Akira Muto

Toho University | Toho-U · Faculty of Medicine

Brain activity and connectivity alter drastically during epileptic seizures. The brain networks shift from a balanced resting state to a hyperactive and hypersynchronous state. It is, however, less clear which mechanisms underlie the state transitions. By studying neural and glial activity in zebrafish models of epileptic seizures, we observe strik...

Brain activity and connectivity alters drastically during epileptic seizures. Throughout this transition, brain networks shift from a balanced resting state to a hyperactive and hypersynchronous state, spreading across the brain. It is however less clear which mechanisms underlie these state transitions. By studying neuronal and glia activity acros...

Background: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amyg...

The visual system plays a major role in food/prey recognition in diurnal animals, and food intake is regulated by the hypothalamus. However, whether and how visual information about prey is conveyed to the hypothalamic feeding centre is largely unknown. Here we perform real-time imaging of neuronal activity in freely behaving or constrained zebrafi...

Pretectal response to prey in a 4 dpf zebrafish larva (Supplement to Figure 2b)

Correlated calcium signals in the pretectal areas and the inferior lobes of the hypothalamus in response to prey in an agarose-embedded larva (Supplement to Figure 3b)

Hypothalamic response to prey in an agarose-embedded larva (Supplement to Figure 1d)

Supplementary Figures

Pretectal activity in the presence of a paramecium in a 6 dpf zebrafish larva embedded in agarose (Supplement to Figure 2d)

Pretectal activity in the presence of a paramecium in a 6 dpf larva embedded in agarose (no eye convergence) (Supplement to Figure 2d)

Activity of the inferior lobes of the hypothalamus (ILH) during prey capture behaviour (Supplement to Figure 1b-c)

Pretectal response to a moving spot (Supplement to Figure 2e and f)

Prey capture is abolished in pretectum-ablated zebrafish (Supplement to Figure 2m)

gSAIzGFFM119B-labelled pretectal cells project to the inferior lobe of the hypothalamus (Supplement to Figure 3a)

The visual system plays a major role in food/prey recognition in diurnal animals, and food intake is regulated by the hypothalamus. However, whether and how visual information about prey is conveyed to the hypothalamic feeding centre is largely unknown. Here we perform real-time imaging of neuronal activity in freely behaving or constrained zebrafi...

Visualization of neuronal activity during animal behavior is a critical step in understanding how the brain generates behavior. In the model vertebrate zebrafish, imaging of the brain has been done mostly by using immobilized fish. Here, we describe a novel method to image neuronal activity of the larval zebrafish brain during prey capture behavior...

Targeted expression by the Gal4-UAS system is a powerful genetic method to analyze the functions of genes and cells in vivo. Although the Gal4-UAS system has been extensively used in genetic studies in _Drosophila_, it had not been applied to genetic studies in vertebrates until the mid-2000s. This was mainly due to the lack of an efficient transge...

The Tol2 element is an active transposon that was found from the genome of the Japanese medaka fish. Since the Tol2 transposition system is active in all vertebrate cells tested so far, it has been applied to germ line transgenesis in various model animals including fish, frog, chicken, and mouse, and to gene transfer in culture cells. In zebrafish...

Significance Voltage-gated sodium channels (Na V ) are known to form clusters at the membranes of excitable cells; however, what governs their transport is largely unknown. We found that the endoplasmic reticulum (ER) and cis -Golgi associated ubiquitin ligase really interesting new gene (RING) finger protein 121 (RNF121) mediates the degradation a...

Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which e...

Glucocorticoids modulate diverse aspects of physiology and behavior, including energy homeostasis, stress response, and memory, through activation of the glucocorticoid receptor (GR). Light perception has profound effects on the production of glucocorticoids via functional connections of the retina to the hypothalamus-pituitary-adrenal axis. We rep...

Prey capture in zebrafish larvae is an innate behavior which can be observed as early as 4~days postfertilization, the day when they start to swim. This simple behavior apparently involves several neural processes including visual perception, recognition, decision-making, and motor control, and, therefore, serves as a good model system to study cog...

To understand how the brain perceives the external world, it is desirable to observe neuronal activity in the brain in real time during perception. The zebrafish is a suitable model animal for fluorescence imaging studies to visualize neuronal activity because its body is transparent through the embryonic and larval stages. Imaging studies have bee...

Channelrhodopsin (ChR)-wide receiver (ChRWR), one of the chimeric molecule of ChR1 and ChR2, has several advantages over ChR2 such as improved expression in the plasma membrane and enhanced photocurrent with small desensitization. Here we generated transgenic zebrafish (Danio rerio) expressing ChRWR as a conjugate of EGFP under the regulation of UA...

Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how...

Supplementary Figure S4 Swim velocity and distance traveled in the novel tank. Velocity was measured for each episode of swimming faster than 1.6 cm/s (the freezing threshold) and averaged. Distances were measured by calculating the length of the trajectories over the 10 min observation period. a, Mutants appear to swim on average more sluggishly t...

Supplementary Figure S6 Effect of bupropion antidepressant and cortisol antagonist, RU486, on fish behavior in the novel tank test. a, Freezing indices of experienced fish before (‘pre treatment’, day 0) and after 3 or 4 days of treatment with bupropion (3μM) or vehicle (‘no drug’) (n=10-36 fish per group). Behavior is apparently unchanged by bupro...

Supplementary Figure S8 serta expression in the pretectum. serta mRNA expression in two consecutive sagital sections (100 μm) showing serta expression in the pretectal diencephalic cluster, the parvocellular preoptic nucleus (PP), of experienced fish. Expression strengths appear similar between the two genotypes in these areas of the brain.

Supplementary Figure S3 Development of fish behavior in the novel tank over three weeks. Freezing and wall avoidance indices were measured in 15 s bins over the 10 min observation period. This measurement was repeated for the same cohort of fish in weekly intervals. a, Freezing index of WT. b, Freezing index of mutants. c, Wall avoidance index of W...

Supplementary Figure S2 Hypothalamic crh expression is increased in response to stress and chronically elevated in grs357 mutants. crh mRNA-expressing cells in two consecutive sagital sections (100μm) showing the nucleus lateralis tuberis (NLT) (blue arrowhead) and posterior tuberal nucleus (PTN) (white arrowhead). crh is undetectable in the NLT of...

Supplementary Figure S1 Transcriptional activation by GR (R443C) is largely abolished in COS-7 cells. Cells were transfected with GR (WT) or GR (R443C) and GRE-luciferase DNAs.

In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important f...

Measurement of the activity of neuronal ensembles is an essential step to understand how the neuronal network is organized and functioning. Electrical excitation of neurons causes calcium influx via voltage-gated calcium ion channels, which can be monitored by calcium imaging using fluorescent calcium probes. DNA-encoded calcium indicators (DECIs)...

Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the geneti...

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We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the ze...

The ability to process temporal information is fundamental to sensory perception, cognitive processing and motor behaviour of all living organisms, from amoebae to humans. Neural circuit mechanisms based on neuronal and synaptic properties have been shown to process temporal information over the range of tens of microseconds to hundreds of millisec...

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that...

Pineal Photoreceptors Are Present in Retinal Photoreceptor Degeneration Mutants Coronal sections of the forebrain at 7 dpf were stained with DAPI (A, C, E, and G) and zpr1, a marker of both retinal and pineal photoreceptors (B, D, F, and H). Pineal photoreceptors (arrow and inset) were consistently present in mutants in which retinal photoreceptors...

Dorsal RGCs Are Present and Properly Differentiated in darl Mutants Sagittal sections of WT (A and C) and darls327 retina (B and D) were stained with DAPI (A and B) and zn5 (C and D), a marker for differentiated RGCs. RGCs are present in the dorsal part of the retina and sending out axons into the optic nerve head in the mutant. The mutant eyes are...

The Tectum of exa Mutants Has an Abnormal Shape RGC axon tracing, following whole-eye DiI fills at 7 dpf, reveals a subtle extension of the tectal neuropil (delineated by DAPI counterstaining) at the ventral-posterior margin (arrow). Scale bar is 50 μm (1.7 MB PDF)

Retinal Axon Outgrowth Is Delayed in shir Mutants Lateral views of the retinal ganglion cell axons labeled with DiO. Anterior to the left, dorsal to the top. (A and B) At 7 dpf, the retinofugal projection in shirs362 (B) appears similar to WT (A), although the anterior portion may be less dense (arrow). (C and D) At 5 dpf, RGC axon outgrowth in shi...

Optokinetic Response The WT larva is on the left, and a zats125 mutant is on the right. For the first 60 s no stimulus is shown, and both fish show spontaneous eye movements. After 60 s, a clockwise-rotating striped pattern is projected on the drum around the fish. The WT fish responds by tracking the pattern slowly to the right and making fast res...

Optomotor Response The movie shows a close-up of part of a racetrack tank during OMR testing. A visible light filter has been used to remove the stimulus, and the fish are visualized using infrared light (Sony TRV-9 video camera, night vision mode). The stimulus is represented below. Initially, a converging grating brings the fish into the field of...

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that...

The visual system adjusts its sensitivity to a wide range of light intensities. We report here that mutation of the zebrafish sdy gene, which encodes tyrosinase, slows down the onset of adaptation to bright light. When fish larvae were challenged with periods of darkness during the day, the sdy mutants required nearly an hour to recover optokinetic...

This chapter describes some of the behavioral screening assays devised in laboratory to isolate mutations affecting the zebrafish nervous system. Zebrafish are ideally suited for a behavioral genetic approach. The larvae show a wide range of interesting behaviors, yet are small and can be produced in large numbers. Importantly, larvae do not need t...

Green fluorescent protein is a sensitive and reliable fluorescent marker for examining levels and subcellular sites of expression of functional proteins in living cells. This chapter reports efficient expression of green fluorescent protein in Xenopus laevis oocytes by direct microinjection of its plasmid DNA into the nucleus. Application of this m...

The inositol 1,4,5-trisphosphate (IP3) receptor is a calcium ion channel involved in the release of free Ca2+ from intracellular stores. For analysis of the role of IP3-induced Ca2+ release (IICR) on patterning of the embryonic body, monoclonal antibodies that inhibit IICR were produced. Injection of these blocking antibodies into the ventral part...

Injection of inositol 1,4,5-trisphosphate (InsP3) into fertilized Xenopus eggs induced transient changes in cell shape. The region around the injected site contracted during the first 2 min, followed by swelling. These changes which initiated at the injected site extended toward the opposite side. Injection of adenophostin B, a potent InsP3 recepto...

To study the role of inositol 1,4,5-trisphosphate (IP3) receptors during early embryogenesis in Xenopus, we examined the temporal-spatial localization of Xenopus IP3 receptor (XIP3R). XIP3R protein is enriched in the animal hemisphere of early cleavage stage embryos and becomes localized in the ectoderm and involuted mesoderm in gastrula stage embr...

In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the puta...

To study the development of the calcium release mechanism, we examined the temporal and spatial expression of inositol 1,4,5-trisphosphate receptor (IP3R) during the oogenesis and meiotic maturation of Xenopus laevis. Observation of a series of fixed samples by immunofluoresence microscopy revealed a relocalization of Xenopus IP3R (XIP3R)-positive...

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium tra...

Calcium signaling is known to be associated with cytokinesis; however, the detailed spatio-temporal pattern of calcium dynamics has remained unclear. We have studied changes of intracellular free calcium in cleavage-stage Xenopus embryos using fluorescent calcium indicator dyes, mainly Calcium Green-1. Cleavage formation was followed by calcium tra...

The coding region of the green fluorescent protein mutant S65T has been fused to the eukaryotic expression vector pMT3 and injected into the nucleus of Xenopus laevis oocytes. The fluorescent gene product is observed within 24-48 h and can be used as an internal control to monitor gene expression without the need for added substrates. Co-injection...

Desensitization of a chemotactic receptor is an adaptive process that terminates inflammation. Although homologous desensitization can be well explained by the action of specific receptor kinases, the mechanisms of heterologous desensitization remain elusive. As an approach to evaluate the roles of Gq pathway in desensitization of calcium signaling...

To study the role of the IP3 receptor (IP3R) upon egg activation, cDNA clones encoding IP3R expressed in the Xenopus oocytes were isolated. By analyses of the primary structure and functional expression of the cDNA, Xenopus IP3R (XIP3R) was shown to have an IP3-binding domain and a putative Ca2+ channel region. Immunocytochemical studies revealed p...