Research report Nitric oxide synthesis inhibition increases proliferation of neural precursors isolated from the postnatal mouse subventricular zone Esperanza R. Matarredona, Maribel Murillo-Carretero, Bernardo Moreno-Lo ´pez, Carmen Estrada * A ´ rea de Fisiologı ´a, Facultad de Medicina, Universidad de Ca ´diz, Ca ´diz, Spain Accepted 2 October 2003 Abstract The subventricular zone (SVZ) of rodents retains the capacity to generate new neurons throughout the entire life of the animal. Neural progenitors of the SVZ survive and proliferate in vitro in the presence of epidermal growth factor (EGF). Nitric oxide (NO) has been shown to participate in neural tissue formation during development and to have antiproliferative actions, mediated in part by inhibition of the EGF receptor. Based on these findings, we have investigated the possible effects of endogenously produced and exogenously added NO on SVZ cell proliferation and differentiation. Explants were obtained from postnatal mouse SVZ and cultured in the presence of EGF. Cells migrated out of the explants and proliferated in culture, as assessed by bromodeoxyuridine (BrdU) incorporation. After 72 h in vitro, the colonies formed around the explants were constituted by cells of neuronal or glial lineages, as well as undifferentiated progenitors. Immunoreactivity for the neuronal isoform of NO synthase was observed in neuronal cells with long varicose processes. Cultures treated with the NOS inhibitor N N -nitro-L-arginine methyl ester (L-NAME) showed an increase in the percentage of BrdU-immunoreactive cells, whereas treatment with the NO donor diethylenetriamine–nitric oxide adduct (DETA-NO) led to a decrease in cell proliferation, without affecting apoptosis. The differentiation pattern was also altered by L-NAME treatment resulting in an enlargement of the neuronal population. The results suggest that endogenous NO may contribute to postnatal neurogenesis by modulating the proliferation and fate of SVZ progenitor cells. D 2003 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Genesis of neurons and glia Keywords: Neural cell differentiation; Neuroblast; Nitric oxide synthase; Postnatal neurogenesis; Subventricular zone 1. Introduction Specific regions of the mammalian brain retain the capacity to generate new neurons throughout the entire life of the animal [13,15,25]. In rodents, one of these regions is the subventricular zone (SVZ), located in the lateral walls of the lateral ventricles. The SVZ is a source of newly formed neuroblasts which, after migration through the rostral mi- gratory stream, reach the olfactory bulb and differentiate as interneurons [26,27]. The fate of the SVZ stem cells and their early progeny is probably regulated by local factors and cell–cell contacts, which determine when and where a cell will proliferate, migrate or differentiate. Although these influences are presently under investigation, the proliferative effect of the epidermal growth factor (EGF) on these precursor cells has been well established, both in vitro [12,20,39] and in vivo [9,23]. Nitric oxide (NO) is an intercellular messenger with multiple functions in the cardiovascular, immunological and nervous systems [18,32]. In the brain, NO is synthe- sized by specific neurons expressing the neuronal isoform of NO synthase (nNOS) [2,40]. During development, the transient expression of nNOS in different brain areas 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.10.010 Abbreviations: BrdU, bromodeoxyuridine; DETA-NO, diethylenetri- amine – nitric oxide adduct; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GFAP, glial fibrillary acidic protein; L- NAME, N N -nitro-L-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; NSE, neuronal specific enolase; PSA-NCAM, polysialy- lated form of the neuronal cell adhesion molecule; SVZ, subventricular zone * Corresponding author. Tel.: +34-956-015252; fax: +34-956-015214. E-mail address: carmen.estrada@uca.es (C. Estrada). www.elsevier.com/locate/brainres Brain Research 995 (2004) 274 – 284