Biology subjects

Yasuda, R.

Publications and source records attributed to Yasuda, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Dynamic visualization of physiological CaMKII activity using sensitive FRET biosensors

Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key mediator of complex physiological processes throughout the body, from the brain to the reproductive system, where CaMKII translates spatiotemporally dynamic calcium elevations into specific biological functions. Directly visualizing CaMKII activity dynamics in living cells using genetically encoded fluorescent biosensors can thus provide crucial insights into the molecular regulation of health and disease. Yet the ability to sensitively and specifically monitor endogenous CaMKII activity in physiologically relevant contexts is limited by the lack of sensors that can achieve robust, quantitative visualization of CaMKII responses. Here, we leveraged a recent serine/threonine kinome-wide substrate atlas to rationally engineer a powerful suite of Forster resonance energy transfer (FRET)-based CaMKII kinase activity reporters with high specificity, sensitivity, and signal-to-noise ratio. Using these biosensors, we were able to sensitively and robustly visualize endogenous CaMKII activity dynamics in both cultured cell lines and primary cells, including cardiomyocytes, oocytes, and neurons. We further utilized 2pFLIM imaging of organotypic hippocampal slices to quantitatively track LTP-induced CaMKII activity within single dendritic spines, highlighting a major advance in the study of physiological CaMKII signaling.

bioengineering↗

bioRxiv2025

Mitochondria structurally remodel near synapses to fuel the sustained energy demands of plasticity

Brain synapses are hotspots of energy consumption, and the synaptic plasticity underlying learning sharply increases this demand. Because most synapses lie far from their cell body, their energy demands must be met locally. A dendritic mitochondrion can extend ~30 m and span many spines, yet ramps up ATP synthesis only near spines undergoing plasticity, providing immediate and sustained energy. Whether structural changes in these juxtaspinal mitochondrial regions (2 m segments at spine bases) support this response is unknown; resolving their energy-producing architecture poses substantial challenges. Compared with the lamellar cristae of axonal mitochondria, dendritic mitochondrial cristae form complex, tubular, and interconnected networks that require multiple high-resolution tomograms to characterize. We therefore developed a correlative light and electron microscopy (CLEM) pipeline with a custom deep-learning model that simultaneously segments five distinct 3D suborganellar structures from limited training data, enabling quantitative analysis across 42 tomograms at 2 nm pixel resolution. We find that juxtaspinal mitochondrial regions are larger than non-juxtaspinal regions within the same mitochondrion and enlarge further during synaptic plasticity. Juxtaspinal mitochondrial ATP synthesis and spine ATP levels rise in parallel; during single-spine plasticity, ATP rises within minutes, before detectable enlargement, indicating that juxtaspinal mitochondrial enlargement sustains rather than initiates the local energy response. The enlarged regions exhibit increased cristae membrane surface area, crista junction number, and membrane area with high curvature, together with increased MIC60 and ATP synthase copies, endoplasmic reticulum contacts, and more proximal ribosomes. Matrix-targeted PKA inhibition reduces MIC60 phosphorylation and impairs juxtaspinal mitochondrial enlargement, plasticity-associated ATP increase, and single-spine plasticity. Together, our findings support a model in which spine-specific remodeling within an individual mitochondrion boosts local metabolic capacity to sustain ATP synthesis and fuel synaptic plasticity.

neuroscience↗

bioRxiv2024

lncRNA ADEPTR loss-of-function elicits sex-specific behavioral and spine deficits

Activity-dependent changes in neuronal connections are fundamental to learning and long-term memory storage. However, the precise contribution of long noncoding RNAs (lncRNAs) to these modifications remains unclear. In this study, we assessed the role of the lncRNA ADEPTR, a cAMP-modulated lncRNA localized in dendrites, which is crucial for synapse morphology. By generating two different mouse models--one with a deletion of ADEPTR (L-ADEPTR) and one with a deletion of its protein interaction region (S-ADEPTR)--we investigated the sex-specific impacts of ADEPTR loss of function on learning, memory, dendritic arborization, and synapse morphology. Our behavioral analyses revealed a reduction in anxiety in adult male mice, while learning and memory remained unaffected in both models. Systematic evaluations of neuronal morphology across various developmental stages ([~]3-day-old postnatal neuronal cultures and postnatal 14- and 42-day-old male and female mice) uncovered substantial deficits in neuronal architecture in both S- and L-ADEPTR male and female neuronal cultures. At postnatal day 42, in contrast to their male counterparts, L-ADEPTR female mice exhibited a significant deficiency in thin spines. Additionally, we found that the expression of plasticity-related gene BDNF, and immediate early gene cFOS were enhanced in both the cortex and hippocampus of adult male and female S- and L-ADEPTR mice, suggesting the activation of a compensatory mechanism protecting against learning and memory deficits. Collectively, these observations underscore the sex-specific role of lncRNA ADEPTR in shaping neuronal morphology and anxiety behavior.

neuroscience↗

bioRxiv2024

Formation of long-term memory without short-term memory by CaMKII inhibition

Most of the current models of long-term memory consolidation require prior establishment of short-term memory. Here, we show that optogenetic or genetic inhibition of CaMKII, a kinase important for synaptic plasticity, in an inhibitory avoidance task impairs short-term memory at 1 h but not long-term memory at 1 d. Similarly, cortico-amygdala synaptic potentiation was more sensitive to CaMKII inhibition at 1 h but not at 1 d. These results strongly suggest that long-term memory does not require the prior formation of short-term memory and that CaMKII-dependent synaptic plasticity specifically regulates short-term memory, but not long-term memory, for avoidance memory.

neuroscience↗

bioRxiv2023

Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

Head-mounted miniscopes have enabled functional fluorescence imaging in freely moving animals. However, current technology is limited to recording at most two spectrally distinct fluorophores, severely restricting the number of identifiable cell types. Here we introduce multiplexed neuronal imaging (Neuroplex), a pipeline combining miniscope Ca2+ recordings with in vivo multiplexed confocal spectral imaging to distinguish nine projection-defined neuronal subtypes through the same GRIN lens. By co-registering defined neurons with fluorophore-specific spectral fingerprints via linear unmixing, we link projection-defined identities to behaviorally relevant neuronal activity. This approach overcomes spectral constraints of miniscopes, enabling circuit-level dissection of behavior in single animals.

neuroscience↗

bioRxiv2023

Matrix Metalloproteinase-9 controls structural synaptic plasticity via BDNF-dependent signaling

Synaptic plasticity underlies learning and memory processes as well as contributes, in its aberrant form, to neuropsychiatric disorders. One of its major forms is structural long-term potentiation (sLTP), an activity-dependent growth of dendritic spines that harbor excitatory synapses. The process depends on the release of brain-derived neurotrophic factor (BDNF), and activation of its receptor, TrkB. Matrix metalloproteinase-9 (MMP-9), an extracellular protease is essential for many forms of neuronal plasticity engaged in physiological as well as pathological processes. Here, we utilized two-photon microscopy and two-photon glutamate uncaging to demonstrate that MMP-9 activity is essential for sLTP and is rapidly ([~]seconds) released from dendritic spines in response to synaptic stimulation. Moreover, we show that either chemical or genetic inhibition of MMP-9 impairs TrkB activation, as measured by fluorescence lifetime imaging microscopy of FRET sensor. Furthermore, we provide evidence for a cell-free cleavage of proBDNF into mature BDNF by MMP-9. Our findings point to the autocrine mechanism of action of MMP-9 through BDNF maturation and TrkB activation during sLTP.

neuroscience↗

bioRxiv2023

Cell-type-specific plasticity shapes neocortical dynamics for motor learning

Neocortical spiking dynamics control aspects of behavior, yet how these dynamics emerge during motor learning remains elusive. Activity-dependent synaptic plasticity is likely a key mechanism, as it reconfigures network architectures that govern neural dynamics. Here, we examined how the mouse premotor cortex acquires its well-characterized neural dynamics that control movement timing, specifically lick timing. To probe the role of synaptic plasticity, we have genetically manipulated proteins essential for major forms of synaptic plasticity, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Cofilin, in a region and cell-type-specific manner. Transient inactivation of CaMKII in the premotor cortex blocked learning of new lick timing without affecting the execution of learned action or ongoing spiking activity. Furthermore, among the major glutamatergic neurons in the premotor cortex, CaMKII and Cofilin activity in pyramidal tract (PT) neurons, but not intratelencephalic (IT) neurons, is necessary for learning. High-density electrophysiology in the premotor cortex uncovered that neural dynamics anticipating licks are progressively shaped during learning, which explains the change in lick timing. Such reconfiguration in behaviorally relevant dynamics is impeded by CaMKII manipulation in PT neurons. Altogether, the activity of plasticity-related proteins in PT neurons plays a central role in sculpting neocortical dynamics to learn new behavior.

neuroscience↗

bioRxiv2023

Dendritic, delayed, and stochastic CaMKII activation underlies behavioral time scale plasticity in CA1 synapses

Behavioral time scale plasticity (BTSP), is a form of non-Hebbian plasticity induced by integrating pre- and postsynaptic components separated by behavioral time scale (seconds). BTSP in the hippocampal CA1 neurons underlies place cell formation. However, the molecular mechanisms underlying this behavioral time scale (eligibility trace) and synapse specificity are unknown. CaMKII can be activated in a synapse-specific manner and remain active for a few seconds, making it a compelling candidate for the eligibility trace during BTSP. Here, we show that BTSP can be induced in a single dendritic spine using 2-photon glutamate uncaging paired with postsynaptic current injection temporally separated by behavioral time scale. Using an improved CaMKII sensor, we saw no detectable CaMKII activation during this BTSP induction. Instead, we observed a dendritic, delayed, and stochastic CaMKII activation (DDSC) associated with Ca2+ influx and plateau 20-40 s after BTSP induction. DDSC requires both pre-and postsynaptic activity, suggesting that CaMKII can integrate these two signals. Also, optogenetically blocking CaMKII 30 s after the BTSP protocol inhibited synaptic potentiation, indicating that DDSC is an essential mechanism of BTSP. IP3-dependent intracellular Ca2+ release facilitates both DDSC and BTSP. Thus, our study suggests that the non-synapse specific CaMKII activation provides an instructive signal with an extensive time window over tens of seconds during BTSP.

neuroscience↗

bioRxiv2022

Disinhibition of Rab10 gates AMPAR insertion and spine enlargement during long-term potentiation

Rab-dependent membrane trafficking is critical for changing the structure and function of dendritic spines during synaptic plasticity. Here, we developed highly sensitive sensors to monitor Rab protein activity in single dendritic spines undergoing structural long-term potentiation (sLTP) in rodent organotypic hippocampal slices. During sLTP, Rab10 was persistently inactivated (>30 min) in the stimulated spines, whereas Rab4 was transiently activated over [~]5 min. Inhibiting or deleting Rab10 enhanced sLTP, electrophysiological LTP and AMPA receptor (AMPAR) trafficking during sLTP. In contrast, disrupting Rab4 impaired sLTP only in the first few minutes, and decreased AMPAR trafficking during sLTP. Thus, our results suggest that Rab10 and Rab4 oppositely regulate AMPAR trafficking during sLTP, and inactivation of Rab10 signaling facilitates the induction of LTP and associated spine structural plasticity.

neuroscience↗

bioRxiv2021

Dual regulation of spine-specific and synapse-to-nucleus signaling by PKCδ during plasticity

The activity-dependent plasticity of synapses is believed to be the cellular basis of learning. These synaptic changes are mediated through the coordination of local biochemical reactions in synapses and changes in gene transcription in the nucleus to modulate neuronal circuits and behavior. The protein kinase C (PKC) family of isozymes has long been established as critical for synaptic plasticity. However, due to a lack of suitable isozyme-specific tools, the role of the novel subfamily of PKC isozymes is largely unknown. Here, through the development of FLIM-FRET activity sensors, we investigate novel PKC isozymes in synaptic plasticity in mouse CA1 pyramidal neurons. We find that PKC{delta} is activated downstream of TrkB and that the spatiotemporal nature of its activation depends on the plasticity stimulation. In response to single spine plasticity, PKC{delta} is activated primarily in the stimulated spine and is required for local expression of plasticity. However, in response to multi-spine stimulation, a long-lasting and spreading activation of PKC{delta} scales with the number of spines stimulated and, by regulating CREB activity, couples spine plasticity to transcription in the nucleus. Thus, PKC{delta} plays a dual functional role in facilitating synaptic plasticity.

neuroscience↗

bioRxiv2021

Rapid generation of conditional knockout mice using the CRISPR-CAS9 system and electroporation for neuroscience research

The Cre/Loxp-based conditional knockout technology is a powerful tool for gene function analyses by allowing region-time-specific gene manipulation. However, inserting a pair of LoxP cassettes for generating conditional knock-out can be technically challenging and thus time- and resource-consuming. This study proposes an efficient, low-cost method to generate floxed mice using the in vitro fertilization and the CRISPR-Cas9 system over two consecutive generations. This method allowed us to produce floxed mice targeting exon 5 to exon 6 of CaMK1 in a short period, 125 days, using only 16 mice. The efficiency of generating floxed mice was 10%, significantly higher than the conventional ES cell-based method. We directly edited the genome of C57BL/6N fertilized eggs, our target genetic background, to eliminate additional backcrossing steps. We confirmed that the genome of this floxed mouse is responsive to Cre protein. This low-cost, highly efficient method for generating conditional knock-out will facilitate comprehensive, tissue-specific genome analyses.

neuroscience↗

bioRxiv2021

Local autocrine signaling of IGF1 synthesized and released by CA1 pyramidal neurons regulates plasticity of dendritic spines

Insulin-like growth factor 1 (IGF1) regulates hippocampal plasticity, learning, and memory. While circulating, liver-derived IGF1 is known to play an essential role in hippocampal function and plasticity, IGF1 is also synthesized in multiple brain regions, including the hippocampus. However, little is known about the role of hippocampus-derived IGF1 in synaptic plasticity, the type of cells that may provide relevant IGF1, and the spatiotemporal dynamics of IGF1 signaling. Here, using a new FRET sensor for IGF1 signaling, we show that IGF1 in the hippocampus is primarily synthesized in CA1 pyramidal neurons and released in an activity-dependent manner in mice. The local IGF1 release from dendritic spines triggers local autocrine IGF1 receptor activation on the same spine, regulating structural and electrophysiological plasticity of the activated spine. Thus, our study demonstrates a novel mechanism underlying synaptic plasticity by the synthesis and autocrine signaling of IGF1 specific to CA1 pyramidal neurons.

neuroscience↗

bioRxiv2020

Action potential-coupled Rho GTPase signaling drives presynaptic plasticity

In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with presynaptic vesicle membranes and negatively regulate synaptic vesicle replenishment at both excitatory and inhibitory synapses. Using optogenetics and fluorescence lifetime imaging, we show this pathway bidirectionally sculpts short-term synaptic depression and that its presynaptic activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study provides a new proteomic framework for understanding presynaptic physiology and uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across cell types.

neuroscience↗

bioRxiv2020

A cAMP/PKA-dependent synaptically targeted lncRNA mediates structural plasticity in hippocampal neurons by functionally interacting with the Spectrin/Ankyrin Network

Activity-dependent structural plasticity at the synapse requires specific changes in the neuronal transcriptome. While much is known about the role of coding elements in this process, the role of the long-noncoding transcriptome remains elusive. Here we report the discovery of an intronic long noncoding RNA (lncRNA)--termed ADEPTR--whose expression is upregulated and is synaptically transported in a cAMP/PKA-dependent manner in hippocampal neurons, independent of its protein-coding host gene. Loss of ADEPTR function suppresses activity-dependent changes in synaptic transmission and structural plasticity of dendritic spines. Mechanistically, dendritic localization of ADEPTR is mediated by molecular motor protein Kif2A. ADEPTR physically binds to actin-scaffolding regulators Ankyrin (AnkB) and Spectrin (Sptn1) and is required for their dendritic localization. Taken together, this study demonstrates that ADEPTR regulates the dendritic Spectrin-Ankyrin network for structural plasticity at the synapse and illuminates a novel role for lncRNAs at the synapse. One Sentence SummaryWe have uncovered an intronic long noncoding RNA that is synaptically transported in a cAMP-dependent manner and is linked to cytoskeletal components of structural plasticity in hippocampal neurons.

neuroscience↗

bioRxiv2019

Rapid Ultrastructural Changes of PSD and Extrasynaptic Axon-spine Interface Membrane during LTP Induced in Single Dendritic Spine

Structural plasticity of dendritic spines is considered to be the basis of synaptic plasticity, learning and memory. Here, we performed ultrastructural analysis of spines undergoing LTP using a novel high throughput correlative light-electron microscopy approach. We found that the PSD displays rapid (< 3 min) reorganization of its nanostructure, including perforation and segmentation. This increased structural complexity is maintained over intermediate and late phases of LTP (20 and 120 min). In a few spines, segmented PSDs are connected to different presynaptic terminals, producing a multi-innervated spine in the intermediate and late phases. In addition, the area of extrasynaptic axon-spine interface (eASI) displayed a pronounced, rapid and sustained increase. Finally, presynaptic vesicle number increased slowly and became significantly higher at late phases of LTP. These rapid ultrastructural changes in PSD and surrounding membrane, together with the slow increase in presynaptic vesicle number, likely support the rapid and sustained increase in synaptic transmission during LTP.

neuroscience↗

bioRxiv2019

Rac1 is a downstream effector of PKCα in structural synaptic plasticity

Structural and functional plasticity of dendritic spines is the basis of animal learning. The calcium-dependent protein kinase C isoform, PKC, has been suggested to be critical for this actin-dependent plasticity. However, mechanisms linking PKC and structural plasticity of spines are unknown. Here, we examine the spatiotemporal activation of actin regulators, including small GTPases Rac1, Cdc42 and Ras, in the presence or absence of PKC during single-spine structural plasticity. Removal of PKC expression in the postsynapse attenuated Rac1 activation during structural plasticity without affecting Ras or Cdc42 activity. Moreover, disruption of a PDZ binding domain within PKC led to impaired Rac1 activation and deficits in structural spine remodeling. These results demonstrate that PKC positively regulates the activation of Rac1 during structural plasticity.

neuroscience↗

bioRxiv2018

RGS14 restricts plasticity in hippocampal CA2 by limiting postsynaptic calcium signaling

Pyramidal neurons in hippocampal area CA2 are distinct from neighboring CA1 in that they resist synaptic long-term potentiation (LTP) at CA3 Schaffer Collateral synapses. Regulator of G Protein Signaling 14 (RGS14) is a complex scaffolding protein enriched in CA2 dendritic spines that naturally blocks CA2 synaptic plasticity and hippocampus-dependent learning, but the cellular mechanisms by which RGS14 gates LTP are largely unexplored. A previous study has attributed the lack of plasticity to higher rates of calcium (Ca2+) buffering and extrusion in CA2 spines. Additionally, a recent proteomics study revealed that RGS14 interacts with two key Ca2+-activated proteins in CA2 neurons: calcium/calmodulin, and CaMKII. Here, we investigate whether RGS14 regulates Ca2+ signaling in its host CA2 neurons. We find the nascent LTP of CA2 synapses due to genetic knockout (KO) of RGS14 in mice requires Ca2+-dependent postsynaptic signaling through NMDA receptors, CaMK, and PKA, revealing similar mechanisms to those in CA1. We report RGS14 negatively regulates the long-term structural plasticity of dendritic spines of CA2 neurons. We further show that wild-type (WT) CA2 neurons display significantly attenuated spine Ca2+ transients during structural plasticity induction compared with the Ca2+ transients from CA2 spines of RGS14 KO mice and CA1 controls. Finally, we demonstrate that acute overexpression of RGS14 is sufficient to block spine plasticity, and elevating extracellular Ca2+ levels restores plasticity to RGS14-expressing neurons. Together, these results demonstrate for the first time that RGS14 regulates plasticity in hippocampal area CA2 by restricting Ca2+ elevations in CA2 spines and downstream signaling pathways.\n\nSignificance StatementRecent studies of hippocampal area CA2 have provided strong evidence in support of a clear role for this apparently plasticity-resistant subregion of the hippocampus in social, spatial, and temporal aspects of memory. Regulator of G Protein Signaling 14 (RGS14) is a critical factor that inhibits synaptic plasticity in CA2, but the molecular mechanisms by which RGS14 limits LTP remained unknown. Here we provide new evidence that RGS14 restricts spine calcium (Ca2+) in CA2 neurons and that key downstream Ca2+-activated signaling pathways are required for CA2 plasticity in mice lacking RGS14. These results define a previously unrecognized role for RGS14 as a natural inhibitor of postsynaptic Ca2+ signaling in hippocampal area CA2.

neuroscience↗

bioRxiv2018

An Open-source Tool for Analysis and Automatic Identification of Dendritic Spines Using Machine Learning

Synaptic plasticity, the cellular basis for learning and memory, is mediated by a complex biochemical network of signaling proteins. These proteins are compartmentalized in dendritic spines, the tiny, bulbous, post-synaptic structures found on neuronal dendrites. The ability to screen a high number of molecular targets for their effect on dendritic spine structural plasticity will require a high-throughput imaging system capable of stimulating and monitoring hundreds of dendritic spines in various conditions. For this purpose, we present a program capable of automatically identifying dendritic spines in live, fluorescent tissue. Our software relies on a machine learning approach to minimize any need for parameter tuning from the user. Custom thresholding and binarization functions serve to \"clean\" fluorescent images, and a neural network is trained using features based on the relative shape of the spine perimeter and its corresponding dendritic backbone. Our algorithm is rapid, flexible, has over 90% accuracy in spine detection, and bundled with our user-friendly, open-source, MATLAB-based software package for spine analysis.

neuroscience↗