Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis Qi-Gang Zhou,* ,1 Yao Hu,* ,1 Yao Hua,* ,1 Mei Hu,* Chun-Xia Luo,* Xiao Han, Xin-Jian Zhu,* Bin Wang,* Jin-Shu Xu* and Dong-Ya Zhu* ,   *Department of Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing, China  Key Laboratory of Mammalian Genes, Nanjing Medical University, Nanjing, China Abstract Increasing evidence suggests that depression may be asso- ciated with a lack of hippocampal neurogenesis. It is well established that neuronal nitric oxide synthase (nNOS)- derived NO exerts a negative control on the hippocampal neurogenesis. Using genetic and pharmacological methods, we investigated the roles of nNOS in depression induced by chronic mild stress (CMS) in mice. Hippocampal nNOS over-expression was first observed 4 days and remained elevated 21 and 56 days after exposure to CMS. The mice exposed to CMS exhibited behavioral changes typical of depression, and impaired neurogenesis in the hippocampus. The CMS-induced behavioral despair and hippocampal neurogenesis impairment were prevented and reversed in the null mutant mice lacking nNOS gene (nNOS)/)) and in the mice receiving nNOS inhibitor. Disrupting hippocampal neu- rogenesis blocked the antidepressant effect of nNOS inhibi- tion. Moreover, nNOS)/) mice exhibited antidepressant-like properties. Our findings suggest that nNOS over-expression in the hippocampus is essential for chronic stress-induced depression and inhibiting nNOS signaling in brain may represent a novel approach for the treatment of depressive disorders. Keywords: depression, hippocampal, neurogenesis, neuro- nal nitric oxide synthase, nitric oxide, stress. J. Neurochem. (2007) 103, 1843–1854. Major depression is a serious mental disorder, with a lifetime risk of 10–30% for women and 7–15% for men (Blendy 2006). It is estimated by the World Health Organization that depression will be the most important cause of disability in the world by the year 2020 (Murray and Lopez 1997), yet the mechanisms underlying its pathophysiology are still poorly understood. Monoamine hypothesis is an early milestone in the field of depression (Hindmarch 2002). The hippocampus is one of the several limbic brain structures implicated in the pathophysiology and treatment of mood disorders (Warner- Schmidt and Duman 2006). Pre-clinical and clinical studies demonstrate that stress and depression lead to reductions of the total volume and atrophy and loss of neurons in the adult hippocampus (Tsankova et al. 2006). Persistent neurogenesis occurs in the adult mammalian hippocampus throughout life, including humans (Altman and Das 1965; Kempermann et al. 1997; Eriksson et al. 1998; Gould et al. 1998). Stress, which triggers bouts of depression (Tsankova et al. 2006), suppresses adult hippocampal neurogenesis (Jacobs et al. 2000; van Praag et al. 2002). Various chronic antidepressant treatments increase adult hippocampal neurogenesis (Santar- elli et al. 2003; Malberg 2004). Thus, the depression may be associated with a lack of hippocampal neurogenesis although it is a complex disorder that targets more than one region of the brain. Nitric oxide, a free radical with signaling functions in the central and peripheral nervous system, is produced by nitric oxide synthase (NOS) during the conversion of L-arginine to citrulline. Three isoforms of NOS exist and include neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) (Paakkari and Lindsberg 1995). Of the three isoforms of NOS described, nNOS is the main form expressed in the brain (Moreno-Lo ´ pez et al. 2000). Neuronal NOS is local- ized to the sites of neuronal proliferation and migration in the hippocampal dentate gyrus (DG) (Islam et al. 2003), fore- brain subventricular zone (SVZ), rostral migratory stream Received May 23, 2007; revised manuscript received July 16, 2007; accepted July 17, 2007. Address correspondence and reprint requests to Dr Dong-Ya Zhu, Department of Pharmacology, School of Pharmacy, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China. E-mail: dyzhu@njmu.edu.cn 1 Qi-Gang Zhou, Yao Hu, and Yao Hua contributed equally to this work. Abbreviations used: 5-HT, 5-hydroxytryptamine or serotonin; 7-NI, 7- nitroindazole; AZT, 3¢-azido-deoxythymidine; BrdU, 5-bromo-2¢- deoxyuridine; CMS, chronic mild stress; DG, dentate gyrus; eNOS, endothelial nitric oxide synthase; FST, forced swimming test; GR, glu- cocorticoid receptor; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase; PBS, phos- phate-buffered saline; PS, physical state; SVZ, subventricular zone; TBST, Tris-Buffered Saline Tween-20; TST, tail suspension test. Journal of Neurochemistry , 2007, 103, 1843–1854 doi:10.1111/j.1471-4159.2007.04914.x Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 1843–1854 1843