<i>In</i><i>vivo</i> transduction with control sindMu-ZsGreen lentiviruses that display isotype IgG on their surface.

<div><p>To demonstrate specificity of the lentiviral targeting approach, <i>in </i><i>vivo</i> brain sections were transduced with sindMu-ZsGreen lentiviruses that displayed isotype IgG on their surfaces (sindMu-ZsGreen/ IgG isotype). <b>image a</b> - ZsGreen lentiviral expression; <b>image b</b> - GFAP staining for astrocytes; <b>image c</b> – NeuN staining for neurons; <b>image d</b> – merged image for ZsGreen, GFAP and NeuN expression. Indicated scale bar - 50µm(zoom x40).</p> <p><b>B</b>. Quantitation of in-vivo lentiviral targeting - Analysis of the relative percentage of ZsGreen-positive cells that also stained positively for NeuN or GFAP, following transduction with either VSV-G-ZsGreen or sindMu-ZsGreen/GLAST IgG recombinant lentiviruses. As shown, similar relative targeting index values for both astrocytes and neurons were observed when the VSV-G-ZsGreen was used. However, using sindMu-ZsGreen/GLAST IgG lentivirus, the NeuN-ZsGreen co-staining was significantly decreased, while the relative percentage of GFAP/ZsGreen-positive cells was significantly higher.</p></div

Preferential <i>in vitro</i> targeting of primary astrocytes by engineered lentiviruses sindMu-ZsGreen/GLAST IgG.

<div><p><b>A</b>. <i>In </i><i>vitro</i> targeting of astrocytes by lentiviruses – mixed primary glia cells isolated from the brains of one day old mice and were cultured <i>in </i><i>vitro</i>. Cultures were transduced with the indicated engineered lentiviruses, and 48h post transduction cells were harvested and stained for GFAP to specifically detect astrocytes. Cells were then analyzed by FACS for ZsGreen expression and for specific GFAP staining.</p> <p><b>Panel (a)</b> - transduction of cells with lentiviruses that did not incorporate IgG (sindMu-ZsGreen).</p> <p><b>Panel (b)</b> - transduction of cells with lentiviruses that incorporated an IgG isotype (sindMu-ZsGreen/IgG isotype).</p> <p><b>Panel (c)</b> - transduction of cells with lentiviruses that incorporated soluble GLAST IgG (sindMu-ZsGreen/GLAST IgG) and demonstrated preferential targeting for astrocytes.</p> <p><b>Panel (d)</b> - transduction of cells with lentiviruses that were pseudotyped with VSV-G and exhibited broad non-specific transduction to cells in the culture (VSV-G-ZsGreen).</p> <p><b>B</b>. FACS analysis demonstrating the composition of the mixed microglia culture. Cells of mixed microglia cultures were transduced with sindMu-ZsGreen/IgG isotype and stained for CD11b antigen as specific marker of microglia. Cells were then washed and analyzed for ZsGreen expression and CD11b staining by FACS.</p> <p><b>C</b>. A summary of quantitation of the <i>in </i><i>vitro</i> targeting of mixed microglia cell cultures. Cells were transduced with the indicated lentiviruses. 48h post transduction, cells were harvested and stained for astrocyte staining (GFAP) or microglia (CD11b). Cells were then analyzed by FACS for ZsGreen expression and for cell specific antigen. Values are presented relatively to the transduction efficiencies by the sindMu-ZsGreen/isotype IgG lentivirus. Results are representative of the means of triplicate wells; error bars show the standard deviation of the means.</p> <p><b>D</b>. Increasing amounts of the sindMu-ZsGreen/GLAST IgG lentivirus were used to transduce 3x10⁵ cells/well primary glia cultures. 48h post transduction, cells were harvested and stained for astrocyte staining (GFAP) or microglia (CD11b). Cells were then analyzed by FACS for ZsGreen expression and for cell specific antigen. Results are representative of the means of triplicate wells; error bars show the standard deviation of the means.</p></div

<i>In vivo</i> selective targeting of astrocytes by SindMu-ZsGreen/GLAST lentiviruses.

<div><p><b>A</b>. Broad-field images of brain sections following transduction with sindMu-ZsGreen/GLAST IgG lentivirus. The figure shows defined areas in the brain and cell types that were transduced and expressed viral-mediated ZsGreen. <b>panel a</b> - ZsGreen lentiviral expression; <b>panel b</b> - GFAP staining for astrocytes; <b>panel c</b> - NeuN staining for neurons; <b>panel d</b> - merged image for ZsGreen expression in neurons (NeuN) and astrocytes (GFAP). Indicated scale bar, <u>200µm (zoom x10)</u>.</p> <p><b>B</b>. Higher magnification of sindMu-ZsGreen/GLAST IgG transduction - engineered lentiviruses were injected into the hippocampus and thalamus of mice, and 14 days post injection brain slices were generated and stained with the appropriate antibodies. <b>panel a</b> - ZsGreen lentiviral expression; <b>panel b</b> - GFAP staining for astrocytes; <b>panel c</b> - NeuN staining for neurons; <b>panel d</b> – merged image for ZsGreen, GFAP and NeuN expression. Arrows present cells were GFAP and ZsGreen are co expressed. Indicated scale bar, 50µm (<u>zoom x40</u>).</p> <p><b>C</b>. Single cell imaging of the above transductions with sindMu-ZsGreen/GLAST IgG lentiviruses. Analysis shows preferential targeting of astrocytes in the CNS of mice by SindMu-ZsGreen/GLAST-1 IgG. <b>panel a</b> - ZsGreen lentiviral expression; <b>panel b</b> - GFAP staining for astrocytes; <b>panel c</b> – merged image for ZsGreen and GFAP expression at the level on a single cell. Indicated scale bar - 20µm (zoom x100).</p></div

Salivary cortisol levels.

<p>ANOVA repeated measures indicated a significant increase in cortisol levels in the TSST group (n = 11) during the task compared to before the task <i>p <</i> .<i>01</i> but, not in the control group (n = 5), <i>n</i>.<i>s</i>.. The bars represent standard errors (SE).</p

Source localization by sLORTA in four time windows.

<p>Graphical representations of the sLORETA results comparing the amplitudes of ERPs elicited by picture under high load condition in control and TSST group, in the four time-windows. Areas with significantly increased activity (yellow) and significantly decreased activity (light blue) due to picture presence, are presented (<i>p</i> < .05). Less activation in V1 was observed in the TSST group due to picture presence early at ~100ms after stimulus onset.</p

The emotional perceptual load task.

<p>The emotional perceptual load task.</p

Grand averaged representative potentials.

<p>Event-related potentials at frontal (upper; F1, F2, F3, F4, F5, F6, F7, F8 and Fz) and occipito-parietal (lower: O1, O2, Oz, POz, PO3, PO4 and PO8) areas elicited by each of the four conditions: low load no picture, low load picture, high load no picture and high load picture, are depicted for participates in Control (left; <i>n</i> = 17) and TSST (right; <i>n</i> = 17) groups. Arrow represents the beginning of the trial. Latency and amplitude peak were calculated for four occipito-parietal components: P1, N1, P2, LPP, and for four frontal components: N1, P1, N2 and LPP.</p

Behavioral response time results.

<p>ANOVA repeated measures indicated a significant three-way interaction picture valence X load X group, <i>p <</i> .05. In the control group (n = 17), negative pictures under low perceptual load increased RTs compared to neutral pictures, but not under high perceptual load. In addition, picture presence (negative and neutral) increased RTs in both low and high perceptual load. In the TSST group (n = 17), RTs were not increased due to negative pictures under both low and high perceptual loads; however, picture presence increased RTs in low load but not in high load conditions. In both groups RTs were generally slower under high load than in low load condition. The error bars represent SE. <i>Note</i>: <i>RT = response time in ms</i>.</p

Means and SE of amplitude’s peak and latency for each ERP component.

<p><b>A</b>: The mean amplitudes' peak for occipito-perietal P1, N1, P2 and LPP, (lower) and frontal N1, P1, N2 and LPP (upper), component as a function of perceptual load and picture presence in each group. <b>B:</b> The mean amplitudes’ latency for the occipito-perietal P1, N1, P2 and LPP, (lower) and frontal N1, P1, N2 and LPP (upper), component as a function of perceptual load and picture presence in each group. Bars represent SE.</p

F-statistics of each effect analysis in control and TSST groups.

<p>F-statistics of each effect analysis in control and TSST groups.</p