The Journal of Neuroscience, March 14, 2007, ():
Status Epilepticus-Induced Somatostatinergic Hilar Interneuron Degeneration Is Regulated by Striatal Enriched Protein Tyrosine Phosphatase
J. Neurosci. Choi et al.
27: 2999
Supplemental Data
Files in this Data Supplement:
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Supplemental Methods
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Supplementary Figure 1. Quantitation of STEP- and somatostatin-positive cell density in the hilus. Density of both STEP- and somatostatin (SST)-positive cells was higher in the deep hilus than in the border area. Data were obtained from 6 mice for each group. Symbols represent values for individual mice; Horizontal bars represent mean values. DH: Deep hilus, BD: Border area. Please refer to the Supplementary Methods section for a description of the stereological counting technique.
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Supplemental Figure 2. FK506 does not attenuate SE-induced cell death in the endopiriform nucleus. A, Left panel: Cresyl violet-stained section denoting the location of the endopiriform nucleus. Scale bar: 500 µm. Right panel: immunolabeling revealed limited STEP expression in the endopiriform nucleus. Scale bar: 100 µm. B, Mice were injected with pilocarpine and FK506 as described in the main text and killed 6 hr after SE onset. Representative images of Fluoro-Jade B labeling reveal that SE elicited marked cell death in the endopiriform nucleus, and, in contrast to the hilus, FK506 did not increase cell survival. AG: amygdala, EPN: endopiriform nucleus. Scale bar: 100 µm. C, Quantitative analysis of Fluoro-Jade B-(FJB) positive cells in mice injected with pilocarpine and either FK506 or vehicle. Data were obtained from 6 mice in each group and represented by mean ± SEM.
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Supplemental Figure 3. TAT-STEP blocks pERK nuclear translocation. A, TAT-STEP (50 nM, 0.5 µL) was infused into the motor cortex, then 1.5 hrs later, mice were injected with pilocarpine and sacrificed 15 min after SE onset. Immunolabeling for His revealed the cytoplasmic localization of TAT-STEP (red). TAT-STEP infusion suppressed pERK (green) translocation into nucleus (arrows). In the contralalateral motor cortex marked pERK nuclear translocation was observed. B, TAT-STEP inhibits ERK activity without effect on the activity of wild type STEP. Phosphatase activity is expressed as a percentage of wild-type activity. Five µg of wild-type (WT) STEP46 fusion protein, 5 µg of TAT-STEP46 (C-S) and a combination of 5 µg WT STEP46 and TAT-STEP46 (C-S) (WT+CS) were assayed. Data are the mean (± S.D.) of three independent experiments. T test values= TAT-STEP vs WT-STEP; p< 0.00005; TAT-STEP vs WT-STEP + TAT-STEP; p< 0.0005 ; WT-STEP vs WT-STEP + TAT-STEP; p> 0.84.
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Supplemental Figure 4. Microinfusion into the dentate gyrus. A, U0126 (100 µM, 0.5 µL) was unilaterally infused (arrow denotes approximate location of infusion) 1.5 hours before pilocarpine injection, and animals were killed 15 min following SE onset. Immunohistochemical labeling for pERK revealed that U0126 effectively inhibited SE-induced ERK activation in the infused dentate gyrus. In contrast, robust ERK activation was observed in the contralateral hemisphere. Note the robust pERK expression in the ipsilateral mossy fibers. Mossy fibers express high basal levels of pERK; the inability of the MEK1/2 inhibitor U0126 to block mossy fiber pERK expression indicates that ERK was activated prior to U0126 infusion. B, TAT-STEP was infused into the hilus and detected using immunofluorescent labeling for the His tag (red). Note the intracellular localization of TAT-STEP at 2 hrs-post infusion. Tissue was counter labeled with Hoechst 33258 (blue). Arrow denotes approximate location of infusion. GCL: granule cell layer, Hil: hilus.
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Supplemental Figure 5. Neuroprotection and the MAPK pathway in the hilus. Mice were unilaterally infused with FK506 (100 µM, 0.5 µL), U0126 (100 µM, 0.5 µL) and/or TAT-STEP (100 nM, 0.5 µL), injected with pilocarpine, and sacrificed 6 hr after SE onset. A, Representative images of Fluoro-Jade B labeling. Boxed regions are magnified below. Compared to the contralateral hilus, cell death was attenuated by FK506. (Below) Representative images reveal that the neuroprotective effects of FK506 were blocked by U0126 and TAT-STEP. In contrast, the control peptide TAT-myc (100 nM, 0.5 µL) did not attenuate FK506-mediated neuroprotection. B, Quantitative analysis of Fluoro-Jade B-(FJB) positive cells in the hilus. Data are presented as the mean ± SEM. ## p < 0.01 compared with vehicle-injected mice. ** p < 0.01 compared with FK506-injected mice. Ψ Ψ p<0.01 compared with TAT-STEP-injected mice.
