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Fig. 1. (A) NOS activity in hippocampal extracts from wild-type (wt, black), tPA^–/– (mid-gray) and tPA^–/– mice treated with 1 µg/µl tPA (light gray), with or without KA. NOS activity was assessed by quantitation of [³H]L-arginine converted to [³H]L-citrulline. One unit enzyme activity produces 1 nmole NO per minute at 37°C. Statistical comparison was conducted comparing individual time points to their corresponding non-treated controls using a two-tailed t test. **P<0.01, ***P <0.001. Statistical comparison comparing all treatment groups within time points was conducting using one-way ANOVA followed by a Bonferroni-Dunn test for multiple comparisons. P<0.05; n=6-7 per treatment group. (B) NOS activity in wild-type mice treated with tPA Stop and MK-801 as well as NOS activity in tPA^–/– mice infused with the catalytically inactive tPA mutant S481A. Statistical comparison was conducted as in A. (C) Western blot analysis for anti-nTyr in extracts from wild-type and tPA^–/– hippocampi injected with KA and sacrificed at day 5. Arrows indicate a band that exhibits increased staining on the injected side in wild-type mice. Equal loading of protein was verified using anti-actin. n=6. (D) Coronal sections of wild-type and tPA^–/– mice injected with KA and sacrificed at day 5 were stained with Cresyl Violet to assess neurodegeneration (a,b, asterisks). Immunohistochemical staining for nTyr identified scattered positive cells in all hippocampal regions, and a strongly immunoreactive focus that coincided with degenerating pyramidal layer neurons on the injected side of wild-type mice only (arrows indicate region of interest, c-g). Insets show high magnification of nTyr-stained neurons in the CA1 region of the hippocampus. Bar, 50 µm.

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