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Handa, T.

Publications and source records attributed to Handa, T..

13 recordsLinked to original sources

Open-source modular FPGA system for two-photon mesoscope enabling multi-layer, multi-depth neural activity recording and lifetime imaging

Large field-of-view (FOV) two-photon microscopy makes it possible to record a large number of neural activities from multiple brain regions simultaneously. However, the larger the field of view, the longer it takes to scan the entire FOV. To increase imaging speed, we have developed open-source software to digitize analogue signals from a photomultiplier tube using a field-programmable gate array (FPGA) at a rate of 3.2 GS/s. By combining this with a newly developed a circular delay-path module for a custom two-photon mesoscope (Diesel2p), we succeeded in simultaneous recording of >10,000 neurons from the entire bilateral dorsal cortex at up to four depths. We also demonstrated large FOV lifetime imaging using the same system. Our modular, open-source FPGA system can be readily integrated into any type of two-photon microscope and will accelerate the biomedical application of multi-scale two-photon imaging in a wide range of pathophysiological investigations.

neuroscience↗

Decomposed frontal corticostriatal ensemble activity changes across trials, revealing distinct features relevant to outcome-based decision making

The frontal cortex-striatum circuit plays a pivotal role in adaptive goal-directed behaviours. However, the mediation of decision-related signals through cross-regional transmission between the medial frontal cortex and the striatum by neuronal ensembles remains unclear. We analysed neuronal ensemble activity obtained through simultaneous multiunit recordings in the secondary motor cortex (M2) and dorsal striatum (DS) while the rats performed an outcome-based choice task. Tensor component analysis (TCA), an unsupervised dimensionality reduction approach at the single-trial level, was adopted for concatenated ensembles of M2 and DS neurons. We identified distinct three spatiotemporal neural dynamics (TCA components) at the single-trial level specific to task-relevant variables. Choice-position selective neural dynamics was correlated with the trial-to-trial fluctuation of behavioural variables. This analytical approach unveiled choice-pattern selective neural dynamics distinguishing whether the incoming choice was a repetition or switch from the previous choice. Other neural dynamics was selective to outcome. Choice-pattern selective within-trial activity increased before response choice, whereas outcome selective within-trial activity increased following response. These results suggest that the concatenated ensembles of M2 and DS process distinct features of decision-related signals at various points in time. The M2 and DS may collaboratively monitor action outcomes and determine the subsequent choice, whether to repeat or switch, for coordinated action selection.

neuroscience↗

Cholinergic modulation of interhemispheric inhibition in the mouse motor cortex

Interhemispheric inhibition (IHI) of the homotopic motor cortex is believed to be effective for accurate unilateral motor function. However, the cellular mechanisms underlying IHI during unilateral motor behavior remain unclear. Furthermore, the impact of the neuromodulator acetylcholine (ACh) on IHI and the associated cellular mechanisms are not well understood. To address this knowledge gap, we conducted recordings of neuronal activity from the bilateral motor cortex of mice during the paw-reaching task. Subsequently, we analyzed interhemispheric spike correlation at the cell-pair level, classifying putative cell types to explore the underlying cellular circuitry mechanisms of IHI. We found a cell-type pair-specific enhancement of the interhemispheric spike correlation when the mice were engaged in the reaching task. We also found that the interhemispheric spike correlation was modulated by pharmacological ACh manipulation. The local field responses to contralateral excitation differed along the cortical depths, and muscarinic receptor antagonism enhanced the inhibitory component of the field response in deep layers. The muscarinic subtype M2 receptor is predominantly expressed in deep cortical neurons, including GABAergic interneurons. These results suggest that GABAergic interneurons expressing muscarinic receptors in deep layers mediate the neuromodulation of IHI in the homotopic motor cortex.

neuroscience↗

Drug repurposing approach for potential Pfmrk inhibitors as antimalarial agents: an In-silico analysis

Malaria is a major global health issue due to the emergence of resistance to most of the available antimalarial drugs. There is an urgent need to discover new antimalarials to tackle the resistance issue. A CDK-like protein, Pfmrk from Plasmodium falciparum, plays a crucial role in regulating cell proliferation and shares 36.28% homology with humans CDK (hCDK7). Pfmrk complex with Pfcyc-1 and stimulates kinase activity. Also, Pfcyc-1 from P. falciparum, which has the highest sequence homology with human cyclin (Cyclin H), binds to and activates Pfmrk in a cyclin-dependent way. This is the first indication that cyclin subunits regulate human and plasmodial CDKs in a similar manner. In this study, molecular docking analysis of Pfmrk against the selected FDA-approved drugs acquired from the ZINC15 database. The top five drugs, Lurasidone, Vorapaxar, Donovex, Alvesco, and Orap, were screened based on binding energies of best-docked scores ranging between -8 kcal/mol and -12 kcal/mol. Based on Molecular dynamics simulations for 100ns, Lurasidone showed the highest binding affinity (-105.90 {+/-} 57.72 kJ/mol), followed by Donovex (-92.877 {+/-} 17.872 kJ/mol) and exhibited stable interactions with the amino acid residues present in the active site of Pfmrk. The outcomes of in silico investigation putatively suggested that Lurasidone and Donovex exhibit antimalarial potency and could be translated as potential Pfmrk inhibitors and developing new drugs based on further in-vitro studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/547694v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1e14204org.highwire.dtl.DTLVardef@1b522e8org.highwire.dtl.DTLVardef@106df75org.highwire.dtl.DTLVardef@389899_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsWe investigated the potential FDA-approved drugs for repurposing against the modelled protein Pfmrk. Alvesco, Donovex, Lurasidone, Orap, and Vorapaxar are potential FDA-approved drugs firmly binding with Pfmrk. Molecular docking and simulation studies show that Donovex and Lurasidone are potential inhibitors of the modelled Pfmrk protein. Donovex and Lurasidone are potential drugs that act as kinase inhibitors by binding to the ATP -binding site of the enzyme Pfmrk.

biochemistry↗

Global monitoring of soil animal communities using a common methodology

Here we introduce the Soil BON Foodweb Team, a cross-continental collaborative network that aims to monitor soil animal communities and food webs using consistent methodology at a global scale. Soil animals support vital soil processes via soil structure modification, direct consumption of dead organic matter, and interactions with microbial and plant communities. Soil animal effects on ecosystem functions have been demonstrated by correlative analyses as well as in laboratory and field experiments, but these studies typically focus on selected animal groups or species at one or few sites with limited variation in environmental conditions. The lack of comprehensive harmonised large-scale soil animal community data including microfauna, mesofauna, and macrofauna, in conjunction with related soil functions, limits our understanding of biological interactions in soil communities and how these interactions affect ecosystem functioning. To provide such data, the Soil BON Foodweb Team invites researchers worldwide to use a common methodology to address six long-term goals: (1) to collect globally representative harmonised data on soil micro-, meso-, and macrofauna communities; (2) to describe key environmental drivers of soil animal communities and food webs; (3) to assess the efficiency of conservation approaches for the protection of soil animal communities; (4) to describe soil food webs and their association with soil functioning globally; (5) to establish a global research network for soil biodiversity monitoring and collaborative projects in related topics; (6) to reinforce local collaboration networks and expertise and support capacity building for soil animal research around the world. In this paper, we describe the vision of the global research network and the common sampling protocol to assess soil animal communities and advocate for the use of standard methodologies across observational and experimental soil animal studies. We will use this protocol to conduct soil animal assessments and reconstruct soil food webs on the sites included in the global soil biodiversity monitoring network, Soil BON, allowing us to assess linkages among soil biodiversity, vegetation, soil physico-chemical properties, and ecosystem functions. In the present paper, we call for researchers especially from countries and ecoregions that remain underrepresented in the majority of soil biodiversity assessments to join us. Together we will be able to provide science-based evidence to support soil biodiversity conservation and functioning of terrestrial ecosystems.

ecology↗

Synergistic information in the frontal cortex-striatal pathway

Across the cortico-basal ganglia circuit, the medial frontal cortex (MFC) communicates with the dorsal striatum (DS) during learning and planning. How these two brain regions communicate with each other is, however, not fully understood. Here we report the presence of synergistic information during information transfer across the frontal cortex-striatal pathway. Synergistic information emerges from the positive interaction of DS and MFC neurons and provides the DS with additional cortical information. This information is held latent in neuronal signals. To reveal it, we simultaneously record neuronal activities from the MFC and DS of rats trained on an outcome-based decision-making task and determined whether past neuronal activities of the DS positively influence communication rates. We detect a neuronal synergy that enables the MFC to boost its communication rate to the DS. Our results suggest that past neuronal activities of the DS help decode MFC signals. This ability is not attributed to the inherent autocorrelation of DS spiking activities.

neuroscience↗

Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody probe exhibited numerous foci, possibly representing transcription "factories" in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters. SummaryThe authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.

cell biology↗

Euchromatin factors HULC and Set1C affect heterochromatin organization for mating-type switching in fission yeast Schizosaccharomyces pombe

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1{Delta}. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant indicated that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistently, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C might control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

genetics↗

Modeling population size independent tissue epigenomes by ChIL-seq with single-thin sections

Recent advances in omics studies have enabled analysis at the single-cell level; however, methods for analyzing the whole cell of large organs and tissues remain challenging. Here, we developed a method named tsChIL to understand the diverse cellular dynamics at the tissue level using high-depth epigenomic data. tsChIL allowed the analysis of a single tissue section and could reproducibly acquire epigenomic profiles from several types of tissues, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation of cells in regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analysis by tsChIL can elucidate in vivo cell-type dynamics of tissues.

molecular biology↗

Concomitant processing of choice and outcome in frontal corticostriatal ensembles correlates with performance of rats

The frontal cortex-basal ganglia network plays a pivotal role in adaptive goal-directed behaviors. Medial frontal cortex (MFC) encodes information about choices and outcomes into sequential activation of neural population, or neural trajectory. While MFC projects to the dorsal striatum (DS), whether DS also displays temporally coordinated activity remains unknown. We studied this question by simultaneously recording neural ensembles in the MFC and DS of rodents performing an outcome-based alternative choice task. We found that the two regions exhibited highly parallel evolution of neural trajectories, transforming choice information into outcome-related information. When the two trajectories were highly correlated, spike synchrony was task-dependently modulated in some MFC-DS neuron pairs. Our results suggest that neural trajectories concomitantly process decision-relevant information in MFC and DS with increased spike synchrony between these regions.

neuroscience↗

Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

The carboxyl-terminal domain of RNA polymerase II is dynamically phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to unphosphorylated and phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as clusters of Serine 5 phosphorylated RNAP2 that form around active genes and are separated in both space and time from nascent mRNA synthesis. Computational models fit to our data determine that 5 to 40 RNAP2 cluster around the promoter of a gene during typical transcriptional bursts. Rapid imaging demonstrates nearly all RNAP2 in the cluster acquire Serine 5 phosphorylation within 3-6 seconds of arrival. Transcription from the cluster appears to be highly efficient, with nearly half of the clustered RNAP2 ultimately escaping the promoter in 1.5 minutes on average to elongate a full-length mRNA in approximately five minutes. The highly dynamic and spatially organized concentrations of RNAP2 we observe support the notion of highly efficient transcription clusters that form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

molecular biology↗

Accurate action potential inference from a calcium sensor protein through biophysical modeling

Multiphoton imaging of genetically encoded calcium indicators is routinely used to report activity from populations of spatially resolved neurons in vivo. However, since the relationship between fluorescence and action potentials (APs) is nonlinear and varies over neurons, quantitatively inferring AP discharge is problematic. To address this we developed a biophysical model of calcium binding kinetics for the indicator GCaMP6s that accurately describes AP-evoked fluorescence changes in vivo. The models physical interpretation allowed the same parameters to describe GCaMP6s binding kinetics for both in vitro binding assays and in vivo imaging. Using this model, we developed an algorithm to infer APs from fluorescence and measured its accuracy with cell-attached electrical recordings. This approach consistently inferred more accurate AP counts and times than alternative methods for firing rates from 0 to >20 Hz, while requiring less training data. These results demonstrate the utility of quantitative, biophysically grounded models for complex biological data.

neuroscience↗

Individual Differences In Perceptual Decision Making Reflect Neural Variability In Medial Frontal Cortex

Decision making obeys common neural mechanisms, but there is considerable variability in individuals decision making behavior particularly under uncertainty. How individual differences arise within common decision making brain systems is not known. Here, we explored this question in the medial frontal cortex (MFC) of rats performing a sensory-guided choice task. When rats trained on familiar stimuli were exposed to unfamiliar stimuli, choice responses varied significantly across individuals. We examined how variability in MFC neural processing could mediate this individual difference and constructed a network model to replicate this. Our model suggested that susceptibility of neural trajectories is a crucial determinant of the observed choice variability. The model predicted that trial-by-trial variability of trajectories are correlated with the susceptibility, and hence also correlated with the individual difference. This prediction was confirmed by experiment. Thus, our results suggest that variability in neural dynamics in MFC networks underlies individual differences in decision making.

neuroscience↗