Fig 1: Serine racemase is localized in both neurons and astrocytes in the MEA.a Representative images of epileptic tissue acquired on a confocal microscope showing anti-NeuN (N), anti-serine racemase (SR), and anti-GFAP (A) immunoreactivity in neurons and astrocytes within different regions of the MEA (see schematic, row 6, column 3). Fluorophores for N (red), A (red), and SR (green) have been pseudo colored to show colocalization (yellow) in the merged (M) images. Panels in rows 3 through 6 are high magnification images of the lettered boxed regions identified above or within the same row. Text (top-right) in the high magnification panels identifies the immunoreactivity shown. For example, expression of serine racemase in neurons (box A) is shown in high mag through panels in row 3 (columns 1–3) and in astrocytes through panels in row 4 (columns 1–2). Panels in row 5 showcase racemase-positive (*) and racemase-negative neurons (red arrows) within the same section. bv blood vessels. b Representative images from control tissue showing colocalization of serine racemase with neurons and astrocytes. The panel in row 3, column 2 is a composite high mag image of N, A, and SR in the boxed region E (row 3, column 1). c A secondary-only control for SR with N and A (in control tissue) highlighting the specificity of the antibody used. In the absence of the SR antibody there is neither a signal nor colocalization with N or A. Each experiment shown in a–c was repeated at least twice independently with similar results.
Fig 2: Focal application of D-serine limits microglia proliferation and/or infiltration into layer 3 of the MEA.a Immunofluorescence images of the MEA in non-status (controls) and post-status rats treated with D-serine or aCSF (vehicle). Neurons immunoassayed with fluorescently tagged antibodies against NeuN (blue) and microglia with the anti-CD11b antibody, OX42 (green), shown merged (leftmost panels) and separately as high magnification images of the boxed areas in panels to the left (yellow). b Quadruple immunostaining of neurons (blue), astrocytes (green), microglia (magenta), and nuclei (blue) highlighting complete neuroglia pathology within MEA of post-status rats treated with D-serine or aCSF (panels on right are high magnification images of the boxed regions in panels on left). c Section of the brain slice containing the MEA that was micro dissected out for use in immunoblotting (DG dentate gurus, Hip hippocampus). Roughly four sections (600 μm thick) per hemisphere were harvested from each animal and pooled. Cerebellar tissue from these brains was used as control. d Representative immunoblots for NeuN (neurons), GFAP (astrocytes), and Iba1 (microglia) for MEA and cerebellum harvested from non-status (ns) and post-status (ps) rats 1, 5, 12, and 29 days post insult. GAPDH was used as loading control. Relative positions of standard molecular weight markers indicated to the right of each panel (for full scans see Supplementary Fig. 5). e Time course of changes in relative abundance of NeuN (neurons), GFAP (astrocytes), and Iba1 (microglia) in MEA and cerebellum quantified from the immunoblots (n = 3). Error bars represent SEM.
Fig 3: Focal application of D-serine minimizes astrocytic density in layer 3 of the MEA.a Immunofluorescence images of the MEA (inset, bottom left panel) in non-status (controls) and post-status rats treated with D-serine or aCSF (vehicle). Neurons immunoassayed with fluorescently tagged antibodies against NeuN (red), astrocytes with antibodies against GFAP (green), and nuclei with DAPI (blue) shown merged (leftmost panels) and separately, as high magnification images of the boxed areas in panels to the left (yellow). Raw data and histograms of astrocytic (b) and microglia (c, immunoassayed with the anti-CD11b antibody, OX42, and shown in Fig. 4a) densities in layers 2 and 3 of MEA in animals under the conditions indicated (color codes show segregation of cohorts based on treatment regimen and final outcomes). Data within bar plots indicates number of animals used (numerator) and the total number of sections assayed for each condition (denominator). Astrocyte dendritic complexity (quantified using Sholl analysis d, f) and volume (e) in animals under the conditions indicated. Note differences in dendritic morphology of representative cells (left; arrowheads in d point to somata, purple and dendrites, yellow) and their tracings (middle), color coded for changes in the number of branches at concentric circles of increasing radii (right). Raw data and histogram of astrocytic cell volumes (e) and plot of the number of crossings as a function of radial distance (f), averaged across 10–14 cells from three animals in each cohort. g Scatter plot of volume versus maximum crossings for astrocytes in epileptic (pink), nonepileptic (green), and D-serine infused animals (blue). ***p < 0.001, ****p < 0.0001, one-way ANOVA with a Dunnett’s post-hoc correction. Error bars represent SEM.
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