Haruo Mizutani

5-HT1B receptor-mediated presynaptic inhibition at the calyx of Held of immature rats

5‐Hydroxytryptamine (5‐HT) inhibits transmitter release via activating GTP‐binding proteins, but the target of 5‐HT receptors in the nerve terminal is not determined. We addressed this question at the calyx of Held synapse in the brainstem slice of immature rats. Bath‐application of 5‐HT attenuated the amplitude of nerve‐evoked excitatory postsynaptic currents (EPSCs) associated with an increase in the paired‐pulse ratio, whereas it had no effect on the amplitude of spontaneous miniature EPSCs. The 5‐HT1B receptor agonist CP93129 mimicked the inhibitory effect of 5‐HT, but the 5‐HT1A agonist (R)‐(+)‐8‐hydroxy‐DPAT (8‐OHDPAT) had no effect. The 5‐HT1B receptor antagonist NAS‐181 blocked the inhibitory effect of 5‐HT. These results suggest that 5‐HT activated 5‐HT1B receptors in calyceal nerve terminals, thereby inhibiting transmitter release. In direct whole‐cell recordings from calyceal nerve terminals, 5‐HT attenuated voltage‐dependent Ca2+ currents, but had no effect on voltage‐dependent K+ currents. When EPSCs were evoked by presynaptic Ca2+ currents during simultaneous pre‐ and postsynaptic recordings, the magnitude of the 5‐HT‐induced inhibition of Ca2+ currents fully explained that of EPSCs. Upon repetitive applications, 5‐HT showed tachyphylaxis, with its effect on both EPSCs and presynaptic Ca2+ currents becoming weaker in the second application. 1,2‐bis(o‐aminophenoxy)ethane‐N‐N′‐N′‐N′‐tetraacetic acid (BAPTA; 10 mm) loaded into the nerve terminal abolished this tachyphylaxis. The presynaptic inhibitory effect of 5‐HT was prominent at postnatal day 5, but became weaker as animals matured. We conclude that activation of 5‐HT1B receptors inhibits voltage‐gated Ca2+ channels, thereby inhibiting transmitter release at immature calyceal nerve terminals, and that 5‐HT1B receptors undergo Ca2+‐dependent tachyphylaxis on repetitive activations.

Hard-X-ray Phase-Difference Microscopy with a Low-Brilliance Laboratory X-ray Source

We have developed a hard-X-ray phase-imaging microscopy method using a low-brilliance X-ray source. The microscope consists of a sample, a Fresnel zone plate, a transmission grating, and a source grating creating an array of mutually incoherent X-ray sources. The microscope generates an image exhibiting twin features of the sample with opposite signs separated by a distance, which is processed to generate a phase image. The method is quantitative even for non-weak-phase objects that are difficult to be quantitatively examined by the widely used Zernike phase-contrast microscopy, and it has potentially broad applications in the material and biological science fields.

High throughput and wide field of view EUV microscope for blur-free one-shot imaging of living organisms

We present and demonstrate the use of an extreme ultraviolet (EUV) microscope that was developed in-house. Images are acquired using Bragg reflection multilayer optics and a laser-produced plasma light source. The upper-limit spatial resolution of the EUV microscope is 130 nm with a 10 ns exposure time and 250 x 250 microm(2) field of view. Resolution is superior to that of visible microscopes with the same size of field of view, and the exposure time is short enough to observe fine structures in-vivo. Observation of the cerebral cortex of a mouse is demonstrated.

X-ray microscopy for neural circuit reconstruction

Neural circuits in the central nervous system build our various higher brain functions. However, little is known about mechanisms underlying neuronal information processing in the brain. Anatomical graph structures of real neural networks will provide us with fundamental views to elucidate them. We aim at developing a three-dimensional atlas of neural circuits using high resolution hard X-ray microscopy by synchrotron radiation. We stained neurons of a complete brain from a mouse by the Golgi-Cox method. The heavy metals used in our procedure enhanced X-ray absorption and phase contrast. 3D images of fibriform axons and dendrites of various neurons were reconstructed by back projection. X-ray microscopy with a Talbot interferometer demonstrated finer histological structures of pyramidal neurons in the hippocampus and the cerebral cortex. This observation probably serves as a foundation for achieving a mammalian Connectome Project (identifying complete wiring diagrams of the brain) with X-ray nano-tomography in the near future.

Nano‐Resolution X‐ray Tomography for Deciphering Wiring Diagram of Mammalian Brain

Neural circuits in the central nervous system are the substrate of various high‐order brain functions. However, little is known about the mechanisms underlying neuronal information processing in the brain. Anatomical and functional graph structures of neural networks with actual connections will provide us with perspectives to elucidate the brain complexity. Here, we aim to develop a three‐dimensional mouse brain atlas of neural circuits using nano‐resolution x‐ray tomography by synchrotron radiation. In addition to identifying a large number of synapses, our research will also clarify the structure of neuronal networks for understanding the most complex organ in the body. In this study, we observed metal‐stained biological tissues of the mouse brain using hard x‐ray Zernike‐type phase‐contrast microscopy with 60‐nm resolution at SPring‐8. As a result, the nano‐resolution hard x‐ray phase‐contrast microscope revealed nerve fibers and organelles including mitochondria and synapses in the neural tissue. In ...

Bridging the Knowledge Gap between Research and Education through Textbooks

We describe a web-based system that helps undergraduate students learn about research at their university through the medium of textbooks. The system, called University on Textbooks, links textbooks to research articles written by university researchers, enabling people to navigate between introductory materials in textbooks and specific topics reported in research articles. Bidirectional links are generated from semantic statements created manually for topical passages from textbooks and abstracts of articles. The semantic statements are created using an ontology that is based on a description logic, so a reasoner can infer the semantic similarity between pairs of statements. To demonstrate the feasibility and effectiveness of our approach, we present a prototype containing 100 semantic statements describing passages from an undergraduate life sciences textbook in Japanese that have been linked to semantic statements created for 392 research articles from life sciences.

Three-dimensional reconstruction of neurons in the brain by using X-ray micro-CT technology

To study cellular functions in situ in realtime, we developed a fibercoupled confocal microscope, (FCM) and observed fluorescently labeled cells in the organs of animal. The microscope system consisted of microlens-attached Nipkow disk (Yokogawa), and an imaging fiber (IF, FiberTech). The IF had 10,000 of single mode flexible fiber of 3.75 m in diameter, which was scanned with multiple laser beams to form a confocal image. The tip of IF was 0.75 mm thick, for easier approach to the target inside the organ without a large physical damage. When the incidental face of IF was placed below the surface of organs, many spots (50 cells) were clearly visible in the field of 300 m2, showing fluctuation in fluorescence intensity (breathing and some cellular activities). Such an in situ imaging by FCM is very promising for detailed studies on the relationship between cell structure and functions.

X‐ray Phase Imaging and Tomography Using a Fresnel Zone Plate and a Transmission Grating

We report hard x‐ray phase imaging and tomography consisting of an objective lens and a transmission grating. Our simple microscopy system provides a quantitative phase image even for a strong phase object, and it works by partially coherent illumination on the objective lens. We successfully performed phase imaging and tomography using our method. Our approach may prove to be a powerful way for biologists and material scientists to quantitatively visualize internal structures even in relatively thick samples with high spatial resolution and high sensitivity.

Development of Computer Tomography System for the Soft X‐ray Microscope at Ritsumeikan University

A synchrotron‐based full‐field imaging soft x‐ray microscope was tuned appropriately to perform computer tomography. The contrast and focal depth of the optical system were evaluated by using a Fresnel zone plate as a test object of variable spatial frequency. A focal depth of 15 μm was obtained at the spatial frequency of 4.3 μm−1 according to Rayleigh’s criterion. As a first trial of three‐dimensional observation using this system, the cerebral cortex of the brain of a mouse, trimmed to a columnar shape by focused ion beam milling, was studied using a wavelength of 1.87‐nm.