Tuning of the Zernike phase-plate for visualization of detailed ultrastructure in complex biological specimens

In order to acquire phase-contrast images with adequate contrast, conventional TEM requires large amount of defocus. Increasing the defocus improves the low-frequency components but attenuates the high-frequency ones. On the other hand, Zernike phase-contrast TEM (ZPC-TEM) can recover low-frequency components without losing the high-frequency ones under in-focus conditions. ZPC-TEM however, has another problem, especially in imaging of complex biological specimens such as cells and tissues; strong halos appear around specimen structures, and these halos hinder the interpretation of images. Due to this problem, the application of ZPC-TEM has been restricted to imaging of smaller particles. In order to improve the halo appearance, we fabricated a new quarter-wave thin film phase-plate with a smaller central hole and tested it on vitreous biological specimens. ZPC-TEM with the new plate could successfully visualize, in in-focus images, the intracellular fine features of cultured cells and brain tissues. This result indicates that reduction of the central hole diameter makes ZPC-TEM applicable on size scales ranging from protein particles to tissue sections. The application of ZPC-TEM to vitreous biological specimens will be a powerful method to advance the new field of imaging science for ultrastructures in close-to-physiological state.     

Organic pollution in dammed river water in a low-precipitation region of Japan

Organic pollution in eutrophic river water dammed by estuary weirs was studied in the Shin and Kasuga rivers of the Shikoku region, Japan. Both rivers are located in a low-precipitation region of Japan. The investigation was performed at intervals of 3–14 days from 12 July 2002 to 13 July 2003. The annual averages of chlorophyll a concentration were approximately 150 and 40 μg L⁻¹ in the Shin and Kasuga rivers, respectively. The concentration often exceeded 500 μg L⁻¹ in the Shin River in winter. Dissolved oxygen saturation exhibited marked variation. High and low values, i.e., >150% in the surface water layer and 0% in the bottom water layer, were occasionally observed. Dissolved methane concentrations >2,000 nM were often observed from spring to fall. In both rivers, the serious organic substance pollution was confirmed through 1 year.     

Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line,Pv11

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5–34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

     

Comparison of Optic Disc Morphology of Optic Nerve Atrophy between Compressive Optic Neuropathy and Glaucomatous Optic Neuropathy

Objectives

To compare the optic nerve head (ONH) structure between compressive optic neuropathy (CON) and glaucomatous optic neuropathy (GON), and to determine whether selected ONH quantitative parameters effectively discriminate between GON and CON, especially CON cases presenting with a glaucoma-like disc.

Methods

We prospectively assessed 34 patients with CON, 34 age-matched patients with moderate or severe GON, and 34 age-matched healthy control subjects. The quantitative parameters of ONH structure were compared using the Heidelberg Retina Tomograph 2 (HRT2) and Spectralis optical coherence tomography with an enhanced depth imaging method.

Results

The mean and maximum cup depths of CON were significantly smaller than those with GON (P<0.001 and P<0.001, respectively). The distance between Bruch''s membrane opening and anterior surface of the lamina cribrosa (BMO-anterior LC) of CON was also significantly smaller than that of glaucoma but was similar to that of the healthy group (P<0.001 and P = 0.47, respectively). Based on Moorfields regression analysis of the glaucoma classification of HRT2, 15 eyes with CON were classified with a glaucoma-like disc. The cup/disc area ratio did not differ between cases of CON with a glaucoma-like disc and cases of GON (P = 0.16), but the BMO-anterior LC and mean and maximum cup depths of CON cases with a glaucoma-like disc were smaller than those in GON (P = 0.005, P = 0.003, and P = 0.001, respectively).

Conclusions

Measurements of the cup depths and the LC depth had good ability to differentiate between CON with a glaucoma-like disc and glaucoma. There was no laminar remodeling detected by laminar surface position in the patients with CON compared to those with GON.     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.