Ryuhei Higashi

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder.

In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca2+ transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder.

Helical arrangement of filaments in microvillar actin bundles.

Actin filament arrays in in vivo microvillar bundles of rat intestinal enterocyte were re-evaluated using electron tomography (ET). Conventional electron microscope observation of semi-thin cross sections (300nm thick) of high-pressure freeze fixed and resin embedded brush border has shown a whirling pattern in the center of the microvilli instead of hexagonally arranged dots, which strongly suggests that the bundle consists of a non-parallel array of filaments. A depth compensation method for the ET was developed to estimate the actual structure of the actin bundle. Specimen shrinkage by beam irradiation during image acquisition was estimated to be 63%, and we restored the original thickness in the reconstruction. The depth compensated tomogram displayed the individual actin filaments within the bundles and it indicated that the actin filaments do not lie exactly parallel to each other: instead, they are twisted in a clockwise coil with a pitch of ∼120°/μm. Furthermore, the lattice of actin filaments was occasionally re-arranged within the bundle. As the microvillar bundle mechanically interacts with the membrane and is thought to be compressed by the membranes faint tensile force, we removed the shrouding membrane using detergents to eliminate the mechanical interaction. The bared bundles no longer showed the whirling pattern, suggesting that the bundle had released its coiled property. These findings indicate that the bundle has not rigid but elastic properties and a dynamic transformation in its structure caused by a change in the mechanical interaction between the membrane and the bundle.

Autonomic and sensory nerve modulation of peristalsis in the upper urinary tract.

The primary function of the upper urinary tract is to propel urine and various water-soluble toxic compounds from the kidneys to the bladder for storage and evacuation to maintain body ionic balance and contribute to the regulation of blood volume and pressure. The mechanism by which the upper urinary tract propels urine has long been considered to be myogenic in origin as peristaltic contractions in vivo and in vitro (pyeloureteric peristalsis) propagate in a manner little affected by drugs that block nerve conduction or the sympathetic and parasympathetic transmission. However, it is now well established that the release of intrinsic prostaglandins and neuropeptides from primary sensory nerves (PSNs) helps to maintain pyeloureteric peristalsis. Electrical field stimulation of PSNs evokes species-specific positive inotropic and chronotropic effects that have been attributed to release of excitatory tachykinins superimposed on negative inotropic and chronotropic effects associated with the release of calcitonin gene related peptide (CGRP), a rise in cellular cyclic-adenosine monophosphate (cAMP) and a protein kinase A-dependent activation of glibenclamide-sensitive ATP-dependent K+ (KATP) channels. This review summarises the existing evidence of the nervous control of the upper urinary tract and recent evidence suggesting that the autonomic innervation may indirectly modulate pyeloureteric peristalsis via the activation of PSN nicotinic receptors and via the modulation of KV7 channels located on interstitial cells within the renal pelvis wall.

Role of mucosa in generating spontaneous activity in the guinea pig seminal vesicle

The mucosa may have neuron‐like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+. These spontaneous events primarily depend on Ca2+ handling by sarco‐endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco‐endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances.

Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy

IntroductionMitochondrial fission and fusion events are fundamental mechanisms for quality control of mitochondrial functions. Mitochondrial DNA (mtDNA) usually divides in offspring mitochondria after fission and mtDNA dynamics are thought to be coordinated with mitochondrial turnover. Recently, several candidate mechanisms for the relationship between mtDNA division and mitochondrial fission have been suggested ([1], 2012). The dynamics of mtDNA or nucleoids can be observed using fluorescent microscopy, but the ultrastructural aspects of their coordination remain unclear. Although visualization of mtDNA at the electron microscopic level is an important step in understanding how mtDNA division and mitochondrial division are coordinated, it is quite difficult to observe using conventional electron microscopic methods. In the present study, we attempted to establish correlative light and electron microscopy (CLEM) observation to visualize the three-dimensional localization of the mtDNA /nucleoid within mitochondria at electron microscopic resolution using a combination of immuno-electron and focused-ion beam scanning electron microscopy (FIB/SEM) tomography methods. Materials & methodsHeLa cells were fixed using 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer, and then immunohistochemically labeled with anti-TFAM IgG antibody (Abnova, USA) or anti-DNA IgM antibody (Progen, Germany). The cells were then reacted with biotin-labeled secondary antibodies. The immunoreactivities were visualized using two methods: the ABC method and streptavidin Fluoro-Nanogold. Immunohistochemically labeled specimens were then observed using light microscopy. These specimens were then developed using a Gold Enhancement kit (Nanoprobe, USA) for 150 s. Specimens for electron microscopy were stained using the ROTO method, embedded in resin, and subjected to FIB/SEM tomography (Quanta 3D FEG, FEI). 3D reconstruction was performed using the software Amira (FEI). Results & discussionWe were not able to identify a nucleoid-like structure within mitochondria, even in a complete 3D reconstruction using FIB/SEM with conventional staining. In CLEM observations, immunoreaction (IR) products were correlatively observed under LM and EM. Pre-embedding immuno-electron microscopy showed DAB and gold IR in the matrix of some mitochondria. Interestingly, IR products were observed in the globular region of the mitochondrial matrix (approximately 0.4 µm in diameter), frequently localizing in the peripheral end of the mitochondrial matrix, adjacent to the inner membrane. Using the post-embedding immunogold method, gold labels were also observed in a portion of the matrix adjacent to the mitochondrial inner membrane. These immunocytochemical results were concordant with our fluorescent microscopic observations.

En bloc staining with hydroquinone treatment for block face imaging.

IntroductionBecause recent three-dimensional (3D) ultrastructural reconstruction techniques such as serial block face scanning electron microscopy (SBFSEM), obtain their images directly from the flat surface of specimens via material contrast[1], specimens should be strongly stained with heavy metals prior to resin embedding in order to obtain higher material contrast using backscattered electrons (BSEs). To enhance membrane contrast for block face imaging (BFI), we usually stain specimens using the method published by Deerinck[2], and the images obtained show TEM-like contrast.However, recently, our research subjects have required reconstruction of a much larger volume, increasing the total image acquisition time. To reduce the total acquisition time, both high sensitivity detectors and a new specimen preparation method that provides much higher contrast are required. Takahashi et al.[3] have reported that hydroquinone (HQ) treatment during traditional electro-conductive staining increases specimen conductivity and drastically reduces the charge problem for SEM observation. They concluded that HQ treatment might increase the efficiency of secondary electron (SE) generation. Because BFI can be performed using SE as well as BSE, we examined whether addition of HQ treatment to en bloc staining protocols increased the contrast for BFI using SE. Materials & methodsMouse liver tissue was used. Mice were deeply anesthetized by diethyl ether and sodium pentobarbital, and tissues were fixed by transcardial perfusion of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) through the left ventricle, followed by heparin-containing saline. After perfusion, liver tissues were removed and cut into small cubes approximately 1 mm(3) in the fixative, and were further fixed in the same fixative for 2 h at 4°C. Subsequently, en blocstaining was performed as follows: the specimens were treated using a reduced-OTO staining method (1.5% potassium ferrocyanide-2% OsO4, 1% thiocarbohydrazide, and then 2% OsO4). Subsequently, specimens were treated with 1% HQ solution. Some specimens were exempted from this step and used as controls. Specimens were further stained with 4% uranyl acetate and Waltons lead aspartate solution.After staining, specimens were dehydrated using an ethanol series and embedded in epoxy resin (EPON812, TAAB). Surface of specimens block were cut with a diamond knife, and the newly created flat surfaces of the specimens were coated with evaporated carbon (50 Å) and observed using a SEM (Quanta 3D FEG, FEI).ResultsThe HQ-treated specimens generated a larger amount of SEs than control specimens when subjected to irradiation with the same beam, although BSE numbers were not evidently increased by the treatment. The present results suggest that HQ treatment increases SE generation efficiency, but does not enhance the recruitment of heavy metals into specimens. HQ treatment increased the contrast-to-noise ratio of BFI for images obtained using SEs, and may reduce the total image acquisition time of recently developed 3D reconstruction methods based on SEM.