Diagrams.

<p>(<i>Upper panel</i>): Flow diagram of Exp. #1 regarding times and type of treatment with pentylenetetrazole. <i>(Lower panel):</i> Schematic diagram of the distribution of proliferative (BrdU-positive) cells in the brain at day 25 after a single administration of a convulsive dose of PTZ. Note the change from a widespread distribution (<b>A</b>) to a more restricted distribution at 25 days post-seizure (<b>B</b>). In control rats, there were only a few BrdU-positive cells, often in a duplex, mitosis-like state (<b>C, D, E</b>). Many proliferative cells in PTZ-treated animals appeared to enter the brain from the circulation via leptomeningeal blood vessels (<b>F</b>, arrow points to a mitosis-like state). (<b>G</b>, <b>H</b>): Quantitation of BrdU-positive cells. One episode of convulsive seizure causes, at day 3, dramatically increased BrdU-positive cell numbers in the hippocampus (15-fold over controls; p = 0.001) (<b>G</b>) and temporal neocortex (22.5-fold over controls; p = 0.001) (<b>H</b>). Although the numbers of BrdU-positive cells decreased dramatically by day 25, their number remained, nonetheless, at relatively high levels in the hippocampus (4.8-fold; p = 0.001)(<b>G</b>) and temporal neocortex (5.6-fold; p = 0.001) (<b>H</b>) over control levels. N = 15 rats for each timepoint. <i>Abbreviations</i>: <i>Te-II</i>, temporal neocortex layer II; <i>Ent</i>, entorhinal neocortex; <i>HC</i>, hippocampus; <i>lep</i>, leptomeninx. Bars: (<b>C, D, E</b>), 20 µm; (<b>F</b>), 30 µm.</p

Localization and quantification of DCX in the rat brain during kindling development.

<p>(<b>A, B</b>) Overview of DCX staining in the ventral (<b>A,</b> arrows) and dorsal (<b>B,</b> arrows) hippocampal hilus of the kindled animals. The dorsal hippocampus of kindled animals, was highly significant (p = 0.001) enriched in DCX⁺-cells (<b>C</b>). Note that the DCX antigens were localized both in cell bodies and extensions penetrating the densely packed granule cell neurons (<b>D</b>, arrows). (<b>E-F</b>): Phenotyping of DCX-cells. After 2× PTZ some DCX⁺ positive cells (green) in the dorsal hippocampus along the hilar border with the granule cell layer had a NeuN nucleus (red) (<b>E</b>, arrows). In kindled animals some of the DCX (green)/BrdU (red) double-labeled cells had a clonal appearance (<b>F</b>, inset, 3D-image) while other DCX⁺ cells (green) sometimes displayed a fragmented BrdU-positivity (<b>F</b>, arrow). By quantitative RT-PCR there was a 3-fold increase (p = 0.01) in the relative amount of DCX transcripts in kindled animals over that of controls (<b>G</b>). Note that the number of DCX⁺ cells also is maximal when PTZ is administered every 25^th day (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039302#pone-0039302-g004" target="_blank">Fig. 4</a>H, filled circles) as opposed to every 30^th day (<b>H</b>, open circles). <i>Abbreviations</i>: <i>gcl</i>, granule cell layer; <i>hl</i>, hilus; <i>pml</i>, polymorphic layer. Bars: (<b>A,B</b>), 200 µm; (<b>D</b>), 100 µm.</p

L-NAME treatment increased neurogenesis and seizure susceptibility.

<p>Daily treatment with L-NAME for 24 days (<b>A</b>) resulted in a significant increase in the number of fully seizing animals after the second PTZ administration on day 25 (<b>B</b>). In control animals, nestin immunoreactivity was detected in capillary walls (<b>C</b>). By quantitative RT-PCR there was a 7.8-fold (p = 0.001) in nestin mRNA levels at day 3 post-seizure (<b>D</b>). L-NAME-treated animals had significantly more (2.7-fold; p = 0.001) nestin mRNA than did animals treated with PTZ alone at day 25 (<b>D</b>) whereas kindled animals did not show an increased level of nestin mRNA (<b>D</b>). At the tissue level, nestin immunoreactivity at day3 in PTZ-treated animals was confined to radial glia-like cells in the inner molecular layer of the dentate gyrus, the polymorphic layer and, interestingly, to the CA2 region (arrow) (<b>E</b>, and inset). After 25 days, the nestin-like immunoreactivity was restricted to the polymorphic layer (<b>F</b> and inset). <i>Abbreviations</i>: <i>DG</i>, dentate gyrus; <i>pml</i>, polymorphic layer; <i>CA2</i>, hippocampal region.</p

Flow diagrams of the experimental design regarding times and type of treatment and EEG recording.

<p>After Exp. #2 (<b>A</b>) the proportion of rats that achieved full kindling status reached 80% (<b>C</b>; p = 0.001). By comparison, 31% of rats administered a subconvulsive dose of PTZ at all times (Exp. #3)(<b>B</b>) reached full kindling status after two injections and up to 81% of animals reached full kindling status after the fourth treatment (<b>C;</b> p = 0.001). (<b>D</b>–<b>F</b>): A subconvulsive PTZ treatment elicited intermittent non-ictal events that are typically dependent on the behavioral state of the animal (active or passive wakefulness)(<b>D</b>). After the second PTZ injection, the animals usually demonstrated mild multifocal body jerks (<b>E</b>). After the third PTZ injection, the animals showed typical seizure activity associated with motor arrest (<b>F</b>).</p

Number and phenotyping of BrdU-positive cells after seizure activity.

<p>Each PTZ treatment led to an accumulation of BrdU⁺ cells in the dentate gyrus of the kindled rats (9-fold, p = 0.0001; <b>A</b>). (<b>B–E</b>): 3D projections of confocal BrdU(red)/NeuN(green) double-labeled images from PTZ-treated animals. A single episode of seizure activity led to the appearance of BrdU-positive cells in the polymorphic layer that were in a mitosis-like state (<b>B</b>). Occasionally some neurons in layers II and III of the temporal neocortex also displayed BrdU⁺ cells in close apposition to neurons (<b>B</b>, insets). After 2× PTZ some BrdU-positive cells have differentiated into neurons, particularly in the granule cell layer (<b>C</b>, arrows). In addition, some BrdU-positive nuclei were detected in the walls of large blood vessels (<b>C</b>, inset). The number of double-labeled BrdU(red)/NeuN(green) increased with the number of PTZ injections and reached a maximum in the granule cells layer of kindled animals (<b>D</b>, low power; <b>E</b>, higher power). <i>Abbreviations</i>: <i>Te</i>, temporal neocortex; <i>GCL</i>, granule cell layer; <i>BV</i>, Blood Vessel.</p

Table_1.XLSX

<p>Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy. Thus, an imbalance in mitochondrial turnover can contribute to the diminution of cellular function seen during aging. Mitochondrial biogenesis and mitophagy are mediated by a set of proteins including MFN1, MFN2, OPA1, DRP1, FIS1 as well as DMN1l and DNM1, all of which are required for mitochondrial fission. Using N15 labeling, we report that the turnover rates for DMN1l and FIS1 go in opposite directions in the cerebellum of 22-month-old C57BL6j mice as compared to 3-month-old mice. Previous studies have reported decreased turnover rates for the mitochondrial respiratory complexes of aged rodents. In contrast, we found increased turnover rates for mitochondrial proteins of the oxidative phosphorylation chain in the aged mice as compared to young mice. Furthermore, the turnover rate of the components that are most affected by aging –complex III components (ubiquinol cytochrome C oxidoreductase) and complex IV components (cytochrome C oxidase)– was significantly increased in the senescent cerebellum. However, the turnover rates of proteins involved in mitophagy (i.e., the proteasomal and lysosomal degradation of damaged mitochondria) were not significantly altered with age. Overall, our results suggest that an age-related imbalance in the turnover rates of proteins involved in mitochondrial biogenesis and mitophagy (DMN1l, FIS1) in conjunction with an age-related imbalance in the turnover rates of proteins of the complexes III and IV of the electron transfer chain, might impair cerebellar mitochondrial bioenergetics in old mice.</p

Table_2.XLSX

<p>Brain structures differ in the magnitude of age-related neuron loss with the cerebellum being more affected. An underlying cause could be an age-related decline in mitochondrial bioenergetics. Successful aging of mitochondria reflects a balanced turnover of proteins involved in mitochondrial biogenesis and mitophagy. Thus, an imbalance in mitochondrial turnover can contribute to the diminution of cellular function seen during aging. Mitochondrial biogenesis and mitophagy are mediated by a set of proteins including MFN1, MFN2, OPA1, DRP1, FIS1 as well as DMN1l and DNM1, all of which are required for mitochondrial fission. Using N15 labeling, we report that the turnover rates for DMN1l and FIS1 go in opposite directions in the cerebellum of 22-month-old C57BL6j mice as compared to 3-month-old mice. Previous studies have reported decreased turnover rates for the mitochondrial respiratory complexes of aged rodents. In contrast, we found increased turnover rates for mitochondrial proteins of the oxidative phosphorylation chain in the aged mice as compared to young mice. Furthermore, the turnover rate of the components that are most affected by aging –complex III components (ubiquinol cytochrome C oxidoreductase) and complex IV components (cytochrome C oxidase)– was significantly increased in the senescent cerebellum. However, the turnover rates of proteins involved in mitophagy (i.e., the proteasomal and lysosomal degradation of damaged mitochondria) were not significantly altered with age. Overall, our results suggest that an age-related imbalance in the turnover rates of proteins involved in mitochondrial biogenesis and mitophagy (DMN1l, FIS1) in conjunction with an age-related imbalance in the turnover rates of proteins of the complexes III and IV of the electron transfer chain, might impair cerebellar mitochondrial bioenergetics in old mice.</p

L-NAME treatment increased doublecortin levels in the rat hippocampus.

<p>L-NAME treatment increased DCX immunoreactivity on Western blots (<b>A</b>) by 2.2-fold (p = 0.02) at day 50 post-seizure (<b>B</b>). By immunohistochemistry, numerous DCX-positive cells were detected in the subgranular zone of the dorsal hippocampus by day 50 post-seizure (<b>C</b>). Quantitatively, L-NAME elicited significant increases (1.5-fold, p = 0.01) in the number of DCX-positive cells (<b>D</b>). <i>Abbreviations</i>: <i>gcl</i>, granule cell layer.</p

Correspondence analysis of differentially expressed genes and samples grouped by animal age.

<p>The left panel depicts the Eigenvalues of the correspondence analysis and shows that the major factors contributing to the variance of stroketomics analysis were stroke (52%), post-stroke time (25%) and age (12%). (Right panel): The first two sources of variability, stroke and post-stroke time formed the coordinates of the right panel. The graph shows the distribution of transcripts (black dotes) as a function of treatment (stroke) and post-stroke time. Samples from young (green) and aged (red) animals particularly differ in their post-stroke response (illustrated by ellipses that form non-parallel planes). Transcripts with characteristic expression in naive samples are encircled in black.</p

Patterns of gene expression after stroke.

<p>There were several distinct patterns of gene regulation: persistently upregulated (black line), transiently upregulated, (orange line), “late-upregulated” (red line), “late-downregulated” (yellow line), transiently downregulated (blue line), and persistently downregulated (green line). Aged animals showed larger numbers than young of genes that were late-upregulated, persistently upregulated and persistently downregulated. The young rats, in contrast, had a much larger number of transiently upregulated and delayed downregulated genes. Note that this representation does not take into account the fold changes for individual genes but the relative change in gene expression at days 3 and 14 post-stroke.</p