深澤 元晶

PURPOSE: As we are exposed to stress on a daily basis, it is important to detect and treat stress during the subclinical period. However, methods to quantify and confirm stress are currently unavailable, and the detection of subclinical stressors is difficult. This study aimed to determine whether manganese-enhanced magnetic resonance imaging (MEMRI) could be used to assess stress in rat brains. METHODS: We exposed male Wistar/ST rats bred in a specific pathogen-free environment to ultrasound stimuli (22 kHz and 55 kHz) for 10 days and then assessed brain activities using MEMRI, the light/dark box test, and ΔFosB immunohistochemical staining. RESULTS: In the MEMRI assessments, exposure at 22 kHz activated the periaqueductal gray, while exposure at 55 kHz specifically enhanced activity in the nucleus accumbens core and the orbitofrontal cortex. The exploratory behavior of the 55-kHz group increased sharply, while that of the 22-kHz group showed a lower exploratory value. ΔFosB expression increased in the orbitofrontal cortex, nucleus accumbens, periaqueductal gray, and amygdaloid nucleus in the 22-kHz group. CONCLUSION: Ultrasound stimuli at 22 kHz suppressed weight gain in rats and excessive ΔFosB induction in the nucleus accumbens caused excessive sensitization of the neural circuit, thereby contributing to pathological behavior. We thus demonstrated that MEMRI can be useful to objectively assess the pathophysiology of stress-related disorders.

The Golgi apparatus, which plays a role in various biosynthetic pathways, is usually identified in electron microscopy by the morphological criteria of lamellae. A 3-dimensional analyses with SBF-SEM, a volume-SEM proficient in obtaining large volumes of data at the whole-cell level, could be a promising technique for understanding the precise distribution and complex ultrastructure of Golgi apparatus, although optimal methods for such analyses remain unclear since the observation can be hampered with sample charging and low image contrast, and manual segmentation often requires significant manpower. The present study attempted the whole-cell observation and semi-automatic classification and segmentation of the Golgi apparatus in rat hepatocytes for the first time by SBF-SEM via ZIO staining, a classical osmium impregnation. The staining electron-densely visualized individual Golgi lamellae and their ultrastructure could stably be observed without any noticeable charging. The simple thresholding of the serial images enabled the efficient reconstruction of the labeled Golgi apparatus, which revealed plural Golgi apparatus in one hepatocyte. The combination of the heavy metal-based histochemistry of ZIO staining and SBF-SEM was useful in the 3-dimensional observation of the Golgi apparatus at the whole-cell level because of two technical advantages: 1) visualization of the Golgi apparatus without any heavy metal staining and efficient acquisition of the block-face images without additional conductive staining or any devices for eliminating charging; 2) easy identification of the staining and hassle-free, semi-automatic classification and segmentation by simple thresholding of the images. This novel approach could elucidate the topographic characteristics of the Golgi apparatus in hepatocytes.

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INTRODUCTION: We previously demonstrated that cyclic AMP-dependent protein kinase (PKA) phosphorylates neuronal nitric oxide synthase (nNOS) at Ser1412 in the hippocampal dentate gyrus after forebrain ischemia; this phosphorylation event activates NOS activity and might contribute to depression after cerebral ischemia. In this study, we revealed chronological and topographical changes in the phosphorylation of nNOS at Ser1412 immediately after subarachnoid hemorrhage (SAH). METHODS: In a rat single-hemorrhage model of SAH, the hippocampus and adjacent cortex were collected up to 24 h after SAH. Samples from rats that were not injected with autologous blood were used as controls. NOS was partially purified from crude samples via an ADP-agarose gel. Levels of nNOS, nNOS phosphorylated at Ser1412 (p-nNOS), PKA, and p-PKA at Thr197 were studied in the rat hippocampus and cortex using Western blot analyses and immunohistochemistry. RESULTS: According to the Western blot analysis, levels of p-nNOS at Ser1412 were significantly increased in the hippocampus, but not in the cortex, between 1 and 3 h after SAH. Immunohistochemistry revealed the phosphorylation of nNOS at Ser1412 and PKA at Thr197 in the dentate gyrus, but not in the CA1 area, 1 h after SAH. An injection of saline instead of blood also significantly increased levels of p-nNOS at Ser1412 in the hippocampus 1 h after the injection. CONCLUSIONS: An immediate increase in intracranial pressure (ICP) might induce transient cerebral ischemia and promote the PKA-mediated phosphorylation of nNOS at Ser1412 in the dentate gyrus. This signal transduction pathway induces the excessive production of nitric oxide (NO) and might be involved in cognitive dysfunction after SAH.