cre-independent expression specifically of the transgene positioned 3' of the 2A peptide. We observed similar results in vivo by viral transduction of the adult mouse cerebral cortex with AAV-mutagenesis of putative transcription factor binding sites that the 5' transgene confers promoter-like activity that drives expression of the 3' transgene. Finally, we demonstrate that inclusion of an additional lox-STOP-lox cassette between the 2A sequence and 3' transgene dramatically reduces the extent of Cre-independent leaky gene expression. Our findings highlight that caution should be applied to the use of Cre-dependent bicistronic constructs when tight regulation of transgene expression is desired and provide a guide to preventing leaky gene expression when the expression of more than one protein is required.

molecular biology↗

bioRxiv2022

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology

BackgroundCell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. New MethodWe aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. ResultsWe detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH expression level and catecholamine accumulation in the brain regions, and spontaneous locomotion, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. ConclusionsTH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

neuroscience↗

bioRxiv2022

Highly selective transgene expression through double-floxed inverted orientation system by using a unilateral spacer sequence

The double-floxed inverted orientation (DIO) system with adeno-associated viral (AAV) vector provides a beneficial approach to express transgenes in specific cell populations having Cre recombinase. A significant issue with this system is the protection against non-specific expression of transgenes in tissues after vector injection. We here show that Cre-independent recombination in AAV genome carrying the DIO sequence occurs during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress recombination during the vector production, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our DIO/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

neuroscience↗

bioRxiv2022

Reinstating olfactory bulb derived limbic gamma oscillations alleviates depression

Although the etiology of major depressive disorder remains poorly understood, impairment of gamma oscillations recently emerged as a potential biomarker for major depression. The olfactory bulb (OB) is a major source of brain wide gamma oscillations and bulbectomy is an animal model for depression. Here we demonstrate that chemogenetic suppression of OB neuronal activity or temporally suppressing the OB to pyriform cortex synaptic transmission decreased gamma oscillation power in multiple brain areas associated with depression-like behaviors. To assess the hypothesized link between depression and diffuse depression of gamma oscillations, we employed gamma phase-dependent closed loop neuromodulation of cortical areas, paced by the native OB output. This procedure alleviated depressive-like behaviors in animals and suggests that restoring gamma oscillations may improve depression in humans. One Sentence SummaryRole of limbic gamma oscillations in depression

neuroscience↗

bioRxiv2021

Prediction of broad chemical toxicities using induced pluripotent stem cells and gene networks by transfer learning from embryonic stem cell data

The assessment of toxic chemicals using animals has limited applicability to humans. Moreover, from the perspective of animal protection, effective alternatives are also desired. Previously, we developed a method that combines developmental toxicity testing based on undifferentiated human embryonic stem (ES) cells (KhES-3) and gene networks. We showed that [≥] 95% accurate predictions could be achieved for neurotoxins, genotoxic carcinogens, and non-genotoxic carcinogens. Here, we expanded this method to predict broad toxicities and predicted the toxicity of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, nephrotoxins [glomerular nephrotoxins/tubular nephrotoxins], and non-genotoxic carcinogens) and achieved high prediction accuracy (AUC = 0.90-1.00) in all categories. Moreover, to develop a testing system with fewer ethical issues, we screened for an induced pluripotent stem (iPS) cell line on the basis of cytotoxic sensitivity and used this line to predict toxicity in the six categories based on the gene networks of iPS cells using transfer learning from the ES cell gene networks. We successfully predicted toxicities in four toxin categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) at high accuracy (AUC = 0.82-0.99). These results demonstrate that the prediction of chemical toxicity is possible even with iPS cells by transfer learning once a gene expression database has been developed from an ES cell line. This method holds promise for tailor-made safety evaluations using individual iPS cells.

pharmacology and toxicology↗

bioRxiv2021

Structural insights into human excitatory amino acid transporter EAAT2

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity, that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. This is the first characterization of molecular features of EAAT2 and the selective inhibition mechanism, underlying structure-based drug design for EAAT2.

molecular biology↗

bioRxiv2021

Phosphatidylglycerol synthesis facilitates plastid gene expression and light induction of nuclear photosynthetic genes

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development with decreased chlorophyll accumulation, impaired thylakoid formation, and downregulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects the gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated the growth and transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species-responsive genes were upregulated in pgp1-2. Decreased growth light did not alleviated the impaired leaf development and the downregulation of photosynthesis-associated genes in pgp1-2, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered downregulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor regulating chloroplast development, in pgp1-2 upregulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in the plastid gene expression and plant development.

plant biology↗

bioRxiv2021

Novel System to Monitor In Vivo Neural Graft Activity After Spinal Cord Injury

Expectations for neural stem/progenitor cell (NS/PC) transplantation as a treatment for spinal cord injury (SCI) are increasing. However, whether and how grafted cells are incorporated into the host neural circuit and contribute to motor function recovery remain unknown. The aim of this project was to establish a novel non-invasive in vivo imaging system to visualize the activity of neural grafts by which we can simultaneously demonstrate the circuit-level integration between the graft and host, and the contribution of graft neuronal activity to host behaviour. We introduced Akaluc, a newly engineered luciferase, under control of a potent neuronal activity-dependent synthetic promoter, E-SARE, into NS/PCs and engrafted the cells into SCI model mice. Through the use of this system, we reveal that the activity of grafted cells was integrated with host behaviour and driven by host neural circuit inputs. This non-invasive system is expected to help elucidate the therapeutic mechanism of cell transplantation treatment for SCI and determine better therapy techniques that maximize the function of cells in the host circuit.

neuroscience↗

bioRxiv2021

Neural representations of stereotype content predict social decisions

To guide social interaction, people often rely on expectations about the traits of other people based on markers of social group membership, i.e., stereotypes. Although the influence of stereotypes on social behavior is widespread, key questions remain about how traits inferred from social group membership are instantiated in the brain and incorporated into neural computations that guide social behavior. Here, we show that the human lateral orbitofrontal cortex (OFC) represents the content of stereotypes about members of different social groups in the service of social decision-making. During fMRI scanning, participants decided how to distribute resources across themselves and members of a variety of social groups in a modified Dictator Game. Behaviorally, we replicated our recent finding that perceptions of others traits, captured by a two-dimensional framework of stereotype content (warmth and competence), biased participants monetary allocation choices in a context-dependent manner: recipients warmth increased advantageous inequity aversion and their competence increased disadvantageous inequity aversion. Neurally, representational similarity analysis (RSA) revealed that perceptions of others traits in the two-dimensional space were represented in the temporoparietal junction and superior temporal sulcus, two regions associated with mentalizing, and in the lateral OFC, known to represent latent environmental features during goal-directed outcome inference outside the social domain. Critically, only the latter predicted individual choices, suggesting that the effect of stereotypes on behavior is mediated by inference-based, domain-general decision-making processes in the OFC.

neuroscience↗

bioRxiv2021

Recording and neural circuit-based manipulation of the maternal oxytocin pulses in mice

Pulsatile release of the hormone oxytocin (OT) mediates uterine contraction during parturition and milk ejection during lactation1-3. These pulses are generated by unique activity patterns of the central neuroendocrine OT neurons located in the paraventricular and supraoptic hypothalamus. Classical studies have characterized putative OT neurons by in vivo extracellular recording techniques in rats and rabbits under anesthesia1, 4-7 or awake8-10. Due to technical limitations, however, the identity of OT neurons in these previous studies was speculative based on their electrophysiological characteristics and axonal projection to the posterior pituitary, not on OT gene expression. To pinpoint OT neural activities among other hypothalamic neurons that project to the pituitary11, 12 and make better use of cell-type-specific neuroscience toolkits13, a mouse model needs to be developed for studies of parturition and lactation. We herein introduce viral genetic approaches in mice to characterize the maternal activities of OT neurons by fiber photometry. During lactation, a sharp photometric peak of OT neurons appeared at approximately 520 s following simultaneous suckling stimuli from three pups. The amplitude of the peaks increased as the mother mice experienced lactation, irrespective of the age of the pups, suggesting the intrinsic plasticity of maternal OT neurons. Based on a mono-synaptic input map to OT neurons, we pharmacogenetically activated the inhibitory neurons in the bed nucleus of the stria terminalis and found suppression of the activities of OT neurons. Collectively, our study illuminates temporal dynamics in the maternal neural activities of OT neurons and identifies one of its modulatory circuits. Highlights- Pulsatile activities of genetically-defined OT neurons in mother mice were recorded in vivo. - The maternal experience-dependent plasticity of the OT neural activities was found. - Input-mapping of OT neurons in mother mice was performed by rabies-mediated trans-synaptic tracing. - Photometric peaks of OT neurons were suppressed by the activation of BST inhibitory neurons.

neuroscience↗

bioRxiv2021

The sperm protein SPACA4 is required for efficient fertilization in mice

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncers homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely sub-fertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

developmental biology↗

bioRxiv2021

Genomic imprinting drives eusociality

The origin of eusociality, altruistically foregoing personal reproduction to help others, has been a long-standing paradox ever since Darwin. Most eusocial insects and rodents likely evolved from subsocial precursors, in which older offspring "helpers" contribute to the development of younger siblings without a permanent sterile caste. The driving mechanism for the transition from subsociality (with helpers) to eusociality (with lifelong sterile workers) remains an enigma because individuals in subsocial groups are subject to direct natural selection rather than kin selection. Our genomic imprinting theory demonstrates that natural selection generates eusociality in subsocial groups when parental reproductive capacity is linked to a delay in the sexual development of offspring due to sex-antagonistic action of transgenerational epigenetic marks. Focusing on termites, our theory provides the missing evolutionary link to explain the evolution of eusociality from their subsocial wood-feeding cockroach ancestors, and provides a novel framework for understanding the origin of eusociality.

evolutionary biology↗