BACKGROUND: To determine brain areas involved in the antidepressant-related behavioral effects of the selective neuronal nitric oxide synthase inhibitor 1-(2-Trifluoro-methyl-phenyl) imidazole (TRIM) and experimental test compound 4-((3,5-dichloro-2-hydroxybenzyl)amino)-2-hydroxybenzoic acid (ZL006), an inhibitor of the PSD of 95 kDa/neuronal nitric oxide synthase interaction in the N-methyl-D-aspartic acid receptor signalling pathway, regional specific expression of the neuronal activation marker c-FOS was assessed following exposure to the forced swimming test in the Wistar Kyoto rat. METHODS: Wistar Kyoto rats were subjected to a 15-minute swim pretest (pre-forced swimming test) period on day 1. At 24, 5, and 1 hour prior to the 5-minute test, which took place 24 hours following the pre-forced swimming test, animals were treated with TRIM (50 mg/kg; i.p.), ZL006 (10 mg/kg; i.p.), or saline vehicle (1 mL/kg i.p). Behavior was recorded during both pretest and test periods. RESULTS: Both TRIM and ZL006 decreased immobility time in Wistar Kyoto rats in the forced swimming test. Exposure to the forced swimming test increased c-FOS immunoreactivity in the lateral septum, paraventricular nucleus of the hypothalamus, periaqueductal grey, dentate gyrus, and ventral CA1 of the hippocampus compared with non-forced swimming test-exposed controls. Forced swimming test-induced c-FOS immunoreactivity was further increased in the lateral septum, periaqueductal gray, and paraventricular nucleus of the hypothalamus following treatment with TRIM or ZL006. By contrast, forced swimming test-induced c-FOS immunoreactivity was reduced in dorsal dentate gyrus and ventral CA1 following treatment with TRIM or ZL006. Exposure to the forced swimming test resulted in an increase in NADPH diaphorase staining in the paraventricular nucleus of the hypothalamus. This forced swimming test-induced increase was attenuated following treatment with ZL006 and points to the paraventricular nucleus as a brain region where ZL006 acts to attenuate forced swimming test-induced neuronal nitric oxide synthase activity while concomitantly regulating region specific neuronal activation associated with an antidepressant-related response. CONCLUSIONS: This study identified a pattern of enhanced and reduced forced swimming test-related c-FOS immunoreactivity indicative of a regulated network where inhibition of nitric oxide coupled to the N-methyl-D-aspartic acid receptor leads to activation of the lateral septum, periaqueductal gray, and paraventricular nucleus of the hypothalamus with concomitant inhibition of the hippocampus.