Age-Dependent Effect of Nitric Oxide on Subventricular Zone and Olfactory Bulb Neural Precursor Proliferation CARMEN ROMERO-GRIMALDI, BERNARDO MORENO-LO ´ PEZ, AND CARMEN ESTRADA A ´ rea de Fisiologı ´a, Facultad de Medicina, Universidad de Ca ´ diz, 11003 Ca ´ diz, Spain ABSTRACT Nitric oxide (NO) synthase (NOS) is developmentally regulated in the embryonic brain, where NO participates in cell proliferation, survival, and differentiation. In adults, NO inhibits neurogenesis under physiological conditions. This work investigates whether the NO action is preserved all along development up to adulthood or whether its effects in adults are a new feature acquired during brain maturation. The relationship between nitrergic neurons and precursors, as well as the functional consequences of pharmacological NOS inhibition, were comparatively analyzed in the subventricular zone (SVZ) and olfactory bulb (OB) of postnatal (P7) and adult (P60) mouse brains. The SVZ was markedly reduced between P7 and adults, and, at both ages, neurons expressing neuronal NOS (nNOS) were found in its striatal limits. In postnatal mice, these nitrergic neurons contained PSA-NCAM, and their projections were scarce, whereas, in adults, mature nitrergic neurons, devoid of PSA-NCAM, presented abundant neuropil. In the OB, local proliferation almost disappeared in the transition to adulthood, and periglomerular nitrergic neurons, some of which were PSA- NCAM positive, were found in postnatal and adult mice. Administration of the NOS inhibitor L-NAME did not affect cell proliferation in the SVZ or in the OB of postnatal mice, whereas it significantly enhanced the number of mitotic cells in both regions in adults. Thus, the NO action on SVZ neurogenesis is a phenomenon that appears after the postnatal age, which is probably due to the germinal layer size reduction, allowing exposure of the NO-sensitive neural precursors to the NO produced in the SVZ-striatum limits. J. Comp. Neurol. 506: 339 –346, 2008. © 2007 Wiley-Liss, Inc. Indexing terms: EGF receptor; neural stem cells; neurogenesis; nitric oxide synthase; PSA- NCAM, NADPH-diaforase Nitric oxide (NO), a gaseous intercellular messenger synthesized in the brain by specific neurons expressing the neuronal isoform of NO synthase (nNOS), has been demonstrated to play a role in both embryonic nervous tissue formation and adult neurogenesis (for review see Estrada and Murillo-Carretero, 2005; Gibbs, 2003). In embryos, NO inhibits neural precursor cell division and promotes neuronal differentiation in developing Drosoph- ila imaginal disks (Kuzin et al., 1996) and Xenopus tad- pole optic tectum (Peunova et al., 2001). A similar anti- proliferative effect of NO has been reported in vitro in mammalian tumoral cell lines of neural origin (Murillo- Carretero et al., 2002; Obregon et al., 1997; Peunova and Enikolopov, 1995; Phung et al., 1999) and in neuronal precursors isolated from the embryonic (Cheng et al., 2003) and neonatal (Ciani et al., 2004) brain. Additionally, several groups have shown that NO is a negative regula- tor of neurogenesis in the adult SVZ. Significant increases in SVZ cell proliferation have been demonstrated in nNOS knockout mice (Packer et al., 2003; Sun et al., 2005) as well as after pharmacological inhibition of nNOS activity (Cheng et al., 2003; Moreno-Lo ´pez et al., 2004; Packer et al., 2003; Romero-Grimaldi et al., 2006). The NO target Grant sponsor: Fondo de Investigaciones Sanitarias; Grant number: 00/1080; Grant sponsor: Ministerio de Ciencia y Tecnologı ´a; Grant number: SAF2002-02131; Grant sponsor: Junta de Andalucı ´a; Grant number: CTS- 2005/883. *Correspondence to: Carmen Estrada, A ´ rea de Fisiologı ´a, Facultad de Medicina, Universidad de Ca ´ diz, Plaza Falla 9, 11003 Ca ´ diz, Spain. E-mail: carmen.estrada@uca.es Received 31 March 2007; Revised 11 June 2007; Accepted 2 October 2007 DOI 10.1002/cne.21556 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 506:339 –346 (2008) © 2007 WILEY-LISS, INC.