Manganese-enhanced magnetic resonance imaging detects activation of limbic structures in response to auditory stimuli of different frequencies.

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.

Whole-cell observation of ZIO-stained Golgi apparatus in rat hepatocytes with serial block-face scanning electron microscope, SBF-SEM.

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 serial block-face scanning electron microscope (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 zinc, iodine and osmium (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: (i) 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; (ii) 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.

Subarachnoid hemorrhage induces neuronal nitric oxide synthase phosphorylation at Ser1412 in the dentate gyrus of the rat brain.

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.

Expression of 3ß-hydroxysteroid dehydrogenase (hsd3b), star and ad4bp/sf-1 during gonadal development in medaka (Oryzias latipes).

In most vertebrates, sex steroids play a critical role in gonadal development, maturation of germ cells, and development of secondary sexual characteristics. Sex steroids are synthesized in steroid-producing cells (SPCs) in the testis known as Leydig cells, as well as in thecal and granulosa cells in the ovary. In SPCs, cholesterol is sequentially catalyzed by a set of steroidogenic factors and enzymes in order to produce sex steroids. Therefore, integrated expression of the genes involved in steroidogenesis is critical for the proper production of sex steroids. In the present study, regulatory mechanisms of steroidogenic factors and enzymes were examined. We focused on hsd3b, star and ad4bp/sf-1 as well as the description of temporal and spatial expression of these genes during gonadal development in medaka (Oryzias latipes). During testicular development, hsd3b, star and ad4bp/sf-1 were co-expressed in the interstitial somatic cells subsequent to the formation of the seminiferous tubule precursor, suggesting that ad4bp/sf-1 regulated the transcription of both hsd3b and star. During ovarian development, the expression pattern of hsd3b coincided with that of cyp11a1, but not with that of aromatase. Although ad4bp/sf-1 was mainly expressed in presumptive follicular cells, it was also detected in hsd3b positive interstitial cells in the developing ovary. Contrary to our expectations, the onset of star expression occurred during a later stage of ovarian development than the expression of other steroidogenic enzymes. Thus, the regulation mechanism of star transcription appears to differ from that of the other steroidogenic enzymes in the developing ovary, but not in the developing testis.

Immunohistochemical localization of mitochondrial fatty acid ß-oxidation enzymes in Müller cells of the retina.

The presence of a mitochondrial fatty acid ß-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid ß-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid ß-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid ß-oxidation enzymes. A mitochondrial fatty acid ß-oxidation system was thus shown to be present in the retina heterogeneously.

Cloning and expression of medaka cholesterol side chain cleavage cytochrome P450 during gonadal development.

Cholesterol side chain cleavage cytochrome P450 (P450scc, Cyp11a) is responsible for the first step in steroidogenesis, catalyzing the conversion of cholesterol to prognenolone. To investigate the differentiation of steroid-producing cells and the function of sex steroids during gonadal differentiation in the teleost fish, medaka (Oryzias latipes), we isolated the full length cDNA of medaka P450scc and analyzed the expression pattern of P450scc mRNA during gonadal development using in situ hybridization. At hatching, and just after the initiation of morphological sex differentiation, we did not detect any P450scc expression in both sexes. In male gonads, expression of P450scc was detected in the interstitial somatic cells 15 days after hatching following the formation of the seminiferous tubule precursor, and was maintained in the interstitial somatic cells throughout testicular development. In the female gonad, expression of P450scc was initially detected in interstitial somatic cells 5 days after hatching. Subsequently, the expression of P450scc was continuously detected in the interstitial somatic cells of the developing ovary. This expression pattern of P450scc differed from that of female specific steroidogenic enzyme P450arom. Both P450scc and P450arom expressing cells, only P450scc expressing cells, and only P450arom expressing cells were observed. Our results suggest that expression of steroidogenic enzymes is regulated by various mechanisms during ovarian development.

Immunohistochemical localization of mitochondrial fatty acid ß-oxidation enzymes in rat testis.

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid ß-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid ß-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid ß-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis.

High-contrast imaging of plastic-embedded tissues by phase contrast electron microscopy.

Phase contrast electron microscopy utilizing phase plates has been considered suitable for high-contrast observation of weak phase objects. This novel technique was newly applied to histochemically stained strong phase objects of osmificated biological specimens. Sections of various thicknesses, specifically stained for the Golgi apparatus by the ZIO technique using the heavy metals Zn and Os, were observed with a phase contrast electron microscope in Zernike and Hilbert imaging modes. Quantitative analysis of image contrast in real space and the power spectrum in Fourier space showed a high-contrast gain even for strong phase objects. This result clearly indicates that phase contrast electron microscopy can be effectively used not only for weak phase objects but also for strong phase objects in biology.