Image_1_In Vivo Two Photon Imaging of Astrocytic Structure and Function in Alzheimer’s Disease.tif

<p>The physiological function of the neurovascular unit is critically dependent upon the complex structure and functions of astrocytes for optimal preservation of cerebral homeostasis. While it has been shown that astrocytes exhibit aberrant changes in both structure and function in transgenic murine models of Alzheimer’s disease (AD), it is not fully understood how this altered phenotype contributes to the pathogenesis of AD or whether this alteration predicts a therapeutic target in AD. The mechanisms underlying the spatiotemporal relationship between astrocytes, neurons and the vasculature in their orchestrated regulation of local cerebral flow in active brain regions has not been fully elucidated in brain physiology and in AD. As there is an incredible urgency to identify therapeutic targets that are well-tolerated and efficacious in protecting the brain against the pathological impact of AD, here we use the current body of literature to evaluate the hypothesis that pathological changes in astrocytes are central to the pathogenesis of AD. We also examine the current tools available to assess astrocytic calcium signaling in the living murine brain as it has an important role in the complex interaction between astrocytes, neurons and the vasculature. Furthermore, we discuss the altered function of astrocytes in their interaction with neurons in the preservation of glutamate homeostasis and additionally address the role of astrocytes at the vascular interface and their contribution to functional hyperemia within the living murine brain in health and in AD.</p

β55 Staining of Amyloid Plaques in Ex Vivo Human AD Brain Tissue.

<p>Merged red and green channel confocal images of frozen-section human AD brain tissue stained with biotinylated β55 (a) and β55rc (b). β55 positive plaques (green) were clearly visible, while only a few very faint β55rc positive plaques were observed. Background auto-fluorescence, observed in both red and green channels, is shown in yellow. (c) Fluorescence images of human AD brain tissue costained with biotinylated-β55 (red) and Thioflavin-S (green). β55 colocalized with Thioflavin-S positive plaques. (Scale bars: 50 µm).</p

In Vivo Imaging of β55 Positive Amyloid Plaques.

<p><i>In vivo</i> 2-photon microscopy images from an 18 month old APP/PS1 transgenic mouse obtained 1 hour after topical application of fluorescein-labeled β55 (a,b). Texas Red labeled dextran was intravenously injected for visualization of blood vessels. β55 positive plaques and cerebral amyloid angiopathy are clearly visible in the cortex (a) and vasculature (b), respectively. (Scale bars: 20 µm).</p

β55 Staining of Dot Blots of Synthetic Aβ Aggregates.

<p>(a) Dot blot of synthetic Aβ_1–42 and Aβ_1–40 aggregates probed with biotinylated-β55. (b) Western blot of the synthetic Aβ_1–42 and Aβ_1–40 aggregates probed with 6E10 antibody. The increased staining of Aβ_1–42 aggregates in the dot blot relative to Aβ_1–40 aggregates is consistent with the greater fibril and high molecular weight oligomer composition of Aβ_1–42 aggregates observed in the western blot.</p

Predicted Secondary Structure of RNA Aptamers.

<p>Predicted secondary structure of the β55 (left) and β55rc (right) aptamer probes with base pair probability indicated by the color scale bar. Minimum free energy structures were determined using the RNAfold Webserver suite of programs.</p

DNA sequence for the β55 aptamer including the T7 polymerase primer, highlighted in bold text.

<p>DNA sequence for the β55 aptamer including the T7 polymerase primer, highlighted in bold text.</p

Contrast-to-Noise Ratio for β55 and β55rc Positive Amyloid Plaques.

<p>(a) Representative <i>in vivo</i> 2-photon microscopy images from 7.5 month old APP/PS1 transgenic mice acquired 1 hour (left column) and 24 hours (right column) after topical application of either fluorescein-labeled β55 (top row) or β55rc (bottom row). Most β55 plaques were still visible 24 hours after topical application. In contrast, only a small number of very faint β55rc plaques were still visible after 24 hours. (b) Average plaque contrast-to-noise ratio (CNR) observed 1 hour and 24 hours following topical application of fluorescein-labeled β55 (n = 2) or β55rc (n = 2). β55 positive plaques had a significantly greater CNR than β55rc plaques (p<0.01) at both time points. (Scale bars: 50 µm).</p

Contrast-to-noise ratio (CNR) distribution of amyloid plaques observed in 2-photon <i>in vivo</i> images of 7.5 month-old APP/PS1 transgenic mice with β55 and β55rc.

<p>Contrast-to-noise ratio (CNR) distribution of amyloid plaques observed in 2-photon <i>in vivo</i> images of 7.5 month-old APP/PS1 transgenic mice with β55 and β55rc.</p

Colocalization of β55 and Methoxy-XO4 Positive Amyloid Plaques.

<p><i>In vivo</i> 2-photon microscopy plaque images from a 7 month old APP/PS1 transgenic mouse acquired 1 hour after topical application of fluorescein-labeled β55 (a,d) and 1 day after intraperitoneal injection of methoxy-XO4 (b,e). While methoxy-XO4 stains only the dense core of the plaque, β55 stains both the plaque core and a diffuse halo surrounding the plaque (c,f). (Scale bars: 20 µm).</p

Communicating Functional Agents and their Application to Graphical User Interfaces

We demonstrate how concepts of communicating agents can be integrated into purely functional languages by an orthogonal extension of the usual I/O monad. These agents communicate via so-called service access points and support programming in the style of client-server architectures. We then show the feasibility of the approach by applying it to the example of graphical user interfaces, which we consider to be a typical instance of reactive systems. For this purpose we develop the concept of so-called gates, which serve as a mediator between user events and the application logic. It turns out that the combination of functional expressiveness and concurrency yields a powerful framework for the realization of reactive systems such as graphical user interfaces. All concepts discussed in this paper are represented in the functional language Opal and have been implemented in the Opal programming environment