Biology subjects

Yamagata, T.

Publications and source records attributed to Yamagata, T..

16 recordsLinked to original sources

bioRxiv2026

CRISPR-mediated Stxbp1 gene activation ameliorates epileptic and aggressive phenotypes in Stxbp1-haploinsufficient mice

Mutations in the syntaxin-binding protein 1 (STXBP1) gene, which encodes the presynaptic protein Munc18-1, cause a spectrum of severe epileptic encephalopathies and neurodevelopmental disorders, including Ohtahara syndrome, for which no curative treatment is currently available. Because the disease pathomechanism is thought to be driven by haploinsufficiency, restoring expression of the wild-type allele to physiological levels could provide therapeutic benefit. Here, we evaluated CRISPR-mediated transcriptional activation (CRISPR-ON), based on a dCas9-VPR transcriptional activator, as a strategy to upregulate endogenous Stxbp1 expression in a Stxbp1-haploinsufficient (Stxbp1+/-) mouse model. Screening of guide RNAs (gRNAs) targeting the Stxbp1 promoter in Neuro2A cells identified a multiplexed four-gRNA cassette that elevated Stxbp1 mRNA approximately six-fold. AAV-PHP.eB vectors co-expressing this 4xgRNA cassette and Cre recombinase under the EF1a promoter were administered intracerebroventricularly to neonatal Stxbp1+/-/dCas9-VPRfl/+ mice. CRISPR-ON treatment restored not only brain Stxbp1 mRNA but also Munc18-1 protein levels to those of wild-type controls. Electrocorticographic recordings revealed an approximately 50% reduction in the frequency of spike-wave discharges in CRISPR-ON-treated Stxbp1+/- mice compared with untreated Stxbp1+/- controls, and aggressive behavior in the resident-intruder test was also partially attenuated. In contrast, locomotor activity remained unaffected, indicating that CRISPR-ON treatment achieves selective rescue of disease-related phenotypes without inducing motor side effects. Together, these findings demonstrate that CRISPR-ON-mediated activation of endogenous Stxbp1 is a promising therapeutic strategy for STXBP1-related encephalopathies and support endogenous gene activation as a broadly applicable platform for haploinsufficiency disorders.

neuroscience↗

bioRxiv2026

Reduction of Nav1.1 in the dorsal striatum preferentially increases hyperthermia-induced generalized seizures compared with the neocortex and nucleus accumbens

Mutations in SCN1A, which encodes the voltage-gated sodium channel Nav1.1 (I subunit), are the major cause of Dravet syndrome, a severe developmental and epileptic encephalopathy. Although Nav1.1 haploinsufficiency preferentially impairs inhibitory interneuron function, the region-specific contributions of distributed brain circuits to seizure susceptibility, particularly in subcortical structures that have received less attention than the neocortex and hippocampus, remain unclear. Here, we examined the effects of region-specific Nav1.1 deficiency on hyperthermia-induced seizures by selectively deleting Scn1a in the neocortex, nucleus accumbens (NAc), and dorsal striatum (caudate-putamen, CPu) of adult mice using an adeno-associated virus-mediated Cre-loxP approach. Contrary to expectations based on prior cortical studies, homozygous Scn1a deletion in the neocortex produced only modest effects on seizure generalization. In contrast, homozygous deletion in the NAc and CPu induced generalized seizures to varying degrees. Notably, heterozygous Scn1a deletion in the CPu alone was sufficient to trigger generalized seizures, whereas similar manipulations in the neocortex or NAc were not. Seizure threshold temperatures were largely comparable across regions. These findings identify the dorsal striatum as particularly vulnerable to partial Nav1.1 loss and reveal functional heterogeneity within striatal circuits. Our results underscore a previously underappreciated role of striatal inhibitory networks in hyperthermia-induced seizure susceptibility and provide new insights into the circuit mechanisms underlying Dravet syndrome.

neuroscience↗

bioRxiv2026

Reduced cortico-accumbal excitatory input due to Nav1.2 haploinsufficiency impairs sociability independently of dopamine

Mutations in SCN2A, which encodes the voltage-gated sodium channel Nav1.2, are associated with a wide spectrum of neurodevelopmental and neuropsychiatric disorders, including epilepsy, autism spectrum disorder (ASD), and schizophrenia. Although dysfunction of SCN2A-dependent neural circuits has been implicated in these disorders, the circuit mechanisms underlying social behavioral abnormalities remain poorly understood. Here, we investigated the neural circuit basis of social behavioral deficits associated with Scn2a dysfunction, focusing on the nucleus accumbens (NAc), a key hub in cortico-limbic circuits that regulates emotional and motivational behaviors. Using conditional genetic and chemogenetic approaches in mice, we examined the roles of dorsal telencephalic excitatory neurons, including those in the cerebral cortex, hippocampus, and amygdala, as well as parvalbumin-positive fast-spiking interneurons (PV FSIs) in the NAc. Mice with Scn2a haploinsufficiency in dorsal telencephalic excitatory neurons (Scn2afl/+/Emx1-Cre) exhibited reduced sociability in the three-chamber social interaction test. Similarly, chemogenetic inhibition of NAc PV FSIs decreased sociability without affecting locomotor activity or anxiety-like behavior. Scn2afl/+/Emx1-Cre mice also showed a trend toward reduced prepulse inhibition of the acoustic startle response. Notably, dopamine release into the NAc in the Scn2afl/+/Emx1-Cre and systemic Scn2a heterozygous knockout (Scn2a+/-) mice was largely comparable to that in control mice. Together, these findings indicate that reduced activity of dorsal telencephalic excitatory neurons or NAc PV FSIs is sufficient to impair sociability independently of mesolimbic dopamine hypofunction. Our results highlight a potential role of cortico-accumbal circuits in social behavioral deficits associated with SCN2A dysfunction.

neuroscience↗

bioRxiv2026

Parallel analysis of voltage-gated sodium channel subunits reveals preferential colocalizations of beta-1/Nav1.1 and beta-2/Nav1.2

Voltage-gated sodium channels (VGSCs) are conventionally described as heterotrimers composed of one alpha and two beta subunits. However, the patterns of co-expression of alpha- and beta-subunits in neurons remain unclear. In the present study, we report that alpha- (Nav1.1, Nav1.2, and Nav1.6) and beta- (beta-1 and beta-2) subunits are densely expressed in axon initial segments (AISs) of neurons in the neocortex, hippocampus and cerebellum at postnatal days 14-15 (P14-15) and 8-9 weeks (8-9W). These distributions are largely unique and partially overlapping among brain regions. Notably, in the neocortex and hippocampus, AISs of presumptive parvalbumin-positive inhibitory neurons are positive for Nav1.1 and beta-1, whereas those of excitatory ones are positive for Nav1.2 and beta-2. Similarly, AISs of cerebellar basket cells, which are inhibitory neurons, are positive for Nav1.1 and beta-1, whereas those of granule cells, which are excitatory neurons, are positive for Nav1.2 and beta-2. Nav1.6 is expressed in many of these neurons. Some subunits exhibited distinct distribution patterns at the two postnatal stages analyzed, possibly because of their developmental changes of subcellular localizations. Taken together, these results indicate that combinations of VGSC subunits are largely unique among different neuronal subpopulations. These findings provide a useful reference for understanding the distribution and interactions of VGSC subunits in the brain.

neuroscience↗

bioRxiv2026

Reduced activity of nucleus accumbens parvalbumin-expressing fast- spiking inhibitory neurons causes convulsive seizures

Pathogenic mutations in STXBP1, which encodes Munc18-1, a synaptic protein essential for synaptic vesicle exocytosis and neurotransmission, and in SCN2A, which encodes the voltage-gated sodium channel Nav1.2 (II subunit), have been identified in patients with epilepsy. Although haploinsufficiency of either Stxbp1 or Scn2a in cortical excitatory neurons induces epileptic phenotypes in mice and a reduction of cortico-striatal, especially cortico-striatal parvalbumin-expressing fast-spiking interneurons (FSIs), excitatory transmission has been suggested to be the basis, the subcortical circuits remain poorly understood. In this study, we investigated which striatal subregions FSIs are responsible for the epileptic seizures. Using chemogenetic approach, we selectively suppressed FSI activity in either the nucleus accumbens (NAc) or the dorsal striatum (caudate-putamen, CPu) of mice. Suppression of FSIs in the NAc induced outwardly-recognized convulsive seizures accompanied by epileptiform discharges in the electrocorticographic (ECoG) analysis, whereas inhibition of FSIs in the CPu resulted in epileptiform discharges without overt convulsions. Notably, focal suppression of FSIs in either the anterior or medial region of the NAc shell (NAcSh), but not in other NAc subregions, was sufficient to trigger convulsive seizures. These findings identify FSIs in the anteromedial shell of the NAc as a critical hub for convulsive seizure generation and provide new insights into the striatal circuit mechanisms underlying STXBP1- or SCN2A-associated epilepsies.

neuroscience↗

bioRxiv2024

Cortical layer 6b mediates state-dependent changes in brain activity and effects of orexin on waking and sleep

One of the most distinctive features of the mammalian cerebral cortex is its laminar structure. Of all cortical layers, layer 6b (L6b) is by far the least-studied, despite exhibiting direct sensitivity to orexin and having widespread connectivity, suggesting an important role in regulating cortical oscillations and brain state. We performed chronic electroencephalogram (EEG) recordings in mice in which a subset of L6b neurons was conditionally "silenced", during undisturbed conditions, after sleep deprivation (SD), and after intracerebroventricular (ICV) administration of orexin. While the total amount of waking and sleep or the response to SD were not altered, L6b-silenced mice showed a slowing of theta frequency (6-9 Hz) during wake and REM sleep, and a marked reduction of total EEG power, especially in NREM sleep. The infusion of orexin A increased wakefulness in both genotypes, but subsequent levels of EEG slow-wave activity during NREM sleep were lower in L6b-silenced animals in the occipital derivation. In summary, these results demonstrate a role for cortical L6b in state-dependent brain oscillations and in the response to orexinergic neurotransmission. Our findings provide new insights in functions of L6b neurons and could inform the understanding of abnormal regulation of brain states in neurodevelopmental and psychiatric disorders.

neuroscience↗

bioRxiv2024

Efficient and durable gene activation by Cas9-mediated epigenome editing in vivo

Epigenome editing technology holds great promise for treating diverse genetic disorders. While a series of advances has been made on epigenetic silencing using programmable editors, little progress has been made in leveraging epigenetic activation for therapeutic application. Here we demonstrate epigenetic activation of the LAMA1 gene for the treatment of LAMA2-CMD, a severe congenital muscle dystrophy (CMD) caused by biallelic mutations in the LAMA2 gene. LAMA1 is a sister homologue that is known to compensate for the function of LAMA2. However, supplementing LAMA1 or LAMA2 gene via viral platform is not feasible due to the large size of their coding sequences. Through a single administration of our (Adeno-associated virus) AAV vector encoding all the necessary elements for epigenetic activation, a platform termed CRISPR guide-nucleotide directed modulation (GNDM), we observed significant LAMA1 gene upregulation and phenotype improvements in DyW mice, a severe disease model of LAMA2-CMD. Notably, sustained expression of the GNDM gene and subsequent activation of the LAMA1 gene persisted beyond analyzed period of one year despite immune recognition of the GNDM protein by the host immune system. Regulatory T (Treg) cells appeared to facilitate tolerance to GNDM in the transduced muscle tissue. The muscle-tropic AAV capsid exhibited desired vector biodistribution and promising pharmacodynamics with good safety profiles in adult non-human primates (NHPs). Moreover, administration to juvenile NHPs demonstrated superior pharmacodynamics compared to adults, even at half the adult dose, suggesting safer and more effective therapeutic outcomes in mostly pediatric LAMA2-CMD patients. Our approach holds broad applicability for a range of loss-of-function genetic disorders and could offer a therapeutic breakthrough where active epigenome brings clinical benefit.

bioengineering↗

bioRxiv2023

Deficient synaptic neurotransmission results in a persistent sleep-like cortical activity across vigilance states in mice

Growing evidence suggests that brain activity during sleep, as well as sleep regulation, are tightly linked with synaptic function and network excitability at the local and global levels. We previously reported that a mutation in synaptobrevin 2 (Vamp2) in restless (rlss) mice results in a marked increase of wakefulness and suppression of sleep, in particular REM sleep (REMS) as well as increased consolidation of sleep and wakefulness. In the current study, using finer-scale in vivo electrophysiology recordings, we report that spontaneous cortical activity in rlss mice during NREM sleep (NREMS) is characterised by an occurrence of abnormally prolonged periods of complete neuronal silence (OFF-periods), often lasting several seconds, similar to the burst suppression pattern typically seen under deep anaesthesia. Increased incidence of prolonged network OFF-periods was not specific to NREMS, but also present in REMS and wake in rlss mice. Slow-wave activity (SWA) was generally increased in rlss mice, while higher frequencies including theta-frequency activity were decreased, further resulting in diminished differences between vigilance states. The relative increase in SWA after sleep deprivation was attenuated in rlss mice, suggesting either that rlss mice experience persistently elevated sleep pressure, or, alternatively, that the intrusion of sleep-like patterns of activity into awake state diminishes the accumulation of sleep drive. We propose that deficit in global synaptic neurotransmitter release leads to state inertia, reflected in an abnormal propensity of brain networks to enter and remain in a persistent default state resembling coma or deep anaesthesia.

neuroscience↗

bioRxiv2022

Phylogeny and Genetic Diversity of Philippine Native Pigs (Sus scrofa) as Revealed by Mitochondrial DNA Analysis

Philippine native pigs (PhNP) are small black pigs domesticated in rural communities in the Philippines. They are valued locally for their various sociocultural roles. Recently, considerable literature has accumulated in the field of native pig production and marketing. However, there is limited research on the genetic diversity of PhNP. No previous study has investigated the evolutionary relatedness among native pigs from various islands and provinces in Luzon and Visayas, Philippines. In addition, a much-debated question is whether the PhNP were interbreeding with or even domesticated from endemic wild pigs. This study aims to clarify some of the uncertainties surrounding the identity and classification of PhNP based on mitochondrial DNA (mtDNA) signatures. Native pig samples (n=157) were collected from 10 provinces in Luzon and the Visayas, Philippines. Approximately 650 base pairs of the mtDNA d-loop control region were sequenced and analyzed together with publicly available sequences. Pairwise-distance analysis showed genetic separation of North and South Luzon (SL) and the clustering of SL with Visayan pigs. Phylogenetic analysis showed that the PhNP clustered within 3 recognized Asian pig domestication centers: D2 (East Asia), D7 (Southeast Asia) and the Cordillera clade (sister to the Lanyu). We identified 19 haplotypes (1-38 samples each), forming 4 haplogroups i.e. North Luzon, South Luzon and Visayas, Asian mix and the Cordillera cluster. No endemic wild pig mtDNA was detected in the native pig population, but the use of wild pig for interspecific hybridization was observed. This study showed that the Philippine native pigs have ancestral origin from at least 3 Sus scrofa lineage and that they were not domesticated from the endemic wild pigs of the Philippines.

genetics↗

bioRxiv2022

Supramodal representation of the sense of body ownership in the human parieto-premotor and extrastriate cortices

The sense of body ownership, defined as the sensation that ones body belongs to oneself, is a fundamental component of bodily self-consciousness. Several studies have shown the importance of multisensory integration for the emergence of the sense of body ownership, together with the involvement of the parieto-premotor and extrastriate cortices in bodily awareness. However, whether the sense of body ownership elicited by different sources of signal, especially visuotactile and visuomotor inputs, is represented by common neural patterns remains to be elucidated. We used functional magnetic resonance imaging (fMRI) to investigate the existence of neural correlates of the sense of body ownership independent of the sensory modalities. Participants received tactile stimulation or executed finger movements while given synchronous and asynchronous visual feedback of their hand. We used multi-voxel patterns analysis (MVPA) to decode the synchronous and asynchronous conditions with cross-classification between two modalities: the classifier was first trained in the visuotactile sessions and then tested in the visuomotor sessions and vice versa. Regions of interest-based and searchlight analyses revealed significant above-chance cross-classification accuracies in the bilateral intraparietal sulcus (IPS), the bilateral ventral premotor cortex (PMv), and the left extrastriate body area (EBA). Moreover, we observed a significant positive correlation between the cross-classification accuracy in the left PMv and the difference in subjective ratings of the sense of body ownership between the synchronous and asynchronous conditions. Our findings revealed the neural representations of the sense of body ownership in the IPS, PMv, and EBA that is invariant to the sensory modalities. Significance StatementPrevious studies have shown neural correlates of the sense of body ownership in parieto-premotor and extrastriate cortices. However, whether the sense of body ownership induced by different sensory inputs is represented in common neural patterns remains unelucidated. Using functional magnetic resonance imaging (fMRI) with multi-voxel pattern analysis (MVPA), we investigated neural representations of the sense of body ownership invariant to modalities. Decoding neural patterns for visuotactile and visuomotor modalities revealed successful cross-classification accuracies in intraparietal sulcus (IPS), ventral premotor cortex (PMv), and extrastriate body area (EBA). Furthermore, cross-classification accuracy in PMv was positively correlated with subjective ratings of the sense of body ownership. These findings demonstrate that supramodal representations in parieto-premotor and extrastriate cortices underlie the sense of body ownership.

neuroscience↗

bioRxiv2021

Somnotate: A robust automated sleep stage classifier that exceeds human performance and identifies ambiguous states in mice

Electrophysiological recordings from freely behaving animals are a widespread and powerful mode of investigation in sleep research. These recordings generate large amounts of data that require sleep stage annotation (polysomnography), in which the data is parcellated according to three vigilance states: awake, rapid eye movement (REM) sleep, and non-REM (NREM) sleep. Manual and computational annotation methods currently ignore intermediate states because the classification features become ambiguous. However, these intermediate states contain important information regarding vigilance state dynamics. Here, we present a new classifier, "Somnotate", which produces automated annotation accuracies that exceed human expert performance on mouse electrophysiological data, is robust to errors in the training data, compatible with different recording configurations, and maintains high performance during experimental interventions. Somnotate is a probabilistic classifier based on a combination of linear discriminant analysis (LDA) with a hidden Markov model (HMM). A unique feature of Somnotate is that it quantifies and reports the certainty of its annotations, enabling the experimenter to identify ambiguous recording periods in a principled manner. We leverage this feature to identify epochs that exhibit intermediate vigilance states, revealing that many of these cluster around state transitions, whereas others correspond to failed attempts to transition. We show that the success rates of different transitions can be experimentally manipulated and explain previously observed sleep patterns. Somnotate can thus facilitate the study of sleep stage transitions and offers new insight into the mechanisms underlying sleep-wake dynamics. Author summaryTypically, the three different vigilance states - awake, REM sleep, and non-REM sleep - exhibit distinct features that are readily recognised in electrophysiological recordings. However, particularly around vigilance state transitions, epochs often exhibit features from more than one state. These intermediate vigilance states pose challenges for existing manual and automated classification methods, and are hence often ignored. Here, we present Somnotate - an open-source, highly accurate and robust sleep stage classifier, which supports research into intermediate states and sleep stage dynamics. Somnotate quantifies and reports the certainty of its annotations, enabling the experimenter to identify abnormal epochs in a principled manner. We use this feature to identify intermediate states and to detect unsuccessful attempts to switch between vigilance states. This provides new insights into the mechanisms of vigilance state transitions in mice, and creates new opportunities for future experiments.

neuroscience↗

bioRxiv2021

Intraneuronal chloride levels encode tiredness in cortex

Continuous periods of wakefulness are associated with reduced performance levels due to the build-up of sleep pressure in active regions of the brain. These effects manifest as use-dependent changes in cortical network activity and the mechanisms underlying these changes represent targets for overcoming the cognitive effects of tiredness. Here we reveal a central role for intraneuronal chloride levels, which increase in a use-dependent manner during waking, and reduce the strength of local synaptic inhibition in mouse cortex. Activity-dependent increases in chloride account for spatial and temporal features of sleep pressure, they underlie cortical network oscillations in the sleep-deprived state, and targeting chloride regulation in cortex can rescue performance levels when tired. These findings provide a missing link between sleep-wake history, synaptic transmission and cortical dynamics. One-Sentence SummaryThe effects of sleep pressure on cortical function are caused by use-dependent changes in chloride-mediated synaptic inhibition.

neuroscience↗

bioRxiv2021

Scn1a-GFP transgenic mouse revealed Nav1.1 expression in neocortical pyramidal tract projection neurons

Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly-generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually-exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS) and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

neuroscience↗

bioRxiv2020

Waking experience modulates sleep need in mice

Homeostatic regulation of sleep is reflected in the maintenance of a daily balance between sleep and wake. Although numerous internal and external factors can influence sleep, it is unclear whether and to what extent the process that keeps track of time spent awake is determined by the content of the waking experience. We hypothesised that alterations in environmental conditions may elicit different types of wakefulness, which will in turn influence both the capacity to sustain continuous wakefulness as well the rates of accumulating sleep pressure. To address this, we performed two experiments, where we compared wakefulness dominated by novel object exploration with either (i) the effects of voluntary wheel running (Experiment 1) or (ii) performance in a simple touchscreen task (Experiment 2). We find that voluntary wheel running results in longer wake episodes, as compared with exploratory behaviour; yet it does not lead to higher levels of EEG slow wave activity (SWA) during subsequent sleep. On the other hand, engagement in a touchscreen task, motivated by a food reward, results in lower SWA during subsequent sleep, as compared to exploratory wakefulness, even though the total duration of wakefulness was similar. Overall, our study suggests that sleep-wake behaviour is highly flexible within an individual, and that the homeostatic process that keeps track of time spent awake is sensitive to the nature of the waking experience. We therefore conclude that sleep dynamics are determined, to a large degree, by the interaction between the organism and the environment.

neuroscience↗

bioRxiv2020

The role of the hypothalamus in cortical arousaland sleep homeostasis

Sleep and wakefulness are not simple homogenous all-or-none states, but instead are characterized by rich dynamics of brain activity across many temporal and spatial scales. Rapid global state transitions between waking and sleeping are believed to be controlled by hypothalamic circuits, but the contribution of the hypothalamus to within-state changes of sleep and wake "intensity" remains largely unexplored. Here we show that stimulation of inhibitory neurons in the preoptic hypothalamus does not merely trigger awakening from sleep, but the resulting awake state is also characterized by increased cortical activity. This activation is associated with a faster build-up of sleep pressure, proportional to the arousal level. These findings show that hypothalamic systems thought to exclusively control global state switching, also regulate within-state "intensity", which we propose as a key intrinsic variable in shaping the architecture of sleep/wake states across the 24h day.

neuroscience↗

bioRxiv2020

A role for the cortex in sleep-wake regulation

The cortex and subcortical circuitry are thought to play distinct roles in the generation of sleep oscillations and global control of vigilance states. Here we silenced a subset of cortical layer 5 pyramidal and dentate gyrus granule cells in mice using a cell-specific ablation of the key t-SNARE protein SNAP25. We found a marked increase in wakefulness accompanied by a reduced rebound of EEG slow-wave activity after sleep deprivation. Our data illustrates an important role for the cortex in both global state control and sleep homeostasis.

neuroscience↗