Publications by authors named "Sachiko Tsuda"

20 Publications

  • Page 1 of 1

Two-Photon Laser Ablation and In Vivo Wide-Field Imaging of Inferior Olive Neurons Revealed the Recovery of Olivocerebellar Circuits in Zebrafish.

Int J Environ Res Public Health 2021 08 6;18(16). Epub 2021 Aug 6.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.

The cerebellum, a brain region with a high degree of plasticity, is pivotal in motor control, learning, and cognition. The cerebellar reserve is the capacity of the cerebellum to respond and adapt to various disorders via resilience and reversibility. Although structural and functional recovery has been reported in mammals and has attracted attention regarding treatments for cerebellar dysfunction, such as spinocerebellar degeneration, the regulatory mechanisms of the cerebellar reserve are largely unidentified, particularly at the circuit level. Herein, we established an optical approach using zebrafish, an ideal vertebrate model in optical techniques, neuroscience, and developmental biology. By combining two-photon laser ablation of the inferior olive (IO) and long-term non-invasive imaging of "the whole brain" at a single-cell resolution, we succeeded in visualization of the morphological changes occurring in the IO neuron population and showed at a single-cell level that structural remodeling of the olivocerebellar circuit occurred in a relatively short period. This system, in combination with various functional analyses, represents a novel and powerful approach for uncovering the mechanisms of the cerebellar reserve, and highlights the potential of the zebrafish model to elucidate the organizing principles of neuronal circuits and their homeostasis in health and disease.
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http://dx.doi.org/10.3390/ijerph18168357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8391264PMC
August 2021

In vivo wide-field voltage imaging in zebrafish with voltage-sensitive dye and genetically encoded voltage indicator.

Dev Growth Differ 2021 Aug 19. Epub 2021 Aug 19.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan.

The brain consists of neural circuits, which are assemblies of various neuron types. For understanding how the brain works, it is essential to identify the functions of each type of neuron and neuronal circuits. Recent advances in our understanding of brain function and its development have been achieved using light to detect neuronal activity. Optical measurement of membrane potentials through voltage imaging is a desirable approach, enabling fast, direct, and simultaneous detection of membrane potentials in a population of neurons. Its high speed and directness can help detect synaptic and action potentials and hyperpolarization, which encode critical information for brain function. Here, we describe in vivo voltage imaging procedures that we have recently established using zebrafish, a powerful animal model in developmental biology and neuroscience. By applying two types of voltage sensors, voltage-sensitive dyes (VSDs, Di-4-ANEPPS) and genetically encoded voltage indicators (GEVIs, ASAP1), spatiotemporal dynamics of voltage signals can be detected in the whole cerebellum and spinal cord in awake fish at single-cell and neuronal population levels. Combining this method with other approaches, such as optogenetics, behavioral analysis, and electrophysiology would facilitate a deeper understanding of the network dynamics of the brain circuitry and its development.
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http://dx.doi.org/10.1111/dgd.12744DOI Listing
August 2021

Involvement of Oct4-type transcription factor Pou5f3 in posterior spinal cord formation in zebrafish embryos.

Dev Growth Differ 2021 Aug 18;63(6):306-322. Epub 2021 Aug 18.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama City, Japan.

In vertebrate embryogenesis, elongation of the posterior body is driven by de novo production of the axial and paraxial mesoderm as well as the neural tube at the posterior end. This process is presumed to depend on the stem cell-like population in the tail bud region, but the details of the gene regulatory network involved are unknown. Previous studies suggested the involvement of pou5f3, an Oct4-type POU gene in zebrafish, in axial elongation. In the present study, we first found that pou5f3 is expressed mainly in the dorsal region of the tail bud immediately after gastrulation, and that this expression is restricted to the posterior-most region of the elongating neural tube during somitogenesis. This pou5f3 expression was complementary to the broad expression of sox3 in the neural tube, and formed a sharp boundary with specific expression of tbxta (orthologue of mammalian T/Brachyury) in the tail bud, implicating pou5f3 in the specification of tail bud-derived cells toward neural differentiation in the spinal cord. When pou5f3 was functionally impaired after gastrulation by induction of a dominant-interfering pou5f3 mutant gene (en-pou5f3), trunk and tail elongation were markedly disturbed at distinct positions along the axis depending on the stage. This finding showed involvement of pou5f3 in de novo generation of the body from the tail bud. Conditional functional abrogation also showed that pou5f3 downregulates mesoderm-forming genes but promotes neural development by activating neurogenesis genes around the tail bud. These results suggest that pou5f3 is involved in formation of the posterior spinal cord.
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http://dx.doi.org/10.1111/dgd.12742DOI Listing
August 2021

A globin-family protein, Cytoglobin 1, is involved in the development of neural crest-derived tissues and organs in zebrafish.

Dev Biol 2021 04 11;472:1-17. Epub 2021 Jan 11.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan. Electronic address:

The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1, established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs.
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http://dx.doi.org/10.1016/j.ydbio.2020.12.016DOI Listing
April 2021

Involvement of an Oct4-related PouV gene, pou5f3/pou2, in neurogenesis in the early neural plate of zebrafish embryos.

Dev Biol 2020 01 11;457(1):30-42. Epub 2019 Sep 11.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan. Electronic address:

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos.
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http://dx.doi.org/10.1016/j.ydbio.2019.09.002DOI Listing
January 2020

Horizontal Boundary Cells, a Special Group of Somitic Cells, Play Crucial Roles in the Formation of Dorsoventral Compartments in Teleost Somite.

Cell Rep 2019 04;27(3):928-939.e4

Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

Establishment of robust gene expression boundary is crucial for creating elaborate morphology during development. However, mechanisms underlying boundary formation have been extensively studied only in a few model systems. We examined the establishment of zic1/zic4-expression boundary demarcating dorsoventral boundary of the entire trunk of medaka fish (Oryzias latipes) and identified a subgroup of dermomyotomal cells called horizontal boundary cells (HBCs) as crucial players for the boundary formation. Embryological and genetic analyses demonstrated that HBCs play crucial roles in the two major events of the process, i.e., refinement and maintenance. In the refinement, HBCs could serve as a chemical barrier against Wnts from the neural tube by expressing Hhip. At later stages, HBCs participate in the maintenance of the boundary by differentiating into the horizontal myoseptum physically inhibiting cell mixing across the boundary. These findings reveal the mechanisms underlying the dorsoventral boundary in the teleost trunk by specialized boundary cells.
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http://dx.doi.org/10.1016/j.celrep.2019.03.068DOI Listing
April 2019

4D imaging identifies dynamic migration and the fate of gbx2-expressing cells in the brain primordium of zebrafish.

Neurosci Lett 2019 01 14;690:112-119. Epub 2018 Sep 14.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan; Saitama University Brain Science Institute, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan.

One of the pivotal events in neural development is compartmentalization, wherein the neural tissue divides into domains and undergoes functional differentiation. For example, midbrain-hindbrain boundary (MHB) formation and subsequent isthmus development are key steps in cerebellar development. Although several regulatory mechanisms are known to underlie this event, little is known about cellular behaviors. In this study, to examine the cellular dynamics around the MHB region, we performed confocal time-lapse imaging in zebrafish embryos to track cell populations in the neural tube via 4D analysis. We used a transgenic line wherein enhanced green fluorescent protein (EGFP) expression is driven by the gastrulation brain homeobox 2 (gbx2) enhancer, which is involved in MHB maintenance. 4D time-lapse imaging of 5-20 h revealed a novel pattern in cell migration: a dynamic ventrocaudally directed migration from the MHB region toward the hindbrain. Furthermore, in the hindbrain region, these EGFP-positive cells altered their shapes and extended the axons. Immunohistochemical analysis and retrograde labeling showed that these cells in the hindbrain were in the process of neuronal differentiation, including reticulospinal neurons. These results revealed the dynamic and two-step behavior and possible fate of the cell population, which are linked to brain compartmentalization, leading to a deeper understanding of brain development and formation of neuronal circuits.
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http://dx.doi.org/10.1016/j.neulet.2018.09.027DOI Listing
January 2019

Optical measurement of neuronal activity in the developing cerebellum of zebrafish using voltage-sensitive dye imaging.

Neuroreport 2018 11;29(16):1349-1354

Graduate School of Science and Engineering, Division of Life Science.

Voltage-sensitive dye (VSD) imaging enables fast, direct, and simultaneous detection of membrane potentials from a population of neurons forming neuronal circuits. This enables the detection of hyperpolarization together with depolarization, whose balance plays a pivotal role in the function of many brain regions. Among these is the cerebellum, which contains a significant number of inhibitory neurons. However, the mechanism underlying the functional development remains unclear. In this study, we used a model system ideal to study neurogenesis by applying VSD imaging to the cerebellum of zebrafish larvae to analyze the neuronal activity of the developing cerebellum, focusing on both excitation and inhibition. We performed in-vivo high-speed imaging of the entire cerebellum of the zebrafish, which was stained using Di-4-ANEPPS, a widely used VSD. To examine whether neuronal activity in the zebrafish cerebellum could be detected by this VSD, we applied electrical stimulation during VSD imaging, which showed that depolarization was detected widely in the cerebellum upon stimulation. These responses mostly disappeared following treatment with tetrodotoxin, indicating that Di-4-ANEPPS enabled optical measurement of neuronal activity in the developing cerebellum of zebrafish. Moreover, hyperpolarizing signals were also detected upon stimulation, but these were significantly reduced by treatment with picrotoxin, a GABAA receptor inhibitor, indicating that these responses represent inhibitory signals. This approach will enable a detailed analysis of the spatiotemporal dynamics of the excitation and inhibition in the cerebellum along its developmental stages, leading to a deeper understanding of the functional development of the cerebellum in vertebrates.
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http://dx.doi.org/10.1097/WNR.0000000000001113DOI Listing
November 2018

β-Oxidation in ghrelin-producing cells is important for ghrelin acyl-modification.

Sci Rep 2018 06 15;8(1):9176. Epub 2018 Jun 15.

Area of Regulatory Biology, Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakuraku, Saitama, 338-8570, Japan.

Ghrelin is a unique fatty acid-modified peptide hormone produced in the stomach and has important roles in energy homeostasis and gastrointestinal motility. However, the medium-chain fatty acid source for ghrelin acyl-modification is not known. We found that a fat-free diet and the removal of intestinal microbiota did not decrease acyl-ghrelin production in the stomach or plasma acyl-ghrelin levels in mice. RT-PCR analysis showed that genes involving fatty acid synthesis, metabolism, and transport were expressed in pancreas-derived ghrelinoma (PG-1) cells. Treatment with an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) strongly decreased acylated ghrelin levels but did not affect ghrelin or ghrelin o-acyl transferase (GOAT) mRNA levels in PG-1 cells. Our results suggest that the medium-chain fatty acid used for the acyl-modification of ghrelin is produced in ghrelin-producing cells themselves by β-oxidation of long-chain fatty acids provided from the circulation.
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http://dx.doi.org/10.1038/s41598-018-27458-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003948PMC
June 2018

Optical interrogation of neuronal circuitry in zebrafish using genetically encoded voltage indicators.

Sci Rep 2018 04 16;8(1):6048. Epub 2018 Apr 16.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama, 338-8570, Japan.

Optical measurement of membrane potentials enables fast, direct and simultaneous detection of membrane potentials from a population of neurons, providing a desirable approach for functional analysis of neuronal circuits. Here, we applied recently developed genetically encoded voltage indicators, ASAP1 (Accelerated Sensor of Action Potentials 1) and QuasAr2 (Quality superior to Arch 2), to zebrafish, an ideal model system for studying neurogenesis. To achieve this, we established transgenic lines which express the voltage sensors, and showed that ASAP1 is expressed in zebrafish neurons. To examine whether neuronal activity could be detected by ASAP1, we performed whole-cerebellum imaging, showing that depolarization was detected widely in the cerebellum and optic tectum upon electrical stimulation. Spontaneous activity in the spinal cord was also detected by ASAP1 imaging at single-cell resolution as well as at the neuronal population level. These responses mostly disappeared following treatment with tetrodotoxin, indicating that ASAP1 enabled optical measurement of neuronal activity in the zebrafish brain. Combining this method with other approaches, such as optogenetics and behavioural analysis may facilitate a deeper understanding of the functional organization of brain circuitry and its development.
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http://dx.doi.org/10.1038/s41598-018-23906-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902623PMC
April 2018

The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos.

Differentiation 2018 Jan - Feb;99:28-40. Epub 2017 Dec 21.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan; Saitama University Brain Science Institute, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan. Electronic address:

During vertebrate brain development, the gastrulation brain homeobox 2 gene (gbx2) is expressed in the forebrain, but its precise roles are still unknown. In this study, we addressed this issue in zebrafish (Danio rerio) first by carefully examining gbx2 expression in the developing forebrain. We showed that gbx2 was expressed in the telencephalon during late somitogenesis, from 18h post-fertilization (hpf) to 24 hpf, and in the thalamic primordium after 26 hpf. In contrast, another gbx gene, gbx1, was expressed in the anterior-most ventral telencephalon after 36 hpf. Thus, the expression patterns of these two gbx genes did not overlap, arguing against their redundant function in the forebrain. Two-color fluorescence in situ hybridization (FISH) showed close relationships between the telencephalic expression of gbx2 and other forebrain-forming genes, suggesting that their interactions contribute to the regionalization of the telencephalon. FISH further revealed that gbx2 is expressed in the ventricular region of the telencephalon. By using transgenic fish in which gbx2 can be induced by heat shock, we found that gbx2 induction at 16 hpf repressed the expression of emx3, dlx2a, and six3b in the ventral telencephalon. Among secreted factor genes, bmp2b and wnt1 were repressed in the vicinity of the gbx2 domain in the telencephalon. The expression of forebrain-forming genes was examined in mutant embryos lacking gbx2, showing emx3 and dlx2a to be upregulated in the subpallium at 24 hpf. Taken together, these findings indicate that gbx2 contributes to the development of the subpallium through its repressive activities against other telencephalon-forming genes. We further showed that inhibiting FGF signaling and activating Wnt signaling repressed gbx2 and affected the regionalization of the telencephalon, supporting a functional link between gbx2, intracellular signaling, and telencephalon development.
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http://dx.doi.org/10.1016/j.diff.2017.12.005DOI Listing
November 2018

Comprehensive analysis of target genes in zebrafish embryos reveals gbx2 involvement in neurogenesis.

Dev Biol 2017 10 26;430(1):237-248. Epub 2017 Jul 26.

Division of Life Science, Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan; Saitama University Brain Science Institute, Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan. Electronic address:

It is well established that the gbx2 homeobox gene contributes to the positioning of the midbrain-hindbrain boundary (MHB) governing the development of adjacent brain regions in vertebrate embryos, but the specific aspects of the gene regulatory network regulated by gbx2 during brain development remain unclear. In the present study, we sought to comprehensively identify gbx2 target genes in zebrafish embryos by microarray analysis around the end of gastrulation, when the MHB is established, using transgenic embryos harboring heat-inducible gbx2. This analysis revealed that a large number of genes were either upregulated or downregulated following gbx2 induction, and the time course of induction differed depending on the genes. The differences in response to gbx2 were found by functional annotation analysis to be related to the functions and structures of the target genes. Among the significantly downregulated genes was her5, whose expression in the midbrain was precisely complementary to gbx2 expression around the MHB, suggesting that gbx2 expression in the anterior hindbrain restricts her5 expression to the midbrain. Because her5 represses neurogenesis, gbx2 may positively regulate neural development in its expression domain. Indeed, we showed further that gbx2 induction upregulated neural marker expression in the midbrain. Quantitative PCR analysis revealed that gbx2 upregulated the expression of the zebrafish proneural gene ebf2, whereas it repressed notch1a, which generally represses neurogenesis. Taken together, these results demonstrate that gbx2 not only functions to position the MHB but also regulates neurogenesis in the anterior hindbrain.
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http://dx.doi.org/10.1016/j.ydbio.2017.07.015DOI Listing
October 2017

Underlying mechanism of the cyclic migrating motor complex in Suncus murinus: a change in gastrointestinal pH is the key regulator.

Physiol Rep 2017 Jan;5(1)

Department of Life Nano-Bio, Strategic Research Division, Graduate School of Science and Engineering, Saitama University, Saitama, Japan

In the fasted gastrointestinal (GI) tract, a characteristic cyclical rhythmic migrating motor complex (MMC) occurs in an ultradian rhythm, at 90-120 min time intervals, in many species. However, the underlying mechanism directing this ultradian rhythmic MMC pattern is yet to be completely elucidated. Therefore, this study aimed to identify the possible causes or factors that involve in the occurrence of the fasting gastric contractions by using Suncus murinus a small model animal featuring almost the same rhythmic MMC as that found in humans and dogs. We observed that either intraduodenal infusion of saline at pH 8 evoked the strong gastric contraction or continuously lowering duodenal pH to 3-evoked gastric phase II-like and phase III-like contractions, and both strong contractions were essentially abolished by the intravenous administration of MA 2029 (motilin receptor antagonist) and D-Lys3-GHRP6 (ghrelin receptor antagonist) in a vagus-independent manner. Moreover, we observed that the prostaglandin E2-alpha (PGE2α) and serotonin type 4 (5HT4) receptors play important roles as intermediate molecules in changes in GI pH and motilin release. These results suggest a clear insight mechanism that change in the duodenal pH to alkaline condition is an essential factor for stimulating the endogenous release of motilin and governs the fasting MMC in a vagus-independent manner. Finally, we believe that the changes in duodenal pH triggered by flowing gastric acid and the release of duodenal bicarbonate through the involvement of PGE2α and 5HT4 receptor are the key events in the occurrence of the MMC.
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http://dx.doi.org/10.14814/phy2.13105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256163PMC
January 2017

Optogenetic mapping of cerebellar inhibitory circuitry reveals spatially biased coordination of interneurons via electrical synapses.

Cell Rep 2014 Jun 22;7(5):1601-1613. Epub 2014 May 22.

Lee Kong Chian School of Medicine, Nanyang Technological University, 50 Nanyang Drive, Research Techno Plaza, Singapore 637553, Singapore; Laboratory of Synaptic Circuitry, Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore; A(∗)STAR/Duke-NUS Neuroscience Research Partnership, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore; Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore; Marine Biological Laboratory, Woods Hole, MA 02543, USA; Center for Functional Connectomics, Korea Institute of Science and Technology, 39-1 Hawolgokdong, Seongbukgu, Seoul 136-791, Republic of Korea; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599, Singapore. Electronic address:

We used high-speed optogenetic mapping technology to examine the spatial organization of local inhibitory circuits formed by cerebellar interneurons. Transgenic mice expressing channelrhodopsin-2 exclusively in molecular layer interneurons allowed us to focally photostimulate these neurons, while measuring resulting responses in postsynaptic Purkinje cells. This approach revealed that interneurons converge upon Purkinje cells over a broad area and that at least seven interneurons form functional synapses with a single Purkinje cell. The number of converging interneurons was reduced by treatment with gap junction blockers, revealing that electrical synapses between interneurons contribute substantially to the spatial convergence. Remarkably, gap junction blockers affected convergence in sagittal slices, but not in coronal slices, indicating a sagittal bias in electrical coupling between interneurons. We conclude that electrical synapse networks spatially coordinate interneurons in the cerebellum and may also serve this function in other brain regions.
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http://dx.doi.org/10.1016/j.celrep.2014.04.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107211PMC
June 2014

Next-generation transgenic mice for optogenetic analysis of neural circuits.

Front Neural Circuits 2013 26;7:160. Epub 2013 Nov 26.

Department of Neurobiology, Duke University Medical Center Durham, NC, USA.

Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice.
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http://dx.doi.org/10.3389/fncir.2013.00160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840435PMC
April 2014

Probing the function of neuronal populations: combining micromirror-based optogenetic photostimulation with voltage-sensitive dye imaging.

Neurosci Res 2013 Jan 17;75(1):76-81. Epub 2012 Dec 17.

Laboratory of Synaptic Circuitry, Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore.

Recent advances in our understanding of brain function have come from using light to either control or image neuronal activity. Here we describe an approach that combines both techniques: a micromirror array is used to photostimulate populations of presynaptic neurons expressing channelrhodopsin-2, while a red-shifted voltage-sensitive dye allows optical detection of resulting postsynaptic activity. Such technology allowed us to control the activity of cerebellar interneurons while simultaneously recording inhibitory responses in multiple Purkinje neurons, their postsynaptic targets. This approach should substantially accelerate our understanding of information processing by populations of neurons within brain circuits.
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http://dx.doi.org/10.1016/j.neures.2012.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594342PMC
January 2013

Optogenetic probing of functional brain circuitry.

Exp Physiol 2011 Jan 5;96(1):26-33. Epub 2010 Nov 5.

Laboratory of Synaptic Circuitry, Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, 2 Jalan Bukit Merah, Singapore 169547, Singapore.

Recently developed optogenetic technologies offer the promise of high-speed mapping of brain circuitry. Genetically targeted light-gated channels and pumps, such as channelrhodopsins and halorhodopsin, allow optical control of neuronal activity with high spatial and temporal resolution. Optogenetic probes of neuronal activity, such as Clomeleon and Mermaid, allow light to be used to monitor the activity of a genetically defined population of neurons. Combining these two complementary sets of optogenetic probes will make it possible to perform all-optical circuit mapping. Owing to the improved efficiency and higher speed of data acquisition, this hybrid approach should enable high-throughput mapping of brain circuitry.
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http://dx.doi.org/10.1113/expphysiol.2010.055731DOI Listing
January 2011

FAK-mediated extracellular signals are essential for interkinetic nuclear migration and planar divisions in the neuroepithelium.

J Cell Sci 2010 Feb 12;123(Pt 3):484-96. Epub 2010 Jan 12.

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

During the development of the vertebrate nervous system, mitosis of neural progenitor cells takes place near the lumen, the apical side of the neural tube, through a characteristic movement of nuclei known as interkinetic nuclear migration (INM). Furthermore, during the proliferative period, neural progenitor cells exhibit planar cell divisions to produce equivalent daughter cells. Here, we examine the potential role of extracellular signals in INM and planar divisions using the medaka mutant tacobo (tab). This tab mutant shows pleiotropic phenotypes, including neurogenesis, and positional cloning identified tab as laminin gamma1 (lamc1), providing a unique framework to study the role of extracellular signals in neurogenesis. In tab mutant neural tubes, a number of nuclei exhibit abnormal patterns of migration leading to basally mislocalized mitosis. Furthermore, the orientation of cell division near the apical surface is randomized. Probably because of these defects, neurogenesis is accelerated in the tab neural tube. Detailed analyses demonstrate that extracellular signals mediated by the FAK pathway regulate INM and planar divisions in the neuroepithelium, possibly through interaction with the intracellular dynein-motor system.
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http://dx.doi.org/10.1242/jcs.057851DOI Listing
February 2010

Phenotypic analysis of a novel chordin mutant in medaka.

Dev Dyn 2007 Aug;236(8):2298-310

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan.

We have isolated and characterized a ventralized mutant in medaka (the Japanese killifish; Oryzias latipes), which turned out to have a mutation in the chordin gene. The mutant exhibits ventralization of the body axis, malformation of axial bones, over-bifurcation of yolk sac blood vessels, and laterality defects in internal organs. The mutant exhibits variability of phenotypes, depending on the culture temperature, from embryos with a slightly ventralized phenotype to those without any head and trunk structures. Taking advantages of these variable and severe phenotypes, we analyzed the role of Chordin-dependent tissues such as the notochord and Kupffer's vesicle (KV) in the establishment of left-right axis in fish. The results demonstrate that, in the absence of the notochord and KV, the medaka lateral plate mesoderm autonomously and bilaterally expresses spaw gene in a default state.
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http://dx.doi.org/10.1002/dvdy.21245DOI Listing
August 2007

Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle.

Dev Growth Differ 2007 Jun;49(5):395-405

Department of Biological Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.
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http://dx.doi.org/10.1111/j.1440-169X.2007.00937.xDOI Listing
June 2007
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