VCS infusion completely degrades GD1a and b-series gangliosides.

<p>Coronal sections were stained with antibodies to the appropriate gangliosides as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029285#s2" target="_blank">Methods</a> section. VCS completely degraded three of the four major brain gangliosides (GD1a, GD1b, and GT1b) throughout the hippocampus, including the CA1 and CA3 subfields and the dentate gyrus (DG). The apoptogenic GD3 ganglioside was also hydrolyzed.</p

VCS is neuroprotective against kainate-induced lesions.

<p>Top panels: Cresyl-violet-stained sections of the dorsal hippocampus (DH), CA3 subfield, and dentate gyrus (DG). Robust neuronal death is largely restricted to the CA3 hippocampal subfield, and to a lesser extent in the dentate hilar region, 3 days following kainic acid injection in mice treated chronically with aCSF. In contrast, mice infused with VCS were protected from neuronal loss. Bottom panels: Fluorojade C immunofluorescence demonstrates ongoing neurodegeneration in CA3 and dentate hilar regions in aCSF-infused mice 3 days following kainic-acid injection. Kainate-injected mice treated with VCS exhibited almost no neurodegeneration.</p

VCS does not affect locomotor activity or anxiety.

<p>Anxiety was assessed in the open-field and elevated plus maze tasks. (<b>a</b>) In the elevated plus maze, VCS- and aCSF-treated mice both spent approximately 80% of the time in the closed arms, which is normal for mice upon first exposure to the plus maze. Closed-arm entries also did not differ by treatment. (<b>b,c</b>) In the open field, VCS-and aCSF-treated mice spent the same amounts of time in the periphery, center, and intermediate zone. Their latencies to first exit the periphery and enter the central zone also did not differ. (<b>d</b>) Locomotor activity, measured by the number of cm traversed over the 5-min. session, did not differ by treatment in either the elevated plus maze or the open field.</p

GM1 levels are significantly elevated following VCS infusion.

<p>Expression of GM1 ganglioside is largely restricted to white matter in the central nervous system, as exemplified by strong immunostaining in the corpus callosum of aCSF-treated mice. After 25 days of VCS infusion, GM1 expression was increased in white matter and the cortex, and to a lesser extent in the hippocampus.</p

Effects of <i>V. cholerae</i> sialidase (VCS) and GD3S deletion on ganglioside biosynthesis and hydrolysis.

<p>(<b>a</b>) The ganglioside biosynthetic pathway; the four major brain gangliosides are circled. Gangliosides are synthesized by sequential addition of sialic acid residues to a sphingosine backbone. GD3 synthase (GD3S) converts GM3 to GD3, and is ultimately responsible for synthesis of all of the b-series gangliosides. GD3S−/− mice lack the b-series gangliosides including the apoptogenic GD3 and two of the four major brain gangliosides. Levels of GM1 and GD1a are elevated in GD3S null mice, as constitutively high levels of Lac-Cer are converted to a-series rather than b-series gangliosides <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029285#pone.0029285-Bernardo1" target="_blank">[15]</a>. (<b>b</b>) <i>Vibrio cholerae</i> sialidase (VCS) hydrolyzes the sialic acid α2–8 (red) linkages, and terminal α2–3 linkages (red, underlined). Internal α2–3 linkages (blue) are unaffected by VCS. Thus GD1b, GT1b, and GD1a, are converted to GM1. In addition, the apoptogenic GD3 is degraded. (<b>c</b>) Ganglioside degradative pathway; the four major brain gangliosides are circled. VCS hydrolyzes three of the four major brain gangliosides into GM1. In addition, GD3 ganglioside is degraded. The resulting brain ganglioside profile is similar to that induced by GD3S elimination except that GD1a is also hydrolyzed and levels of GM1 ganglioside are much higher <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029285#pone.0029285-Yamashita1" target="_blank">[21]</a>. Abbreviations: Gal, galactose; Glc-Cer, glucosylceramide; Lac-Cer, UDP-galactose-glucosylceramide (lactosyl ceramide); GalNac, N-acetylgalatosamine; NeuAc, N-acetylneuraminic acid (sialic acid).</p

VCS does not affect spatial learning or memory.

<p>Spatial reference memory and repeated acquisition were assessed in a series of water-maze tasks. (<b>a</b>) The water maze was divided virtually into zones that allowed us to determine in which quadrant the mice swam as well as distance from the platform and time in the periphery. (<b>b</b>) Mice in both treatment groups learned to find the hidden platform proficiently. (<b>c, d</b>) Three annular zones were used to assess memory during the probe trial—10, 15, and 40 cm in diam., all of which were outside the periphery but inside the target quadrant. The 10-cm annulus represented the exact size and location of the platform during training. Chronic VCS treatment did not adversely affect spatial memory on the probe trial, either measured by the traditional quadrant divisions (<b>d</b>) or the more sensitive annular analysis (<b>c</b>). (<b>e</b>) Following the probe trial mice were re-trained to find the platform in a different location every day, in 10 trials. This repeated reversals testing did not reveal any treatment differences, either at baseline or under saline. The low dose of scopolamine did not affect learning in either group, but both groups were impaired by the 3.2 mg/kg dose. An equivalent dose of the quaternary control scopolamine methylbromide, which does not cross the blood-brain barrier, did not significantly affect performance in either group, demonstrating that the performance under scopolamine 3.2 mg/kg can be attributed to centrally-mediated cognitive impairments and not non-mnemonic performance factors.</p

VCS prevents the neuroinflammatory response following kainic acid injection.

<p>GFAP immunofluorescence is significantly increased 3 days following kainic acid inject in aCSF-treated mice, indicating a massive inflammatory response in the hippocampus. The reactive astrogliosis was nearly absent in kainate-treated mice that had received chronic infusions of VCS.</p

supplement_appendix - The Turnout Gap in Surveys: Explanations and Solutions

<p>supplement_appendix for The Turnout Gap in Surveys: Explanations and Solutions by Matthew DeBell, Jon A. Krosnick, Katie Gera, David S. Yeager and Michael P. McDonald in Sociological Methods & Research</p