Serial EM Images, and Reconstruction, of a Dendritic Spine from a C2 Barrel Hollow

<div><p>(A–D) Show four micrographs from a series of 18 that were used to reconstruct the entire dendritic spine (S); making an asymmetric synaptic contact with a bouton (B) (arrow in [A] and [B]). The astrocytic element that surrounds this spine is marked with an asterisk (*) and can be seen to be closely associated with the interface between the spine head and the axonal bouton. Scale bar in (D) indicates 0.5 μm.</p> <p>(E) Shows the corresponding 3D reconstruction of this spine (green), bouton (grey), PSD (red), and astrocyte (blue) in three images below. The left-hand image shows the spine in the same orientation as the above micrographs, with a transparent astrocyte revealing the shape of the spine beneath; the middle image is in the same orientation, but the astrocyte is now opaque, showing the degree to which the spine is covered. The right-hand image shows the spine and covering astrocyte, viewed after a 180° rotation around the <i>y</i> axis.</p></div

Up-Regulation of GLAST and GLT1 Protein Levels after Whisker Stimulation

<div><p>(A) A single barrel column (C2) was removed by aspiration through a glass micropipette, under sodium pentobarbital anesthesia.</p> <p>(B) Tangential section of the barrel cortex, Nissl stained, shows the location of the excised barrel column. A clear hole can be seen in the section in the region of barrel C2, with the neighboring barrels intact.</p> <p>(C) Representative immunoblot microassay of C2 columns dissected immediately after 24 h of C2 whisker stimulation (stim), 4 d after stimulation (4 d post stim), and from unstimulated mice (unstim). Blot was probed for GLAST, GLT1, and actin, and indicates an increase in GLAST and GLT1 levels after 24 h of whisker stimulation, but not 4 d later.</p> <p>(D) These changes were quantified using densitometry with the values being normalized against the actin levels. Results were expressed as percentages of levels in unstimulated mice (100%) and statistically analyzed with a Tukey studentized range test, <i>p</i> < 0.01; error bars indicate SD. Scale bar in (B) indicates 0.5 mm.</p> <p>(E) Representative immunoblots from animals treated as in (C), and analyzed for protein levels of EAAC1, tubulin, and actin.</p> <p>(F) Quantification of the immunoblot signals revealed no significant alteration in EAAC1 levels in stimulated animals. EAAC1 and tubulin values were normalized on actin levels, and expressed as % of level in control animals. Note that the relative level of tubulin was unchanged by the stimulation. Error bars indicate SD.</p></div

Sensory Stimulation Increases Percentage of Spines Whose Bouton–Spine Interface Is Surrounded by Astrocyte

<p>The histogram shows the distribution of four classes of spines, sorted according to their degree of contact with the astrocyte (see examples of classes I–IV), expressed as mean ± standard error of the mean (SEM) (unstimulated, <i>n</i> = 6 mice; stimulated. <i>n</i> = 6 mice). The percentage of spines in class IV, whose bouton–spine interface is completely surrounded by an astrocytic element, was increased significantly in stimulated mice (<i>p</i> < 0.03). Dendritic spines were classified into four classes, I–IV, based on the arrangement of the astrocyte at their surface. Electron micrographs of spines of each class are shown, as well as the 3D reconstruction of the whole spine to the right. (spines are indicated with an S and axonal boutons, B). Examples of spines in classes I–IV, and their reconstructions, are shown. Scale bar in lower micrograph represents 200 nm.</p

Whisker Stimulation Increases the Astrocytic Participation at the Bouton–Spine Interface

<div><p>(A) Shows a 3D reconstruction of a spine head (green), its PSD (red), and the associated astrocyte (blue). The orientation of the structure shows the region occupied by the axonal bouton (removed). The line drawing below shows the parameters measured: the total perimeter of the interface between the bouton and the spine, and the part of this perimeter that is occupied by the astrocyte, the astrocytic perimeter.</p> <p>(B) Stimulation did not change the degree of contact between bouton and spine, measured by the total perimeter (<i>p</i> > 0.5). However, the amount of the perimeter occupied by the astrocyte was significantly increased (<i>p</i> < 0.0001), using mean values per animal.</p> <p>(C) Correlation between the length of the perimeter that is occupied by astrocytic membrane and the PSD surface area on spines in unstimulated (light grey diamonds, <i>n</i> = 271; <i>p</i> < 0.001, <i>R²</i> = 0.68) and stimulated neuropil (dark grey diamonds, <i>n</i> = 340; <i>p</i> < 0.001, <i>R²</i> = 0.73).</p></div

CRT N-terminal fragments are re-localized at the cell surface.

<p>(<b>A</b>) The kinetics of the appearance of the F protein and CRT C-terminal and N-terminal fragments at the surface of infected Vero cells were monitored by flow cytofluorimetry using the corresponding specific antibodies. Control cell cultures were not infected (grey lines). From infected cell cultures, infected cells (red line; GFP-positive) and non-infected cells (blue line, GFP-negative) were identified by virus-encoded GFP fluorescence. Cell surface immuno-labelling of cells (unifxed and nonpermeablized) with F and CRT C-terminal and N-terminal were performed with specific antibodies as indicated at the top of the panels. The mean fluorescence intensity (MFI) of the labelling was determined within each cell population regularly over 48 hours of infection (top panels). The three bottom panels represent the distribution of the MFI within each population at 48 hours post-infection. Here is shown one representative experiment out of three independent experiments. (<b>B</b>) Membrane localisation of CRT N-terminal fragment following CDV infection. At 24 hours post-infection in Vero cells, cultures were immuno-labelled for the viral F protein to identify infected cells (Ba; fluorochrome: FITC), C-terminal specific anti-CRT antibody (Bb; fluorochrome: CY3), and N-terminal specific anti-CRT antibody (Bc; fluorochrome: CY5). In panel Bd, cell membranes were stained with alexa-405-conjugated wheat germ agglutinin (WGA). The merges images b and d (Be) reveal little co-localisation of CRT C-terminal fragment with the cell surface, while the merges images c and d (Bf) indicate partial surface localization of the CRT N-terminal fragment. Panel g is a merge between images b and c. Scale bar, 30 µm. Immunofluorescence analyses were performed in fixed and permeabilized cells.</p

Mechanistic model of neurodegenerative processes induced by CDV infection.

<p>The F and H CDV proteins are accumulating in the ER. This event induces an early ER stress event. In early ER stress, the quantities of CRT chaperon increase, the Ca²⁺ homeostasis is altered and Ca²⁺ is depleted from ER stores. Increase of cytosolic Ca²⁺ can have as consequence a glutamate release during CDV infection as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032803#pone.0032803-Brunner1" target="_blank">[3]</a>. Glutamate release could induce, in the neighbouring neurons, Ca²⁺ entry followed by an ER stress induction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032803#pone.0032803-Chen1" target="_blank">[56]</a>. During ER stress, the infected cells show enhance expression of the chaperons CRT, calnexin and GRP94 and relocalisation of the transcription factor ATF-6 in the nucleus followed by the expression of the proapoptotic factor CHOP/GADD 153. More importantly, infected cells show CRT fragmentation in a CDV-dependent manner. C-terminal fragments are retained in the ER by the KDEL signal whereas CRT N-terminal fragments are present after 24 hours at the cell surface. Cell surface exposition of CRT N-terminal fragment may contribute to CDV-mediated neurodegenerative auto-immunity. In <b><i>grey italic</i></b> are events described in previous publications.</p

CDV infection of Vero cells causes CRT fragmentation with vasostatin formation.

<p>(<b>A</b>) Schematic representation of the 60 kDa CRT protein. The globular 27 kDa N-terminal domain (N-term) is the most important antigenic site of the protein. This domain, as well as the P domain, possesses the chaperon function. C-terminal domain (C-term) is important for Ca²⁺ storage and possesses the KDEL ER retention signal. P and C-terminal domains have together an estimated mass of 30 kDa. (<b>B</b>) The Vero cells were either left non-infected or infected with CDV. At 48 h post-infection cells were lysed and analyzed by Western blot using C-terminal-specific (left) or N-terminal-specific (right) antibodies. Note the C-terminal 30 kDa fragment, and the 27 kDa N-terminal fragment. GAPDH is used as an internal control. (<b>C</b>) Impact of the ER stress inducing drugs dithiothreitol (DTT) and thapsigargin (Th) on CRT expression during CDV infection. Red fluorescence in all panels corresponds to CRT immunostaining, which increases in a DTT concentration-dependent manner (top panels) or in a thapsigargin concentration-dependent manner (white arrow heads). For comparison, calreticulin staining is shown in infected cells probed by immunostaining of the F protein (bottom left and insert panels). Immunofluorescence analyses were performed in fixed and permeabilized cells. (<b>D</b>) Western blot using the antibody recognizing either the N- terminal domain of CRT (27 kDa, right panel) or the C- terminal domain (30 kDa; left panel). Both antibodies recognize the full-length CRT (top line, 60 kDa). Cellular extracts come from Vero cells exposed to DTT or thapsigargin (Th), or CDV infected as indicated. GAPDH was used as an internal control. CRT cleavage was specifically mediated by CDV infection (line 11) and not by exposure to DTT or thapsigargin.</p

Infection of Vero cells by rgA75/17-V (CDV) induces ER stress.

<p>(<b>A, B, C, D</b>) Representative photomicrographs of non-infected Vero cells (A) and infected with a Vero cell-adapted canine distemper virus (CDV) strain (rgA75/17-V). The former recombinant CDV expresses the enhanced green fluorescence protein (e-GFP) for easier identification of infected cells (B, C, D). Cultures were infected 1 day after seeding. Cells were then fixed and permeabilized and subsequently analysed by immunofluorescence at 24 hours (A, B and D) or 48 hours (C) after infection. Antibodies against the protein F of CDV (F), calreticulin, CHOP-GADD and Calnexin are as indicated in the panels. Merged images are shown on bottom panels, including labelling with 4′6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 30 µm. Calreticulin and calnexin expression are increased in infected cells that expressed the F protein (B and D) and at 48 hours post-infection, infected cells also express strongly the nuclear proapoptotic CHOP/GADD 153 (C). (<b>E</b>) Increase of CRT, CHOP/GADD 153 and calnexin during culture infection, as determined by flow cytofluorimetry. Each sample was analysed in triplicate on three separate experiments, and one representative experiment is shown here.</p