We investigated the role of neuronal (type I) nitric oxide synthase (nNOS) in NMDA-mediated excitotoxicity in wild-type (SV129 and C57BL/6J) and type I NOS knock-out (nNOS^−/−) mice and examined its relationship to apoptosis. Excitotoxic lesions were produced by intrastriatal stereotactic NMDA microinjections (10–20 nmol). Lesion size was dose- and time-dependent, completely blocked by MK-801 pretreatment, and smaller in nNOS knock-out mice compared with wild-type littermates (nNOS^+/+, 11.7 ± 1.7 mm³; n = 8; nNOS^−/−, 6.4 ± 1.8 mm³;n = 7). The density and distribution of striatal NMDA binding sites, determined by NMDA receptor autoradiography, did not differ between strains. Pharmacological inhibition of nNOS by 7-nitroindazole (50 mg/kg, i.p.) decreased NMDA lesion size by 32% in wild-type mice (n = 7). Neurochemical and immunohistochemical measurements of brain nitrotyrosine, a product of peroxynitrite formation, were increased markedly in wild-type but not in the nNOS^−/− mice. Moreover, elevations in 2,3- and 2,5-dihydroxybenzoic acid levels were significantly reduced in the mutant striatum, as a measure of hydroxyl radical production.

The importance of apoptosis to NMDA receptor-mediated toxicity was evaluated by DNA laddering and by quantitative histochemistry [terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate–biotin nick end-labeling (TUNEL) staining]. DNA laddering was first detected within lesioned tissue after 12–24 hr. TUNEL-positive cells were first observed at 12 hr, increased in number at 48 hr and 7 d, and were located predominantly in proximity to the lesion border. The density was significantly lower in nNOS^−/− mice. Hence, oligonucleosomal DNA breakdown suggesting apoptosis develops as a late consequence of NMDA microinjection and is reduced in nNOS mutants. The mechanism of protection in nNOS^−/− mice may relate to decreased oxygen free radical production and related NO reaction products and, in part, involves mechanisms of neuronal death associated with the delayed appearance of apoptosis.