Biology subjects

Inoue, W.

Publications and source records attributed to Inoue, W..

16 recordsLinked to original sources

bioRxiv2026

An anatomically distinct dopaminergic cell population of the zona incerta as evidence for the human A13 nucleus

The zona incerta (ZI) is a functionally and molecularly diverse deep brain region increasingly recognized as an integrative hub. The rodent ZI contains the dopaminergic A13 nucleus, with studies supporting a central role in modulating sensorimotor integration, nociception, and confers resilience in models of neurodegeneration; however, to date, no clear human homologue has been identified. Here, we identified a tyrosine hydroxylase-positive subregion in the human ZI consistent with the A13, and refined its borders using ex vivo MRI and histology. We translate these findings to in vivo MRI, demonstrating the A13 is distinguishable from surrounding regions by elevated T1 and T2* values. We further revealed a principal rostral-caudal axis of molecular and structural connectivity variation in the ZI, and identified the A13 as a localized molecular specialization. Altogether, we report a distinct ZI subregion corresponding to the A13 nucleus, providing an anatomical foundation for further mechanistic and translational investigation.

neuroscience↗

bioRxiv2025

Hypothalamic recurrent inhibition regulates functional states of stress effector neurons.

Stress triggers rapid and reversible shifts in vital physiological functions from homeostatic operation to emergency response. However, the neural mechanisms regulating such functional stress states remain poorly understood. Here we identify a novel recurrent inhibitory circuit governing functional states of key stress regulatory neurons: corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN). Microendoscopic calcium imaging in freely behaving mice revealed synchronized low-activity state at baselines and a reversible high-activity state during mild stress. Ensemble analysis indicated increased dimensionality of network dynamics during high-activity state. Computational modeling of calcium ensemble data, together with independent modeling of single-unit CRHPVN neurons spiking dynamics, converged to show that recurrent inhibition is a key circuit motif for stress-induced functional state transitions. Guided by model predictions, chemogenetic manipulations of PVN-projecting GABAergic neurons (PVN[->]GABA) revealed their roles in constraining CRHPVN neurons to low-activity state at baselines via a prolonged feedback inhibition. Unexpectedly, slow CRHergic excitation was dispensable for driving this prolonged feedback, whereas glutamatergic transmission predominated at CRH[->]PVN[->]GABA excitatory synapses. Incorporating these findings, we refined our computational model to include fast excitation and slow inhibition, yielding new predictions for circuit operation. Together, our results establish recurrent inhibition as a fundamental circuit motif controlling CRHPVN neurons functional states and highlight the value of iterative experiment-model integration in advancing understanding of neural circuits functions.

neuroscience↗

bioRxiv2025

Astrocytic Chromatin Remodeler ATRX Gates Hippocampal Memory Consolidation through Metabolic and Synaptic Regulation

Astrocytes are increasingly recognized as active regulators of synaptic transmission and memory, yet the epigenetic mechanisms underlying their contribution to cognitive processes remain poorly defined. Here, we investigated the role of the chromatin remodeler ATRX in astrocytes by generating mice with inducible, astrocyte-specific Atrx deletion (aiKO) using tamoxifen administration at postnatal days 10-12, resulting in ATRX loss in approximately half of hippocampal and cortical astrocytes. Transcriptomic profiling of hippocampal tissue at one and three months revealed a progressive increase in differentially expressed genes, with early enrichment for cytoskeletal and immune pathways and later dysregulation of energy metabolism, ion transport, and synaptic gene sets. Electrophysiological recordings from CA1 pyramidal neurons in aiKO slices demonstrated increased neuronal excitability, reduced membrane capacitance, and decreased frequency of spontaneous excitatory postsynaptic currents, indicating non-cell-autonomous neuronal dysfunction. Morphological analysis identified a transient reduction in dendritic branching at one month and a selective loss of thin dendritic spines by three months, without changes in total dendrite length or overall spine density. Behaviorally, aiKO mice displayed normal locomotion, anxiety, and short-term memory, but exhibited deficits in 24-hour novel object recognition and long-term spatial memory in the Morris water maze. These findings demonstrate that ATRX-mediated chromatin remodeling in astrocytes is essential for maintaining hippocampal transcriptional homeostasis, neuronal function, and long-term memory. Our results highlight a critical role for astrocytic epigenetic regulation in cognitive processes and suggest that astrocyte dysfunction may contribute to the pathogenesis of ATR-X syndrome and related intellectual disability disorders, underscoring the importance of targeting multiple cell types for therapeutic intervention. HighlightsO_LIAstrocytic ATRX loss causes non-cell-autonomous neuronal hyperexcitability C_LIO_LIInducing ATRX deficiency in astrocytes causes selective long-term memory deficits C_LIO_LIDynamic transcriptomic changes reveal metabolic and synaptic pathway dysregulation C_LI

neuroscience↗

bioRxiv2025

Second-Scale Neural Dynamics Shape Hormonal Outputs in Hypothalamic CRH Neurons

The elevation of glucocorticoids is a hallmark of stress, arising from the integration of rapid neuronal signals into sustained hormonal outputs. A key interface for this neuroendocrine signal translation is corticotropin releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN), which release CRH at the median eminence (ME). We recently discovered that CRHPVN neurons exhibit a characteristic shift in firing patterns from rhythmic short-bursts (low-activity state) to tonic firing (high-activity state) in response to stress. This raised a critical question: how are these distinct firing patterns are integrated into slower, sustained hormonal outputs at neuroendocrine terminals? Here, we implemented optical approaches to detect CRH release ex vivo and in vivo. Using newly developed sniffer cells for CRH, we measured CRH release at ME ex vivo, triggered by the distinct, in vivo-like firing patterns. Our results demonstrated that the primary determinant of neuroendocrine CRH release was the firing rate sustained over the timescale of seconds, with little contribution from specific firing patterns. These results collaborated with second-scale increase in firing rate triggered by stress stimuli. Additionally, we recorded the dynamics of CRH at the ME in the freely moving mice using genetically-encoded GPCR-activation based (GRAB) sensors for CRH. Foot shock stress triggered transient, time-locked increases in CRH release on the timescale of seconds. Importantly, these second-scale CRH pulses, when elicited during repeated foot shocks, were integrated over minutes to scale downstream hormone releases. Together, our data revealed critical roles of second-scale dynamics in CRHPVN neuron activity for the neuroendocrine translation of stress signals.

neuroscience↗

bioRxiv2024

Single neuron diversity supports area functional specialization along the visual cortical pathways

Humans and other primates have specialized visual pathways composed of interconnected cortical areas. The input area V1 contains neurons that encode basic visual features, whereas downstream in the lateral prefrontal cortex (LPFC) neurons acquire tuning for novel complex feature associations. It has been assumed that each cortical area is composed of repeatable neuronal subtypes, and variations in synaptic strength and connectivity patterns underlie functional specialization. Here we test the hypothesis that diversity in the intrinsic make-up of single neurons contributes to area specialization along the visual pathways. We measured morphological and electrophysiological properties of single neurons in areas V1 and LPFC of marmosets. Excitatory neurons in LPFC were larger, less excitable, and fired broader spikes than V1 neurons. Some inhibitory fast spiking interneurons in the LPFC had longer axons and fired spikes with longer latencies and a more depolarized action potential trough than in V1. Intrinsic bursting was found in subpopulations of both excitatory and inhibitory LPFC but not V1 neurons. The latter may favour temporal summation of spikes and therefore enhanced synaptic plasticity in LPFC relative to V1. Our results show that specialization within the primate visual system permeates the most basic processing level, the single neuron.

neuroscience↗

bioRxiv2024

In Search of Transcriptomic Correlates of Neuronal Firing-Rate Adaptation across Subtypes, Regions and Species: A Patch-seq Analysis

Can the transcriptomic profile of a neuron predict its physiological properties? Using a Patch-seq dataset of the primary visual cortex, we addressed this question by focusing on spike rate adaptation (SRA), a well-known phenomenon that depends on small conductance calcium (Ca)-dependent potassium (SK) channels. We first show that in parvalbumin-expressing (PV) and somatostatin-expressing (SST) interneurons, expression levels of genes encoding the ion channels underlying action potential generation are correlated with the half-width (HW) of spikes. Surprisingly, the SK encoding genes are not correlated with the degree of SRA (dAdap). Instead, genes that encode proteins upstream from the SK current, as well as the low-voltage-activated Calcium channel, are correlated with dAdap. This general principle is supported by datasets from the mouse primary motor cortex and multiple macaque brain regions. Finally, we generate testable predictions by constructing a minimal model.

neuroscience↗

bioRxiv2024

Spike frequency adaptation in primate lateral prefrontal cortex neurons results from interplay between intrinsic properties and circuit dynamics

Recordings of cortical neurons isolated from brain slices and dissociated from their networks, display intrinsic spike frequency adaptation (I-SFA) to a constant current input. Interestingly, extracellular recordings in behaving subjects also show extrinsic-SFA (E-SFA) in response to sustained visual stimulation. Because neurons are isolated from brain networks in slice recordings, it is challenging to infer how I-SFA contributes to E-SFA in interconnected brains during behavior. To investigate this, we recorded responses of macaque lateral prefrontal cortex neurons in vivo during a visually guided saccade task and in acute brain slices in vitro. Broad spiking (BS) putative pyramidal cells and narrow spiking (NS) putative inhibitory interneurons exhibited E-SFA in vivo. In acute brain slices, both cell types displayed I-SFA though their magnitudes differed. To investigate how in vitro I-SFA contributes to in vivo E-SFA, we developed a data-driven hybrid circuit model in which local NS neurons are driven by BS input. We observed that model NS cell responses show longer SFA than observed in vivo. Introducing inhibition of NS cells to the model circuit removed this discrepancy. Our results indicate that both I-SFA and inhibitory circuit dynamics contribute to E-SFA in LPFC neurons. They highlight the contribution of single neuron and network dependent computations to neural activity underlying behavior.

neuroscience↗

bioRxiv2024

Dopamine D1 receptor expression in dlPFC inhibitory parvalbumin neurons may contribute to higher visuospatial distractibility in marmosets versus macaques

Marmosets and macaques are common non-human primate models of cognition, yet marmosets appear more distractible and perform worse in cognitive tasks. The dorsolateral prefrontal cortex (dlPFC) is pivotal for sustained attention, and prior macaque research suggests that dopaminergic modulation and inhibitory parvalbumin (PV) neurons could contribute to distractibility. Thus, we compared the two species using a visual fixation task with distractors, performed molecular and anatomical analyses in dlPFC, and linked functional microcircuitry with cognitive performance using computational modeling. We found that marmosets are more distractible than macaques, and that marmoset dlPFC PV neurons contain higher levels of dopamine-1 receptor (D1R) transcripts and protein, similar to their levels in mice. The modeling indicated that higher D1R expression in marmoset dlPFC PV neurons may increase distractibility by making dlPFC microcircuits more vulnerable to disruptions of their task-related persistent activity, especially when dopamine is released in dlPFC in response to unexpected salient stimuli. Declaration of InterestsThe authors have nothing to declare. Author ContributionsAFTA, MKPJ, TGI, and SFW designed the study; MKPJ collected and analyzed anatomical data; TGI designed the computational framework with guidance from RG and SFW; TGI performed the modeling simulations and data analysis; FM and SAM collected and analyzed transcriptomics data; GGB and DAL provided macaque brain tissue for transcriptomics performed by FM; SM and NS collected and analyzed phylogenetic transcriptomics dataset; JFM and ASN performed and analyzed the behavioral testing; IW and JMT provided the marmoset tissue for immunofluorescence; MKPJ, TGI, SFW, and AFTA wrote the first draft and all authors revised and edited subsequent drafts of the article.

neuroscience↗

bioRxiv2024

Direct modulation of CRH nerve terminal function by noradrenaline and corticosterone.

Nerve terminals are the final point of regulation before neurosecretion. As such, neuromodulators acting on nerve terminals can exert significant influence on neural signalling. Hypothalamic corticotropin-releasing hormone (CRH) neurons send axonal projections to the median eminence where CRH is secreted to stimulate the hypothalamic-pituitary-adrenal (HPA) axis. Noradrenaline and corticosterone are two of the most important neuromodulators of HPA axis function; noradrenaline excites CRH neurons and corticosterone inhibits CRH neurons by negative feedback. Here, we used GCaMP6f Ca2+ imaging and measurement of nerve terminal CRH secretion using sniffer cells to determine whether these neuromodulators act directly on CRH nerve terminals. Contrary to expectations, noradrenaline inhibited action potential-dependent Ca2+ elevations in CRH nerve terminals and suppressed evoked CRH secretion. This inhibitory effect was blocked by 2-adrenoreceptor antagonism. Corticosterone also suppressed evoked CRH peptide secretion from nerve terminals, independent of action potential-dependent Ca2+ levels. This inhibition was prevented by the glucocorticoid receptor antagonist, RU486, and indicates that CRH nerve terminals may be a site of fast glucocorticoid negative feedback. Together these findings establish median eminence nerve terminals as a key site for regulation of the HPA axis. SignificanceCorticotropin-releasing hormone (CRH) neurons control the stress axis. Noradrenaline and corticosterone are two signalling molecules that control CRH neuron cell body excitability. However, their effect on CRH nerve terminal function is unknown. To examine this, we performed live Ca2+ imaging and measured CRH secretion. We found that noradrenaline suppressed nerve terminal Ca2+ levels and inhibited nerve terminal CRH secretion. Corticosterone had no effect on nerve terminal Ca2+, but inhibited nerve terminal CRH secretion. This suggests that CRH nerve terminals may be a site of fast corticosteroid negative feedback. Together, these data demonstrate that CRH nerve terminals are a critical point of regulation in the control of the stress axis.

neuroscience↗

bioRxiv2024

Viral mimicry and memory deficits upon microglial deletion of ATRX

The importance of chromatin-mediated processes in neurodevelopmental and intellectual disability disorders is well recognised. However, how chromatin dysregulation in glial cells impacts cognitive abilities is less well understood. Here, we demonstrate that targeted loss of the ATRX chromatin remodeler in microglia alters chromatin accessibility profiles, leading to the de-repression of endogenous retroelements, triggering viral mimicry. Functionally, we find that ATRX microglial deficiency alters the electrophysiological properties of hippocampal neurons and causes deficits in object recognition and spatial memory. Overall, these findings demonstrate that ATRX is required in microglia to preserve chromatin structure and maintain microglial homeostasis. Disruption of these functions elicit neuroinflammation and cognitive deficits and potentially contribute to the pathology of human neurological disorders caused by ATRX mutations.

neuroscience↗

bioRxiv2022

Investigating microglia-neuron crosstalk by characterizing microglial contamination in human and mouse Patch-seq datasets

Microglia are dynamic immune cells with diverse functional roles, including the regulation of neuronal excitability. Here, we leveraged an inconvenient truth of neuronal Patch-seq datasets -- that they routinely display evidence of contamination by surrounding microglia -- to better understand aspects of microglia-neuronal crosstalk. We first quantified the presence of microglial transcripts in three Patch-seq datasets of human and mouse neocortical neurons and observed extensive off-target contamination by microglia in each. Variation in microglial contamination was explained foremost by donor identity, especially in human samples, and neuronal cell type identity. Differential expression testing and enrichment analyses suggest that microglial contamination in Patch-seq is reflective of activated microglia and that these transcriptional signatures are distinct from those captured via single-nucleus RNAseq. Finally, neurons with greater microglial contamination differed markedly in their electrophysiological characteristics, including lowered input resistances and more depolarized action potential thresholds. Our results suggest microglial contamination contributes to cell- and donor-related electrophysiological variability and sheds light on how microglia might impact neurons in vivo.

neuroscience↗

bioRxiv2022

State-dependent activity dynamics of hypothalamic stress effector neurons

The stress response necessitates an immediate boost in vital physiological functions from their homeostatic operation to elevated emergency response. However, neural mechanisms underlying this state-dependent change remain largely unknown. Using a combination of in vivo and ex vivo electrophysiology with computational modeling, we report that corticotropin releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN), the effector neurons of hormonal stress response, rapidly transition between distinct activity states through recurrent inhibition. Specifically, in vivo optrode recording shows that under non-stress conditions, CRHPVN neurons often fire with rhythmic brief bursts (RB), which, somewhat counterintuitively, constrains firing rate due to long ([~]2 s) inter-burst intervals. Stressful stimuli rapidly switch RB to continuous single spiking (SS), permitting a large increase in firing rate. A spiking network model shows that recurrent inhibition can control this activity-state switch, and more broadly the gain of spiking responses to excitatory inputs. In biological CRHPVN neurons ex vivo, the injection of whole-cell currents derived from our computational model recreates the in vivo-like switch between RB and SS, providing a direct evidence that physiologically relevant network inputs enable state-dependent computation in single neurons. Together, we present a novel mechanism for state-dependent activity dynamics in CRHPVN neurons.

neuroscience↗

bioRxiv2021

Hippocampal-hypothalamic circuit controls context-dependent innate defensive responses

Preys use their memory - where they sensed a predatory threat and whether a safe shelter is nearby - to dynamically control their survival instinct to avoid harm and reach safety. However, it remains unknown which brain regions are involved, and how such top-down control of innate behaviour is implemented at the circuit level. Here, we show that the anterior hypothalamic nucleus (AHN) is best positioned to perform this task as an exclusive target of the hippocampus (HPC) within the medial hypothalamic defense system. Selective optogenetic stimulation and inhibition of hippocampal inputs to the AHN revealed that the HPC[->]AHN pathway not only mediates the contextual memory of predator threats but also controls the goal-directed escape by transmitting information about the surrounding environment. These results reveal a new mechanism for experience-dependent, top-down control of innate defensive behaviours.

neuroscience↗

bioRxiv2020

Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice

Microglia express muscarinic G protein-coupled receptors (GPCRs) that sense cholinergic activity and are activated by acetylcholine to potentially regulate microglial functions. Knowledge about how distinct types of muscarinic GPCR signaling regulate microglia function in vitro and in vivo is still poor, partly due to the fact that some of these receptors are also present in astrocytes and neurons. We generated mice expressing the hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in microglia to investigate the role of muscarinic M3Gq-linked signaling. We show that activation of hM3Dq using clozapine N-oxide (CNO) elevated intracellular calcium levels and increased phagocytosis of FluoSpheres in vitro. Acute treatment with CNO in vivo did not affect male mouse behavior, however chronic CNO treatment decreased sickness behavior triggered by lipopolysaccharide (LPS) treatment. Interestingly, whereas acute treatment with CNO increased synthesis of cytokine mRNA, chronic treatment attenuated LPS-induced cytokine mRNA changes in the brain, likely explaining the improvement in sickness behavior by chronic hM3Dq activation. No effect of CNO was observed in DREADD-negative mice. These results suggest that chronic activation of M3 muscarinic receptors (the hM3Dq progenitor) in microglia, and potentially other Gq-coupled GPCRs, preconditions microglia to decrease their response to further immunological challenges. Our results indicate that hM3Dq can be a useful tool to modulate neuroinflammation and study microglial immunological memory in vivo, which may be applicable for manipulations of neuroinflammation in neurodegenerative and psychiatric diseases. HighlightsO_LIMicroglial function was manipulated by specific activation of hM3Dq signaling. C_LIO_LIChronic hM3Dq activation prevented LPS-induced sickness behavior in mice. C_LIO_LIMicroglial hM3Dq signaling modulated expression of pro-inflammatory cytokines. C_LI

neuroscience↗

bioRxiv2019

Stress-induced neuronal hypertrophy decreases the intrinsic excitability in stress habituation

A rapid activation of the hypothalamic-pituitary-adrenal (HPA) axis is a hallmark stress response to an imminent threat, but its chronic activation can be detrimental. Thus, the long-term survival of animals requires experience-dependent fine-tuning of the stress response. However, the cellular mechanisms underlying the ability to decrease the stress responsiveness of the HPA axis remain largely unsolved. Using a stress habituation model in male mice and slice patch-clamp electrophysiology, we studied hypothalamic corticotropin-releasing hormone neurons that form the apex of the HPA axis. We found that the intrinsic excitability of these neurons substantially decreased after daily repeated restraint stress in a time course that coincided with their loss of stress responsiveness in vivo. This plasticity of intrinsic excitability co-developed with an expansion of surface membrane area, resulting in an increase in input conductance with minimal changes in conductance density. Moreover, multi-photon and electron microcopy data found that repeated stress augmented ruffling of the plasma membrane, suggesting an ultrastructural plasticity that efficiently accommodates membrane area expansion with proportionally less expansion of gross cell volume. Overall, we report a novel structure-function relationship for intrinsic excitability plasticity that correlates with habituation of the neuroendocrine stress response.\n\nSignificance statementThe long-term survival of animals requires experience-dependent fine-tuning of stress response. Using a mouse model of repeated stress that develops habituation of the hypothalamic-pituitary-adrenal (HPA) axis, our study demonstrates a robust decrease in the intrinsic excitability of the output neuroendocrine neurons of the HPA axis. Mechanistically, we show that repeated stress increases the cell size of these neurons (i.e. surface membrane area). This cell-size change increases input conductance, and hence decreases excitability. Our findings challenge a conventional view that plasticity of intrinsic excitability relies on changes on membrane excitability resulting from up- and down-regulation of various voltage-gated ion channels. Our study reports a novel structure-function relationship for intrinsic excitability plasticity that correlates with habituation of the neuroendocrine stress response.

neuroscience↗

bioRxiv2017

Evaluation of variability in human protein X-ray structures

Systematic analysis of statistical and dynamical properties of proteins is critical to understanding cellular events. Extraction of biologically relevant information from a set of high-resolution structures is important because it can provide mechanistic details behind the functional properties of protein families, enabling rational comparison between families. Most of the current structure comparisons are pairwise-based, which hampers the global analysis of increasing contents in the Protein Data Bank. Additionally, pairing of protein structures introduces uncertainty with respect to reproducibility because it frequently accompanies other settings for superimposition. This study introduces intramolecular distance scoring, for the analysis of human proteins, for each of which at least several high-resolution are available. We show that the results are comprehensively used to overview advances at the atomic level exploration of each protein and protein family. This method, and the interpretation based on model calculations, provide new criteria for understanding specific and non-specific structure variation in a protein, enabling global comparison of the dynamics among a vast variety of proteins from different species.

biophysics↗