Akinobu Togo

Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus.

A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission.

Three-dimensional organization of the endoplasmic reticulum membrane around the mitochondrial constriction site in mammalian cells revealed by using focused-ion beam tomography

The endoplasmic reticulum (ER) and mitochondria associate at multiple contact sites to form specific domains known as mitochondria-ER associated membranes (MAMs) that play a role in the regulation of various cellular processes such as Ca2+ transfer, autophagy, and inflammation. Recently, it has been suggested that MAMs are also involved in mitochondrial dynamics, especially fission events. Cytological analysis showed that ER tubules were frequently located close to each other in mitochondrial fission sites that accumulate fission-related proteins. Three-dimensional (3D) imaging of ER-mitochondrial contacts in yeast mitochondria by using cryo-electron tomography also showed that ER tubules were attached near the constriction site, which is considered to be a fission site1). MAMs have been suggested to play a role in the initiation of mitochondrial fission, although the molecular relationships between MAMs and the mitochondrial fission process have not been established. Although an ER-mitochondrial membrane association has also been observed at the fission site in mammalian mitochondria, the detailed organization of MAMs around mammalian mitochondria remains to be established. To visualize the 3D distribution of the ER-mitochondrial contacts around the mitochondria, especially around the constriction site in mammalian cells, we attempted 3D structural analysis of the mammalian cytoplasm using high-resolution focused ion-beam scanning electron microscopy (FIB-SEM) tomography, and observed the distribution pattern of ER contacts around the mammalian mitochondrial constriction site.Rat hepatocytes and HeLa cells were used. Liver tissue was obtained from male rats (Wistar, 6W) fixed by transcardial perfusion of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) under deep anesthesia. HeLa cells were fixed with the same fixative. The specimens were then stained en bloc to enhance membrane contrast and embedded in epoxy resin2). The surface of the specimens was freshly exposed using an ultramicrotome and examined by FIB/SEM (Quanta 3D FEG, FEI, USA). Ion-beam milling and image acquisition cycles were performed under the following conditions. The milling was performed with a gallium ion beam at 30 kV with a current of 100 pA, with a milling pitch of 10 nm/step. Material contrast images using backscattered electrons (BSE) were acquired at a landing energy of 2 keV with a bias voltage of 1.5-2.5 kV using a vCD detector. The remaining acquisition parameters were as follows: beam current = 11 pA, dwell time = 6-30 µs/pixel, image size = 1024 × 883 pixel (5.9 × 5.1 µm), pixel size = 5.8 nm/pixel. The resultant image stack was processed using Avizo 6.3 and Amira 5.4(FEI, USA).Reconstructed volume showed the existence of several constriction sites on mitochondria in both chemically fixed normal hepatocytes and HeLa cells. Each material contrast image of specimen surfaces showed two types of membrane associations between the ER and mitochondria. The first was an osmiophilic bridge-like structure; these bridges were approximately 50 nm in length, and they connected the ER membrane and the mitochondrial outer membrane (OMM). The second was a close apposition (< 20 nm) of the ER membrane and the OMM. Membrane segmentation revealed the 3D distribution of the membrane contacts; 10 to 20% of the mitochondrial surface was occupied by ER contacts. No fundamental difference was observed between hepatocytes and HeLa cells in the distribution pattern of the contacts. Although ER-contacts and bridge-like structures were occasionally found to accumulate around the mitochondrial constriction area, we did not observe any ring-like ER tubules around the mammalian mitochondrial constriction site, as in yeast. These results suggest that the role of ER-membrane associations in the mitochondrial fission process may differ between mammals and yeast.

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

Three-dimensional ultrastructural analysis of cells in the periodontal ligament using focused ion beam/scanning electron microscope tomography

The accurate comprehension of normal tissue provides essential data to analyse abnormalities such as disease and regenerative processes. In addition, understanding the proper structure of the target tissue and its microenvironment may facilitate successful novel treatment strategies. Many studies have examined the nature and structure of periodontal ligaments (PDLs); however, the three-dimensional (3D) structure of cells in normal PDLs remains poorly understood. In this study, we used focused ion beam/scanning electron microscope tomography to investigate the whole 3D ultrastructure of PDL cells along with quantitatively analysing their structural properties and ascertaining their orientation to the direction of the collagen fibre. PDL cells were shown to be in contact with each other, forming a widespread mesh-like network between the cementum and the alveolar bone. The volume of the cells in the horizontal fibre area was significantly larger than in other areas, whereas the anisotropy of these cells was lower than in other areas. Furthermore, the orientation of cells to the PDL fibres was not parallel to the PDL fibres in each area. As similar evaluations are recognized as being challenging using conventional two-dimensional methods, these novel 3D findings may contribute necessary knowledge for the comprehensive understanding and analysis of PDLs.

Anchoring structure of the calvarial periosteum revealed by focused ion beam/scanning electron microscope tomography

An important consideration in regeneration therapy is the fact that the tissue surrounding an organ supports its function. Understanding the structure of the periosteum can contribute to more effective bone regeneration therapy. As a cellular source, the periosteum also assists bone growth and fracture healing; this further necessitates its direct contact with the bone. However, its anchoring strength appears to be inexplicably stronger than expected. In this study, we used focused ion beam/scanning electron microscope tomography to investigate ultrathin serial sections as well as the three dimensional ultrastructure of the periosteum to clarify the architecture of its anchoring strength, as such assessments are challenging using conventional methods. We discovered perforating fibres that arise from the bone surface at 30 degree angles. Additionally, the fibres across the osteoblast layer were frequently interconnected to form a net-like structure. Fibroblast processes were observed extending into the perforating fibres; their morphologies were distinct from those of typical fibroblasts. Thus, our study revealed novel ultrastructures of the periosteum that support anchorage and serve as a cellular source as well as a mechanical stress transmitter.

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.